The hyponatremic patient: a systematic approach to laboratory diagnosis

HYPONATREMIA (SERUM SODIUM LEVEL LESS THAN 134 MMOL/L) is a common electrolyte disturbance. Its high prevalence and potential neurologic sequelae make a logical and rigorous differential diagnosis mandatory before any therapeutic intervention. A history of concurrent illness and medication use as well as the assessment of extracellular volume status on physical examination may provide useful clues as to the pathogenesis of hyponatremia. Measurement of the effective serum tonicity (serum osmolality less serum urea level) is the first step in the laboratory evaluation. In patients with normal or elevated effective serum osmolality (280 mOsm/kg or greater), pseudohyponatremia should be excluded. In the hypo-osmolar state (serum osmolality less than 280 mOsm/kg), urine osmolality is used to determine whether water excretion is normal or impaired. A urine osmolality value of less than 100 mOsm/kg indicates complete and appropriate suppression of antidiuretic hormone secretion. A urine sodium level less than 20 mmol/L is indicative of hypovolemia, whereas a level greater than 40 mmol/L is suggestive of the syndrome of inappropriate antidiuretic hormone secretion. Levels of hormones (thyroid-stimulating hormone and cortisol) and arterial blood gases should be determined in difficult cases of hyponatremia.Hyponatremia (serum sodium level less than 134 mmol/L) is a common electrolyte disturbance occurring in a broad spectrum of patients, from asymptomatic to critically ill.^1,2 There are serious neurologic sequelae associated with hyponatremia and its treatment. Therefore, a logical, rigorous differential diagnosis is mandatory before therapy can be begun.^3,4 Since hyponatremia is caused primarily by the retention of solute-free water, its cause encompasses disorders associated with limitation in water excretion.⁵ The principal causes of hyponatremia are summarized in Open in a separate windowAs with other electrolyte abnormalities, the history and physical examination can provide important clues toward the correct diagnosis. In most cases the initial laboratory evaluation includes measurement of serum osmolality and urine osmolality (by osmometer if available), urine sodium concentration and serum levels of other electrolytes (potassium, chloride and bicarbonate) as well as serum concentrations of urea, glucose, uric acid, total proteins and triglycerides. In addition, determination of serum levels of thyroid-stimulating hormone and cortisol is important to exclude any associated endocrinopathy (Fig. 1). Measurement of arterial blood gases is also useful in the differential diagnosis of hyponatremia, particularly in patients with abnormal serum bicarbonate concentrations.Open in a separate windowFig. 1: Clinical diagnostic algorithm for hyponatremia. TSH = thyroid-stimulating hormone, EABV = effective arterial blood volume, SIADH = syndrome of inappropriate secretion of antidiuretic hormone, FE = fractional excretion.Table 2Open in a separate windowThe step-by-step diagnostic evaluation of hyponatremia is shown in Fig. 1.     

Streptomyces Development in Colonies and Soils

Streptomyces development was analyzed under conditions resembling those in soil. The mycelial growth rate was much lower than that in standard laboratory cultures, and the life span of the previously named first compartmentalized mycelium was remarkably increased.Streptomycetes are gram-positive, mycelium-forming, soil bacteria that play an important role in mineralization processes in nature and are abundant producers of secondary metabolites. Since the discovery of the ability of these microorganisms to produce clinically useful antibiotics (2, 15), they have received tremendous scientific attention (12). Furthermore, its remarkably complex developmental features make Streptomyces an interesting subject to study. Our research group has extended our knowledge about the developmental cycle of streptomycetes, describing new aspects, such as the existence of young, fully compartmentalized mycelia (5-7). Laboratory culture conditions (dense inocula, rich culture media, and relatively elevated temperatures [28 to 30°C]) result in high growth rates and an orderly-death process affecting these mycelia (first death round), which is observed at early time points (5, 7).In this work, we analyzed Streptomyces development under conditions resembling those found in nature. Single colonies and soil cultures of Streptomyces antibioticus ATCC 11891 and Streptomyces coelicolor M145 were used for this analysis. For single-colony studies, suitable dilutions of spores of these species were prepared before inoculation of plates containing GYM medium (glucose, yeast extract, malt extract) (11) or GAE medium (glucose, asparagine, yeast extract) (10). Approximately 20 colonies per plate were obtained. Soil cultures were grown in petri dishes with autoclaved oak forest soil (11.5 g per plate). Plates were inoculated directly with 5 ml of a spore suspension (1.5 × 10⁷ viable spores ml⁻¹; two independent cultures for each species). Coverslips were inserted into the soil at an angle, and the plates were incubated at 30°C. To maintain a humid environment and facilitate spore germination, the cultures were irrigated with 3 ml of sterile liquid GAE medium each week.The development of S. coelicolor M145 single colonies growing on GYM medium is shown in Fig. ​Fig.1.1. Samples were collected and examined by confocal microscopy after different incubation times, as previously described (5, 6). After spore germination, a viable mycelium develops, forming clumps which progressively extend along the horizontal (Fig. 1a and b) and vertical (Fig. 1c and d) axes of a plate. This mycelium is fully compartmentalized and corresponds to the first compartmentalized hyphae previously described for confluent surface cultures (Fig. 1e, f, and j) (see below) (5); 36 h later, death occurs, affecting the compartmentalized hyphae (Fig. 1e and f) in the center of the colony (Fig. ​(Fig.1g)1g) and in the mycelial layers below the mycelial surface (Fig. 1d and k). This death causes the characteristic appearance of the variegated first mycelium, in which alternating live and dead segments are observed (Fig. 1f and j) (5). The live segments show a decrease in fluorescence, like the decrease in fluorescence that occurs in solid confluent cultures (Fig. ​(Fig.11 h and i) (5, 9). As the cycle proceeds, the intensity of the fluorescence in these segments returns, and the segments begin to enlarge asynchronously to form a new, multinucleated mycelium, consisting of islands or sectors on the colony surfaces (Fig. 1m to o). Finally, death of the deeper layers of the colony (Fig. ​(Fig.1q)1q) and sporulation (Fig. ​(Fig.1r)1r) take place. Interestingly, some of the spores formed germinate (Fig. ​(Fig.1s),1s), giving rise to a new round of mycelial growth, cell death, and sporulation. This process is repeated several times, and typical, morphologically heterogeneous Streptomyces colonies grow (not shown). The same process was observed for S. antibioticus ATCC 11891, with minor differences mainly in the developmental time (not shown).Open in a separate windowFIG. 1.Confocal laser scanning fluorescence microscopy analysis of the development-related cell death of S. coelicolor M145 in surface cultures containing single colonies. Developmental culture times (in hours) are indicated. The images in panels l and n were obtained in differential interference contrast mode and correspond to the same fields as in panels k and m, respectively. The others are culture sections stained with SYTO 9 and propidium iodide. Panels c, d, k, l, p, and q are cross sections; the other images are longitudinal sections (see the methods). Panels h and i are images of the same field taken with different laser intensities, showing low-fluorescence viable hyphae in the center of the colonies that develop into a multinucleated mycelium. The arrows in panels e and s indicate septa (e) and germinated spores (s). See the text for details.Figure ​Figure22 shows the different types of mycelia present in S. coelicolor cultures under the conditions described above, depending on the compartmentalization status. Hyphae were treated with different fluorescent stains (SYTO 9 plus propidium iodide for nucleic acids, CellMask plus FM4-64 for cell membranes, and wheat germ agglutinin [WGA] for cell walls). Samples were processed as previously described (5). The young initial mycelia are fully compartmentalized and have membranous septa (Fig. 2b to c) with little associated cell wall material that is barely visible with WGA (Fig. ​(Fig.2d).2d). In contrast, the second mycelium is a multinucleated structure with fewer membrane-cell wall septa (Fig. 2e to h). At the end of the developmental cycle, multinucleated hyphae begin to undergo the segmentation which precedes the formation of spore chains (Fig. 2i to m). Similar results were obtained for S. antibioticus (not shown), but there were some differences in the numbers of spores formed. Samples of young and late mycelia were freeze-substituted using the methodology described by Porta and Lopez-Iglesias (13) and were examined with a transmission electron microscope (Fig. 2n and o). The septal structure of the first mycelium (Fig. ​(Fig.2n)2n) lacks the complexity of the septal structure in the second mycelium, in which a membrane with a thick cell wall is clearly visible (Fig. ​(Fig.2o).2o). These data coincide with those previously described for solid confluent cultures (4).Open in a separate windowFIG. 2.Analysis of S. coelicolor hyphal compartmentalization with several fluorescent indicators (single colonies). Developmental culture times (in hours) are indicated. (a, e, and i) Mycelium stained with SYTO 9 and propidium iodide (viability). (b, f, and j) Hyphae stained with Cell Mask (a membrane stain). (c, g, and l) Hyphae stained with FM 4-64 (a membrane stain). (d, h, and m) Hyphae stained with WGA (cell wall stain). Septa in all the images in panels a to j, l, and m are indicated by arrows. (k) Image of the same field as panel j obtained in differential interference contrast mode. (n and o) Transmission electron micrographs of S. coelicolor hyphae at different developmental phases. The first-mycelium septa (n) are comprised of two membranes separated by a thin cell wall; in contrast, second-mycelium septa have thick cell walls (o). See the text for details. IP, propidium iodide.The main features of S. coelicolor growing in soils are shown in Fig. ​Fig.3.3. Under these conditions, spore germination is a very slow, nonsynchronous process that commences at about 7 days (Fig. 3c and d) and lasts for at least 21 days (Fig. 3i to l), peaking at around 14 days (Fig. 3e to h). Mycelium does not clump to form dense pellets, as it does in colonies; instead, it remains in the first-compartmentalized-mycelium phase during the time analyzed. Like the membrane septa in single colonies, the membrane septa of the hyphae are stained with FM4-64 (Fig. 3j and k), although only some of them are associated with thick cell walls (WGA staining) (Fig. ​(Fig.3l).3l). Similar results were obtained for S. antibioticus cultures (not shown).Open in a separate windowFIG. 3.Confocal laser scanning fluorescence microscopy analysis of the development-related cell death and hyphal compartmentalization of S. coelicolor M145 growing in soil. Developmental culture times (in days) are indicated. The images in panels b, f, and h were obtained in differential interference contrast mode and correspond to the same fields as the images in panels a, e, and g, respectively. The dark zone in panel h corresponds to a particle of soil containing hyphae. (a, c, d, e, g, i, j, and k) Hyphae stained with SYTO 9, propidium iodide (viability stain), and FM4-64 (membrane stain) simultaneously. (i) SYTO 9 and propidium iodide staining. (j) FM4-64 staining. The image in panel k is an overlay of the images in panels i and j and illustrates that first-mycelium membranous septa are not always apparent when they are stained with nucleic acid stains (SYTO 9 and propidium iodide). (l) Hyphae stained with WGA (cell wall stain), showing the few septa with thick cell walls present in the cells. Septa are indicated by arrows. IP, propidium iodide.In previous work (8), we have shown that the mycelium currently called the substrate mycelium corresponds to the early second multinucleated mycelium, according to our nomenclature, which still lacks the hydrophobic layers characteristic of the aerial mycelium. The aerial mycelium therefore corresponds to the late second mycelium which has acquired hydrophobic covers. This multinucleated mycelium as a whole should be considered the reproductive structure, since it is destined to sporulate (Fig. ​(Fig.4)4) (8). The time course of lysine 6-aminotransferase activity during cephamycin C biosynthesis has been analyzed by other workers using isolated colonies of Streptomyces clavuligerus and confocal microscopy with green fluorescent protein as a reporter (4). A complex medium and a temperature of 29°C were used, conditions which can be considered similar to the conditions used in our work. Interestingly, expression did not occur during the development of the early mycelium and was observed in the mycelium only after 80 h of growth. This suggests that the second mycelium is the antibiotic-producing mycelium, a hypothesis previously confirmed using submerged-growth cultures of S. coelicolor (9).Open in a separate windowFIG. 4.Cell cycle features of Streptomyces growing under natural conditions. Mycelial structures (MI, first mycelium; MII, second mycelium) and cell death are indicated. The postulated vegetative and reproductive phases are also indicated (see text).The significance of the first compartmentalized mycelium has been obscured by its short life span under typical laboratory culture conditions (5, 6, 8). In previous work (3, 7), we postulated that this structure is the vegetative phase of the bacterium, an hypothesis that has been recently corroborated by proteomic analysis (data not shown). Death in confluent cultures begins shortly after germination (4 h) and continues asynchronously for 15 h. The second multinucleated mycelium emerges after this early programmed cell death and is the predominant structure under these conditions. In contrast, as our results here show, the first mycelium lives for a long time in isolated colonies and soil cultures. As suggested in our previous work (5, 6, 8), if we assume that the compartmentalized mycelium is the Streptomyces vegetative growth phase, then this phase is the predominant phase in individual colonies (where it remains for at least 36 h), soils (21 days), and submerged cultures (around 20 h) (9). The differences in the life span of the vegetative phase could be attributable to the extremely high cell densities attained under ordinary laboratory culture conditions, which provoke massive differentiation and sporulation (5-7, 8).But just exactly what are “natural conditions”? Some authors have developed soil cultures of Streptomyces to study survival (16, 17), genetic transfer (14, 17-19), phage-bacterium interactions (3), and antibiotic production (1). Most of these studies were carried out using amended soils (supplemented with chitin and starch), conditions under which growth and sporulation were observed during the first few days (1, 17). These conditions, in fact, might resemble environments that are particularly rich in organic matter where Streptomyces could conceivably develop. However, natural growth conditions imply discontinuous growth and limited colony development (20, 21). To mimic such conditions, we chose relatively poor but more balanced carbon-nitrogen soil cultures (GAE medium-amended soil) and less dense spore inocula, conditions that allow longer mycelium growth times. Other conditions assayed, such as those obtained by irrigating the soil with water alone, did not result in spore germination and mycelial growth (not shown). We were unable to detect death, the second multinucleated mycelium described above, or sporulation, even after 1 month of incubation at 30°C. It is clear that in nature, cell death and sporulation must take place at the end of the long vegetative phase (1, 17) when the imbalance of nutrients results in bacterial differentiation.In summary, the developmental kinetics of Streptomyces under conditions resembling conditions in nature differs substantially from the developmental kinetics observed in ordinary laboratory cultures, a fact that should be born in mind when the significance of development-associated phenomena is analyzed.     

Influence of Ca2+ Ions on the Activity of Lantibiotics Containing a Mersacidin-Like Lipid II Binding Motif

Mersacidin binds to lipid II and thus blocks the transglycosylation step of the cell wall biosynthesis. Binding of lipid II involves a special motif, the so-called mersacidin-lipid II binding motif, which is conserved in a major subgroup of lantibiotics. We analyzed the role of Ca²⁺ ions in the mode of action of mersacidin and some related peptides containing a mersacidin-like lipid II binding motif. We found that the stimulating effect of Ca²⁺ ions on the antimicrobial activity known for mersacidin also applies to plantaricin C and lacticin 3147. Ca²⁺ ions appear to facilitate the interaction of the lantibiotics with the bacterial membrane and with lipid II rather than being an essential part of a peptide-lipid II complex. In the case of lacticin 481, both the interaction with lipid II and the antimicrobial activity were Ca²⁺ independent.Bacteriocins are a heterogeneous group of ribosomally synthesized antibiotic peptides and proteins which were proposed to fall into three classes, the lanthionine-containing bacteriocins (class I), the non-lanthionine-containing bacteriocins (class II), and the bacteriolysins, respectively (for a review, see reference 12).The lanthionine-containing bacteriocins (lantibiotics) are produced by and are effective against a broad spectrum of gram-positive bacteria. They are small, posttranslationally modified antimicrobial peptides containing characteristic thioether ring structures (lanthionine and 3-methyllanthionine) and other unusual amino acids, e.g., d-Ala (3, 49).Mersacidin was the first lantibiotic shown to interact with a defined target molecule, the ultimate cell wall precursor lipid II (6) (Fig. ​(Fig.1).1). Further studies revealed that this molecule is also the target of nisin and many other lantibiotics (19). Lipid II is synthesized on the cytoplasmic side of the membrane and translocated to the outside of the bacterial cell membrane, where the disaccharide pentapeptide part of lipid II is incorporated into the growing peptidoglycan network by the cell wall biosynthesis machinery (for reviews, see references 5 and 45).Open in a separate windowFIG. 1.Primary structure of lantibiotics containing the mersacidin-lipid II binding motif (A) and the structure of the cell wall precursor lipid II (B). The binding motif of mersacidin and identical amino acids in the mersacidin-like lantibiotics are highlighted in gray. Dha, dehydroalanine; Dhb, dehydrobutyrine; Ala-S-Ala, lanthionine; Abu-S-Ala, methyllanthionine; DAla, d-alanine.To date, two different lipid II binding motifs in lantibiotics have been identified, referred to as the nisin-lipid II and mersacidin-lipid II binding motifs, and a classification regarding their interaction with the cell wall precursor was recently proposed by Bierbaum and Sahl (3).The nisin-lipid II binding motif is also found in related lantibiotics, e.g., gallidermin, epidermin (4), mutacin 1140 (40), and subtilin (30). Nisin displays a dual mode of action by binding to lipid II. It prevents lipid II incorporation into the growing murein layer, thereby blocking cell wall biosynthesis (8), and it uses lipid II as an anchor molecule for subsequent pore formation (48). The nisin/lipid II interaction was analyzed by nuclear magnetic resonance spectroscopy and it was shown that the N-terminal part of the peptide forms a cage-like structure encompassing the pyrophosphate group of the lipid II molecule, leading to the formation of five intermolecular hydrogen bonds between the backbone amids of the lantibiotic and pyrophosphate groups (22).The second binding motif occurs in mersacidin and related lantibiotics (Fig. ​(Fig.1).1). The interaction of mersacidin with lipid II leads to inhibition of the peptidoglycan biosynthesis at the level of transglycosylation (7). In contrast to nisin, the activity of mersacidin is influenced by Ca²⁺ ions, since its antimicrobial activity increased twofold in Ca²⁺-containing medium (2). When a Ca²⁺ binding pocket was identified in the mersacidin-like lantibiotic actagardine by crystal structure determination, it was suggested that the deprotonated Glu17 in the mersacidin-lipid II binding motif (Fig. ​(Fig.1)1) is involved in Ca²⁺ binding (24). Furthermore, nuclear magnetic resonance studies revealed that, upon binding of lipid II, mersacidin effectively alters its overall backbone geometry with Ala-12 and Abu-13, acting as a hinge region. The conformational change exposes the amino group of Lys1 and the carboxyl group of Glu17 to the lipid II molecule (21). It was speculated that Ca²⁺ is needed to bridge the mersacidin Glu17 side chain to the negatively charged groups of lipid II; alternatively, a direct salt bridge with the positively charged side chain of Lys3 in lipid II is formed (21). This hypothesis is in good agreement with the observation that replacement of Glu17 by Ala abolished the antimicrobial activity of mersacidin (43).To analyze the impact of Ca²⁺ on the activity of mersacidin-like lantibiotics, we selected four peptides which possess the respective lipid II-binding motif, yet show significant differences in primary structures (Fig. ​(Fig.1).1). Like mersacidin, plantaricin C and the two-component lantibiotic lacticin 3147 have been shown to inhibit cell wall biosynthesis at the level of transglycosylation (46, 47). Additionally, lacticin 3147 shows a dual mode of action and is able to form lipid II-dependent pores (28, 47). The mode of action of lacticin 481 so far has not been characterized in sufficient detail.We found that Ca²⁺ increases the antimicrobial activity of all peptides containing the mersacidin-lipid II binding motif, except for lacticin 481, however, which was also found to bind to lipid II.     

Teaching cases: A patient with loss of vision in the right eye and neurofibromatosis type 1

Abstract: Neurofibromatosis type 1 is a common autosomal dominant condition that affects about 1 in 5000 people. We describe a 75-year-old man who, in addition to many classic developmental changes of the disease in his skin, eyes and nervous system, had blindness in his right eye as a complication.Case: A 75-year-old man with long-standing neurofibromatosis type 1 was admitted because the vision in his right eye had decreased progressively over 3 months. Physical examination showed disseminated cutaneous and subcutaneous neurofibromas of varying size (Figure 1) and café-au-lait spots (Figure 2). The patient had a visual acuity of 6/18 (20/60) in his right eye and Lisch nodules (iris hamartomas) (Figure 3). A neurologic examination showed no abnormalities other than his loss of vision. Axial T₁-weighted magnetic resonance imaging of the brain and orbits (Figure 4) showed an isointense mass lateral to the right optic nerve that appeared atrophic and pushed to the left. The mass showed a hyperintense signal on T₂-weighted images with contrast enhancement. These findings are compatible with glioma of the optic nerve.Open in a separate windowFigure 1: Disseminated cutaneous and subcutaneous neurofibromas of varying size on the torso of a patient with neurofibromatosis type 1.Open in a separate windowFigure 2: A café-au-lait spot on the patient''s right knee.Open in a separate windowFigure 3: Lisch nodules on the left iris.Open in a separate windowFigure 4: T₁-weighted axial magnetic resonance imaging of the brain and orbits, showing an isointense mass lateral to the right optic nerve (white arrow) that appears atrophic and pushed to the left (black arrow on inset).Axial and coronal magnetic resonance imaging (Figure 5) showed a mass in the left parietal lobe with hyperintensity on T₂-weighted images and hypointensity on T₁-weighted images. After a contrast medium was administered, the lesion showed a thickened, enhanced wall with a central necrotic area. These findings are compatible with astrocytoma.Open in a separate windowFigure 5: T₂-weighted axial (left) and coronal (right) magnetic resonance imaging showing a mass with hyperintensity (arrow) in the left temporal lobe. After administration of a contrast medium, the lesion is visible with a thickened enhanced wall and a central necrotic area.Because of slight enlargement and increased hardness of the subcutaneous lesions, an excisional biopsy was performed. Histology showed delicate fascicles consisting of cells with oval or spindle-shaped nuclei, scant cytoplasm and round cells with entrapped axons (Figure 6). Only scattered neoplastic Schwann cells were stained during immunostaining for S-100 protein (Figure 7). This pattern is consistent with neurofibroma. The patient chose not to receive further treatment and was discharged.Open in a separate windowFigure 6: Biopsy specimen of a subcutaneous neurofibroma showing spindle-shaped and round cells with entrapped axons (hematoxylin and eosin, original magnification ×10).Open in a separate windowFigure 7: Only scattered neoplastic Schwann cells (arrow) are stained after immunostaining for S-100 protein. Normally, S-100 protein is present in cells derived from the neural crest, such as Schwann cells. It can be found in melanoma cells, in malignant peripheral nerve sheath tumours and in certain types of sarcomas.Neurofibromatosis type 1, also known as von Recklinghausen disease,¹ is characterized by changes in pigmentation and the growth of tumours along nerves in the skin and other parts of the body. It is caused by a defect in a tumour-suppressing gene on chromosome 17q11.2. Normally the gene produces neurofibromin, a protein that regulates cellular proliferation.² With the gene mutation, the lack of neurofibromin results in overgrowth of cells from neural crest areas in both the central nervous system (causing Schwann cell tumours on virtually every nerve) and the skin. All people who inherit a copy of the mutated gene are affected. As the pattern of inheritance is autosomal dominant, only 1 copy of the defective gene is needed to cause the condition. However, it is not necessary to have an affected parent. About 30%–50% of patients have a new mutation.Neurofibromatosis type 2 is a much rarer form of neurofibromatosis caused by mutations in both alleles of a different tumour suppressor gene on chromosome 22q12.1.About 1 in 3000–5000 individuals are affected by neurofibromatosis type 1, without differences related to ethnic background.³ Pigmented small macules and café-au-lait patches are often present shortly after birth, although neurofibromas are rare in early childhood. In later childhood and adolescence, both neurofibromas and pigmented lesions become common. Clinical manifestations are variable (4Table 1Open in a separate windowA diagnosis of neurofibromatosis type 1 is based on clinical findings. The patient should have 2 or more of the following: 6 or more café-au-lait spots of ≥ 1.5 cm in postpubertal individuals or ≥ 0.5 cm in prepubertal individuals; 2 or more neurofibromas of any type or 1 or more plexiform neurofibroma; and freckling in the underarms and groin.¹ The differential diagnosis includes benign café-au-lait pigmentation (present in up to 10% of the general population), multiple lipomas, and sporadic schwannomas, gliomas and meningiomas in the central nervous system.Most people with mild neurofibromatosis have little disability. People affected by more severe variants have a shortened life expectancy, especially if tumours of the central nervous system or other malignant neoplasms arise during the course of illness.^1,3 The condition can have a serious psychological impact because the accumulation of skin nodules can be quite disfiguring.⁵ Surgical excision and laser treatment of the neurofibromas are possible, but neither treatment is universally effective.⁶ Transplantation with an allograft of composite tissue on the lower and middle parts of a patient''s face was recently reported.⁷Gliomas of the optic nerve are found in up to 15% of pediatric patients with neurofibromatosis type 1. Best detected using magnetic resonance imaging, these gliomas are symptomatic in about 50% of patients at diagnosis. A minority will progress to vision loss.⁸ The high prevalence of gliomas of the optic nerve that are asymptomatic may, however, be biased by referral patterns, Indeed, in patients with neurofibromatosis type 1, the threshold of risk for optic nerve glioma is low.⁹Guidelines are available for the diagnosis and management of neurofibromatosis type 1.^10,11 Physicians who identify patients with neurofibromatosis type 1 should refer them early to facilities where appropriate evaluation and monitoring of lesions can be carried out. Early detection and monitoring may help to prevent disability and death.     

Mutations in the Ectodomain of Newcastle Disease Virus Fusion Protein Confer a Hemagglutinin-Neuraminidase-Independent Phenotype

The entry of enveloped viruses into host cells is preceded by membrane fusion, which in paramyxoviruses is triggered by the fusion (F) protein. Refolding of the F protein from a metastable conformation to a highly stable postfusion form is critical for the promotion of fusion, although the mechanism is still not well understood. Here we examined the effects of mutations of individual residues of the F protein of Newcastle disease virus, located at critical regions of the protein, such as the C terminus of the N-terminal heptad repeat (HRA) and the N terminus of the C-terminal heptad repeat (HRB). Seven of the mutants were expressed at the cell surface, showing differences in antibody reactivity in comparison with the F wild type. The N211A, L461A, I463A, and I463F mutants showed a hyperfusogenic phenotype both in syncytium and in dye transfer assays. The four mutants promoted fusion more efficiently at lower temperatures than the wild type did, meaning they probably had lower energy requirements for activation. Moreover, the N211A, I463A, and I463F mutants exhibited hemagglutinin-neuraminidase (HN)-independent activity when influenza virus hemagglutinin (HA) was coexpressed as an attachment protein. The data are discussed in terms of alterations of the refolding pathway and/or the stability of the prefusion and fusion conformations.Newcastle disease virus (NDV) is an avian enveloped virus belonging to the family Paramyxoviridae. Two viral membrane-associated proteins are responsible for the entry of the virus into the host cell: they are hemagglutinin-neuraminidase (HN), a receptor-binding protein that interacts with sialoglycoconjugates at the cell surface, and F, a trimeric class I fusion protein that, upon activation, triggers the fusion of the viral and target membranes. F protein is activated after the attachment of its homotypic HN protein to the proper receptor; however, how HN activates F is not well understood. F protein is synthesized as an inactive precursor, F₀, that is activated by proteolytic cleavage to the disulfide-linked F₁-F₂ fusion-competent form (Fig. ​(Fig.1)1) (10). The crystal structures of several paramyxoviral fusion proteins, in both the prefusion and postfusion conformations (3, 26, 27), have revealed that these proteins undergo major conformational changes, from a metastable conformation to a highly stable, postfusion form. Several regions in the ectodomain of class I viral fusion proteins are involved in these conformational conversions, including a hydrophobic fusion peptide at the N terminus of the F1 protein and two hydrophobic heptad repeat motifs, HRA and HRB, located at its N and C termini, respectively (Fig. ​(Fig.1).1). In the prefusion form, HRB shows a triple-stranded coiled-coil conformation forming the stalk of the mushroom-like protein (3, 19, 27). Its globular head contains three domains, DI to DIII (Fig. ​(Fig.1),1), with the base of the head being formed by the DI and DII domains, with residues predominantly located between HRA and HRB. The top of the head is formed by DIII, consisting mainly of HRA and the fusion peptide, located on the side of the head sequestered between adjacent subunits. In this prefusion state, HRA is folded as two antiparallel β-strands and four (h1 to h4) helices (27) (see Fig. ​Fig.6).6). The DIII domain undergoes major structural changes from the prefusion to the final postfusion conformation. HRA refolds as an α-helix, propelling the fusion peptide into the target membrane and generating a prehairpin intermediate (see Fig. ​Fig.6).6). The final, stable conformation consists of a six-helical bundle (6HB), comprising a dimer of trimers in which the trimeric HRA coiled coil forms the core, packed along the outside by three antiparallel HRB α-helices (1, 3, 19, 27).Open in a separate windowFIG. 1.Schematic representation of the structure of the NDV fusion protein. (A) Domain structure of F protein (27). (B) Locations of the fusion peptide, HR regions, and sequences studied. Mutated residues are indicated in bold.Open in a separate windowFIG. 6.Scheme of conformational changes in HRA from prefusion to postfusion state. (A) Ribbon model of PIV5 F protein in its metastable prefusion conformation (PDB accession number 2b9b) (27), showing some residues (named in white) from the A subunit and the corresponding residues in the NDV F protein (named in yellow). Subunits B and C are depicted in gray for clarity. (B) In the metastable, prefusion conformation, HRA is folded as a spring-loaded mixture of α-helices, turns, and β-strands, comprising 11 segments in the DIII head domain of the trimer (27). (C) After fusion, HRA is presented as a single long helix that allows the fusion peptide to be buried in the target membrane. The approximate positions of HRC and the core β-sheet are shown as dashed lines for both conformations.The refolding mechanism that triggers F protein activation is still not well understood. Mutational analysis of the HRA and HRB domains of paramyxovirus F proteins (3, 13, 18, 19, 22, 23), as well as the use of HRA- and HRB-derived peptides (6, 17), has led to the proposal of a series of discrete refolding intermediates of the F protein, from the metastable native conformation, through the prehairpin intermediate, and to the final postfusion hairpin structure (6HB) (17, 19, 27). To gain further insight into the individual residues critical for this mechanism, in this work we mutated several residues of the head and stalk of the NDV F protein (Fig. ​(Fig.1).1). The mutations disrupted F protein antibody reactivity, fusogenicity, and HN dependence in different ways. Interestingly, a mutant of the C-terminal h4 α-helix of HRA (N211A mutant) and two mutants of a residue located at the most N-terminal position of HRB (I463A and I463F mutants) exhibited a hyperfusogenic phenotype and HN-independent activity when influenza virus hemagglutinin (HA) was coexpressed as an attachment protein. The data are discussed in terms of alterations of the refolding pathway and/or the stability of the prefusion and fusion conformations.     

Recombinant Yellow Fever Vaccine Virus 17D Expressing Simian Immunodeficiency Virus SIVmac239 Gag Induces SIV-Specific CD8+ T-Cell Responses in Rhesus Macaques

Here we describe a novel vaccine vector for expressing human immunodeficiency virus (HIV) antigens. We show that recombinant attenuated yellow fever vaccine virus 17D expressing simian immunodeficiency virus SIVmac239 Gag sequences can be used as a vector to generate SIV-specific CD8⁺ T-cell responses in the rhesus macaque. Priming with recombinant BCG expressing SIV antigens increased the frequency of these SIV-specific CD8⁺ T-cell responses after recombinant YF17D boosting. These recombinant YF17D-induced SIV-specific CD8⁺ T cells secreted several cytokines, were largely effector memory T cells, and suppressed viral replication in CD4⁺ T cells.None of the vaccine regimens tested in human immunodeficiency virus (HIV) vaccine efficacy trials to date have either reduced the rate of HIV infection or reduced the level of HIV replication. Structural features and the enormous variability of the envelope glycoprotein have frustrated efforts to induce broadly reactive neutralizing antibodies against HIV (10). Investigators have therefore focused their attention on T-cell-based vaccines (40). Simian immunodeficiency virus (SIV) challenge of rhesus macaques vaccinated with T-cell-based vaccines has shown that it is possible to control virus replication after SIV infection (22, 41, 42). The recent STEP trial of a recombinant Ad5-vectored vaccine was widely seen as an important test of this concept (http://www.hvtn.org/media/pr/step111307.html) (9, 25). Unfortunately, vaccinees became infected at higher rates than the controls (9). While it is still not clear what caused the enhanced infection rate in the vaccinated group, future Ad5-based human vaccine trials may be difficult to justify. We therefore need to develop new vaccine vectors for delivering SIV and HIV genes. Several other viral vectors currently under consideration include nonreplicating adenovirus (Ad)-based vectors (1, 21, 22), Venezuelan equine encephalitis (VEE) virus (12, 20), adeno-associated virus (AAV) (19), modified vaccinia virus Ankara (MVA) (3, 4, 13, 15, 18, 38), NYVAC (6), cytomegalovirus (CMV) (16), and replicating Ad (30). However, only a few of these have shown promise in monkey trials using rigorous SIV challenges.We explored whether the small (11-kb) yellow fever vaccine flavivirus 17D (YF17D) might be a suitable vector for HIV vaccines. The YF17D vaccine is inexpensive, production and quality control protocols already exist, and it disseminates widely in vivo after a single dose (27). Importantly, methods for the manipulation of the YF17D genome were recently established (7, 8, 24, 28). This effective vaccine has been safely used on >400 million people in the last 70 years (27). Additionally, the YF17D strain elicits robust CD8⁺ T-cell responses in humans (26). Chimeric YF17D is presently being developed as a vaccine for other flaviviruses, such as Japanese encephalitis virus (28), dengue virus (14), and West Nile virus (29). Inserts expressing a malaria B-cell epitope have been engineered into the E protein of YF17D (7). In murine models, recombinant YF17D viruses have generated robust and specific responses to engineered antigens inserted between the 2B and NS3 proteins in vivo (24, 35).We first used the YF17D vaccine virus to infect four Mamu-A*01-positive macaques. The vaccine virus replicated in these four animals and induced neutralizing antibodies in all four macaques by 2 weeks postvaccination (Fig. 1A and B). To monitor the CD8⁺ T-cell immune response against YF17D, we scanned its proteome for peptides that might bind to Mamu-A*01 using the major histocompatibility complex (MHC) pathway algorithm (31). We synthesized the 52 YF17D-derived peptides most likely to bind to Mamu-A*01 based on their predicted affinity for this MHC class I molecule. We then used a gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay to screen these peptides in YF17D-immunized animals at several time points after vaccination and discovered that four Mamu-A*01-binding peptides, LTPVTMAEV (LV9_1285-1293), VSPGNGWMI (VI9_3250-3258), MSPKGISRM (MM9_2179-2187), and TTPFGQQRVF (TF10_2853-2862), were recognized in vivo (Fig. ​(Fig.1C).1C). Using a previously reported protocol (26), we also observed CD8⁺ T-cell activation in all four animals (Fig. 1D and E). Thus, as was observed previously, the YF17D vaccine virus replicates in Indian rhesus monkeys (36) and induces neutralizing antibodies, yellow fever 17D-specific Mamu-A*01-restricted CD8⁺ T-cell responses, and CD8⁺ T-cell activation.Open in a separate windowFIG. 1.YF17D replicates and induces neutralizing antibodies, virus-specific CD8⁺ T cells, and the activation of CD8⁺ T cells in rhesus macaques. (A) Replication of YF17D during the first 10 days after vaccination with two different doses, as measured by quantitative PCR (Q-PCR) using the following primers: forward primer YF-17D 10188 (5′-GCGGATCACTGATTGGAATGAC-3′), reverse primer YF-17D 10264 (5′-CGTTCGGATACGATGGATGACTA-3′), and probe 6-carboxyfluorescein (6Fam)-5′-AATAGGGCCACCTGGGCCTCCC-3′-6-carboxytetramethylrhodamine (TamraQ). (B) Titer of neutralizing antibodies determined at 2 and 5 weeks after YF17D vaccination. (C) Fresh PBMC from vaccinees (100,000 cells/well) were used in IFN-γ ELISPOT assays (41) to assess T-cell responses against YF17D. We used 4 epitopes (LTPVTMAEV [LV9_1285-1293], VSPGNGWMI [VI9_3250-3258], MSPKGISRM [MM9_2179-2187], and TTPFGQQRVF [TF10_2853-2862]) predicted to bind to Mamu-A*01 as defined by the MHC pathway algorithm (31). All IFN-γ ELISPOT results were considered positive if they were ≥50 SFC/10⁶ PBMC and ≥2 standard deviations over the background. (D) Identification of activated CD8⁺ T cells after vaccination with YF17D based on the expression of the proliferation and proapoptotic markers Ki-67 and Bcl-2, respectively (26). We stained whole blood cells with antibodies against CD3 and CD8. We then permeabilized and subsequently labeled these cells with Bcl-2- and Ki-67-specific antibodies. The flow graphs were gated on CD3⁺ CD8⁺ lymphocytes. (E) Expression kinetics of Ki-67 and Bcl-2 in CD8⁺ T cells after vaccination with YF17D.We next engineered the YF17D vaccine virus to express amino acids 45 to 269 of SIVmac239 Gag (rYF17D/SIVGag_45-269) by inserting a yellow fever codon-optimized sequence between the genes encoding the viral proteins E and NS1. This recombinant virus replicated and induced neutralizing antibodies in mice (data not shown). We then tested the rYF17D/SIVGag_45-269 construct in six Mamu-A*01-positive Indian rhesus macaques. We found evidence for the viral replication of rYF17D/SIVGag_45-269 for five of these six macaques (Fig. ​(Fig.2A).2A). However, neutralizing antibodies were evident for all six animals at 2 weeks postvaccination (Fig. ​(Fig.2B).2B). Furthermore, all animals developed SIV-specific CD8⁺ T cells after a single immunization with rYF17D/SIVGag_45-269 (Fig. ​(Fig.2C).2C). To test whether a second dose of this vaccine could boost virus-specific T-cell responses, we administered rYF17D/SIVGag_45-269 (2.0 × 10⁵ PFU) to four macaques on day 28 after the first immunization and monitored cellular immune responses. With the exception of animal r04091, the rYF17D/SIVGag_45-269 boost did not increase the frequency of the vaccine-induced T-cell responses. This recombinant vaccine virus also induced CD8⁺ T-cell activation in the majority of the vaccinated animals (Fig. ​(Fig.2D2D).Open in a separate windowFIG. 2.rYF17D/SIVGag_45-269 replicates and induces neutralizing antibodies, virus-specific CD8⁺ T cells, and the activation of CD8⁺ T cells in rhesus macaques. (A) Replication of rYF17D/SIVGag_45-269 during the first 10 days after vaccination with two different doses as measured by Q-PCR using the YF17D-specific primers described in the legend of Fig. ​Fig.1.1. (B) Titer of neutralizing antibodies determined at 2 and 5 weeks after rYF17D/SIVGag_45-269 vaccination. The low levels of neutralization for animal r02013 were observed in three separate assays. (C) Fresh PBMC from vaccinees (100,000 cells/well) were used in IFN-γ ELISPOT assays to assess T-cell responses against the YF17D vector (red) and the SIV Gag(45-269) insert (black) at several time points postvaccination. We measured YF17D-specific responses using the same epitopes described in the legend of Fig. ​Fig.1.1. For SIV Gag-specific responses, we used 6 pools of 15-mers overlapping by 11 amino acids spanning the entire length of the SIVmac239 Gag insert. In addition, we measured Mamu-A*01-restricted responses against the dominant Gag_181-189CM9 and subdominant Gag_254-262QI9 epitopes. Four animals received a second dose of rYF17D/SIVGag_45-269 on day 28 after the first vaccination (dashed line). (D) Expression kinetics of Ki-67 and Bcl-2 in CD8⁺ T cells after vaccination with rYF17D/SIVGag_45-269. This assay was performed as described in the legend of Fig. ​Fig.11.We could not detect differences in vaccine-induced immune responses between the group of animals vaccinated with YF17D and the group vaccinated with rYF17D/SIVGag_45-269. There was, however, considerable animal-to-animal variability. Animal r02034, which was vaccinated with YF17D, exhibited massive CD8⁺ T-cell activation (a peak of 35% at day 14) (Fig. ​(Fig.1E),1E), which was probably induced by the high levels of viral replication (16,800 copies/ml at day 5) (Fig. ​(Fig.1A).1A). It was difficult to see differences between the neutralizing antibody responses induced by YF17D and those induced by rYF17D/SIVGag_45-269 (Fig. ​(Fig.1B1B and ​and2B).2B). However, neutralizing antibodies in animal r02013 decreased by 5 weeks postvaccination. It was also difficult to detect differences in the YF17D-specific CD8⁺ T-cell responses induced by these two vaccines. Peak Mamu-A*01-restricted CD8⁺ T-cell responses against YF17D ranged from barely detectable (animal r02110 at day 11) (Fig. ​(Fig.1C)1C) to 265 spot-forming cells (SFCs)/10⁶ peripheral blood mononuclear cells (PBMC) (animal r02034 at day 28) (Fig. ​(Fig.1C).1C). Similarly, three of the rYF17D/SIVGag_45-269-vaccinated animals (animals r04091, r04051, and r02013) made low-frequency CD8⁺ T-cell responses against the Mamu-A*01-bound YF17D peptides, whereas the other three animals (animals r03130, r02049, and r02042) recognized these epitopes with responses ranging from 50 to 200 SFCs/10⁶ PBMC (Fig. ​(Fig.2C).2C). For almost every rYF17D/SIVGag_45-269-vaccinated animal, the Gag_181-189CM9-specific responses (range, 50 to 750 SFCs/10⁶ PBMC) were higher than those generated against the Mamu-A*01-restricted YF17D epitopes (range, 0 to 175 SFCs/10⁶ PBMC), suggesting that the recombinant virus replicated stably in vivo (Fig. ​(Fig.2C).2C). Thus, the recombinant YF17D virus replicated and induced both virus-specific neutralizing antibodies and CD8⁺ T cells that were not demonstrably different from those induced by YF17D alone.Most viral vectors are usually more efficient after a prime with DNA or recombinant BCG (rBCG) (4, 11, 15, 18). We therefore used rYF17D/SIVGag_45-269 to boost two macaques that had been primed with rBCG expressing SIV proteins (Fig. ​(Fig.3A).3A). We detected no SIV-specific responses after either of the two priming rBCG vaccinations. Unfortunately, while the recombinant YF17D virus replicated well in animal r01056, we found evidence for only low levels of replication of rYF17D/SIVGag_45-269 on day 5 postvaccination for animal r01108 (7 copies/ml) (Fig. ​(Fig.3B).3B). Both animals, however, generated neutralizing antibodies at 2 weeks postvaccination (Fig. ​(Fig.3C).3C). Encouragingly, we detected high-frequency CD8⁺ T-cell responses in the Mamu-A*01-positive macaque (animal r01056) after boosting with rYF17D/SIVGag_45-269 (Fig. 3D to F). These responses were directed mainly against the Mamu-A*01-restricted Gag_181-189CM9 epitope, which is contained in the peptide pool Gag E (Fig. ​(Fig.3D).3D). Furthermore, the boost induced a massive activation of animal r01056''s CD8⁺ T cells, peaking at 35% at 17 days postvaccination (Fig. ​(Fig.3E).3E). Of these activated CD8⁺ T cells, approximately 10% were directed against the Gag_181-189CM9 epitope, with a frequency of 3.5% of CD8⁺ T cells (Fig. ​(Fig.3E).3E). These epitope-specific CD8⁺ T cells made IFN-γ, tumor necrosis factor alpha (TNF-α), macrophage inflammatory protein 1β (MIP-1β), and degranulated (Fig. ​(Fig.3F3F and data not shown). Thus, an rBCG prime followed by a recombinant yellow fever 17D boost induced polyfunctional antigen-specific CD8⁺ T cells.Open in a separate windowFIG. 3.rYF17D/SIVGag_45-269 vaccination induced a robust expansion of Gag-specific responses in an rBCG-primed macaque. (A) Vaccination scheme. We immunized two rhesus macaques with rBCG intradermally (i.d.) (2.0 × 10⁵ CFU), rBCG orally (10⁷ CFU), and rYF17D/SIVGag_45-269 subcutaneously (2.0 × 10⁵ PFU) at 6-month intervals. rBCG was engineered to express 18 minigenes containing sequences of Gag, Vif, Nef, Rev, and Tat from SIVmac239. (B) Replication of rYF17D/SIVGag_45-269 during the first 10 days after vaccination as measured by Q-PCR using the YF17D-specific primers described in the legend of Fig. ​Fig.1.1. (C) Titer of neutralizing antibodies determined at 2 and 5 weeks after rYF17D/SIVGag_45-269 vaccination. (D) Fresh PBMC from animal r01056 (100,000 cells/well) were used in IFN-γ ELISPOT assays to assess T-cell responses against the YF17D vector (red) and the SIV Gag(45-269) insert (black) at several time points postvaccination. (E) Kinetics of CD8⁺ T-cell activation (as described in the legend of Fig. ​Fig.1)1) and expansion of Gag_181-189CM9-specific CD8⁺ T cells in animal r01056 after vaccination with rYF17D/SIVGag_45-269. (F) Vaccination with rYF17D/SIVGag_45-269 induced robust CD8⁺ T-cell responses against Gag_181-189CM9 in r01056. CD8⁺ T-cell activation (Ki-67⁺/Bcl-2⁻) for baseline and day 13 are shown. Gag_181-189CM9-specific responses were measured by tetramer staining and intracellular cytokine staining (ICS) with antibodies against MIP-1β and IFN-γ.Vaccine-induced CD8⁺ T cells are usually central memory T cells (T_CM) or effector memory T cells (T_EM). These two subsets of CD8⁺ T cells differ in function and surface markers (23). Repeated boosting drives CD8⁺ T cells toward the T_EM subset (23). We therefore determined whether a rBCG prime followed by a rYF17D/SIVGag_45-269 boost induced T_CM or T_EM CD8⁺ T cells. Staining of PBMC obtained on day 30 postvaccination revealed that the SIV-specific CD8⁺ T cells were largely T_EM cells since the majority of them were CD28 negative (Fig. ​(Fig.4A).4A). Furthermore, these cells persisted with the same phenotype until day 60 after vaccination (Fig. ​(Fig.4B).4B). It was recently suggested that T_EM cells residing in the mucosae can effectively control infection after a low-dose challenge with SIVmac239 (16).Open in a separate windowFIG. 4.rYF17D/SIVGag_45-269 vaccination of animal r01056 induced effector memory Gag_181-189CM9-specific CD8⁺ T cells that suppressed viral replication in CD4⁺ targets. (A and B) Frequency and memory phenotype of tetramer-positive Gag_181-189-specific CD8⁺ T cells in animal r01056 on day 30 (A) and day 60 (B) after rYF17D/SIVGag_45-269 vaccination. CD28 and CD95 expression profiles of tetramer-positive cells show a polarized effector memory phenotype. Cells were gated on CD3⁺ CD8⁺ lymphocytes. (C) Ex vivo Gag_181-189CM9-specific CD8⁺ T cells from animal r01056 inhibit viral replication from SIVmac239-infected CD4⁺ T cells. Gag_181-189CM9-specific CD8⁺ T cells from three SIV-infected Mamu-A*01-positive animals and rYF17D/SIVGag_45-269-vaccinated animal r01056 were tested for their ability to suppress viral replication from SIV-infected CD4⁺ T cells (39). Forty-eight hours after the incubation of various ratios of SIV-infected CD4⁺ T cells and Gag_181-189CM9-specific CD8⁺ T cells, the supernatant was removed and measured for viral RNA (vRNA) copies per ml by Q-PCR. We observed no suppression when effectors were incubated with CD4⁺ targets from Mamu-A*01-negative animals (data not shown). Animal rh2029 was infected with SIVmac239 (viral load, ∼10⁵ vRNA copies/ml) containing mutations in 8 Mamu-B*08-restricted epitopes as part of another study (37). Animal r01080 was vaccinated with a DNA/Ad5 regimen expressing Gag, Rev, Tat, and Nef and later infected with SIVmac239 (viral load, ∼10³ vRNA copies/ml) (42). Animal r95061 was vaccinated with a DNA/MVA regimen containing Gag_181-189CM9 and was later challenged with SIVmac239 (undetectable viral load) (2).We then assessed whether rYF17D/SIVGag_45-269-induced CD8⁺ T cells could recognize virally infected CD4⁺ T cells. We have shown that these vaccine-induced CD8⁺ T cells stain for tetramers and produce cytokines after stimulation with synthetic peptides (Fig. ​(Fig.3).3). None of these assays, however, tested whether these SIV-specific CD8⁺ T cells recognize SIV-infected cells and reduce viral replication. We therefore used a newly developed assay (39) to determine whether vaccine-induced CD8⁺ T cells can reduce viral replication in CD4⁺ T cells. We sorted tetramer-positive (Gag_181-189CM9-specific) lymphocytes directly from fresh PBMC and incubated them for 48 h with SIVmac239-infected CD4⁺ T cells expressing Mamu-A*01. We assessed the percentage of CD4⁺ T cells that expressed SIV Gag p27 (data not shown) and the quantity of virus in the culture supernatant (Fig. ​(Fig.4C).4C). Vaccine-induced CD8⁺ T cells reduced viral replication to the same extent as that seen with Gag_181-189CM9-specific CD8⁺ T cells purified from three SIVmac239-infected rhesus macaques, including an elite controller rhesus macaque, animal r95061 (Fig. ​(Fig.4C4C).The most encouraging aspect of this study is that rBCG primed a high-frequency CD8⁺ T-cell response after boosting with rYF17D/SIVGag_45-269. These CD8⁺ T cells reached frequencies that were similar to those induced by an rBCG prime followed by an Ad5 boost (11). Even without the benefit of the rBCG prime, the levels of CD8⁺ T cells induced by a single rYF17D/SIVGag_45-269 vaccination were equivalent to those induced by our best SIV vaccine, SIVmac239ΔNef. Recombinant YF17D generated an average of 195 SFCs/10⁶ PBMC (range, 100 to 750 SFCs/10⁶ PBMC) (n = 6), whereas SIVmac239ΔNef induced an average of 238 SFCs/10⁶ PBMC (range, 150 to 320 SFCs/10⁶ PBMC) (n = 3) (32). It is also possible that any YF17D/HIV recombinants would likely replicate better in humans than they have in rhesus macaques and thus induce more robust immune responses. Also, rBCG was shown previously to be effective in humans (5, 17, 33, 34) and may be more useful at priming T-cell responses in humans than it has been in our limited study with rhesus macaques. These two vectors have long-distinguished safety and efficacy histories in humans and may therefore be well suited for HIV vaccine development.     

ATP-Dependent Carboxylation of Acetophenone by a Novel Type of Carboxylase

Anaerobic ethylbenzene metabolism in the betaproteobacterium Aromatoleum aromaticum is initiated by anaerobic oxidation to acetophenone via (S)-1-phenylethanol. The subsequent carboxylation of acetophenone to benzoylacetate is catalyzed by an acetophenone-induced enzyme, which has been purified and studied. The same enzyme is involved in acetophenone metabolism in the absence of ethylbenzene. Acetophenone carboxylase consists of five subunits with molecular masses of 70, 15, 87, 75, and 34 kDa, whose genes (apcABCDE) form an apparent operon. The enzyme is synthesized at high levels in cells grown on ethylbenzene or acetophenone, but not in cells grown on benzoate. During purification, acetophenone carboxylase dissociates into inactive subcomplexes consisting of the 70-, 15-, 87-, and 75-kDa subunits (apcABCD gene products) and the 34-kDa subunit (apcE gene product), respectively. Acetophenone carboxylase activity was restored by mixing the purified subcomplexes. The enzyme contains 1 Zn²⁺ ion per αβγδ core complex and is dependent on the presence of Mg²⁺ or Mn²⁺. In spite of the presence of Zn in the enzyme, it is strongly inhibited by Zn²⁺ ions. Carboxylation of acetophenone is dependent on ATP hydrolysis to ADP and P_i, exhibiting a stoichiometry of 2 mol ATP per mol acetophenone carboxylated. The enzyme shows uncoupled ATPase activity with either bicarbonate or acetophenone in the absence of the second substrate. These observations indicate that both substrates may be phosphorylated, which is consistent with isotope exchange activity observed with deuterated acetophenone and inhibition by carbamoylphosphate, a structural analogue of carboxyphosphate. A potential mechanism of ATP-dependent acetophenone carboxylation is suggested.Ethylbenzene belongs to the BTEX (benzene, toluene, ethylbenzene, and xylene) group of petroleum-derived hydrocarbons with extensive industrial and ecological relevance. Anaerobic catabolism of ethylbenzene proceeds via different pathways in denitrifying and sulfate-reducing bacteria. The latter generate a succinate adduct of ethylbenzene as the first intermediate, probably by addition of fumarate to the methylene carbon atom (14). However, denitrifying bacteria are capable of oxygen-independent hydroxylation of the methylene group of ethylbenzene to yield (S)-1-phenylethanol (1, 12, 20), which is catalyzed by the molybdenum enzyme ethylbenzene dehydrogenase (11, 15, 21). The pathway continues with the oxidation of (S)-1-phenylethanol to acetophenone by an alcohol dehydrogenase (10, 16). Acetophenone may also be produced from (R)-1-phenylethanol or directly used as a substrate. The enzymes of further acetophenone catabolism are uncharacterized. From the observed CO₂ dependence of ethylbenzene and acetophenone degradation, acetophenone was proposed to be carboxylated to benzoylacetate, which is then activated to the coenzyme A (CoA) thioester and thiolytically cleaved to benzoyl-CoA and acetyl-CoA (1, 6, 20, 22). On the level of benzoyl-CoA, the pathway of anaerobic ethylbenzene degradation flows into that of anaerobic benzoate degradation (for reviews, see references 2, 8, and 9) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Proposed catabolic pathway of ethylbenzene, phenylethanol, and acetophenone in A. aromaticum strain EbN1.In this communication, we identify and characterize the postulated enzyme responsible for acetophenone carboxylation in Aromatoleum aromaticum strain EbN1. The enzyme is specifically induced in ethylbenzene- and acetophenone-grown cells. Acetophenone carboxylation is shown to be dependent on ATP hydrolysis, reminiscent of but distinct from the related carboxylation of acetone (25).     

Identification of the Biosynthetic Gene Cluster for the Pseudomonas aeruginosa Antimetabolite l-2-Amino-4-Methoxy-trans-3-Butenoic Acid

l-2-Amino-4-methoxy-trans-3-butenoic acid (AMB) is a potent antibiotic and toxin produced by Pseudomonas aeruginosa. Using a novel biochemical assay combined with site-directed mutagenesis in strain PAO1, we have identified a five-gene cluster specifying AMB biosynthesis, probably involving a thiotemplate mechanism. Overexpression of this cluster in strain PA7, a natural AMB-negative isolate, led to AMB overproduction.The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide range of human infections and is considered the main pathogen responsible for chronic pneumonia in cystic fibrosis patients (7, 23). P. aeruginosa also infects other organisms, such as insects (4), nematodes (6), plants (18), and amoebae (20). Its ability to thrive as a pathogen and to compete in aquatic and soil environments can be partly attributed to the production and interplay of secreted virulence factors and secondary metabolites. While the importance of many of these exoproducts has been studied, the antimetabolite l-2-amino-4-methoxy-trans-3-butenoic acid (AMB; methoxyvinylglycine) (Fig. ​(Fig.1)1) has received only limited attention. Identified during a search for new antibiotics, AMB was found to reversibly inhibit the growth of Bacillus spp. (26) and Escherichia coli (25) and was later shown to inhibit the growth and metabolism of cultured Walker carcinosarcoma cells (28). AMB is a γ-substituted vinylglycine, a naturally occurring amino acid with a β,γ-C=C double bond. Other members of this family are aminoethoxyvinylglycine from Streptomyces spp. (19) and rhizobitoxine, made by Bradyrhizobium japonicum (16) and Pseudomonas andropogonis (15) (Fig. ​(Fig.1).1). As inhibitors of pyridoxal phosphate-dependent enzymes (13, 17, 21, 22), γ-substituted vinylglycines have multiple targets in bacteria, animals, and plants (3, 5, 10, 21, 22, 29). However, the importance of AMB as a toxin in biological interactions with P. aeruginosa has not been addressed, as AMB biosynthesis and the genes involved have not been elucidated.Open in a separate windowFIG. 1.Chemical structures of the γ-substituted vinylglycines AMB, aminoethoxyvinylglycine, and rhizobitoxine.     

Low-dose oral vitamin K therapy for the management of asymptomatic patients with elevated international normalized ratios: a brief review

ASYMPTOMATIC ELEVATION OF THE INTERNATIONAL normalized ratio (INR) is a common problem associated with hemorrhage. Evidence from randomized controlled trials supports the use of low-dose oral vitamin K therapy as a treatment that promptly reduces the INR. Vitamin K given orally is more effective than subcutaneous vitamin K injection, and as effective as intravenous administration when INR values are compared 24 hours after administration. A 1.0-mg vitamin K dose is likely most appropriate for patients with INR values between 4.5 and 10. The fear of over-correction of the INR has limited the widespread use of vitamin K; however, our review suggests that this occurs infrequently when small doses are administered orally.Asymptomatic elevation of the international normalized ratio (INR) is a common and important clinical problem encountered by all health care professionals who supervise patients taking warfarin. Patients in typical outpatient practices have INRs outside the desired range 50% of the time.^1,2 One randomized controlled trial (RCT) suggested that, despite measures to ensure an appropriate level of anticoagulation, 14% of total patient-time was spent with INR values above the therapeutic range.³ There is a strong relation between the degree of INR elevation and the risk of hemorrhage. Serious warfarin-associated bleeding usually occurs from the gastrointestinal or genitourinary system;⁴ the risk of such bleeding may as much as double for each 1-point increase in the INR.⁵ Investigators of a prospective cohort study⁶ followed 114 consecutive patients who presented to an anticoagulation clinic with an INR greater than 6 and found that abnormal bleeding developed in 10 (8.8%) of them and life-threatening hemorrhage in 5 (4.4%; 2 fatal) over the 2-week follow-up period. Therefore, interventions leading to a prompt reduction of the INR may reduce the risk of serious bleeding in patients taking warfarin.Most indications for warfarin anticoagulation have a target therapeutic INR range of 2.0 to 3.0. However, some indications, such as mechanical heart valves, require a higher intensity of anticoagulation. 7^,8 An elevated INR is one that is above the therapeutic range. However, most studies that have evaluated interventions for asymptomatic elevation of the INR have examined INRs several points above the upper limit of the therapeutic range, usually selecting a lower limit for intervention between 4.5 and 6.0. In assessing patients with an elevated INR, one should consider potential causes such as noncompliance, inappropriate dosing, fluctuations in vitamin K intake, hepatic dysfunction, laboratory errors, drug interactions (Box 1) and alcohol intake.Table 1Box 1A common strategy for lowering an elevated INR is simply to withhold warfarin. In some cases parenteral vitamin K therapy may be administered. Recent interest has focused on the use of vitamin K orally as a simple, safe and effective way of normalizing an excessively elevated INR. Although no tablet form of vitamin K is currently available in Canada, the intravenous formulation (see Fig. 1) can be given orally, either undiluted or after mixing with orange juice to mask its unpleasant taste. We reviewed the literature to ascertain whether or not oral vitamin K therapy is effective, to identify the degree of INR abnormality that is best managed with oral therapy, to identify the dose that is most appropriate and to identify the relative risks of hemorrhage and thrombosis with this regimen as compared with other management approaches.Fig. 1: Ampule of vitamin K. Because the tablet form of vitamin K is not currently available in Canada, the parenteral formulation can be given orally. It is dispensed in ampules of 0.5 mL (equivalent to 1.0 mg) and 1.0 mL (equivalent to 10.0 mg). The ...     

Oleate Hydratase Catalyzes the Hydration of a Nonactivated Carbon-Carbon Bond

The hydration of oleic acid into 10-hydroxystearic acid was originally described for a Pseudomonas cell extract almost half a century ago. In the intervening years, the enzyme has never been characterized in any detail. We report here the isolation and characterization of oleate hydratase (EC 4.2.1.53) from Elizabethkingia meningoseptica.The ability of cells to convert oleic acid (OA) into 10-hydroxystearic acid (10-HSA) was discovered by Wallen et al. in Pseudomonas sp. strain 3266 in 1962 (Fig. ​(Fig.1)1) (12). In the following years, many other strains were identified that were also able to convert OA into 10-HSA or to further oxidize it to 10-ketostearic acid (2, 3, 5, 7). The Pseudomonas cells generally start to produce optically pure d-10-HSA in the stationary growth phase, and they do not seem to metabolize it any further, since levels of product accumulate in the fermentation broth. The putative enzyme for this conversion is referred to as oleate hydratase (EC 4.2.1.53); however, so far it has not been purified or characterized in any detail.Open in a separate windowFIG. 1.Reaction catalyzed by oleate hydratase; the conversion of OA into 10-HSA.Kinetic studies have been performed with cell extracts, giving some insight into the stereospecificity and the possible mechanism of the reaction. Studies with ¹⁸O-labeled water reported the incorporation of ¹⁸O at the C-10 position of 10-HSA, confirming a hydration mechanism (7). The reaction was shown to be reversible; however, the detected concentration ratio at equilibrium was always in the range of 85:15 in favor of 10-HSA (9).Here we report the isolation and first biochemical characterization of the oleate hydratase protein from Elizabethkingia meningoseptica (formerly known as Pseudomonas sp. strain 3266).The primer set GM3 and GM4 (8) was used for PCR amplification of the Pseudomonas sp. strain 3266 16S-rRNA genes. The product (1,444 bp) was sequenced, and 16S phylogeny analysis resulted in a unanimous determination of the species as Elizabethkingia (Chryseobacterium) meningoseptica with a >99.8% resemblance.     

Simple,Fast, and Sensitive Method for Quantification of Tellurite in Culture Media

A fast, simple, and reliable chemical method for tellurite quantification is described. The procedure is based on the NaBH₄-mediated reduction of TeO₃²⁻ followed by the spectrophotometric determination of elemental tellurium in solution. The method is highly reproducible, is stable at different pH values, and exhibits linearity over a broad range of tellurite concentrations.The tellurium oxyanion tellurite is toxic for most organisms, making important its accurate assessment. Several methods for quantifying tellurite have been described to date. However, most of them are rather complicated and require sophisticated equipment and in some cases the detection is not quite sensitive enough to allow the assessment of TeO₃²⁻ concentrations below 50 μg/ml (200 μM). For example, the analytical determination of tellurium (Te) oxyanions by atomic absorption spectrometry (AAS) is hampered by poor sensitivity. Where flame or electrothermal AAS routinely yields detection limits of less than 10 ppb for iron (16), normal flame AAS tellurium detection limits are 100 to 1,000 times higher and require pretreatment to achieve the +IV oxidation state before analysis (11).On the other hand, hydride generation AAS (HGAAS) is used to achieve ppb-level detection limits for Se and Te as well as arsenic and antimony among others. For Te the volatile hydride gas H₂Te is generated by first converting the metalloid to the +IV oxidation state and then by chemical reduction to the gaseous hydride using—almost universally—sodium borohydride (NaBH₄). In automated HGAAS systems, an inert purge gas sweeps the volatile hydride formed in a glass reaction vessel into a quartz cell heated by the AAS flame where gaseous hydride decomposition and atomization occur. Though tellurite reduction, precipitation, and detection methods have been reported (3, 17), they are temporally relatively unstable and pH dependent.Since tellurium is toxic and environmentally important (7, 8), determining low concentrations in bacterial cultures is very desirable and a simple analysis without pretreatment steps that could quickly establish total metalloid oxyanion content in a liquid sample would be a plus. Here we report a new method for the determination of tellurite in bacterial culture media. This procedure is based on the NaBH₄ reduction of tellurite to the elemental form, which is analyzed spectrophotometrically at 500 nm or 320 nm (see below), by which the light scattered by the particles of elemental metalloid in solution is measured. While the detection limits do not compare to those of HGAAS (14) or capillary electrophoresis (13), they do approach those of old flame AAS but involve a much simpler and quicker procedure requiring only one reagent and a spectrophotometer to determine total content of solutions of +IV oxyanions in solution. Linear calibration range, method development time and probe stability, effect of sample pH, common interferences, and detection limits were investigated.Calibration curves to determine K₂TeO₃ concentrations in routinely used microbiological culture media such as Luria-Bertani (LB) or M9 minimal medium amended with 0.2% glucose (15) were constructed. A set of solutions containing increasing concentrations of K₂TeO₃ (Sigma) were prepared in LB or M9 culture medium, and the tellurium oxyanion was quantitatively reduced using freshly prepared 3.5 mM NaBH₄ (final concentration).The reaction was carried out at 60°C for 10 min (bubbling was overcome by vortexing), and after 5 min at room temperature, the optical density at 500 nm (OD₅₀₀) was determined spectrophotometrically as described previously (4, 5, 9, 12). Blanks contained no borohydride. Figure ​Figure11 shows that in both media good curve linearity was obtained, with r² values of 0.9740 and 0.9963 for LB and M9, respectively. Tellurite concentrations lower than 1 μg/ml or higher than 200 μg/ml were also tested, but OD₅₀₀ values were close to the spectrophotometer error limit at low concentrations or nonlinear above 200 μg/ml (not shown). Thus, the NaBH₄ method allows determination of a wide range of tellurite concentrations in a fast and simple way. Tellurite concentrations lower than 50 μg/ml in both rich and minimal media can be easily determined; the experimental error was about 10%, similar to that reported for the diethyl dithiocarbamate (DDTC) tellurite method (17).Open in a separate windowFIG. 1.Calibration curves to determine K₂TeO₃ concentrations in LB (A) (R² = 0.9963) or M9 minimal (B) (R² = 0.9740) medium. Optical density at 500 nm was determined after reducing the oxyanion with sodium borohydride. Error bars denote 1 standard deviation of three replicates.To analyze the resulting solutions after tellurite reduction by NaBH₄, absorption/scattering spectra were determined. Figure ​Figure22 shows that spectra from LB and those from M9 after tellurite reduction are quite different, which may be a consequence of the different chemical compositions of these culture media. In both cases, absorption spectra showed linearity between optical density at 500 nm and tellurite concentration in the sample. However, high tellurite concentrations (∼100 μg/ml) caused a loss of linearity in LB medium.Open in a separate windowFIG. 2.Absorption spectra after reducing samples of LB (A) or M9 (B) culture medium containing increasing tellurite concentrations with 3.5 mM NaBH₄. Tellurite concentrations used were 20, 40, 60, 80, and 100 (LB) and 2, 4, 6, 8, and 10 (M9) μg/ml. (Inset) Calibration curve in M9 medium using the absorbance maxima at 320 nm.Figure ​Figure2B2B shows that in M9 medium there is a zone around 320 nm exhibiting higher optical density than that at 500 nm, which represents an advantage in the determination of tellurite in chemically defined culture media. This is reflected in a wider range of measurable concentrations at 320 nm (Fig. ​(Fig.2B,2B, inset), as well as in a higher sensitivity of the method as determined by the slope of the calibration curve. The product of tellurite reduction by NaBH₄ showed good stability at both wavelengths in rich and minimal culture media (not shown).Since in M9 medium the method allows the determination of minor tellurite concentrations (1 to 20 μg/ml), it would be of great help in assessing tellurite uptake in tellurite-sensitive microorganisms whose MICs range from 1 to 10 μg/ml. Sulfur-containing salts, commonly present in culture media as sulfites and sulfates, did not interfere with our NaBH₄ method for tellurite assessment at concentrations up to 0.5 M (not shown).As shown in Fig. ​Fig.3,3, tellurite assessment was not affected by the pH of the culture medium. In fact, linearity was observed in a wide pH range with minor slope changes in LB. Similar results were obtained with M9 medium, although tellurite assessment was not possible at pH values higher than 7.0 because of the formation of a precipitate. This may be due to an interaction of the phosphate salts present in the medium and some charged (2⁺) chemical species forming at alkaline pH values, as has been reported earlier (17).Open in a separate windowFIG. 3.Effect of pH in determining tellurite concentrations in LB (A) and M9 minimal (B) media.To date, the most commonly used procedure for determining tellurite in culture media is that involving the spectrophotometric determination (340 nm) of the complex that forms between tellurite and diethyl dithiocarbamate (17). This procedure has been used to assess tellurite uptake by the phototrophic bacterium Rhodobacter capsulatus, which is naturally resistant to K₂TeO₃ (MIC, ∼1.4 mM) (2, 3). However, K₂TeO₃ uptake studies in highly sensitive cells such as Escherichia coli (MIC, ∼4 μM) are difficult to carry out because of the low concentrations of toxicant present in the culture medium, far below the detection limit of the DDTC procedure (17).In this context and for testing the applicability of our method in vivo, we used the tellurite-sensitive bacterium E. coli BW25113 (10) and the tellurite-resistant Aeromonas caviae ST (5, 6). An overnight culture of E. coli BW25113 in M9 minimal medium was diluted 100-fold with fresh M9 supplemented with 0.2% glucose and grown at 37°C with shaking. When the OD₆₀₀ was 0.1, the culture was amended with 20 μg/ml K₂TeO₃ (arrow, Fig. ​Fig.4A).4A). Then aliquots were taken at the indicated times and cells were centrifuged at 8,500 × g for 3 min; supernatants were used to assess extracellular tellurite by our NaBH₄ method. While added tellurite did not affect bacterial growth (Fig. ​(Fig.4A),4A), the remaining tellurite in the supernatant dropped approximately to one-third after 3 h (Fig. ​(Fig.4B).4B). Tellurite determinations were validated using, in parallel, the DDTC method (not shown).Open in a separate windowFIG. 4.Tellurite uptake by Escherichia coli. Time zero in panel B represents the moment of tellurite addition.Regarding the tellurite-resistant bacterium A. caviae ST, a 1:100 dilution of an overnight culture was inoculated into fresh LB medium and the OD₆₀₀ was recorded at the indicated times. When the OD₆₀₀ was ∼0.4, the culture was amended with tellurite (400 μg/ml final concentration) (Fig. ​(Fig.5A,5A, arrow) and the remaining tellurite in the supernatants was assessed as described above. Figure ​Figure5B5B shows that in 4 h ∼27% of the toxic oxyanion was removed from the culture medium.Open in a separate windowFIG. 5.Tellurite uptake by Aeromonas caviae ST. See the text for details.In summary and in comparison to the DDTC procedure, the NaBH₄ method described here allows more sensitive determination of the initial tellurite concentrations as well as the continuous uptake of the toxicant by tellurite-sensitive and tellurite-resistant microorganisms. This method should be of great help in future studies aimed at unveiling the tellurite reductase activity exhibited by some metabolic enzymes such as nitrate reductase (1), catalase (4), and the pyruvate dehydrogenase complex (5, 6). These studies are currently being carried out in our laboratory.     

Effects of rising tuition fees on medical school class composition and financial outlook

Background

Since 1997, tuition has more than doubled at Ontario medical schools but has remained relatively stable in other Canadian provinces. We sought to determine whether the increasing tuition fees in Ontario affected the demographic characteristics and financial outlook of medical students in that province as compared with those of medical students in the rest of Canada.

Methods

As part of a larger Internet survey of all students at Canadian medical schools outside Quebec, conducted in January and February 2001, we compared the respondents from Ontario schools with those from the other schools (control group). Respondents were asked about their age, sex, self-reported family income (as a direct indicator of socioeconomic status), the first 3 digits of their postal code at graduation from high school (as an indirect indicator of socioeconomic status), and importance of financial considerations in choosing a specialty and location of practice. We used logistic regression models to see if temporal changes (1997 v. 2000) among Ontario medical students differed from those among medical students elsewhere in Canada apart from Quebec.

Results

Responses were obtained from 2994 (68.5%) of 4368 medical students. Across the medical schools, there was an increase in self-reported family income between 1997 and 2000 (p = 0.03). In Ontario, the proportion of respondents with a family income of less than $40 000 declined from 22.6% to 15.0%. However, compared with the control respondents, the overall rise in family income among Ontario students was not statistically significant. First-year Ontario students reported higher levels of expected debt at graduation than did graduating students (median $80 000 v. $57 000) (p < 0.001), and the proportion of students expecting to graduate with debt of at least $100 000 more than doubled. Neither of these differences was observed in the control group. First-year Ontario students were also more likely than fourth-year Ontario students to report that their financial situation was “very” or “extremely” stressful and to cite financial considerations as having a major influence on specialty choice or practice location. These differences were not observed in the control group.

Interpretation

At Canadian medical schools, there are fewer students from low-income families in general. However, Ontario medical students report a large increase in expected debt on graduation, an increased consideration of finances in deciding what or where to practise, and increasing financial stress, factors that are not observed among students in other provinces.Since 1997 all 5 Ontario medical schools have increased tuition fees dramatically. At the University of Toronto, for example, tuition nearly tripled in 3 years, from $4844 in 1997/98 to $14 000 in 2000/01. Tuition fees at other Canadian medical schools have been relatively stable (Fig. 1).Open in a separate windowFig. 1: Medical school tuition and ancillary fees in Ontario and elsewhere in Canada (Quebec excepted), 1997–2000. Source: Canadian Medical Education Statistics.²¹The effects of increasing tuition have not been examined systematically in Canada; however, concern has been expressed that accessibility is being compromised.^1,2,3 A small number of studies in the United States have investigated the effects of increasing tuition;^4,5 several US commentators have also argued that high tuition may restrict access to medical education for those from low-income families, underrepresented minority groups and rural areas.^6,7 Moreover, recent editorials have emphasized the advantages of a diverse, representative medical student body.^8,9,10 Aside from issues of accessibility, medical student diversity may also be beneficial for pragmatic reasons. Students from underrepresented groups are more likely to treat ethnic minorities,^11,12 practise in rural communities^13,14 and work in socioeconomically depressed areas.^15,16,17We studied the effects of increases in tuition fees on the demographic characteristics and attitudes of medical students by comparing students in Ontario, where tuition fees increased rapidly, with students from a control group of Canadian medical schools in provinces that did not experience a rapid increase. Our main hypotheses were that the increases in fees would be associated with increases in family income and expected debt at graduation. We also hypothesized that students in Ontario who enrolled after the increase would feel more financial stress than control students and be more likely to feel that financial considerations would affect their choice of specialty or practice location.     

Biosynthesis of Sibiromycin,a Potent Antitumor Antibiotic

Pyrrolobenzodiazepines, a class of natural products produced by actinomycetes, are sequence selective DNA alkylating compounds with significant antitumor properties. Among the pyrrolo[1,4]benzodiazepines (PBDs) sibiromycin, one of two identified glycosylated PBDs, displays the highest affinity for DNA and the most potent antitumor properties. Despite the promising antitumor properties clinical trials of sibiromycin were precluded by the cardiotoxicity effect in animals attributed to the presence of the C-9 hydroxyl group. As a first step toward the development of sibiromycin analogs, we have cloned and localized the sibiromycin gene cluster to a 32.7-kb contiguous DNA region. Cluster boundaries tentatively assigned by comparative genomics were verified by gene replacement experiments. The sibiromycin gene cluster consisting of 26 open reading frames reveals a “modular” strategy in which the synthesis of the anthranilic and dihydropyrrole moieties is completed before assembly by the nonribosomal peptide synthetase enzymes. In addition, the gene cluster identified includes open reading frames encoding enzymes involved in sibirosamine biosynthesis, as well as regulatory and resistance proteins. Gene replacement and chemical complementation studies are reported to support the proposed biosynthetic pathway.Pyrrolo[1,4]benzodiazepines (PBDs) are a class of natural products found in actinomycetes (Fig. ​(Fig.1)1) and defined by a common pyrrolo[1,4]benzodiazepine ring system (41). They are sequence-selective DNA alkylating agents with significant antitumor properties (21). Once in the minor groove of DNA an aminal bond is formed between the electrophilic C-11 of a PBD and the exocyclic N-2 of a guanine base in a double-stranded DNA (20). Formation of the PBD-DNA complex causes very little distortion of the double-helical structure of DNA (20), and as such this complex is less readily repaired by DNA repair proteins compared to DNA adducts with other alkylating agents (4), significantly contributing to the potency of PBDs. Successful syntheses of PBD analogs have been reported, but synthetic procedures for the more chemically diverse PBDs are laborious and have modest yields (1, 44). In addition, a chemical synthesis for glycosylated PBDs has not yet been accomplished. Structure-activity relationship studies on the synthetically and naturally produced PBDs showed that the C-9 hydroxylation present in anthramycin is the source of the cardiotoxic properties of this compound (Fig. ​(Fig.1)1) (3, 17, 26, 38). These studies also showed that O glycosylation at C7 significantly enhanced DNA-binding affinity (Fig. ​(Fig.1)1) (17). The only known glycosylated PBDs are sibiromycin and sibanomicin produced by Streptosporangium sibiricum and Micromonospora sp., respectively, both containing a sibirosamine moiety (16, 35). Only the producer of sibiromycin is commercially available. A loose correlation between DNA binding affinity and cytotoxicity has been shown with naturally and synthetically produced PBDs (42). Sibiromycin has the highest DNA binding affinity and cytotoxicity with 50% inhibitory concentrations varying from 4 to 1.7 pM in leukemia, plasmacytoma, and ovarian cancer cell lines (42). Despite its potency, further testing of sibiromycin is precluded due to the presence of C-9 hydroxyl group responsible for the cardiotoxic properties. In order to generate analogs of glycosylated PBDs by combinatorial biosynthesis and to exploit their potency, we chose to characterize the sibiromycin gene cluster.Open in a separate windowFIG. 1.(A) Pyrrolobenzodiazepine common ring system. (B) Metabolic precursors and chemical structures of sibiromycin, anthramycin, tomaymycin, and lincomycin A.The metabolic precursors of the pyrrolobenzodiazepine ring of three PBDs (anthramycin, sibiromycin, and tomaymycin) were identified by feeding experiments to be l-tryptophan via the kynurenine pathway for the anthranilate moiety and l-tyrosine for the hydropyrrole moiety (11), suggesting a common biosynthetic pathway for these moieties in PBDs. The tyrosine-to-hydropyrrole transformation has been also identified by feeding studies in the biosynthesis of lincomycin, a lincosamide antibiotic (2) (Fig. ​(Fig.1B).1B). Despite the sequencing of the biosynthetic gene clusters of anthramycin (10) and lincomycin (37), limited functional assignment of open reading frames (ORFs) and elucidation of the biosynthetic pathways were reported partly due to the presence of several gene products with no significant similarities to functionally characterized enzymes. We reasoned that we could take advantage of the identification of the sibiromycin gene cluster not only to try to lay the groundwork for the production of analogs of sibiromycin by combinatorial biosynthesis but also to establish the biosynthetic pathways of the anthranilate and the hydropyrrole moieties by a comparative analysis of the PBDs and lincomycin gene clusters. To help in this analysis, we have also utilized the gene cluster of another PBD, tomaymycin, whose characterization is reported in the accompanying study (24a). The comparative analysis takes advantage of the presence of similarity and differences at the anthranilate and hydropyrrole moieties among these natural products (Fig. ​(Fig.1).1). For example, both anthramycin and sibiromycin contain C-8 methyl and C-9 hydroxyl substituents not present in tomaymycin. However, tomaymycin shares with sibiromycin a C-7 hydroxyl substituent. Therefore, homologous proteins involved in C-9 hydroxylation are expected to be present in the anthramycin and sibiromycin gene cluster but absent in the tomaymycin gene cluster. We applied a similar approach for the biosynthesis of the hydropyrrole moiety using also the lincomycin gene cluster.In the present study, we describe the cloning and sequencing of the sibiromycin gene cluster, the first biosynthetic gene cluster for a glycosylated PBD. Gene replacement experiments were used to confirm that the identified gene cluster was involved in sibiromycin biosynthesis, to define the boundaries of the sibiromycin gene cluster, and to elucidate the biosynthesis of the anthranilate moiety. Using the comparative approach, we were able not only to elucidate the sibiromycin biosynthetic pathway with a certain degree of confidence but also to assign ORFs in the anthramycin gene cluster contributing to the determination of the anthramycin biosynthetic pathway. The proposed biosynthetic pathway for the anthranilic moiety was supported by gene replacement and chemical complementation studies. The data reported here provide the basis for future studies on the enzymes involved in the biochemistry present in these pathways and for combinatorial biosynthetic experiments for the production of glycosylated PBDs.     

In Salmonella enterica, 2-Methylcitrate Blocks Gluconeogenesis

Strains of Salmonella enterica serovar Typhimurium LT2 lacking a functional 2-methylcitric acid cycle (2-MCC) display increased sensitivity to propionate. Previous work from our group indicated that this sensitivity to propionate is in part due to the production of 2-methylcitrate (2-MC) by the Krebs cycle enzyme citrate synthase (GltA). Here we report in vivo and in vitro data which show that a target of the 2-MC isomer produced by GltA (2-MC^GltA) is fructose-1,6-bisphosphatase (FBPase), a key enzyme in gluconeogenesis. Lack of growth due to inhibition of FBPase by 2-MC^GltA was overcome by increasing the level of FBPase or by micromolar amounts of glucose in the medium. We isolated an fbp allele encoding a single amino acid substitution in FBPase (S123F), which allowed a strain lacking a functional 2-MCC to grow in the presence of propionate. We show that the 2-MC^GltA and the 2-MC isomer synthesized by the 2-MC synthase (PrpC; 2-MC^PrpC) are not equally toxic to the cell, with 2-MC^GltA being significantly more toxic than 2-MC^PrpC. This difference in 2-MC toxicity is likely due to the fact that as a si-citrate synthase, GltA may produce multiple isomers of 2-MC, which we propose are not substrates for the 2-MC dehydratase (PrpD) enzyme, accumulate inside the cell, and have deleterious effects on FBPase activity. Our findings may help explain human inborn errors in propionate metabolism.Humans have used fermentation as an effective method of preservation for a wide variety of foods (41). Today, the weak short-chain fatty acids (SCFAs) produced by fermentation, such as acetic, propionic, butyric, and lactic acids, are widely used as food preservatives and in pre- and postharvest agricultural processes (34, 38, 45). Propionate, one of the most abundant SCFAs found in the environment (12), is widely used as a preservative of baked goods in the food industry (38).While SCFAs such as propionate are extensively used as food preservatives, our understanding of how microbial growth is prevented by them is incomplete. Early studies argued that growth inhibition either was caused by dissipation of the proton motive force (4, 48) or was due to decreases in intracellular pH (15, 48) or the intracellular accumulation of the propionate anion (46, 47). More recently, the global affects of SCFAs on gene expression (1, 43, 44) and protein synthesis (8, 37, 52, 56) were reported, revealing wide-ranging effects on gene expression in response to propionate in the environment (43). Evidence also suggests that central metabolic processes may be inhibited by SCFAs or their catabolites. An overview of the effects of propionate on the cell can be seen in Fig. ​Fig.11.Open in a separate windowFIG. 1.Overview of propionate metabolism and toxicity in Salmonella.Propionyl coenzyme A (Pr-CoA), an intermediate in propionate metabolism, was shown to inhibit pyruvate dehydrogenase in Rhodobacter sphaeroides (40) and Aspergillus niger (10) and competitively inhibit citrate synthase in Escherichia coli (39). 2-Methylcitrate (2-MC), the product of the condensation of oxaloacetate (OAA) and Pr-CoA, was shown to inhibit growth of Salmonella enterica, but the mechanism of action remained unclear (28) (Fig. ​(Fig.1).1). With such broad negative effects exerted by propionate or its catabolites, the best strategy for microbes to deal with SCFAs such as propionate is to efficiently catabolize them into central metabolites (Fig. ​(Fig.11).S. enterica, like many other enteric bacteria, is exposed to high levels of propionate in human digestive tracts with total SCFA levels varying from 20 to 300 mM and propionate reaching levels as high as 23.1 mmol/kg (9, 17). To cope with such high concentrations of propionate, this bacterium and other enterobacteria like E. coli utilize the 2-methylcitric acid cycle (2-MCC) to convert propionate to pyruvate (31, 53). In S. enterica, the prpBCDE operon encodes most of the 2-MCC enzymes (30). These genes encode a 2-methylisocitrate lyase (PrpB), a 2-methylcitrate synthase (PrpC), a 2-methylcitrate dehydratase (PrpD), and a propionyl coenzyme A (CoA) synthetase (PrpE) (Fig. ​(Fig.1).1). Early work with S. enterica showed that insertion elements placed within the prpBCDE operon greatly increased the sensitivity of S. enterica to propionate (23). Strains carrying insertions in prpE, however, were still able to grow on propionate and were not sensitive to propionate because acetyl-CoA synthetase (Acs) compensates for the lack of PrpE (32).The goal of the studies reported here was to identify a target of 2-MC in S. enterica. Our in vivo and in vitro data support the conclusion that 2-MC inhibits fructose-1,6-bisphosphatase (FBPase), a key enzyme of gluconeogenesis. The inhibition of FBPase blocks the synthesis of glucose, with the concomitant broad negative effects on cell function. We show that while both the 2-MC synthase (PrpC) and citrate synthase (GltA) enzymes synthesize 2-MC, the 2-MC made by GltA (2-MC^GltA) is more toxic to the cell than the 2-MC made by PrpC (2-MC^PrpC), and we suggest that the reason for this toxicity is due to the difference in stereochemistry of the GltA and PrpC reaction products.     

Targeted Transduction via CD4 by a Lentiviral Vector Uses a Clathrin-Mediated Entry Pathway

We recently developed a novel targeting Sindbis virus envelope pseudotyped lentiviral vector, 2.2ZZ, which acquires specific transduction capacity by antibody conjugation and binding with specific antigens on the surface of targeted cells. Here we characterize the virological properties of this vector by examining its targeting to CD4 antigen. Our results show that entry is dependent on CD4 cell surface density and occurs via the clathrin-mediated endocytic pathway. These findings provide insight into the mechanism of infection by a new viral vector with combined properties of Sindbis virus and lentiviruses and infectivity conferred by monoclonal antibody-ligand interactions.Effective gene therapy in clinical settings will require the targeting of such therapies to specific tissues and organs while maintaining stable gene expression. We describe a novel targeting vector which acquires specific transduction capacity by antibody conjugation and binding to specific antigens on the surface of targeted cells. Our lab modified the fusion protein of Sindbis virus envelope, E2, by inserting an Fc-binding portion, the ZZ domain, of protein A (17, 19, 21). The lentiviral vector pseudotyped by this modified Sindbis envelope, which we designated 2.2ZZ vector, binds to and enters cells bearing specific cell surface antigens only when conjugated with the appropriate monoclonal antibody (17, 21). The 2.2ZZ vector has been used to target human leukocyte antigen (HLA) class I, CD4, CD19, CD20, CD34, CD45, CD146, P glycoprotein of melanoma cells, and prostate stem cell antigen successfully (10, 14-17, 20, 21). Wang and coworkers adapted an early form of 2.2ZZ, M168 (17), to generate a modified envelope with membrane-bound antibodies and used it to successfully target CD20 on B cells (27).Here, we characterized the virological properties of this newly generated 2.2ZZ targeting vector, one that bears some properties of Sindbis virus and some properties of lentiviruses and also possesses certain novel properties of infectivity conferred by monoclonal antibody-ligand interactions, by studying the effect of surface receptor concentration on its transduction and its endocytic pathways. Native Sindbis virus exploits the clathrin-mediated pathway to enter cells (6, 8), whereas human immunodeficiency virus (HIV) fuses directly with the plasma membrane (25), although recent evidence suggests that HIV enters cell via endocytosis (13). Our goal was to ascertain the pathway of viral entry for this chimeric virus. We therefore examined 2.2ZZ vector targeting to CD4 antigen, since it is one of the best-characterized cell surface molecules with regard to its clathrin-mediated internalization and signaling pathway (22). We have previously demonstrated the viral specificity by targeting transduction to HLtat/CD4 cells and peripheral blood mononuclear cells via anti-CD4 antibodies (14). In this study, we first tested three antibodies which target different epitopes of CD4: the anti-CDR2-like region of CD4 in domain I (Leu3a) (12), the anti-CDR2-like region of CD4 in domain III (OKT4) (12), and domain II of CD4 (BL4) (2). Each antibody directs similar transduction efficiencies of the 2.2ZZ vector (data not shown). We selected the BL4 antibody for further studies of mechanics of viral transduction.We examined whether surface receptor concentrations have an effect on vector transduction by using a 293 Affinofile cell line that inducibly expresses human CD4 molecules under the control of tetracycline (7, 9). Transduction by vesicular stomatitis virus G (VSV-G) pseudotypes and transduction of 2.2ZZ directed by antibodies to HLA class I showed no significant differences in transduction levels among cells expressing different numbers of CD4 molecules (P > 0.05) (Fig. ​(Fig.1B).1B). On the other hand, enhanced transduction was observed when anti-CD4 antibodies were used, and that enhancement correlated with the increase of CD4 molecules present on cell surfaces (Fig. ​(Fig.1A).1A). These findings show that on this cell line higher receptor concentration results in increased transduction by the 2.2ZZ vector.Open in a separate windowFIG. 1.Higher density of CD4 molecules on cell surfaces led to increased transduction of 2.2ZZ. (A) 293 Affinofile cells (1 × 10⁵) carrying the tetracycline-inducible CD4 expression system were treated with different concentrations of tetracycline (0, 3.125, 6.25, 12.5, and 50 ng/ml) for 8 h. The cells were stained with anti-CD4 antibodies conjugated with PE. The number of CD4 molecules/cell was determined by normalizing the mean fluorescence of the cells to that of commercial PE beads. (B) After 8 h of induction with tetracycline, cells were transduced by 20 ng (HIV-1 p24) VSV-G pseudotyped lentiviral vectors, 2.2ZZ vectors with 0.4 μg anti-HLA or anti-CD4 antibodies, and 2.2ZZ vectors in the absence of antibodies, all for a period of 2 h. Three days postinfection, transductions were monitored by EGFP expression. P values represent significances of differences among cells treated with different concentrations of tetracycline.To determine whether the endocytic pathway is required for entry of 2.2ZZ, we first utilized a dominant-negative mutant of dynamin (dyn^K44A) to block the endocytic pathway (5). The dynamin wild type (dyn^WT) was used as a control. Both plasmids were transiently transfected into the 293T cells stably expressing CD4 molecules (293T/CD4 cells) to acquire at least 65% of dynamin expression (Fig. ​(Fig.2A).2A). We then assessed transduction efficiency of 2.2ZZ vectors with anti-CD4 antibodies in the dyn^WT- and dyn^K44A-transfected cells. VSV-G pseudotypes served as a positive control, since VSV enters cells via the clathrin-mediated pathway (6, 26). Gibbon ape leukemia virus (GALV) pseudotypes served as a negative control, since the fusion of GALV occurs at the plasma membrane (18). For VSV-G pseudotypes and 2.2ZZ vectors with anti-HLA or anti-CD4 antibodies, the transduction efficiency was reduced in the dyn^K44A-transfected cells compared to the dyn^WT-transfected cells (P < 0.05) (Fig. ​(Fig.2A).2A). No difference in transduction by GALV pseudotypes was observed in the dyn^WT- and dyn^K44A-transfected cells (P > 0.05). Since the surface levels of HLA and CD4 are not significantly different between the dyn^WT- and dyn^K44A-transfected cells (P > 0.05) (Fig. ​(Fig.2A),2A), the difference in transduction efficiency does not result from differences in expression levels of the surface receptors. These data show that overexpression of dyn^K44A suppressed the transduction of 2.2ZZ using the CD4 molecule for entry, indicating that endocytosis likely plays a role in the entry of the 2.2ZZ vector.Open in a separate windowFIG. 2.Transduction of 2.2ZZ in 293T/CD4 cells was blocked by the dominant-negative mutant of dynamin and decreased in the CD4 mutant cells. (A) (Top) Three micrograms of hemagglutinin-tagged wild-type dynamin or dominant-negative mutant of dynamin was transfected into 1.2 × 10⁶ 293T cells stably expressing CD4 molecules by FuGENE (Roche). Cells (1 × 10⁵) were stained with antihemagglutinin antibodies 24 h posttransfection. (Bottom left) Forty-eight hours posttransfection, 1 × 10⁵ cells were transduced by 20 ng (HIV-1 p24) VSV-G pseudotyped lentiviral vectors, 2.2ZZ vectors with 0.4 μg anti-HLA or anti-CD4 antibodies, 2.2ZZ vectors in the absence of antibodies, and GALV pseudotyped lentiviral vectors for 2 h. Three days postinfection, transductions were monitored by EGFP expression. P values represent significances of differences between dyn^WT- and dyn^K44A-transfected cells. (Bottom right) Staining of the surface HLA and CD4 molecules. The number of HLA or CD4 molecules/cell was determined by normalizing the mean fluorescence of the cells to that of commercial PE beads. P values represent significances of differences between dyn^WT- and dyn^K44A-transfected cells. (B) (Left) Staining of the surface CD4 molecules on 293T cells stably expressing wild-type CD4, Ser408A CD4, and truncated CD4. The number of CD4 molecules/cell was determined by normalizing the mean fluorescence of the cells to that of commercial PE beads. P value represents significance of difference among cells expressing wild-type CD4, Ser408 CD4, and truncated CD4. (Right) 293T cells (1 × 10⁵) stably expressing wild-type CD4, Ser408A CD4, and truncated CD4 were transduced by 20 ng (HIV-1 p24) VSV-G pseudotyped lentiviral vectors, 2.2ZZ vectors with 0.4 μg anti-HLA or anti-CD4 antibodies, and 2.2ZZ vectors in the absence of antibodies for 2 h. Three days postinfection, transductions were monitored by EGFP expression. P values represent significances of differences among cells expressing wild-type CD4, Ser408 CD4, and truncated CD4.We also tested the role of endocytosis in the entry of 2.2ZZ using CD4 genes that differ in endocytosis properties. Mutation or truncation of the cytoplasmic tail results in mutants of CD4 that internalize at reduced rates (1). While wild-type CD4 molecules internalize at a rate of 5%/min, the mutant CD4 molecules internalize at a rate that is fourfold lower (24). We generated 293T cell lines that stably express wild-type CD4, CD4 with a mutation at Ser408, and cytoplasmic tail-truncated CD4. Staining by phycoerythrin (PE)-conjugated antibodies (clone S3.5; Caltag) of domain I of CD4 showed similar levels of CD4 molecules on the surfaces of cells (P > 0.05) (Fig. ​(Fig.2B).2B). VSV-G pseudotypes and 2.2ZZ vectors with anti-CD4 antibodies were used to transduce cells expressing either wild-type or mutant CD4. For VSV-G pseudotypes and 2.2ZZ vectors with anti-HLA antibodies, transduction efficiency was not significantly different between cells expressing wild-type and those expressing mutant CD4 (P > 0.05) (Fig. ​(Fig.2B).2B). However, transduction efficiency by the 2.2ZZ vector with anti-CD4 antibodies was significantly reduced (P < 0.05) in cells expressing mutant CD4 (Fig. ​(Fig.2B).2B). These data suggest that 2.2ZZ targeted to CD4 on the cell surface enters those cells via endocytosis of the CD4 molecules.The most common endocytic pathway through which viruses enter cells is the clathrin-mediated pathway, the classic low-pH-dependent pathway. To determine whether the clathrin pathway is required for the 2.2ZZ vector, as it is for native Sindbis virus, we first blocked the clathrin pathway by neutralizing the endosome environment using 125 nM bafilomycin A (23) for 30 min. The 293T/CD4 cells were treated with bafilomycin A 30 minutes before, and again during, transduction. VSV-G pseudotypes, 2.2ZZ vectors with anti-HLA or anti-CD4 antibodies, and GALV pseudotypes were examined for transduction efficiency. Transductions by VSV-G pseudotypes and 2.2ZZ vectors with anti-HLA or anti-CD4 antibodies were all blocked in cells treated with bafilomycin A (Fig. ​(Fig.3A),3A), while transductions by GALV pseudotypes were not affected. Since the surface levels of HLA and CD4 are not significantly different between the non-reagent-treated and bafilomycin A-treated cells (P > 0.05) (Fig. ​(Fig.3B),3B), the difference in transduction efficiency does not result from differences in expression levels of the surface receptors. These results are consistent with utilization of the endocytic pathway by native VSV as well as the HLA and CD4 molecules and further indicate that the entry of 2.2ZZ vector is via clathrin-mediated endocytosis.Open in a separate windowFIG. 3.Acidification inhibitors blocked transduction of 2.2ZZ. (A) 293T cells (1 × 10⁵) stably expressing CD4 molecules were pretreated with 125 nM bafilomycin A for 30 min. Cells were then transduced by 20 ng (HIV-1 p24) VSV-G pseudotyped lentiviral vectors, 2.2ZZ vector with 0.4 μg anti-HLA or anti-CD4 antibodies, and GALV pseudotyped lentiviral vectors for 2 h in the presence of bafilomycin A. Two hours later, viruses were removed and cells were washed once with 1× phosphate-buffered saline and cultured in medium for 3 days. Transductions were monitored by EGFP expression. (B) Staining of the surface HLA and CD4 molecules. The number of HLA or CD4 molecules/cell was determined by normalizing the mean fluorescence of the cells to that of commercial PE beads. P values represent significances of differences between non-reagent-treated and bafilomycin A-treated cells.Besides using acidification inhibitors, we applied a more direct means that utilized a dominant-negative mutant of Eps15, EΔ95/295, to demonstrate that infectivity occurs via the clathrin-mediated pathway (3, 4). Eps15 is a protein that binds to the AP-2 adapter required for internalization by clathrin-coated pits. The dominant-negative form of Eps15 inhibits endocytosis by clathrin-coated pits by competing with the endogenous Eps15 for AP-2 (3). The 293T/CD4 cells were transiently transfected with EΔ95/295. Another mutant, DIIIΔ2 (3, 4), which lacks the AP-2 binding domain, was used as a negative control. These two plasmids, EΔ95/295 and DIIIΔ2, contain the enhanced green fluorescent protein (EGFP), and so transfection efficiency could be monitored by measuring the EGFP⁺ cells. Lentiviral vectors (FU11mCherry) that express mCherry protein instead of EGFP were used to generate the VSV-G pseudotypes and 2.2ZZ vectors. The 293T/CD4 cells overexpressing the EGFP-tagged EΔ95/295 and DIIIΔ2 proteins were transduced by VSV-G pseudotypes and 2.2ZZ vectors conjugated with anti-HLA or anti-CD4 antibodies. Amphotropic murine retrovirus fuses at the plasma membrane (11); therefore, lentiviral vectors pseudotyped by amphotropic murine retroviral envelope were included as a negative control. Infectivity was assessed by measuring the mCherry⁺ cells. Our results showed that transductions by VSV-G pseudotypes and 2.2ZZ vectors with anti-HLA or anti-CD4 antibodies were markedly inhibited within the EGFP⁺ cell populations (measured by ratio of mCherry⁺/EGFP⁺ to mCherry⁺/EGFP⁻ cells) overexpressing the EΔ95/295 protein compared to cells overexpressing the DIIIΔ2 protein (P < 0.05) (Fig. 4A and B). Transductions by amphotropic pseudotypes were not significantly affected by the clathrin pathway inhibitor, EΔ95/295. Since the surface levels of HLA and CD4 are not significantly different between the EΔ95/295- and DIIIΔ2-transfected cells (Fig. ​(Fig.4C),4C), the difference in transduction efficiency does not result from differences in expression levels of the surface receptors. These results confirm that 2.2ZZ targeting to the CD4 molecule enters cells via the clathrin-mediated pathway.Open in a separate windowFIG. 4.Transduction of 2.2ZZ in 293T/CD4 cells was blocked by dominant-negative mutant of Eps15. (A) Three micrograms of EGFP-fused dominant-negative mutant of Eps15 (EΔ95/295) and DIIIΔ2, which lacks the AP-2 binding domain, was transfected into 1.2 × 10⁶ 293T cells stably expressing CD4 molecules by FuGENE. Forty-eight hours posttransfection, 1 × 10⁵ cells were transduced by 100 ng (HIV-1 p24) VSV-G pseudotyped lentiviral vectors or 2.2ZZ vector with 0.4 μg anti-HLA or anti-CD4 antibodies for 2 hours and 5 × 10⁴ cells were transduced by 4 μl 100× concentrated amphotropic retroviral envelope pseudotyped lentiviral vectors for 6 h. Three days postinfection, transfections were monitored by EGFP expression and transductions were monitored by mCherry expression. (B) Calculations of the ratios of mCherry⁺/EGFP⁺ to mCherry⁺/EGFP⁻ cells. P values represent significances of differences between EΔ95/295- and DIIIΔ2-transfected cells. (C) Staining of the surface HLA and CD4 molecules. The number of HLA or CD4 molecules/cell was determined by normalizing the mean fluorescence of the cells to that of commercial PE beads. P values represent significances of differences between EΔ95/295- and DIIIΔ2-transfected cells.Our results provide insight into the mechanism of infection by our targeting vectors. The efficiency of transduction increases with greater CD4 receptor density and higher rates of endocytosis. Thus, the properties of any given receptor will be critical for the future application of targeting to specific cells for laboratory or clinical purposes.These results have implications for the evolution of viral entry processes. The clathrin-dependent pathway for endocytosis utilized by ZZ virus entry when directed to CD4 as a receptor is the same pathway utilized by native Sindbis virus envelope, which utilizes heparin sulfate and laminin as receptors. Thus, redirection of viral tropism to utilize completely different binding receptors still maintains the same fundamental pathway for entry. Using antibodies to redirect viral infectivity through different cell surface molecules can be considered to reflect a natural evolutionary process whereby viruses acquire the ability to utilize different cell surface receptors. In nature, this evolution probably occurs through genetic variation rather than through bridging molecules such as antibodies; however, regardless of the mechanism, the first step in viral infection is the acquisition of binding to new cell surface receptors. Our results suggest that the mechanics of subsequent steps of internalization and fusion are conserved and therefore occur independently of the initial ligand-receptor interaction. Thus, at least in the case of Sindbis virus, docking of envelope to the receptor and subsequent internalization and fusion events are likely to have evolved independently.     

Antibody Recognition Force Microscopy Shows that Outer Membrane Cytochromes OmcA and MtrC Are Expressed on the Exterior Surface of Shewanella oneidensis MR-1

Antibody recognition force microscopy showed that OmcA and MtrC are expressed on the exterior surface of living Shewanella oneidensis MR-1 cells when Fe(III), including solid-phase hematite (Fe₂O₃), was the terminal electron acceptor. OmcA was localized to the interface between the cell and mineral. MtrC displayed a more uniform distribution across the cell surface. Both cytochromes were associated with an extracellular polymeric substance.Shewanella oneidensis MR-1 is a dissimilatory metal-reducing bacterium that is well known for its ability to use a variety of anaerobic terminal electron acceptors (TEAs), including solid-phase iron oxide minerals, such as goethite and hematite (8, 10). Previous studies suggest that S. oneidensis MR-1 uses outer membrane cytochromes OmcA and MtrC to catalyze the terminal reduction of Fe(III) through direct contact with the extracellular iron oxide mineral (2, 8, 10, 15, 16, 20, 21, 23). However, it has yet to be shown whether OmcA or MtrC is actually targeted to the external surface of live S. oneidensis MR-1 cells when Fe(III) serves as the TEA.In the present study, we used atomic force microscopy (AFM) to probe the surface of live S. oneidensis MR-1 cells, using AFM tips that were functionalized with cytochrome-specific polyclonal antibodies (i.e., anti-OmcA or anti-MtrC). This technique, termed antibody recognition force microscopy (Ig-RFM), detects binding events that occur between antibodies (e.g., anti-OmcA) on an AFM tip and antigens (e.g., OmcA) that are exposed on a cell surface. While this is a relatively new technique, Ig-RFM has been used to map the nanoscale spatial location of single molecules in complex biological structures under physiological conditions (5, 9, 11, 13).Anti-MtrC or anti-OmcA molecules were covalently coupled to silicon nitride (Si₃N₄) cantilevers (Veeco or Olympus) via a flexible, heterofunctional polyethylene glycol (PEG) linker molecule. The PEG linker consists of an NHS (N-hydroxysuccinimide) group at one end and an aldehyde group at the other end (i.e., NHS-PEG-aldehyde). AFM tips were functionalized with amine groups, using ethanolamine (6, 7). The active NHS ester of the NHS-PEG-aldehyde linker molecule was then used to form a covalent linkage between PEG-aldehyde and the amine groups on the AFM tips (6, 7). Next, anti-MtrC or anti-OmcA molecules were covalently tethered to these tips via the linker molecule''s aldehyde group. This was accomplished by incubating the tips with antibody (0.2 mg/ml) and NaCNBH₃ as described previously (7). The cantilevers were purchased from Veeco and had spring constant values between 0.06 and 0.07 N/m, as determined by the thermal method of Hutter and Bechhoefer (12).Prior to conducting the Ig-RFM experiments, the specificity of each polyclonal antibody (i.e., anti-OmcA and anti-MtrC) for OmcA or MtrC was verified by Western blot analysis as described previously (24, 28). Proteins were resolved by both denaturing and nondenaturing polyacrylamide gel electrophoresis (PAGE). Briefly, 2.5 μg of purified OmcA or MtrC (23) was resolved by sodium dodecyl sulfate-PAGE or native PAGE, transferred to a polyvinylidene difluoride membrane, incubated with either anti-OmcA or anti-MtrC, and then visualized using the Amersham ECL Plus Western blotting detection kit. Anti-OmcA bound exclusively to OmcA, anti-MtrC bound exclusively to MtrC, and neither antibody showed cross-reactivity with the other cytochrome. Antibody specificities of anti-OmcA and anti-MtrC were also validated by immunoblot analysis of S. oneidensis whole-cell lysate (28).To determine if MtrC or OmcA was expressed on the external surface of live bacteria when Fe(III) served as the TEA, Ig-RFM was conducted on wild-type versus ΔomcA ΔmtrC double mutant cells. For these experiments, bacteria were cultivated anaerobically with Fe(III), in the form of Fe(III) chelated to nitrilotriacetic acid (NTA), serving as the TEA (19, 23). Growth conditions have been described elsewhere (3, 15) and were based on previous studies (3, 15, 16, 18) that suggest that S. oneidensis MR-1 targets OmcA and MtrC to the cell surface when Fe(III) serves as the TEA.An Asylum Research MFP-3D-BIO AFM or a Digital Instruments Bioscope AFM (16, 17) was used for these experiments. The z-piezoelectric scanners were calibrated as described previously (17). Cells were deposited on a hydrophobic glass coverslip and immersed in imaging buffer (i.e., phosphate-buffered saline [pH 7.4]). The hydrophobic glass coverslips were made as described previously (17) using a self-assembling silane compound called octadecyltrichlorosilane (OTS; Sigma-Aldrich). S. oneidensis MR-1 cells readily adsorbed onto OTS glass coverslips and remained attached to the coverslips during the entire experiment. No lateral cell movement was observed during the experiment, consistent with previous studies that used OTS glass to immobilize bacteria (15, 17, 18, 27).The AFM tip was brought into contact with the surface of a bacterium, and the antibody-functionalized tip was repeatedly brought into and out of contact with the sample, “fishing” for a binding reaction with cytochrome molecules that were exposed on the external cell surface. Binding events were observed upon separating anti-OmcA- or anti-MtrC-functionalized tips from wild-type S. oneidensis MR-1 cells (Fig. ​(Fig.1).1). For the wild-type cells, we observed both nonspecific and specific interactions (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Retraction force curves for anti-MtrC-functionalized tips (A) and anti-OmcA-functionalized tips (B) that are being pulled away from the surface of living ΔomcA ΔmtrC double mutant (gray dotted line) or wild-type (solid black line) S. oneidensis MR-1. These bacteria were adsorbed onto OTS glass coverslips. (C) Retraction curves exhibiting nonspecific binding, specific binding, or no binding between the AFM tip and the cell surface.The distinction between “specific” and “nonspecific” adhesion is made by observing the change in slope of the force curve during the retraction process (26). During specific binding (Fig. ​(Fig.1C),1C), the cantilever is initially relaxed as it is pulled away from the sample. Upon further retraction, the ligand-receptor complex becomes stretched and unravels, resulting in a nonlinear force profile as noted in references 26 and 16. On the other hand, nonspecific adhesion (Fig. ​(Fig.1C)1C) maintains the same slope during the retraction process because only the cantilever flexes (26).Figure ​Figure22 summarizes the frequency or probability of observing a binding event for both anti-OmcA and anti-MtrC tips. Each bar in Fig. ​Fig.22 represents one experiment in which 500 to 1,000 force curves were collected between one AFM tip and two to four live bacterial cells. This figure does not make a distinction between specific and nonspecific binding. It simply shows the frequency of observing an attractive interaction as the antibody-functionalized tip was pulled away from the surface of S. oneidensis MR-1. Binding events occurred with roughly the same frequency when wild-type S. oneidensis MR-1 cells were probed with anti-MtrC-functionalized tips as when they were probed with anti-OmcA-functionalized tips (Fig. ​(Fig.22).Open in a separate windowFIG. 2.Histograms showing the frequency of observing a binding event for anti-MtrC-functionalized (blue) or anti-OmcA-functionalized (red) AFM tips on live wild-type S. oneidensis MR-1 (solid bars) or ΔomcA ΔmtrC double mutant (diagonally hatched bars) cells. The downward arrows designate injection of free antibody into the imaging buffer. The solid gray bars correspond to results obtained with unbaited AFM tips.A number of control experiments were performed to verify the detection of OmcA and MtrC on the surface of wild-type S. oneidensis MR-1. First, 0.1 μM of free anti-OmcA (or anti-MtrC) was added to the imaging fluid to block binding between the antibody-functionalized AFM tip and surface-exposed cytochromes (11, 16). This decreased the adhesion that was observed between the antibody-functionalized tip and the cell surface (Fig. ​(Fig.22).Second, we performed force measurements on ΔomcA ΔmtrC double mutant S. oneidensis MR-1 cells. This mutant is deficient in both OmcA and MtrC (19, 23, 24) but produces other proteins native to the outer surface of S. oneidensis MR-1. The resulting force spectra showed a noticeable reduction in binding events for the ΔomcA ΔmtrC double mutant cells (Fig. ​(Fig.2).2). The binding events that were observed for the double mutant were only nonspecific in nature (Fig. ​(Fig.1).1). This indicates that the antibodies on the tip do not participate in specific interactions with other proteins on the surface of S. oneidensis MR-1 cells.As a final control experiment, force measurements were conducted on wild-type S. oneidensis MR-1 cells, using Si₃N₄ tips conjugated with the PEG linker but not functionalized with polyclonal antibody (unbaited tips). Like the results with the double mutant, the unbaited tips were largely unreactive with the surface of the bacteria (Fig. ​(Fig.2).2). Those binding events that were observed were nonspecific in nature. Taken together, these results demonstrate that the antibody-coated tips have a specific reactivity with OmcA and MtrC molecules. Furthermore, these force measurements show that MtrC and OmcA are present on the external cell surface when Fe(III) serves as the TEA.To map the distribution of cytochromes on living cells, Ig-RFM was conducted on living S. oneidensis MR-1 cells that were growing on a hematite (α-Fe₂O₃) thin film. The conditions for these experiments were as follows. A hematite film was grown on a 10-mm by 10-mm by 1-mm oxide substrate via oxygen plasma-assisted molecular beam epitaxy (14, 16). The cells were grown anaerobically to mid-log phase with Fe(III)-NTA serving as the TEA. Cells were deposited onto the hematite thin film along with anaerobic growth medium that lacked Fe(III)-NTA. The cells were allowed to attach to the hematite surface (without drying) overnight in an anaerobic chamber. The following day, the liquid was carefully removed and immediately replaced with fresh anaerobic solution (pH 7.4). Ig-RFM was performed on the cells by raster scanning an antibody-functionalized AFM tip across the sample surface, thereby creating an affinity map (1). Force curves were collected for a 32-by-32 array. The raw pixilated force-volume data were deconvoluted using a regularized filter algorithm. The total time to acquire a complete image was approximately 20 min.As noted above, attractive interactions between an antibody tip and cell resulted in relatively short-range, nonspecific and longer-range, specific adhesive forces (Fig. ​(Fig.1C).1C). To distinguish between these two interactions, we integrated each force curve beginning at >20 nm and ending at the full retraction of the piezoelectric motor (∼1,800 nm). This integration procedure quantifies the work of binding, measured in joules, between the antibody tip and a particular position on the sample. While this integration procedure does not totally exclude nonspecific binding, it does select for those events associated primarily with specific antibody-antigen binding. Figure ​Figure33 is the antibody-cytochrome recognition images for MtrC and OmcA. The corresponding height (or topography) images of the bacterial cells are also shown in Fig. ​Fig.33.Open in a separate windowFIG. 3.Ig-RFM of live S. oneidensis MR-1 cells deposited on a hematite (α-Fe₂O₃) thin film. Height image (A) and corresponding Ig-RFM image (B) for a bare unfunctionalized Si₃N₄ tip. Height and corresponding Ig-RFM image for a tip functionalized with anti-MtrC (C and D) or anti-OmcA (E and F). Each panel contains a thin white oval showing the approximate location of the bacterium on the hematite surface. A color-coded scale bar is shown on the right (height in micrometers [μm], and the work required to separate the tip from the surface in attojoules [aJ]).OmcA molecules were concentrated at the boundary between the bacterial cell and hematite surface (Fig. 3E and F). MtrC molecules were also detected at the edge of a cell (Fig. 3C and D). Some MtrC, unlike OmcA, was observed on the cell surface distal from the point of contact with the mineral (Fig. 3C and D). Both OmcA and MtrC were also present in an extracellular polymeric substance (EPS) on the hematite surface (Fig. 3D and F), which is consistent with previous results showing MtrC and OmcA in an EPS produced by cells under anaerobic conditions (19, 24). This discovery is interesting in light of the research by Rosso et al. (22) and Bose et al. (4), who found that Shewanella can implement a nonlocal electron transfer strategy to reduce the surface of hematite at locations distant from the point of cell attachment. Rosso et al. (22) proposed that the bacteria utilize unknown extracellular factors to access the most energetically favorable regions of the Fe(III) oxide surface. The Ig-AFM results (Fig. ​(Fig.3)3) suggest the possibility that MtrC and/or OmcA are the “unknown extracellular factors” that are synthesized by Shewanella to reduce crystalline Fe(III) oxides at points distal from the cell. Additional experiments showing reductive dissolution features coinciding with the extracellular location of MtrC and/or OmcA would need to be performed to test this hypothesis.It is important to note that these affinity maps were collected on only a few cells because it so challenging to produce large numbers of quality images. Future work should be conducted on a population of cells. Until this time, these affinity maps can be used to provide a crude, lowest-order estimate of the number of cytochromes on the outer surface of living S. oneidensis MR-1. For example, there were 236 force curves collected on the bacterium shown in Fig. ​Fig.3D.3D. Thirty-eight of these curves exhibited a distinct, sawtooth-shaped, antibody-antigen binding event. In other words, MtrC molecules were detected in one out of every six force curves (16%) that were collected on the cell surface.This probability can be compared to other independent studies that estimated the density and size of MtrC and OmcA molecules from S. oneidensis MR-1. Lower et al. (16) estimated that S. oneidensis has 4 × 10¹⁵ to 7 × 10¹⁵ cytochromes per square meter by comparing AFM measurements for whole cells to force curves on purified MtrC and OmcA molecules. Wigginton et al. (25) used scanning tunneling microscopy to determine that the diameter of an individual cytochrome is 5 to 8 nm. These values can be used to create a simple, geometric, close-packing arrangement of MtrC or OmcA molecules on a surface. Using this approach, cytochromes could occupy 8 to 34% of the cell surface.This estimate is consistent with the observed number of putative MtrC molecules shown in Fig. ​Fig.3D.3D. Therefore, it appears that these affinity maps can be used as a lowest-order estimate for the number of cytochromes on S. oneidensis MR-1 even though we do not know a priori the exact configuration of the antibody tip (e.g., the concentration of antibody on the tip, the exact shape of the tip, the binding epitopes within the antibody).In summary, the data presented here show that S. oneidensis MR-1 localizes OmcA and MtrC molecules to the exterior cell surface, including an EPS, when Fe(III) is the TEA. Here, the cytochromes presumably serve as terminal reductases that catalyze the reduction of Fe(III) through direct contact with the extracellular iron-oxide mineral.