Splenogonadal Fusion: An Unusual Case of an Acute Scrotum

We highlight a case on a normal left testicle with a fibrovascular cord with three nodules consistent with splenic tissue. The torsed splenule demonstrated hemorrhage with neutrophilic infiltrate and thrombus consistent with chronic infarction and torsion. Splenogonadal fusion (SGF) is a rather rare entity, with approximately 184 cases reported in the literature. The most comprehensive review was that of 123 cases completed by Carragher in 1990. Since then, an additional 61 cases have been reported in the scientific literature. We have studied these 61 cases in detail and have included a summary of that information here.Key words: Splenogonadal fusion, Acute scrotumA 10-year-old boy presented with worsening left-sided scrotal pain of 12 hours’ duration. The patient reported similar previous episodes occurring intermittently over the past several months. His past medical history was significant for left hip dysplasia, requiring multiple hip surgeries. On examination, he was found to have an edematous left hemiscrotum with a left testicle that was rigid, tender, and noted to be in a transverse lie. The ultrasound revealed possible polyorchism, with two testicles on the left and one on the right (Figure 1), and left epididymitis. One of the left testicles demonstrated a loss of blood flow consistent with testicular torsion (Figure 2).Open in a separate windowFigure 1Ultrasound of the left hemiscrotum reveals two spherical structures; the one on the left is heterogeneous and hyperdense in comparison to the right.Open in a separate windowFigure 2Doppler ultrasound of left hemiscrotum. No evidence of blood flow to left spherical structure.The patient was taken to the operating room for immediate scrotal exploration. A normalappearing left testicle with a normal epididymis was noted. However, two accessory structures were noted, one of which was torsed 720°; (Figure 3). An inguinal incision was then made and a third accessory structure was noted. All three structures were connected with fibrous tissue, giving a “rosary bead” appearance. The left accessory structures were removed, a left testicular biopsy was taken, and bilateral scrotal orchipexies were performed.Open in a separate windowFigure 3Torsed accessory spleen with splenogonadal fusion.Pathology revealed a normal left testicle with a fibrovascular cord with three nodules consistent with splenic tissue. The torsed splenule demonstrated hemorrhage with neutrophillic infiltrate and thrombus consistent with chronic infarction and torsion (Figure 4).Open in a separate windowFigure 4Splenogonadal fusion, continuous type with three accessory structures.     

Diversity in spindle morphology in Arabidopsis root tip

A primary function of the spindle apparatus is to segregate chromosomes into two equal sets in a dividing cell. It is unclear whether spindles in different cell types play additional roles in cellular regulation. As a first step in revealing new functions of spindles, we investigated spindle morphology in different cell types in Arabidopsis roots in the wild-type and the cytokinesis defective1 (cyd1) mutant backgrounds. cyd1 provides cells larger than those of the wild type for testing the cell size effect on spindle morphology. Our observations indicate that cell type (shape), not cell size, is likely a factor affecting spindle morphology. At least three spindle types were observed, including small spindles with pointed poles in narrow cells, large barrel-shaped spindles (without pointed poles) in wide cells, and spindles intermediate in pole focus and size in other cells. We hypothesize that the cell-type-associated spindle diversity may be an integral part of the cell differentiation processes.Key words: spindle pole, microtubule, morphogenesis, cell type, metaphaseThe cellular apparatus for chromosome segregation during mitosis is typically described as a spindle composed of microtubules and microtubule-associated proteins. Research on the structure and function of the spindle is usually conducted under the assumption that spindles are structurally the same or alike in different cell types in an organism. If the assumption is true, it would indicate that either the intracellular conditions in different dividing cells are very similar or the assembly and maintenance of the spindle are insensitive to otherwise variable intracellular conditions. But experimental evidence related to this assumption is relatively sparse.The root tip in Arabidopsis, as in other higher plants, contains dividing cells of different shapes and sizes. These cells include both meristem initial and derivative cells, with the former and latter being proximal and distal to the quiescent center, respectively.¹ The diversity in dividing cells in the root tip provides an opportunity for testing whether the spindles also exhibit diversity in morphology. To visualize the spindles at the metaphase stage in the root tip cells, we conducted indirect immunofluorescence labeling of the β-tubulin in single cells prepared from wild-type Arabidopsis (in Col-0 background) root tips as previously described in references ² and ³. The spindles in cells of different morphologies were then observed under a confocal laser scanning microscope.³ Three types of spindle were detected. The first type (Fig. 1A) was the smallest in width and length and had the most-pointed poles among the three types. The second type (Fig. 1B) was wider and longer than the first type but with less-pointed poles than the first type. The third type (Fig. 1C) was similar in height to the second type but lacked the pointed poles. In fact, the third type is shaped more like a barrel than a spindle. The first type was found in cells narrow in the direction parallel to the equatorial plane of the spindle, a situation opposite to that of the third type whose cells were wide in the equatorial direction. The wide cells containing the barrel-shaped spindles likely belonged to the epidermal layer in the root tip.¹ The second type was found in cells intermediate in width. Examples of metaphase spindles morphologically resembling the three types of spindles in Arabidopsis root can also be found in a previous report by Xu et al. even although spindle diversity was not the subject of the report.⁴ In Xu et al.''s report, type 1- or 2-like metaphase spindles can be identified in Figures 2B and 3A, and type 3-like metaphase spindles can be identified in Figures 1A and 3B. These observations indicate that at least three types of spindles exist in the root cells.Open in a separate windowFigure 1Spindles in wild-type root cells. (A) Type-1 spindle. (B) Type-2 spindle. (C) Type-3 spindle. The spots without fluorescence signals in the middle of the spindles are where the chromosomes were located. Scale bar for all the figures = 20 µm.Open in a separate windowFigure 2Spindles in cyd1 root cells. (A) Type-1 spindle. Arrows indicate the upper and lower boundaries of the cell. (B and C) Two type-2 spindles. (D and E) Two type-3 spindles. (F) DAPI-staining image corresponding to (E), showing chromosomes at the equatorial plane. Scale bar for the images = 20 µm.The above observations suggest that either the cell size or the cell type (shape) might be a factor in the type of spindle found in a specific cell. To further investigate the relationship between cell morphology and spindle morphology, we studied metaphase spindles in root cells of the cytokinesis defective1 (cyd1) mutant.⁵ Because the root cells in cyd1 were larger than corresponding cells in the wild type, presumably due to abnormal polyploidization prior to the collection of the root cells,^5,6 this investigation might reveal a relationship between increasing cell size and altered spindle morphology. A pattern of different spindle types in different cell types similar to that in the wild type was observed in cyd1 (Fig. 2). Figures 2A–C show narrow cells that contained spindles with pointed poles even though the spindles differed in size and focus. Figure 2D shows a barrel-shaped spindle in a wide cell, resembling Figure 1C in overall appearance. The large number of chromosomes at metaphase (more than the diploid number of 10) in Figure 2F indicates that the cells in Figure 2 were polyploid. These figures thus demonstrate that the enlargement in cell size did not alter the pattern of types 1 and 2 spindles in narrow cells, as well as type 3 spindles in wide cells. Moreover, the edges of the spindles in Figure 2B and E were similarly distanced to the cell walls in the equatorial plane, and yet they differ greatly in shape with the former being type 2 and the latter being type 3. This finding argues against that the cell width in the equatorial direction dictates the spindle shape. On the other hand, the cells in Figure 2B and E are obviously of different types. Taken together, these observations suggest that the spindle diversity in both wild type and cyd1 is associated with cell-type diversity.It is unclear whether the different spindle types have different functions in their respective cell types, in addition to the usual role for chromosome segregation. One possibility is that, at the ensuing telophase, the pointed spindles result in compact chromosomal congregation at the poles whereas the barrel-shaped spindles result in loose chromosomal congregation at the poles, which in turn may differentially affect the shape of the subsequently formed daughter nuclei and their organization. Different nuclear shape and organization are likely to be integrated into the processes that confer cell differentiation.     

Testicular Sclerosing Sertoli Cell Tumor: A Case Report and Review of the Literature

Sertoli cell tumors are very rare testicular tumors, representing 0.4% to 1.5% of all testicular malignancies. They are subclassified as classic, large-cell calcifying, and sclerosing Sertoli cell tumors (SSCT) based on distinct clinical features. Only 42 cases of SSCTs have been reported in the literature. We present a case of a 23-year-old man diagnosed with SSCT.Key words: Testicular neoplasm, Sertoli cell tumor, Sclerosing Sertoli cell tumorA 23-year-old man was referred to the Cleveland Clinic Department of Urology (Cleveland, OH) for an incidentally detected right testicular mass. The mass was identified during a work-up for transient left testicular discomfort. His only notable medical history was nephrolithiasis. There was no personal or family history of testicular cancer or cryptorchidism. On physical examination, he was a well-nourished, well-masculinized young man without gynecomastia. Testicular examination revealed normal volume and consistency bilaterally without other relevant findings. Testicular ultrasonography demonstrated an 8 mm × 6 mm × 6 mm hypoechoic, solid mass in the posterior right testicle with peripheral flow on color Doppler (Figure 1).Open in a separate windowFigure 1Testicular ultrasound demonstrating an 8 mm × 6 mm × 6 mm hypoechoic, solid mass in the posterior right testicle (blue arrows).The remainder of the ultrasound examination yielded normal results. Lactic dehydrogenase, B-human chorionic gonadotropin, and α-fetoprotein levels were all within the normal range. After a thorough review of the options, the patient was then taken to the operating room for inguinal exploration. Intraoperative ultrasound confirmed a superficial 8-mm hypoechoic testis lesion. A whiteyellow, well-demarcated nodule was widely excised and a frozen section was sent to pathology for examination. The frozen section examination revealed the lesion to be a neoplasm with differential diagnosis including sclerosing Sertoli cell tumor (SSCT), adenomatoid tumor, and a variant of Leydig cell tumor. Because the final diagnosis could not be determined from frozen section, the decision was made to perform a right radical orchiectomy. Pathologic examination revealed a grossly unifocal, well-circumscribed, white, firm mass of 0.8 cm. Microscopically the lesion was composed of solid and hollow tubules and occasional anastomosing cords distributed within the hypocellular, densely collagenous stroma. Although the lesion was somewhat well circumscribed, entrapped seminiferous tubules with Sertoli-only cells were present within the tumor (Figure 2). Tumor cells had pale or eosinophilic cytoplasm with small and dark nuclei with inconspicuous nucleoli. The tumor was confined to the testis and margins were negative. A diagnosis of SSCT was reached, supported by positive immunostain results for steroidogenic factor 1, focal inhibin, and calretinin expression, and negative stain results for cytokeratin AE1/AE3 and epithelial membrane antigen in the tumor (Figure 3). The postoperative course was unremarkable. Computed tomography scan of the abdomen and pelvis and chest radiograph were negative for metastatic disease.Open in a separate windowFigure 2Low-power examination revealing a well-circumscribed tumor composed of solid and hollow tubules and occasional anastomosing cords distributed within the hypocellular, densely collagenous stroma. Hematoxylin and eosin stain, original magnification ×40. (B) High-power examination. Note entrapped seminiferous tubules lacking spermatogenesis. Hematoxylin and eosin stain, original magnification ×100.Open in a separate windowFigure 3Nuclear expression of steroidogenic factor 1 in the tumor as well as benign Sertoli cells in entrapped seminiferous tubules (original magnification ×200). (B) Focal calretinin expression in the tumor (inhibin had a similar staining pattern; original magnification ×100).     

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.     

Efficient Production of 2-Oxobutyrate from 2-Hydroxybutyrate by Using Whole Cells of Pseudomonas stutzeri Strain SDM

2-Oxobutyrate is an important intermediate in the chemical, drug, and food industries. Whole cells of Pseudomonas stutzeri SDM, containing NAD-independent lactate dehydrogenases, effectively converted 2-hydroxybutyrate into 2-oxobutyrate. Under optimal conditions, the biocatalytic process produced 2-oxobutyrate at a high concentration (44.4 g liter⁻¹) and a high yield (91.5%).2-Oxobutyrate (2-OBA) is used as a raw material in the synthesis of chiral 2-aminobutyric acid, isoleucine, and some kinds of medicines (1, 8). There is no suitable starting material for 2-OBA production by chemical synthesis; therefore, the development of innovative biotechnology-based techniques for 2-OBA production is desirable (12).2-Hydroxybutyrate (2-HBA) is cheaper than 2-OBA and can be substituted for 2-OBA in the production of isoleucine, as reported previously (9, 10). The results of those studies also indicated that it might be possible to produce 2-OBA from 2-HBA by a suitable biocatalytic process. In the presence of NAD, NAD-dependent 2-hydroxybutyrate dehydrogenase can catalyze the oxidation of 2-HBA to 2-OBA (4). However, due to the high cost of pyridine cofactors (11), it is preferable to use a biocatalyst that directly catalyzes the formation of 2-OBA from 2-HBA without any requirement for NAD as a cofactor.In our previous report, we confirmed that NAD-independent lactate dehydrogenases (iLDHs) in the pyruvate-producing strain Pseudomonas stutzeri SDM (China Center for Type Culture Collection no. M206010) could oxidize lactate and 2-HBA (6). Therefore, in addition to pyruvate production from lactate, P. stutzeri SDM might also have a potential application in 2-OBA production.To determine the 2-OBA production capability of P. stutzeri SDM, the strain was first cultured at 30°C in a minimal salt medium (MSM) supplemented with 5.0 g liter⁻¹ dl-lactate as the sole carbon source (5). The whole-cell catalyst was prepared by centrifuging the medium and resuspending the cell pellet, and biotransformation was then carried out under the following conditions using 2-HBA as the substrate and whole cells of P. stutzeri SDM as the biocatalyst: 2-HBA, 10 g liter⁻¹; dry cell concentration, 6 g liter⁻¹; buffer, 100 mM potassium phosphate (pH 7.0); temperature, 30°C; shaking speed, 300 rpm. After 4 h of reaction, the mixture was analyzed by high-performance liquid chromatography (HPLC; Agilent 1100 series; Hewlett-Packard) using a refractive index detector (3). The HPLC system was fitted with a Bio-Rad Aminex HPX-87 H column. The mobile phase consisted of 10 mM H₂SO₄ pumped at 0.4 ml min⁻¹ (55°C). Biotransformation resulted in the production of a compound that had a retention time of 19.57 min, which corresponded to the peak of authentic 2-OBA (see Fig. S1 in the supplemental material).After acidification and vacuum distillation, the new compound was analyzed by negative-ion mass spectroscopy. The molecular ion ([M − H]⁻, m/z 101.1) signal of the compound was consistent with the molecular weight of 2-OBA, i.e., 102.1 (see Fig. S2 in the supplemental material). These results confirmed that 2-HBA was oxidized to 2-OBA by whole cells of P. stutzeri SDM.To investigate whether iLDHs are responsible for 2-OBA production in the above-described biocatalytic process, 2-HBA oxidation activity in P. stutzeri SDM was probed by native polyacrylamide gel electrophoresis. After electrophoresis, the gels were soaked in a substrate solution [50 mM Tris-HCl buffer (pH 8.0) containing 0.1 mM phenazine methosulfate, 0.1 mM 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, and 1 mM l-lactate, dl-lactate, or dl-2-HBA] and gently shaken. As shown in Fig. ​Fig.1,1, d- and l-iLDH migrated as two bands with distinct mobilities. The activities responsible for d- and l-2-HBA oxidation were located at the same positions as the d- and l-iLDH activities, respectively. No other bands responsible for d- and l-2-HBA oxidation were detected. Moreover, the dialysis of the crude cell extract did not lead to loss of 2-HBA oxidation activity and the addition of 10 mM NAD⁺ could not stimulate the reaction (see Table S1 in the supplemental material). These results implied that in the biocatalytic system, 2-HBA was oxidized to 2-OBA by iLDHs present in P. stutzeri SDM.Open in a separate windowFIG. 1.Activity staining of iLDHs after native polyacrylamide gel electrophoresis with lactate or 2-HBA as the substrate.Although the SDM strain could not use 2-HBA or 2-OBA for growth (see Fig. S3 in the supplemental material), 2-HBA might induce some of the enzymes responsible for 2-OBA production in the biocatalytic process. To exclude this possibility, the SDM strain was cultured in MSM containing dl-lactate or pyruvate as the sole carbon source. As shown in Fig. ​Fig.2,2, the enzyme activities that catalyzed lactate and 2-HBA oxidation were simultaneously present in the cells cultured on lactate and were absent in those cultured on pyruvate. After the lactate or pyruvate was exhausted, 5.05 g liter⁻¹ dl-2-HBA was added to the medium. It was observed that dl-2-HBA was efficiently converted to 2-OBA in the medium containing dl-lactate (Fig. ​(Fig.2a).2a). No 2-OBA production was detected in the medium containing pyruvate. Because 2-HBA addition did not induce the enzymes involved in 2-HBA oxidation (Fig. 2a and b), we concluded that the iLDHs induced by dl-lactate catalyzed 2-HBA oxidation in this biocatalytic process.Open in a separate windowFIG. 2.Time course of P. stutzeri SDM growth on media containing dl-lactate (a) and pyruvate (b). 2-HBA was added to the medium after the exhaustion of lactate or pyruvate. Symbols: ▴, lactate; ▵, pyruvate; •, 2-HBA; ○, 2-OBA; ▪, cell density; ▧, iLDHs activity with dl-lactate as the substrate; ▒, iLDHs activity with dl-2-HBA as the substrate.iLDHs could catalyze the oxidation of the substrate in a flavin-dependent manner and might use membrane quinone as the electron acceptor. Unlike the oxidases, which directly use the oxygen as the electron acceptor, this substrate oxidation mechanism could prevent the formation of H₂O₂ (see Fig. S4 in the supplemental material). The P. stutzeri SDM strain efficiently converted dl-2-HBA to 2-OBA with high yields (4.97 g liter⁻¹ 2-OBA was produced from 5.05 g liter⁻¹ dl-2-HBA); therefore, 2-OBA production by this strain can be a valuable and technically feasible process. To increase the efficiency of P. stutzeri SDM in the biotechnological production of 2-OBA, the conditions for biotransformation using whole cells of P. stutzeri SDM were first optimized. The influence of the reaction pH and 2-HBA concentration on 2-OBA production was determined in 100 mM phosphate buffer containing whole cells harvested from the medium containing dl-lactate as the sole carbon source. The reaction was initiated by adding the whole cells and 2-HBA at 37°C, followed by incubation for 10 min. After stopping the reaction by adding 1 M HCl, the 2-OBA concentration was determined by HPLC.As shown in Fig. ​Fig.3a,3a, ​,2-OBA2-OBA production was highest at pH 7.0. Under acidic or alkaline conditions, the transformation of 2-HBA to 2-OBA decreased. The optimal 2-HBA concentration was found to be 0.4 M, as shown in Fig. ​Fig.3b.3b. 2-OBA production increased as the 2-HBA concentration increased up to about 0.4 M and decreased thereafter. The concentration of the whole-cell catalyst was then optimized using 0.4 M 2-HBA as the substrate at pH 7.0. As shown in Fig. ​Fig.3c,3c, the highest 2-OBA concentration was obtained with 20 g (dry cell weight [DCW]) liter⁻¹ of P. stutzeri SDM. The 2-OBA concentration decreased with any increase beyond this cell concentration.Open in a separate windowFIG. 3.Optimization of the biocatalysis conditions. (a) Effect of pH on 2-OBA production activity. (b) Effect of 2-HBA concentrations on 2-OBA production activity. (c) Effect of the concentration of P. stutzeri SDM on biotransformation. OD, optical density.After optimizing the biocatalytic conditions, we studied the biotechnological production of 2-OBA from 2-HBA by using the whole-cell catalyst P. stutzeri SDM. As shown in Fig. ​Fig.4,4, when 20 g (DCW) liter⁻¹ P. stutzeri SDM was used as the biocatalyst, 48.5 g liter⁻¹ 2-HBA was biotransformed into 44.4 g liter⁻¹ 2-OBA in 24 h.Open in a separate windowFIG. 4.Time course of production of 2-OBA from 2-HBA under the optimum conditions. Symbols: ▪, 2-OBA; •, 2-HBA.Biocatalytic production of 2-OBA was carried out using crotonic acid, propionaldehyde, 1,2-butanediol, or threonine as the substrate (2, 7, 8, 12). Resting cells of the strain Rhodococcus erpi IF0 3730 produced 15.7 g liter⁻¹ 2-OBA from 20 g liter⁻¹ 1,2-butanediol, which is the highest reported yield of 2-OBA to date (8). By using the whole-cell catalyst P. stutzeri SDM, it was possible to produce 2-OBA at a high concentration (44.4 g liter⁻¹) and a high yield (91.5%). Due to the simple composition of the biocatalytic system (see Fig. S5 in the supplemental material), 2-HBA and 2-OBA could be easily separated on a column using a suitable resin. Separation of 2-OBA from the biocatalytic system was relatively inexpensive. The biocatalytic process presented in this report could be a promising alternative for the biotechnological production of 2-OBA.      

Thigmomorphogenesis in Solanum lycopersicum: Morphological and biochemical responses in stem after mechanical stimulation

The activation of the phenylpropanoid pathway in plants by environmental stimuli is one of the most universal biochemical stress responses known. In tomato plant, rubbing applied to a young internode inhibit elongation of the rubbed internode and his neighboring one. These morphological changes were correlated with an increase in lignification enzyme activities, phenylalanine ammonia-lyase (PAL), cinnamyl alcohol dehydrogenase (CAD) and peroxidases (POD), 24 hours after rubbing of the forth internode. Furthermore, a decrease in indole-3-acetic acid (IAA) content was detected in the rubbed internode and the upper one. Taken together, our results suggest that decrease in rubbed internode length is a consequence of IAA oxidation, increases in enzyme activities (PAL, CAD and POD), and cell wall rigidification associated with induction of lignification process.Key words: Mechanical stimulation, PAL, CAD, POD, IAAIn their environment, plants are constantly submitted to several stimuli such as wind, rain and wounding. The growth response of plants to such stimuli was termed thigmomorphogenesis and was observed in a wide range of plants.^1–3 The most common thigmomorphogenetic response is a retardation of tissue elongation accompanied by an increase in thickness.⁴ The plant response to mechanical perturbation is mainly restricted to the young developing internode, since no influence can be detected when the internode has reached its final length.^5,6 These plant growth modifications, which characterize thigmomorphogenesis, are related to biochemical events associated with lignification process⁷ and ethylene production.^8,9In tomato plant the length of internodes 4 (N4) and 5 (N5) was measured 14 days after rubbing of the fourth internode. Results reported in Figure 1 show that rubbing led to a significant reduction of elongation of the stressed internode (N4) (decrease of N4 length from 4.3 cm in the control plant to 2.9 in the rubbed one). This effect was not limited to the rubbed area but affected also the elongation of the neighboring internodes (N5) that were shorter in rubbed plants than in control ones.Open in a separate windowFigure 1Internode lengths of control and rubbed plants measured 14 day after mechanical stress applied to the fourth internode. Standard errors are indicated by vertical bars.Results reported in Figure 2 show an increase in PAL activity in both internodes N4 and N5, 24 hours after mechanical stress application as compared with corresponding controls. CAD activity was also investigated in N4 and N5, 24 h after rubbing of the fourth internode. Results presented in Figure 3 show that mechanical stress application induces a strong increase of CAD activity in the rubbed internode N4 (5.3 nkatal μg-1 protein) with an approximately two-fold increase when compared to control tomato internodes (2.3 nkatal μg-1 protein). Further, CAD activity in N5 was also increased in the rubbed internode (5.538 nkatal μg-1 protein) as compared with the control one (3.256 nkatal μg-1 protein).Open in a separate windowFigure 2PAL activity of internode 4, and 5 in control and rubbed plants 24 h after rubbing of the fourth internode. Standard errors are indicated by vertical bars.Open in a separate windowFigure 3CAD activity of internode 4, and 5 in control and rubbed plants 24 h after rubbing of the fourth internode. Standard errors are indicated by vertical bars.Syringaldazine (S-POD) and gaïacol (G-POD) peroxidase activities were measured in tomato N4 and N5. Results reported in Figure 4 show an increase in soluble peroxidase activity with both substrates in the rubbed internode N4 as compared with control plant. Enhancement in peroxidase activities in N4 was more pronounced with gaïacol (80.7 U) as an electron donor than syringaldazine (33.8 U). Similar results were observed in internode 5 as compared with control one (Fig. 4).Open in a separate windowFigure 4(A) Syringaldazine-POD (Syr-POD) activity of internode 4 and 5 in control and rubbed plants 24 h after rubbing of the fourth internode. Standard errors are indicated by vertical bars. (B) Gaiacol-POD (G-POD) activity of internode 4 and 5 in control and rubbed plants 24 h after rubbing of the fourth internode. Standard errors are indicated by vertical bars.IAA was quantified in control and rubbed plant internodes 24 h after rubbing of the fourth internode. Results reported in figure 5 show that in control sample and as expected, the content of IAA was found to be higher in the younger internode (N5) as compared to the older one (N4). Rubbing led to a significant decrease in IAA levels in N4 (5.06 nmol g⁻¹ MF⁻¹) as compared with corresponding controls (7.27 nmol g⁻¹ MF⁻¹). Similar results were observed in internode 5, where IAA content was reduced from 16.52 nmol g⁻¹ MF⁻¹ in control internode to 12.35 nmol g⁻¹ MF⁻¹ in the rubbed internode (Fig. 5).Open in a separate windowFigure 5IAA Level of internode 4 and 5 in control and rubbed plants 24 h after rubbing of the fourth internode. Standard errors are indicated by vertical bars.The results reported here establish an evident correlation between growth limitation of the rubbed internode and their degree of lignification, the increase in lignification enzymes activities and auxin degradation after mechanical stress application.Auxin seems to be involved in thigmomorphogenesis.¹⁰ It was proposed that MIS (Mechanically-induced stress) has opposite effects on auxin levels in the two species studied to date, Phaseolus vulgaris¹⁰ and Bryonia dioica.^11,12 Auxin level as measured by bioassay, increased in Phaseolus vulgaris following rubbing of the stem.¹⁰ It was proposed that a build up of auxin may result from the reduced polar transport of IAA at the rubbed internode, causing a build up of IAA in the stem tissue. Exogenous IAA did not reverse the MIS inhibition of growth in Phaseolus vulgaris and high levels of IAA retarded growth in non-stressed plants.¹⁰ Thus, retardation of extension growth in Phaseolus vulgaris may have been caused by high levels of endogenous auxin and the increase in stem diameter by increased ethylene production.⁴ However, ethylene increases radial growth only if auxin is present.¹³Boyer¹¹ reported a decrease in auxinlike activity in Bryonia dioica following MIS and this was confirmed in the same species by Hofinger et al.¹² who reported a decrease in IAA using gas chromatography-mass spectrometry. Auxin catabolism was accompanied with changes in both soluble and ionically bound cell wall basic peroxidases¹⁴ and the appearance of an additional peroxidase. This can suggest that in Bryonia, auxin catabolism is hastened by mechanical stimulated peroxidase. In addition, Boyer et al.¹⁵ reported that lithium pre-treatment prevents both thigmomorphogenesis and appearance of specific cathodic isoperoxidase in Bryonia plants subjected to MIS. This is give further credence to the possibility that the peroxidase-auxin system is involved in Bryonia thigmomorphogenesis. In addition, ethylene increases peroxidase activity which reduces the auxin content in the tissue to a level low enough not to support normal growth. We have evidence that decrease of auxin level contribute to mechanism leading to tomato internode inhibition subjected to mechanical stress.Growth inhibition has been suggested to be the result of tissues lignification.⁶ As the initial enzyme in the monolignol biosynthesis pathway, PAL has a direct influence on lignin accumulation.¹⁶ The characteristics of lignin differ among cell wall tissues and plant organs.¹⁷ It comprises polyphenolic polymers derived from the oxidative polymerization of different monolignols, including p-coumaryl, coniferyl and sinapyl alcohols via a side pathway of phenylalanine metabolism leading to lignin synthesis.¹⁸ The increase in lignin content in the rubbed tomato internode could be a response mechanism to mechanical damage caused by rubbing.³ It is known that plants create a natural barrier that includes lignin and suberin synthesis, components directly linked to support systems.^19,20The increase in lignin content of rubbed tomato internode³ is paralleled by a rise in CAD activity and whilst such direct proportionality between CAD activity and lignin accumulation does not always agree with the results in the literature, it clearly is responding in ways similar to those of the other enzymes in the pathway.²¹Mechanical stress-induced membrane depolarization would generate different species of free radicals and peroxides, which in turn initiate lipid peroxidation.²² The degradation of cell membranes is suggested to bring about rapid changes in ionic flux, especially release of K⁺ which would result in an enhanced endogenous Ca/K ratio and in leakage of solutes, among them electron donors such as ascorbic acid and phenolic substances. The increased intracellular relative calcium level activated secretion of basic peroxidases²³ into the free space where, in association with the electron donors and may be with the circulating IAA, they eliminate the peroxides, and facilitated binding of basic peroxidases to membrane structures allowing a role as 1-aminocyclopropane-1-carboxylic acid (ACC)-oxidases. The resulting IAA and ACC oxidase-mediated changes in ethylene production²⁴ would further induce (this time through the protein synthesis machinery) an increase in activity of phenylalanine ammonia-lyase and peroxidases. The resulting lignification and cell wall rigidification determines the growth response of tomato internode to the mechanical stress.     

Novel Growth Regime of MDCK II Model Tissues on Soft Substrates

It is well established that MDCK II cells grow in circular colonies that densify until contact inhibition takes place. Here, we show that this behavior is only typical for colonies developing on hard substrates and report a new growth phase of MDCK II cells on soft gels. At the onset, the new phase is characterized by small, three-dimensional droplets of cells attached to the substrate. When the contact area between the agglomerate and the substrate becomes sufficiently large, a very dense monolayer nucleates in the center of the colony. This monolayer, surrounded by a belt of three-dimensionally packed cells, has a well-defined structure, independent of time and cluster size, as well as a density that is twice the steady-state density found on hard substrates. To release stress in such dense packing, extrusions of viable cells take place several days after seeding. The extruded cells create second-generation clusters, as evidenced by an archipelago of aggregates found in a vicinity of mother colonies, which points to a mechanically regulated migratory behavior.Studying the growth of cell colonies is an important step in the understanding of processes involving coordinated cell behavior such as tissue development, wound healing, and cancer progression. Apart from extremely challenging in vivo studies, artificial tissue models are proven to be very useful in determining the main physical factors that affect the cooperativity of cells, simply because the conditions of growth can be very well controlled. One of the most established cell types in this field of research is the Madin-Darby canine kidney epithelial cell (MDCK), originating from the kidney distal tube (1). A great advantage of this polarized epithelial cell line is that it retained the ability for contact inhibition (2), which makes it a perfect model system for studies of epithelial morphogenesis.Organization of MDCK cells in colonies have been studied in a number of circumstances. For example, it was shown that in three-dimensional soft Matrigel, MDCK cells form a spherical enclosure of a lumen that is enfolded by one layer of polarized cells with an apical membrane exposed to the lumen side (3). These structures can be altered by introducing the hepatocyte growth factor, which induces the formation of linear tubes (4). However, the best-studied regime of growth is performed on two-dimensional surfaces where MDCK II cells form sheets and exhibit contact inhibition. Consequently, the obtained monolayers are well characterized in context of development (5), mechanical properties (6), and obstructed cell migration (7–9).Surprisingly, in the context of mechanics, several studies of monolayer formation showed that different rigidities of polydimethylsiloxane gels (5) and polyacrylamide (PA) gels (9) do not influence the nature of monolayer formation nor the attainable steady-state density. This is supposedly due to long-range forces between cells transmitted by the underlying elastic substrate (9). These results were found to agree well with earlier works on bovine aortic endothelial cells (10) and vascular smooth muscle cells (11), both reporting a lack of sensitivity of monolayers to substrate elasticity. Yet, these results are in stark contrast with single-cell experiments (12–15) that show a clear response of cell morphology, focal adhesions, and cytoskeleton organization to substrate elasticity. Furthermore, sensitivity to the presence of growth factors that are dependent on the elasticity of the substrate in two (16) and three dimensions (4) makes this result even more astonishing. Therefore, we readdress the issue of sensitivity of tissues to the elasticity of the underlying substrate and show that sufficiently soft gels induce a clearly different tissue organization.We plated MDCK II cells on soft PA gels (Young’s modulus E = 0.6 ± 0.2 kPa), harder PA gels (E = 5, 11, 20, 34 kPa), and glass, all coated with Collagen-I. Gels were prepared following the procedure described in Rehfeldt et al. (17); rigidity and homogeneity of the gels was confirmed by bulk and microrheology (see the Supporting Material for comparison). Seeding of MDCK II cells involved a highly concentrated solution dropped in the middle of a hydrated gel or glass sample. For single-cell experiments, cells were dispersed over the entire dish. Samples were periodically fixed up to Day 12, stained for nuclei and actin, and imaged with an epifluorescence microscope. Details are described in the Supporting Material.On hard substrates and glass it was found previously that the area of small clusters expands exponentially until the movement of the edge cannot keep up with the proliferation in the bulk (5). Consequently, the bulk density increases toward the steady state, whereas the density of the edge remains low. At the same time, the colony size grows subexponentially (5). This is what we denote “the classical regime of growth”. Our experiments support these observations for substrates with E ≥ 5 kPa. Specifically, on glass, colonies start as small clusters of very low density of 700 ± 200 cells/mm² (Fig. 1, A and B), typically surrounded by a strong actin cable (Fig. 1, B and C). Interestingly, the spreading area of single cells (Fig. 1 A) on glass was found to be significantly larger, i.e., (2.0 ± 0.9) × 10⁻³ mm². After Day 4 (corresponding cluster area of 600 ± 100 mm²), the density in the center of the colony reached the steady state with 6,800 ± 500 cells/mm², whereas the mean density of the edge profile grew to 4,000 ± 500 cells/mm². This density was retained until Day 12 (cluster area 1800 ± 100 mm²), which is in agreement with previous work (9).Open in a separate windowFigure 1Early phase of cluster growth on hard substrates. (A) Well-spread single cells, and small clusters with a visible actin cable 6 h after seeding. (B) Within one day, clusters densify and merge, making small colonies. (C) Edge of clusters from panel B.In colonies grown on 0.6 kPa gels, however, we encounter a very different growth scenario. The average spreading area of single cells is (0.34 ± 0.3) × 10⁻³ mm², which is six times smaller than on glass substrates (Fig. 2 A). Clusters of only few cells show that cells have a preference for cell-cell contacts (a well-established flat contact zone can be seen at the cell-cell interface in Fig. 2 A) rather than for cell-substrate contacts (contact zone is diffusive and the shape of the cells appears curved). The same conclusion emerges from the fact that dropletlike agglomerates, resting on the substrate, form spontaneously (Fig. 2 A), and that attempts to seed one single cluster of 90,000 cells fail, resulting in a number of three-dimensional colonies (Fig. 2 A). When the contact area with the substrate exceeds 4.7 × 10⁻³ mm², a monolayer appears in the center of such colonies (Fig. 2 B). The colonies can merge, and if individual colonies are small, the collapse into a single domain is associated with the formation of transient irregular structures (Fig. 2 B). Ultimately, large elliptical colonies (average major/minor axis of e = 1.8 ± 0.6) with a smooth edge are formed (Fig. 2 C), unlike on hard substrates where circular clusters (e = 1.06 ± 0.06) with a ragged edge comprise the characteristic phenotype.Open in a separate windowFigure 2Early phase of cluster growth on soft substrates. (A) Twelve hours after seeding, single cells remain mostly round and small. They are found as individual, or within small, three-dimensional structures (top). The latter nucleate a monolayer in their center (bottom), if the contact area with the substrate exceeds ∼5 × 10⁻³ mm². (B) Irregularly-shaped clusters appear due to merging of smaller droplets. A stable monolayer surrounded by a three-dimensional belt of densely packed cells is clearly visible, even in larger structures. (C) All colonies are recorded on Day 4.Irrespective of cluster size, in the new regime of growth, the internal structure is built of two compartments (Fig. 2 B):

• 1.The first is the edge (0.019 ± 0.05-mm wide), a three-dimensional structure of densely packed cells. This belt is a signature of the new regime because on hard substrates the edge is strictly two-dimensional (Fig. 1 C).
• 2.The other is the centrally placed monolayer with a spatially constant density that is very weakly dependent on cluster size and age (Fig. 3). The mean monolayer density is 13,000 ± 2,000 cells/mm², which is an average over 130 clusters that are up to 12 days old and have a size in the range of 10⁻³ to 10 mm², each shown by a data point in Fig. 3. This density is twice the steady-state density of the bulk tissue in the classical regime of growth.Open in a separate windowFigure 3Monolayer densities in colonies grown on 0.6 kPa substrates, as a function of the cluster size and age. Each cluster is represented by a single data point signifying its mean monolayer density. (Black lines) Bulk and (red dashed lines) edge of steady-state densities from monolayers grown on glass substrates. Error bars are omitted for clarity, but are discussed in the Supporting Material.

Until Day 4, the monolayer is very homogeneous, showing a nearly hexagonal arrangement of cells. From Day 4, however, defects start to appear in the form of small holes (typical size of (0.3 ± 0.1) × 10⁻³ mm²). These could be attributed to the extrusions of viable cells, from either the belt or areas of increased local density in the monolayer (inset in Fig. 4). This suggests that extrusions serve to release stress built in the tissue, and, as a consequence, the overall density is decreased.Open in a separate windowFigure 4Cell nuclei within the mother colony and in the neighboring archipelago of second-generation clusters grown on 0.6 kPa gels at Day 12. (Inset; scale bar = 10 μm) Scar in the tissue, a result of a cell-extrusion event. (Main image; scale bar = 100 μm) From the image of cell nuclei (left), it is clear that there are no cells within the scar, whereas the image of actin (right) shows that the cytoplasm of the cells at the edge has closed the hole.Previous reports suggest that isolated MDCK cells undergo anoikis 8 h after losing contact with their neighbors (18). However, in this case, it appears that instead of dying, the extruded cells create new colonies, which can be seen as an archipelago surrounding the mother cluster (Fig. 4). The viability of off-cast cells is further evidenced by the appearance of single cells and second-generation colonies with sizes varying over five orders of magnitude, from Day 4 until the end of the experiment, Day 12. Importantly, no morphological differences were found in the first- and second-generation colonies.In conclusion, we show what we believe to be a novel phase of growth of MDCK model tissue on soft PA gels (E = 0.6 kPa) that, to our knowledge, despite previous similar efforts (9), has not been observed before. This finding is especially interesting in the context of elasticity of real kidneys, for which a Young’s modulus has been found to be between 0.05 and 5 kPa (19,20). This coincides with the elasticity of substrates studied herein, and opens the possibility that the newly found phase of growth has a particular biological relevance. Likewise, the ability to extrude viable cells may point to a new migratory pathway regulated mechanically by the stresses in the tissue, the implication of which we hope to investigate in the future.     

Glutathione signaling acts through NPR1-dependent SA-mediated pathway to mitigate biotic stress

Glutathione (GSH) has widely been known to be a multifunctional molecule especially as an antioxidant up until now, but has found a new role in plant defense signaling. Research from the past three decades indicate that GSH is a player in pathogen defense in plants, but the mechanism underlying this has not been elucidated fully. We have recently shown that GSH acts as a signaling molecule and mitigates biotic stress through non-expressor of PR genes 1 (NPR1)-dependent salicylic acid (SA)-mediated pathway. Transgenic tobacco with enhanced level of GSH (NtGB lines) was found to synthesize more SA, was capable of enhanced expression of genes belonging to NPR1-dependent SA-mediated pathway, were resistant to Pseudomonas syringae, the biotrophic pathogen and many SA-related proteins were upregulated. These results gathered experimental evidence on the mechanism through which GSH combats biotic stress. In continuation with our previous investigation we show here that the expression of glutathione S-transferase (GST), the NPR1-independent SA-mediated gene was unchanged in transgenic tobacco with enhanced level of GSH as compared to wild-type plants. Additionally, the transgenic plants were barely resistant to Botrytis cinerea, the necrotrophic pathogen. SA-treatment led to enhanced level of expression of pathogenesis-related protein gene (PR1) and PR4 as against short-chain dehydrogenase/reductase family protein (SDRLP) and allene oxide synthase (AOS). These data provided significant insight into the involvement of GSH in NPR1-dependent SA-mediated pathway in mitigating biotic stress.Key words: GSH, signaling molecule, biotrophic pathogen, NPR-1, PR-1, PR-4, transgenic tobaccoPlant responses to different environmental stresses are achieved through integrating shared signaling networks and mediated by the synergistic or antagonistic interactions with the phytohormones viz. SA, jasmonic acid (JA), ethylene (ET), abscisic acid (ABA) and reactive oxygen species (ROS).¹ Previous studies have shown that in response to pathogen attack, plants produce a highly specific blend of SA, JA and ET, resulting in the activation of distinct sets of defense-related genes.^2,3 Regulatory functions for ROS in defense, with a focus on the response to pathogen infection occur in conjunction with other plant signaling molecules, particularly with SA and nitric oxide (NO).^4–6 Till date, numerous physiological functions have been attributed to GSH in plants.^7–11 In addition to previous studies, recent study has also shown that GSH acts as a signaling molecule in combating biotic stress through NPR1-dependent SA-mediated pathway.^12,13Our recent investigation involved raising of transgenic tobacco overexpressing gamma-glutamylcysteine synthetase (γ-ECS), the rate-limiting enzyme of the GSH biosynthetic pathway.¹² The stable integration and enhanced expression of the transgene at the mRNA as well as protein level was confirmed by Southern blot, quantitative RT-PCR and western blot analysis respectively. The transgenic plants of the T₂ generation (Fig. 1), the phenotype of which was similar to that of wild-type plants were found to be capable of synthesizing enhanced amount of GSH as confirmed by HPLC analysis.Open in a separate windowFigure 1Transgenic tobacco of T₂ generation, (A) three-week-old plant, (B) mature plant.In the present study, the expression profile of GST was analyzed in NtGB lines by quantitative RT-PCR (qRT-PCR) and found that the expression level of this gene is unchanged in NtGB lines as compared to wild-type plants (Fig. 2). GST is known to be a NPR1-independent SA-related gene.¹⁴ This suggests that GSH does not follow the NPR1-independent SA-mediated pathway in defense signaling.Open in a separate windowFigure 2Expression pattern of GST in wild-type and NtGB lines.Disease test assay with NtGB lines and wild-type plants was performed using B. cinerea and the NtGB lines showed negligible rate of resistance to this necrotrophic pathogen (Fig. 3). SA signaling has been known to control defense against biotrophic pathogen in contrast, JA/ET signaling controls defense against necrotrophic pathogen.^1,15 Thus it has again been proved that GSH is not an active member in the crosstalk of JA-mediated pathway, rather it follows the SA-mediated pathway as has been evidenced earlier.¹²Open in a separate windowFigure 3Resistance pattern of wild-type and NtGB lines against Botrytis cinerea.Additionally, the leaves of wild-type and NtGB lines were treated with 1 mM SA and the expression of PR1, SDRLP, AOS and PR4 genes were analyzed and compared to untreated plants to simulate pathogen infection. The expression of PR1 increased after exogenous application of SA. In case of PR4, the ET marker, the expression level increased in NtGB lines. On the other hand, the level of SDRLP was nearly the same. However, the expression of AOS was absent in SA-treated leaves (Fig. 4). PR1 has been known to be induced by SA-treatment¹⁶ which can be corroborated with our results. In addition, ET is known to enhance SA/NPR1-dependent defense responses,¹⁷ which was reflected in our study as well. AOS, the biosynthetic pathway gene of JA, further known to be the antagonist of SA, was downregulated in SA-treated plants.Open in a separate windowFigure 4Gene expression pattern of PR1, SDRLP, PR4 and AOS in untreated and SA-treated wildtype and NtGB lines.Taken together, it can be summarized that this study provided new evidence on the involvement of GSH with SA in NPR1-dependent manner in combating biotic stress. Additionally, it can be claimed that GSH is a signaling molecule which takes an active part in the cross-communication with other established signaling molecules like SA, JA, ET in induced defense responses and has an immense standpoint in plant defense signaling.     

Assessing Social – Ecological Trade-Offs to Advance Ecosystem-Based Fisheries Management

Modern resource management faces trade-offs in the provision of various ecosystem goods and services to humanity. For fisheries management to develop into an ecosystem-based approach, the goal is not only to maximize economic profits, but to consider equally important conservation and social equity goals. We introduce such a triple-bottom line approach to the management of multi-species fisheries using the Baltic Sea as a case study. We apply a coupled ecological-economic optimization model to address the actual fisheries management challenge of trading-off the recovery of collapsed cod stocks versus the health of ecologically important forage fish populations. Management strategies based on profit maximization would rebuild the cod stock to high levels but may cause the risk of stock collapse for forage species with low market value, such as Baltic sprat (Fig. 1A). Economically efficient conservation efforts to protect sprat would be borne almost exclusively by the forage fishery as sprat fishing effort and profits would strongly be reduced. Unless compensation is paid, this would challenge equity between fishing sectors (Fig. 1B). Optimizing equity while respecting sprat biomass precautionary levels would reduce potential profits of the overall Baltic fishery, but may offer an acceptable balance between overall profits, species conservation and social equity (Fig. 1C). Our case study shows a practical example of how an ecosystem-based fisheries management will be able to offer society options to solve common conflicts between different resource uses. Adding equity considerations to the traditional trade-off between economy and ecology will greatly enhance credibility and hence compliance to management decisions, a further footstep towards healthy fish stocks and sustainable fisheries in the world ocean.Open in a separate windowFigure 1Summary of multispecies management options in the Baltic.(A) Profit maximum. (B) Economic optimum while respecting sprat B_PA. (C) Equitable optimum while respecting sprat B_PA. Central numbers indicate total profits (million €/year) as well as an equity measure (in brackets). Area of each pie slice is relative to status quo values 2008-2010 (black circle), with error bars from sensitivity analysis.     

Overexpression of the groESL Operon Enhances the Heat and Salinity Stress Tolerance of the Nitrogen-Fixing Cyanobacterium Anabaena sp. Strain PCC7120

The bicistronic groESL operon, encoding the Hsp60 and Hsp10 chaperonins, was cloned into an integrative expression vector, pFPN, and incorporated at an innocuous site in the Anabaena sp. strain PCC7120 genome. In the recombinant Anabaena strain, the additional groESL operon was expressed from a strong cyanobacterial P_psbA1 promoter without hampering the stress-responsive expression of the native groESL operon. The net expression of the two groESL operons promoted better growth, supported the vital activities of nitrogen fixation and photosynthesis at ambient conditions, and enhanced the tolerance of the recombinant Anabaena strain to heat and salinity stresses.Nitrogen-fixing cyanobacteria, especially strains of Nostoc and Anabaena, are native to tropical agroclimatic conditions, such as those of Indian paddy fields, and contribute to the carbon (C) and nitrogen (N) economy of these soils (22, 30). However, their biofertilizer potential decreases during exposure to high temperature, salinity, and other such stressful environments (1). A common target for these stresses is cellular proteins, which are denatured and inactivated during stress, resulting in metabolic arrest, cessation of growth, and eventually loss of viability. Molecular chaperones play a major role in the conformational homeostasis of cellular proteins (13, 16, 24, 26) by (i) proper folding of nascent polypeptide chains; (ii) facilitating protein translocation and maturation to functional conformation, including multiprotein complex assembly; (iii) refolding of misfolded proteins; (iv) sequestering damaged proteins to aggregates; and (v) solubilizing protein aggregates for refolding or degradation. Present at basal levels under optimum growth conditions in bacteria, the expression of chaperonins is significantly enhanced during heat shock and other stresses (2, 25, 32).The most common and abundant cyanobacterial chaperones are Hsp60 proteins, and nitrogen-fixing cyanobacteria possess two or more copies of the hsp60 or groEL gene (http://genome.kazusa.or.jp/cyanobase). One occurs as a solitary gene, cpn60 (17, 21), while the other is juxtaposed to its cochaperonin encoding genes groES and constitutes a bicistronic operon groESL (7, 19, 31). The two hsp60 genes encode a 59-kDa GroEL and a 61-kDa Cpn60 protein in Anabaena (2, 20). Both the Hsp60 chaperonins are strongly expressed during heat stress, resulting in the superior thermotolerance of Anabaena, compared to the transient expression of the Hsp60 chaperonins in Escherichia coli (20). GroEL and Cpn60 stably associate with thylakoid membranes in Anabaena strain PCC7120 (14) and in Synechocystis sp. strain PCC6803 (15). In Synechocystis sp. strain PCC6803, photosynthetic inhibitors downregulate, while light and redox perturbation induce cpn60 expression (10, 25, 31), and a cpn60 mutant exhibits a light-sensitive phenotype (http://genome.kazusa.or.jp/cyanobase), indicating a possible role for Cpn60 in photosynthesis. GroEL, a lipochaperonin (12, 28), requires a cochaperonin, GroES, for its folding activity and has wider substrate selectivity. In heterotrophic nitrogen-fixing bacteria, such as Klebsiella pneumoniae and Bradyrhizobium japonicum, the GroEL protein has been implicated in nif gene expression and the assembly, stability, and activity of the nitrogenase proteins (8, 9, 11).Earlier work from our laboratory demonstrated that the Hsp60 family chaperonins are commonly induced general-stress proteins in response to heat, salinity, and osmotic stresses in Anabaena strains (2, 4). Our recent work elucidated a major role of the cpn60 gene in the protection from photosynthesis and the nitrate reductase activity of N-supplemented Anabaena cultures (21). In this study, we integrated and constitutively overexpressed an extra copy of the groESL operon in Anabaena to evaluate the importance and contribution of GroEL chaperonin to the physiology of Anabaena during optimal and stressful conditions.Anabaena sp. strain PCC7120 was photoautotrophically grown in combined nitrogen-free (BG11⁻) or 17 mM NaNO₃-supplemented (BG11⁺) BG11 medium (5) at pH 7.2 under continuous illumination (30 μE m⁻² s⁻¹) and aeration (2 liters min⁻¹) at 25°C ± 2°C. Escherichia coli DH5α cultures were grown in Luria-Bertani medium at 37°C at 150 rpm. For E. coli DH5α, kanamycin and carbenicillin were used at final concentrations of 50 μg ml⁻¹ and 100 μg ml⁻¹, respectively. Recombinant Anabaena clones were selected on BG11⁺ agar plates supplemented with 25 μg ml⁻¹ neomycin or in BG11⁻ liquid medium containing 12.5 μg ml⁻¹ neomycin. The growth of cyanobacterial cultures was estimated either by measuring the chlorophyll a content as described previously (18) or the turbidity (optical density at 750 nm). Photosynthesis was measured as light-dependent oxygen evolution at 25 ± 2°C by a Clark electrode (Oxy-lab 2/2; Hansatech Instruments, England) as described previously (21). Nitrogenase activity was estimated by acetylene reduction assays, as described previously (3). Protein denaturation and aggregation were measured in clarified cell extracts containing ∼500 μg cytosolic proteins treated with 100 μM 8-anilino-1-naphthalene sulfonate (ANS). The pellet (protein aggregate) was solubilized in 20 mM Tris-6 M urea-2% sodium dodecyl sulfate (SDS)-40 mM dithiothreitol for 10 min at 50°C. The noncovalently trapped ANS was estimated using a fluorescence spectrometer (model FP-6500; Jasco, Japan) at a λ_excitation of 380 nm and a λ_emission of 485 nm, as described previously (29).The complete bicistronic groESL operon (2.040 kb) (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ608815","term_id":"222354886","term_text":"FJ608815"}}FJ608815) was PCR amplified from PCC7120 genomic DNA using specific primers (Table ​(Table1)1) and the amplicon cloned into the NdeI-BamHI restriction sites of plasmid vector pFPN, which allows integration at a defined innocuous site in the PCC7120 genome and expression from a strong cyanobacterial P_psbA1 promoter (6). The resulting construct, designated pFPNgro (Table ​(Table1),1), was electroporated into PCC7120 using an exponential-decay wave form electroporator (200 J capacitive energy at a full charging voltage of 2 kV; Pune Polytronics, Pune, India), as described previously (6). The electroporation was carried out at 6 kV cm⁻¹ for 5 ms, employing an external autoclavable electrode with a 2-mm gap. The electroporation buffer contained high concentrations of salt (10 mM HEPES, 100 mM LiCl, 50 mM CaCl₂), as have been recommended for plant cells (23) and other cell types (27). The electrotransformants, selected on BG11⁺ agar plates supplemented with 25 μg ml⁻¹ neomycin by repeated subculturing for at least 25 weeks to achieve complete segregation, were designated AnFPNgro.

TABLE 1.

Plasmids, strains, and primers used in this study

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 ...     

Negative effects of desiccation on the protein sorting and post-translational modification

Bryophytes as the first land plants are believed to have colonized the land from a fresh water origin, requiring adaptive mechanisms that survival of dehydration. Physcomitrella patens is such a non-vascular bryophyte and shows rare desiccation tolerance in its vegetative tissues. Previous studies showed that during the course of dehydration, several related processes are set in motion: plasmolysis, chloroplast remodeling and microtubule depolymerization. And proteomic alteration supported the cellular structural changes in respond to desiccation stress.¹ In this addendum, we report that Golgi bodies are absent and adaptor protein complex AP-1 large subunit is downregulated during the course of dehydration. Those phenomena may be adverse in protein posttranslational modification, protein sorting and cell walls synthesis under the desiccation condition.Key words: AP-1 protein, cell ultrastructure, desiccation, golgi bodies, physcomitrella, proteomeThe plant Golgi apparatus is composed of many small stacks of cisternae, sometimes known as dictyosomes. The Golgi is a complex polarized organelle consisting of both a cis and trans side, containing compartments with functionally different capacities for directing cellular components. The plant Golgi apparatus synthesis a wide range of cell wall polysaccharides and proteoglycans, and also carries out O-linked glycosylation and N-linked glycan processing.^2–5 Moreover, the Golgi is involved in returning escaped proteins back to the endoplasmic reticulum, sorting of proteins and polysaccharides to the cell wall or vacuoles, and in organizing the compartmentation of its own enzymes by retention or retrieval mechanisms.⁶ In conclusion, The Golgi apparatus is central to the growth and division of the plant cell through its roles in protein glycosylation, protein sorting and cell wall synthesis.The transit of proteins and lipids from the trans-Golgi network (TGN) and the plasma membrane to endosomes within eucaryotic cells occurs via the budding and fusion of clathrin-coated vesicles (CCVs).^7,8 At the TGN, this process is mediated by the heterotetrameric AP-1 adaptor complex, which consists of two large subunits, β and γ1; a medium subunit, µ1; and a small σ1 subunit. Recruitment of AP-1 to the TGN membrane is regulated by a small GTPase, ADP-ribosylation factor 1 (ARF1), which cycles between an inactive GDP-bound form in cytosol and an active GTP-bound form that associates with the membrane like other small GTPase.⁹ There is also evidence that phosphorylation/dephosphorylation events are involved in the regulation of the function of AP-1. Ghosh and Kornfeld demostrated that AP-1 recruitment onto the membrane is associated with protein phosphatase 2A (PP2A)-mediated dephosphorylation of its β1 subunit, which enables clathrin assembly. This Golgi-associated isoform of PP2A exhibits specificity for phosphorylated β1 compared with phosphorylated µ1. Once on the membrane, the µ1 subunit undergoes phosphorylation, which results in a conformation change. This conformational change is associated with increased binding to sorting signals on the cytoplasmic tails of cargo molecules. Dephosphorylation of µ1 (and µ2) by another PP2A-like phosphatase reversed the effect and resulted in adaptor release from CCVs. Cyclical phosphorylation/dephosphorylation of the subunits of AP-1 regulate its function from membrane recruitment until its release into cytosol.¹⁰Plants experience desiccation stress either as part of a developmental programme, such as during seed maturation, or because of reductions in air humid and water availability in the soil. Underlying the ability of bryophytes to withstand periods of desiccation are morphological and biochemical adaptations. Plants respond to stress as individual cells and synergistically as a whole organism. Scanning electron microscopy observation showed that the P. patens gametophore cells were shrunk upon the treatment of desiccation, and the shrinking started from the edge of the leaves (Fig. 1). We could clearly observe some dark granula in the untreated cells, but these granula disappeared post-desiccation treatment (Fig. 1). Transmission electron microscopy also revealed that the large stacks of Golgi bodies and numerous coated vesicles are typically visible in the hydrated cells (Fig. 2), but these are absent in the desiccative cells (data not shown). The plant Golgi apparatus plays an important role in protein glycosylation and sorting. Therefore, this event means that the protein sorting and the cargo transporting are disrupted by desiccation stress. During desiccation, the absentness of Golgi bodies reduce the leaf activities of cell, and this is expected to similar to plant dormancy which is a phenomenon in resurrection plants and some drought-tolerant plants. In addition, through two-dimensional gel electrophoresis (2-DE) and LC-MS/MS analysis, AP-1 large subunit was identified as downregulated protein during the course of dehydration (Fig. 3). AP-1 is ubiquitously expressed and participates in the budding of clathrin-coated vesicles from the trans-Golgi network (TGN) and endosomes. AP-1 also recognizes sorting motifs in cargo molecules. Our results suggested that desiccation led to a marked disrupt in protein posttranslational modification, protein sorting and cell walls synthesis.Open in a separate windowFigure 1Scanning Electron microscopy images of normal and dehydrated P. patens gametophores. (A) the fresh leaf; (B) enlargement of the rectangle area of (A); (C) dehydrated gametophores of P. patens. Bar = 5 µm.Open in a separate windowFigure 2Transmission electron microscopy images of cell in fresh game-tophores. The arrows indicate Golgi body, Bar = 2 µm.Open in a separate windowFigure 3Part protein profile of the control and desiccation plants. The arrows indicate the AP-1 large subunit.     

A report on the zombie outbreak of 2009: how mathematics can save us (no,really)

An outbreak of zombification wreaked havoc recently in Canada and the rest of the world. Mathematical models were created to establish the speed of zombie infection and evaluate potential scenarios for intervention, mainly because mathematicians don’t have anything better to do with their time. We review the development of these models and their effect on the undead.In August 2009, a new disease emerged in North America and quickly made its way around the world.¹ Media reports suggest the outbreak began in Ottawa² but rapidly spread across Canada^3,4 and was transported thereafter to the United States⁵ and the United Kingdom.^6,7The infection resulted in a new species of human, classified as nonmortuus contagio, but known in the popular press as “zombie”, from the Congolese nzambi, meaning “spirit of a dead person.” As the name implies, the outbreak resulted in a resurgence of the previously deceased. Clinical signs included discoloration around the eyes, open wounds and rotting flesh, with organs and bodily functions operating at minimal levels.Initial studies reported that the zombies did not feel pain, but these findings could not be verified because of the zombification of the researchers in question. When asked for comment, the lead author of one such study said, “Grrrnn, aaarghhh, huuuuuungry!” When questioned in more detail, he replied, “Braaaaiiiinnnnsss!” No further information is available from the interview.The cause of the virus remains unknown. Anecdotal evidence suggests that zombies can be defeated by guns, the army, eventual starvation or Dire Straits records.New diseases are generally investigated using experiments on infected people, clinical trials or medical observation. Unfortunately, because of the rapid zombification of scientists, epidemiologists and doctors, society was left with only one group of technocrats who remained uninfected: mathematicians. Fortunately, during the time at which the outbreak occurred, members of this group had not been invited to parties and thus remained entirely uninfected.A mathematical model for zombie infection⁸ was quickly designed (Figure 1). As shown by the model, humans could be infected by contact with a zombie, whereas zombies could be created either through the conversion of humans or reanimation of the dead. The model assumed that zombies could be killed in encounters with humans, as often happened when humans ran over zombies in their cars. Initially, such deaths were assumed by authorities to be part of a concerted effort at eradication of zombies, but were later revealed to be simply a result of rush hour. Some drivers were surprised when these recently deceased zombies returned to life and attacked them. Other drivers simply handed the zombies some spare change and waited for the reanimated creatures to clean their windshields.Open in a separate windowFigure 1Schematic diagram of the basic mathematical model (black arrows). Humans (friendly Canadians, in this example) can either die naturally or be converted into zombies — which is not terribly pleasant, but does come with that nifty jacket and tie. Zombies can reanimate the dead or be killed by humans (although it must be said that the latter doesn’t bother them too much). Possible intervention include quarantine of the zombies (green arrow), a potential cure (blue arrow) or impulsive attacks (red arrow).Open in a separate windowThe model showed two equilibria: the disease-free equilibrium (with no zombies) and the doomsday equilibrium (where everyone is a zombie). The application of a linear stability analysis showed that — in the absence of further interventions — the disease-free equilibrium was unstable and the doomsday equilibrium was stable. This finding was not promising.Simulations based on a city of roughly 500 000 people demonstrated that an entire such city would be replaced by zombies after about four days (Figure 2A). Were this mass replacement of a population to occur in a city such as Ottawa, it may be unlikely anyone would notice. Nevertheless, nearby cities such as Montreal would no longer be in the bagel-supplying business in such a scenario, and that result would be serious.Open in a separate windowFigure 2Projected population dynamics (based on type of intervention) in the context of a zombie outbreak. In the basic model (A), zombies eradicate humans after 4 days, leaving nobody to host daytime variety television shows or stop you from entering nightclubs (i.e., no loss there). (B): Quarantine delays the inevitable. Slightly. (C): A cure allows zombies to live in harmony with humans, which would be more fun for zombies than humans. (D): Only episodes of blind, aggressive, unfeeling violence are effective. And that’s just on the part of humans.Given this model, even a small outbreak would lead to a collapse of society as we know it. Explaining to the mathematicians that this outcome might be a bad thing took time because, initially, they were not able to see the downside. However, they were quickly mobilized after realizing their supply of caffeine and science fiction DVDs would dry up.Three interventions were proposed. The first was quarantine (Figure 1, green arrow), whereby a small proportion of the zombies would be kept in isolation. But given that the infection was so virulent, even leaving a few zombies in the wild would result in a restart of the outbreak. Including quarantine thus made no difference to the stability of the doomsday equilibrium (Figure 2B). That was a bit of a downer, to be honest.The second intervention was a theoretical cure that would convert zombies back into humans (Figure 1, blue arrow). Although the mathematicians were reminded that such a cure was entirely theoretical and likely could not be developed within four days, they were quite taken with the idea of proving results based on things that couldn’t possibly exist. This response was annoying, because they should have been concentrating on zombies instead.With a cure, humans and zombies could coexist. But unless the cure were 100% effective, humans would survive only in small numbers (Figure 2C) — most likely in shopping malls, abandoned farmhouses or the Winchester pub.Finally, the idea of impulsive attacks was considered (Figure 1, red arrow). This intervention would involve an escalating series of discrete attacks on the zombies, using an advanced mathematical theory called impulsive differential equations. These equations are similar to ordinary differential equations, except that sometimes they jump up onto tables, paint themselves purple and start singing show tunes for no reason whatsoever.The projected outcome of this intervention was more promising. At regular intervals, humans would mobilize their resources and attack the zombies. Each attack would be carried out with more force than the last one. The humans would keep fighting with increasing intensity until either the zombies were destroyed or the humans were torn apart from limb to limb and their flesh consumed by the ghoulish undead. Still, you’ve got to laugh, haven’t you? If humans could manage these impulsive attacks, the zombies could be destroyed after 10 days (Figure 2D).The overall model had limitations, of course. The numerical contributions of natural births and deaths had been ignored because of the brief timescale involved and the unlikelihood that mathematicians would be engaged in breeding. Inclusion of population demographics in the model suggests a limitless supply of new bodies would be available for zombies to infect, resurrect and convert. We must therefore act quickly and decisively to eradicate zombies before they eradicate us.