Comparison of Switching and Biofilm Formation between MTL-Homozygous Strains of Candida albicans and Candida dubliniensis

Candida albicans and Candida dubliniensis are highly related species that share the same main developmental programs. In C. albicans, it has been demonstrated that the biofilms formed by strains heterozygous and homozygous at the mating type locus (MTL) differ functionally, but studies rarely identify the MTL configuration. This becomes a particular problem in studies of C. dubliniensis, given that one-third of natural strains are MTL homozygous. For that reason, we have analyzed MTL-homozygous strains of C. dubliniensis for their capacity to switch from white to opaque, the stability of the opaque phenotype, CO₂ induction of switching, pheromone induction of adhesion, the effects of minority opaque cells on biofilm thickness and dry weight, and biofilm architecture in comparison with C. albicans. Our results reveal that C. dubliniensis strains switch to opaque at lower average frequencies, exhibit a far lower level of opaque phase stability, are not stimulated to switch by high CO₂, exhibit more variability in biofilm architecture, and most notably, form mature biofilms composed predominately of pseudohyphae rather than true hyphae. Therefore, while several traits of MTL-homozygous strains of C. dubliniensis appear to be degenerating or have been lost, others, most notably several related to biofilm formation, have been conserved. Within this context, the possibility is considered that C. dubliniensis is transitioning from a hypha-dominated to a pseudohypha-dominated biofilm and that aspects of C. dubliniensis colonization may provide insights into the selective pressures that are involved.     

Indene-based frameworks targeting the 5-HT6 serotonin receptor: Ring constraint in indenylsulfonamides using cyclic amines and structurally abbreviated counterparts

Further studies in quest of 5-HT₆ serotonin receptor ligands led to the design and synthesis of a few selected examples of N-(inden-5-yl)sulfonamides with a ring-constrained aminoethyl side chain at the indene 3-position, some of which exhibited a high binding affinity, such as the pyrrolidine analogue 28 (K_i = 3 nM). Moreover, the structurally abbreviated N-(inden-5-yl)sulfonamides showed K_i values ?43 nM, which indicates that neither the N,N-aminoethyl nor the conformationally restricted aminoethyl side arm at the indene 3-position are required for binding. Selected compounds were then tested in a functional cAMP stimulation assay and found to act as 5-HT₆ antagonists, although with moderate potency at the micromolar level.     

A key role for the peroxisomal ABCD2 transporter in fatty acid homeostasis

Peroxisomes are essential organelles exerting key functions in fatty acid metabolism such as the degradation of very long-chain fatty acids (VLCFAs). VLCFAs accumulate in X-adrenoleukodystrophy (X-ALD), a disease caused by deficiency of the Abcd1 peroxisomal transporter. Its closest homologue, Abcd2, exhibits a high degree of functional redundancy on the catabolism of VLCFA, being able to prevent X-ALD-related neurodegeneration in the mouse. In the search for specific roles of Abcd2, we screened fatty acid profiles in organs and primary neurons of mutant knockout mice lacking Abcd2 in basal conditions and under dietary challenges. Our results indicate that ABCD2 plays a role in the degradation of long-chain saturated and omega9-monounsaturated fatty acids and in the synthesis of docosahexanoic acid (DHA). Also, we demonstrated a defective VLCFA beta-oxidation ex vivo in brain slices of Abcd1 and Abcd2 knockouts, using radiolabeled hexacosanoic acid and the precursor of DHA as substrates. As DHA levels are inversely correlated with the incidence of Alzheimer's and several degenerative conditions, we suggest that ABCD2 may act as modulator/modifier gene and therapeutic target in rare and common human disorders.     

The Effect of Ongoing Exposure Dynamics in Dose Response Relationships

Characterizing infectivity as a function of pathogen dose is integral to microbial risk assessment. Dose-response experiments usually administer doses to subjects at one time. Phenomenological models of the resulting data, such as the exponential and the Beta-Poisson models, ignore dose timing and assume independent risks from each pathogen. Real world exposure to pathogens, however, is a sequence of discrete events where concurrent or prior pathogen arrival affects the capacity of immune effectors to engage and kill newly arriving pathogens. We model immune effector and pathogen interactions during the period before infection becomes established in order to capture the dynamics generating dose timing effects. Model analysis reveals an inverse relationship between the time over which exposures accumulate and the risk of infection. Data from one time dose experiments will thus overestimate per pathogen infection risks of real world exposures. For instance, fitting our model to one time dosing data reveals a risk of 0.66 from 313 Cryptosporidium parvum pathogens. When the temporal exposure window is increased 100-fold using the same parameters fitted by our model to the one time dose data, the risk of infection is reduced to 0.09. Confirmation of this risk prediction requires data from experiments administering doses with different timings. Our model demonstrates that dose timing could markedly alter the risks generated by airborne versus fomite transmitted pathogens.     

Molecular Characterization of Sewage-Borne Pathogens and Detection of Sewage Markers in an Urban Stream in Caracas,Venezuela

Molecular characterization of two sewage-borne pathogens identified hepatitis A virus (HAV) subgenotype IA and Giardia duodenalis assemblages A and B as predominant genotypes circulating in an urban area of Venezuela. This study reveals epidemiological features of human pathogens of worldwide distribution and the efficacy of molecular methods for accurate assessment of sewage pollution.Multiple microbial pathogens may frequently be found in surface waters that receive uncontrolled municipal sewage discharges. The range and diversity of sewage-borne pathogens in surface waters are geographically specific and strongly dependent on the burden of infectious diseases in the population, the seasonal patterns of infectious diseases in the community, and the availability of sewage treatment processing (5, 7). The metropolitan city of Caracas, the capital and largest city of Venezuela, is located in northern South America near the Caribbean coast. Water pollution is a big issue in Caracas, like in many other cities in South America, where most of the human sewage (∼97%) from overpopulated urbanized areas is discharged without any treatment into nearby rivers and coastal environments. Despite these facts, the seriousness of sewage-related health issues is not at the forefront of public concern in this country.Giardia is the protozoan parasite most frequently detected in human fecal samples submitted to diagnostic laboratories from major cities in Venezuela. The frequency of giardiasis reported in the population varies between 21% and 45% but may increase up to 75% among school age children (8). Notwithstanding, the epidemiology of giardiasis in Venezuela remains unknown, and no previous studies have documented the distribution of species and genotype assemblages associated with human infections.Hepatitis A virus (HAV), the etiological agent of hepatitis A in humans, has distinguishable epidemiological patterns of distribution and endemicity closely related to socioeconomic development (9, 12). Water- and food-borne outbreaks of HAV have been well documented worldwide (6, 19). Seroepidemiological studies conducted in selected populations in Venezuela have demonstrated high endemicity of hepatitis A infection among low socioeconomic population strata, with seroprevalences between 48% and 98% (13). Nevertheless, studies on HAV genotype circulation in major urban areas of the country are scarce, as is research on predominant exposure routes and potential transmission patterns through the environment.The analysis of nucleic acid sequences of sewage-borne pathogens may provide relevant information on predominant species and genotypes of human-pathogenic viruses and parasites circulating in specific geographical areas (11, 12, 15). The molecular approach may be of relevance for countries lacking reliable disease surveillance programs and proper understanding of the potential transmission of specific human pathogens through the environment. In this research, Giardia cysts and HAV recovered from an urban stream were characterized by multiple molecular methods along with nucleotide sequence analysis to identify predominant genotypes circulating in a major urban area of Venezuela''s capital. The strength and efficacy of multiple molecular methods for accurate assessment of human sewage pollution and risks of exposure to sewage-borne pathogens were also investigated.Dry season sampling (October through March) was conducted in a heavily polluted urban stream (>10⁶ fecal coliforms/100 ml) that flows in a southeast direction through the metropolitan city of Caracas (16). Water sample volumes of 100 ml were collected in three sterile centrifuge tubes two to four times per month. Giardia cysts were concentrated by centrifugation (100 ml at 1,500 × g for 15 min) followed by DNA extraction by the freeze-thaw method in the presence of Chelex-100 (3) for sucrose-purified cysts. Human-pathogenic assemblage occurrence was determined by nested PCR amplification and sequence analysis of the triosephosphate isomerase (tpi) gene (18). Multiple sequence alignments were performed with ClustalW (21), and phylogenetic analyses were conducted using MEGA4 software (20). The genetic diversity of Giardia isolates was inferred by the neighbor-joining method (17) using a bootstrap test of 1,000 replicates. Giardia cysts counts were obtained by fluorescence microscopy using BTF EasyStain monoclonal antibody stain (BTF Precise Microbiology, Inc.) and 4′6-diamidino-2-phenylindole (DAPI). The recovery efficiency of cysts was determined in five experiments using ColorSeed C&G spike suspensions as internal quality controls (14).HAV particles were concentrated from 35 ml by ultracentrifugation and elution with 0.25 N glycine buffer following procedures previously described (16). Viral RNA was extracted from sample concentrates with Trizol (Invitrogen, Inc., Carlsbad, CA) following the manufacturer''s instructions. General detection of HAV was based on amplification of the 5′ nontranslated region (5′ NTR), while analysis of genetic diversity involved sequencing and phylogenetic analysis of the VP1 amino terminus and the full VP1 gene (2, 10). Sequence alignment was conducted with the DNAman software 5.2.2 (Lynnon BioSoft, Quebec, Canada) followed by phylogenetic analysis.Molecular detection of sewage pollution was accomplished by PCR amplification of Bacteroidales human-specific 16S rRNA genes, Bacteroides thetaiotamicron 16S rRNA genes, and the nifH gene of Methanobrevibacter smithii using primers and PCR conditions originally described by Field et al. (4), Carson et al. (1), and Ufnar et al. (22), respectively.Giardia duodenalis tpi nucleotide sequences amplified directly from urban stream waters were included after phylogenetic analysis into two well-defined clusters of assemblages A and B. These results were supported by high bootstrap values, as indicated in Fig. ​Fig.1.1. The level of cysts recovered from these samples ranged from 10,400 to 62,000 cysts/liter; however, mean percent recoveries varied from 20% to 50%, which suggests that the urban stream may harbor and receive much higher loads of cysts.Open in a separate windowFIG. 1.Phylogenetic tree of G. duodenalis assemblages A (VPW2, VPW3, and VPW7) and B (VPW1, VPW4, VPW5, and VPW6) from urban stream samples forming two clusters in a neighbor-joining analysis of tpi nucleotide sequences. Only bootstrap values >80% are shown in the tree.Three genomic regions used for detection and characterization of HAV revealed the predominance of HAV strains belonging to subgenotype IA, the most frequent genotype associated with human disease worldwide (9). A neighbor-joining tree constructed from the alignment of nucleotide sequences from urban stream samples and sequences of HAV strains from case patients (unpublished data) was used to investigate the relationship between genotypes present in environmental and clinical samples. The comparative analysis indicated a high degree of identity (98 to 99%) between nucleotide sequences from the urban stream and the strains from sporadic HAV cases. The phylogenetic analysis grouped all of these sequences into two unique clades within subgenotype IA, strongly supported by significant bootstrap values (Fig. 2A, B, and C).Open in a separate windowFIG. 2.Phylogenetic analysis of the HAV 5′ NTR (A; 284 nucleotides [nt]), VP1 amino terminus (B; 172 nt), and complete VP1 (C; 820 nt) regions. Nucleotide sequences of HAV reference strains are designated by their GenBank accession number, including the name of the country of origin, except for Venezuelan isolates, which are shown in bold. S, isolates derived from human sporadic cases; W, urban stream isolates. Phylogenetic analysis was performed by neighbor joining, and phylogenetic distances were calculated by the Kimura two-parameter test. Bootstrap values ≥90% are shown in the trees. Letters in bold indicate the subtype.Three reliable published assays for detection of human-specific markers of fecal pollution identified and confirmed the predominant point source of water pollution. Sequence analysis of three randomly selected PCR products from each marker revealed ≥99% sequence identity with published sequences (GenBank) derived from different geographical areas, thus indicating the validity and specificity of the molecular markers as reliable indicators of human sewage pollution in Venezuela.The results of this research demonstrate that the molecular assays applied for detection and characterization of sewage-borne pathogens in surface waters may have practical applications for epidemiological investigations on distribution of predominant human-specific genotypes circulating in urban populations. Previous studies identified the most predominant waterborne gastroenteritis viruses circulating in Metropolitan Caracas (16).The molecular-based monitoring approach for rapid and precise identification of sewage-borne pathogens and sewage markers in surface waters has important implications for sewage-related health issues that require special attention in Venezuela and South America. Deficient sewerage coverage and lack of municipal wastewater treatment, commonly associated with informal settlements around densely populated urban areas, are responsible for many of the environmental degradation and public health problems that occur in these countries. The precise identification of human pathogens in the environment offers an appropriate and alternative approach for initial assessment of risks of exposure to waterborne pathogens. Current bacterial indicators of fecal pollution (fecal coliforms, Escherichia coli, and enterococci) do not allow identification of the relative sources of impacts (i.e., sewage, urban runoff, and agricultural waste) on surface waters. Thus, the molecular detection of sewage-borne pathogens and sewage markers in surface waters may be more effective than the bacterial indicator approach for forecasting pathogen distribution and for managing and reducing risks associated with inappropriate sewage disposal into natural waters in Venezuela and South America.     

The acute response of practical occlusion in the knee extensors

Training at low intensities with moderate vascular occlusion results in increased muscle hypertrophy, strength, and endurance. Elastic knee wraps, applied to the proximal portion of the target muscle, might elicit a stimulus similar to the KAATSU Master Apparatus. The purpose of this study was to test the hypothesis that intermittently occluding the leg extensors with elastic knee wraps would increase whole-blood lactate (WBL) over control (CON). Twelve healthy men and women participated in this study (age 21.2 ± 0.35 years, height 168.9 ± 2.60 cm, and body mass 71.2 ± 4.16 kg). One repetition maximum (1RM) testing for the leg extensors was performed on a leg extension machine for the first trial, followed by occlusion (OCC) and CON trials. Four sets of leg extension exercise (30-15-15-15) were completed with 150-second rest between sets at 30% 1RM. Whole-blood lactate, heart rate (HR), and ratings of perceived exertion (RPEs) were measured after every set of exercise and 3 minutes postexercise. Data were analyzed using repeated-measures analysis of variance with statistical significance set at p ≤ 0.05. Whole-blood lactate increased in response to exercise (p = 0.01) but was not different between groups (OCC 6.28 ± 0.66 vs. CON 5.35 ± 0.36 mmol·L, p = 0.051). Heart rate (OCC 128.86 ± 4.37 vs. CON 119.72 ± 4.10 b·min?1) was higher with OCC from sets 2-4 (p ≤ 0.03), with no difference 3 minutes postexercise (p = 0.29). Rating of perceived exertion was higher with OCC after every set (OCC 15.10 ± 0.31 vs. CON 12.16 ± 0.50, p = 0.01). In conclusion, no differences exist for WBL between groups, although there was a trend for higher levels with OCC. The current protocol for practical occlusion did not significantly increase metabolic stress more than normal low-intensity exercise. This study does not support the use of knee wraps as a mode of blood-flow restriction.     

Bcr/Abl interferes with the Fanconi anemia/BRCA pathway: implications in the chromosomal instability of chronic myeloid leukemia cells

Chronic myeloid leukemia (CML) is a malignant clonal disorder of the hematopoietic system caused by the expression of the BCR/ABL fusion oncogene. Although it is well known that CML cells are genetically unstable, the mechanisms accounting for this genomic instability are still poorly understood. Because the Fanconi anemia (FA) pathway is believed to control several mechanisms of DNA repair, we investigated whether this pathway was disrupted in CML cells. Our data show that CML cells have a defective capacity to generate FANCD2 nuclear foci, either in dividing cells or after DNA damage. Similarly, human cord blood CD34(+) cells transduced with BCR/ABL retroviral vectors showed impaired FANCD2 foci formation, whereas FANCD2 monoubiquitination in these cells was unaffected. Soon after the transduction of CD34(+) cells with BCR/ABL retroviral vectors a high proportion of cells with supernumerary centrosomes was observed. Similarly, BCR/ABL induced a high proportion of chromosomal abnormalities, while mediated a cell survival advantage after exposure to DNA cross-linking agents. Significantly, both the impaired formation of FANCD2 nuclear foci, and also the predisposition of BCR/ABL cells to develop centrosomal and chromosomal aberrations were reverted by the ectopic expression of BRCA1. Taken together, our data show for the first time a disruption of the FA/BRCA pathway in BCR/ABL cells, suggesting that this defective pathway should play an important role in the genomic instability of CML by the co-occurrence of centrosomal amplification and DNA repair deficiencies.     

A new approach for in vitro regeneration of tomato plants devoid of exogenous plant growth hormones

Many available methodologies for in vitro regeneration of commercial tomato varieties promote not only the production of normal shoots but also individual leaves, shoots without apical meristems and vitrified structures. All these abnormal formations influence and diminish the regeneration efficiency. At the basis of this phenomenon lies callus development. We optimized an alternative procedure by which the regeneration occurs without abnormal shoot formation. The portion including the proximal part of hypocotyls and the radicle was cultured on medium consisting of Murashige and Skoog salts, 4 mg/L thiamine, 100 mg/L mio-inositol and 3% sucrose. After two-three weeks, 60% explants showed adventitious shoot formation. No changes in the morphological characteristics of regenerated plants and fruits were observed as compared with parents. Karyotypic analysis of regenerated plants showed no variations in chromosome number. The optimized procedure offers the advantage of tomato plant regeneration avoiding callus formation, which enables normal plant recovery with an efficiency ranging from 1.45 +/- 0.05 to 2.57 +/- 0.06 shoots per explant in Campbell-28, Amalia, Lignon, and Floradel cultivars.     

Increased secretion of leukemia inhibitory factor by immature airway smooth muscle cells enhances intracellular signaling and airway contractility

Airway smooth muscle cells (ASMC) play a major role in airway inflammation, hyperresponsiveness, and obstruction in asthma. However, very little is known regarding the relation between inflammatory mediators and cytokines and immature ASMC. The aim of this study was to evaluate 1) the secretion of leukemia inhibitory factor (LIF) (an IL-6 family neurotrophic cytokine) by ASMC; 2) intracellular calcium concentration ([Ca(2+)](i)) signaling; and 3) the effect of LIF on mast cell chemotaxis and rat airway contractility. Immature and adult human ASMC were cultured. ELISA and real-time PCR were performed to assess LIF protein secretion and mRNA production, [methyl-(3)H]thymidine incorporation to quantify ASMC DNA synthesis, a Boyden chamber to evaluate the effect of LIF on mast cell chemotaxis, microspectroflurimetry using indo-1 (at baseline and after stimulation bradykinin, U-46619, histamine, and acetylcholine, in the presence or absence of LIF or TNF-alpha) for [Ca(2+)](i) signaling, and isolated rat pup tracheae to determine the effect of LIF on airway contractility to ACh. TNF-alpha-stimulated immature ASMC produce more LIF mRNA and protein than adult ASMC, although this cytokine induces a moderate increase in DNA synthesis (+20%) in adult ASMC only. Human recombinant LIF exerts no chemotactic effect on human mast cells. In immature ASMC, ACh-induced [Ca(2+)](i) response was enhanced twofold after incubation with LIF, whereas TNF-alpha increased the [Ca(2+)](i) to U-46619 threefold. In TNF-alpha-exposed adult ASMC, [Ca(2+)](i) responses to ACh were of greater magnitude (sixfold increase) than in immature ASMC. Human recombinant LIF increased contractility to ACh by 50% in immature, isolated rat tracheae. Stimulated immature human ASMC greatly secrete LIF, thus potentially contributing to neuroimmune airway inflammation and subsequent remodeling. Increased LIF secretion enhances airway reactivity and [Ca(2+)](i) signaling.     

Serum insulin-like growth factor levels do not predict functional recovery after hospitalization in nonagenarian patients

AIM: To analyze the insulin-like growth factor 1 (IGF-1) serum levels in nonagenarian patients and to investigate the predictive capacity of this measure to assess the functional recovery of this population following hospitalization. METHODS: We performed a prospective study in 60 consecutive nonagenarian patients admitted for medical or surgical diseases. We assessed IGF-1 serum levels and nutritional status. The functional status was assessed using the Barthel index. Thirty-four patients were reinvestigated 3 months after discharge from hospital. RESULTS: The mean levels of IGF-1 were lower in nonagenarians than in younger patients. No relationship was found between IGF-1 levels and nutritional status. The decline in Barthel index values 3 months after discharge from hospital did not correlate with serum levels of IGF-1 on admission. CONCLUSION: IGF-1 serum levels in nonagenarian patients do not predict functional recovery after hospitalization.