Relatedness of Vibrio cholerae O1/O139 Isolates from Patients and Their Household Contacts,Determined by Multilocus Variable-Number Tandem-Repeat Analysis

The genetic relatedness of Vibrio cholerae O1/O139 isolates obtained from 100 patients and 146 of their household contacts in Dhaka, Bangladesh, between 2002 and 2005 was assessed by multilocus variable-number tandem-repeat analysis. Isolate genotypes were analyzed at five loci containing tandem repeats. Across the population, as well as within households, isolates with identical genotypes were clustered in time. Isolates from individuals within the same household were more likely to have similar or identical genotypes than were isolates from different households, but even within a household, isolates from different individuals often had different genotypes. When household contacts were sampled regularly for 3 weeks after the illness of the household index patient, isolates with genotypes related to the index patient appeared in contacts, on average, ∼3 days after the index patient, while isolates with unrelated genotypes appeared in contacts ∼6 days after. Limited data revealed that multiple isolates from the same individual collected within days of each other or even from a single stool sample may have identical, similar, or unrelated genotypes as well. Our results demonstrate that genetically related V. cholerae strains cluster in local outbreaks but also suggest that multiple distinct strains of V. cholerae O1 may circulate simultaneously within a household.Vibrio cholerae is the etiologic agent of cholera, a secretory diarrheal disease with a high mortality rate in humans if untreated (25). Serogroups of V. cholerae, a motile, Gram-negative, curved rod, can be defined serologically by the O side chain of the lipopolysaccharide (LPS) component of the outer membrane (9). V. cholerae is found in a variety of forms in aquatic ecosystems (41, 42), and more than 200 different serogroups have been isolated, mostly from environmental sources (45). However, the vast majority of V. cholerae strains that cause the clinical disease cholera belong to serogroup O1 or O139 (37, 42). V. cholerae O1, the historical agent of epidemic and pandemic cholera and the current leading cause of cholera both globally and in Bangladesh (42), is classified into two major biotypes, classical and El Tor (44), and two major serotypes, Ogawa and Inaba (48). The current global pandemic is caused by V. cholerae O1 El Tor. A second pathogenic serogroup, O139, emerged in the Bengal region in 1992 by horizontal transfer of new LPS biosynthesis-encoding genes into the El Tor biotype (1, 4). This new serogroup continues to cocirculate with El Tor V. cholerae O1 serotypes Ogawa and Inaba as a cause of disease in humans, although it accounts for a smaller proportion of all cholera now than in its first years of circulation (16, 20). Recently, comparative genomics has revealed an extensive amount of lateral gene transfer between strains, suggesting that genomic classification may be an alternative to serogrouping for classifying pathogenic V. cholerae strains (11).Toxigenic V. cholerae may be present in environmental sources in regions of endemicity and emerge, often seasonally, to cause cholera in humans (12, 18). Once an outbreak has begun, organisms from one infected individual are more infectious for the next individual, a property termed hyperinfectivity, and these forms may be able to pass directly from human to human through fecal-oral contamination (35). However, because vibrio organisms are difficult to isolate from implicated environmental or domestic water sources (28, 29), little is known about the diversity of V. cholerae in inocula that cause human infection.Established laboratory methods for differentiating V. cholerae strains, apart from serogrouping and serotyping, include rRNA restriction fragment length polymorphism (ribotyping), pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). These methods, however, have a limited capacity to differentiate between pathogenic V. cholerae strains, as clinical isolates are relatively genetically monomorphic. For instance, V. cholerae O1 comprises approximately 30 ribotypes (39); however, only a few ribotypes are common in clinical isolates, ribotypes evolve slowly, and all isolates of a given pathogenic V. cholerae serotype in a local area over a period of multiple years often belong to a single ribotype (8, 14, 17). In a broad sampling of 154 V. cholerae isolates from Bangladesh and worldwide over several decades, only 15 ribotypes were identified, and of these, many were found in nonpathogenic environmental isolates only; only five ribotypes were associated with the V. cholerae O1 El Tor biotype that currently predominates as the cause of clinical disease, while pathogenic isolates of serogroup O139 were indistinguishable from each other by ribotype (19).PFGE, in which restriction endonuclease digestion of genomic DNA generates mutation-sensitive banding patterns, is often more sensitive than ribotyping in detecting strain variation (7, 34, 51) and detects extensive genetic variation within nonpathogenic V. cholerae serogroups (3, 46). However, PFGE types change slowly and are useful primarily for distinguishing between strains in different pandemics or between different continental branches of those pandemics. In an analysis of 180 mostly western-hemisphere isolates (7), PFGE differences had developed from a prior pandemic strain over the 30 years since its arrival in Latin America, but a new strain that had been causing disease for 2 years still had only a single PFGE type across the 64 isolates analyzed. Similarly, in a Japanese study (2), although 19 PFGE types were identified among O1 isolates, the majority of the domestic isolates, along with several imported isolates, belonged to a single PFGE type.Further differentiation between V. cholerae isolates is achievable by MLST, which characterizes isolates by internal DNA sequences in selected housekeeping genes (32). Nevertheless, epidemic strains also cluster tightly in this typing scheme (5, 32) and the method has been useful primarily for determining relationships between nontoxigenic strains (36) or for linking regional outbreaks (which typically appear monoclonal by these methods) with the pandemic strain responsible (5, 33).Although these methods have distinguished major pandemic clones from other nonpathogenic human and environmental isolates of V. cholerae, the near clonality of pathogenic O1 and O139 strains means that established methods may not provide sufficiently robust differentiation of these genetically similar pathogenic strains to answer important epidemiological questions. Therefore, there is a need for other methods that can distinguish among clinical O1 and O139 isolates and track the epidemiology of outbreaks in a restricted geographic area on a shorter time scale.Multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) is one method that may be useful for differentiating between pathogenic V. cholerae O1 and O139 strains that would be indistinguishable by other techniques (15). This method examines short repeating DNA segments at various locations in the genome that can vary in number at each location and uses the number of repeats at each varying locus as a fingerprint to distinguish between isolates.Escherichia coli is the paradigm organism for demonstrating the value of the MLVA method. Noller et al. (38) showed that E. coli O157 isolates that were indistinguishable by MLST could be distinguished to some extent by PFGE but that MLVA distinguished between isolates that had the same PFGE type and did so in a manner consistent with the known epidemiology of the isolates (38a). In addition, machine-scored VNTR assays have been demonstrated to be robust and portable and to discriminate clearly between isolates by using relatively few loci, therefore limiting the effect of compounding genotyping errors (6).For V. cholerae, five VNTR loci have been identified (15), and the initial application of MLVA at those loci has demonstrated distinct populations of clinical isolates of V. cholerae in different geographic regions within Bangladesh and India (23, 47). Predominant isolates in each of two rural Bangladeshi regions varied gradually over a time scale of months to years (47), and isolates collected from India over a 15-year period varied widely, with individual MLVA types clustering in time and place—some with widespread dissemination and others with limited local occurrence only (23). MLVA has also been used to classify hybrid and altered V. cholerae variants and to demonstrate their genetic distance from the pandemic El Tor strain (10). Use of the MLVA method for epidemiologic study of cholera requires that V. cholerae VNTR alleles remain reasonably stable during bacterial replication in patients or in laboratory culture after isolation. Some degree of stability of two of the five loci used in V. cholerae MLVA has been demonstrated previously by serial passage in vitro through four overnight cultures (15). In this study, we used MLVA to examine V. cholerae O1 and O139 isolates obtained from infected patients and their household contacts—including multiple isolates from the same individual and isolates from multiple individuals within the same household—in a large city where cholera is endemic.     

2-D protein maps of rat gastrocnemius and soleus muscles: a tool for muscle plasticity assessment

Functional characterization of muscle fibers relies on ATPase activity and on differential measurements of metabolic proteins, including mitochondrial and glycolytic enzymes, glucose, lactate and lactic acid transporters, calcium cycling proteins and components of the contractile machinery. The recent introduction of microarray technology has enabled detailed gene expression studies under different physiological and pathological conditions, thus generating novel hypotheses on muscle function. However, microarray approaches are limited by the incomplete genome coverage of currently available chips, and by poor correlation between mRNA concentration and protein expression level. We have used 2-DE and MS to build a reference map of proteins from rat mixed gastrocnemius and soleus muscle, and to assess qualitative and quantitative differences in protein distribution between these two functionally dissimilar muscles. More than 800 spots on each gel were detected by silver staining, of which 167 were excised, digested in-gel with trypsin and analyzed by ESI-MS/MS. One hundred and twenty eight distinct gene products were identified, including metabolic, transport and contractile proteins. Forty one spots displayed differences in relative expression level between mixed gastrocnemius and soleus samples. These data not only enable differentiation of functionally distinct slow-twitch and fast-twitch fiber types, but also provide tools for investigating muscle plasticity in response to physiological and environmental conditions such as aging or hypoxia.     

Antitumor activity of curcumin is mediated through the induction of apoptosis in AK-5 tumor cells

Curcumin, the yellow pigment of turmeric (Curcuma longa), used commonly as a spice, has been shown to possess anticarcinogenic activity. Curcumin inhibited AK-5 tumor growth and induced apoptosis in AK-5 cells. Curcumin induced apoptosis is mediated through the activation of caspase-3, which is specifically inhibited by the tetrapeptide Ac-DEVD-CHO. In addition, curcumin induced tumor cell death is caused through the generation of reactive oxygen intermediates which is inhibited by N-acetyl-L-cysteine. Our studies suggest that the apoptotic process induced by curcumin is the mechanism mediating AK-5 tumor cell death.     

WGEF is a novel RhoGEF expressed in intestine, liver, heart, and kidney

Rho family GTPases regulate multiple cellular processes through their downstream effectors, where their activities are stimulated by the guanine nucleotide exchange factors. Here, we report a new member of RhoGEF, WGEF, which has the classical structure of DH-PH domain and a C-terminal SH3 domain. WGEF was shown to activate RhoA, Cdc42, and Rac1 by pulldown assay, and forced expression of WGEF resulted in marked rearrangement of the actin cytoskeleton, which is typically seen by the activation of RhoA, Cdc42, and Rac1. WGEF was highly expressed in intestine and also in liver, heart and kidney, which may suggest the involvement of WGEF in the development and functions of these organs. The expression pattern may also suggest the possible importance of WGEF in the understanding of diseases based on metabolic disorder.     

In situ extension as an approach for identifying novel alpha-amylase inhibitors

A new approach for the discovery and subsequent structural elucidation of oligosaccharide-based inhibitors of alpha-amylases based upon autoglucosylation of known alpha-glucosidase inhibitors is presented. This concept, highly analogous to what is hypothesized to occur with acarbose, is demonstrated with the known alpha-glucosidase inhibitor, d-gluconohydroximino-1,5-lactam. This was transformed from an inhibitor of human pancreatic alpha-amylase with a K(i) value of 18 mm to a trisaccharide analogue with a K(i) value of 25 mum. The three-dimensional structure of this complex was determined by x-ray crystallography and represents the first such structure determined with this class of inhibitors in any alpha-glycosidase. This approach to the discovery and structural analysis of amylase inhibitors should be generally applicable to other endoglucosidases and readily adaptable to a high throughput format.     

Brain tumor inhibition in experimental model by restorative immunotherapy with a corpuscular antigen

In view of the advances in our understanding of anti-tumor immune response, it is now tempting to contemplate the development of immunotherapies for malignant brain tumors, for which no effective treatment exists. Immunotherapy, with agents known as biological response modifiers (BRMs) are thus gaining increasing interest as the fourth modality of treatment. A non-specific BRM, sheep erythrocytes (SRBC) when administered (ip, 7% PCV/V, 0.5 ml) in a group of animals at the end of seventh month of ethylnitrosourea administration, resulted in significant increase in the mean survival time (> 350 days). Studies conducted for growth kinetics pattern with proliferation index and fluorochrome (HO-33342) uptake techniques at the tissue culture level exhibited a regulatory inhibition of the cells isolated from tissue excised from the tumor susceptible area of brain of SRBC treated animals. Moreover, histological examination of brain from animals showed immunomodulatory role of SRBC in experimentally induced brain tumor. Further probe into the mechanisms involving immunological investigations at the cellular level in these animals indicated an augmented and potentiated cell mediated immune response (CMI) as evidenced by enhanced spontaneous rosette forming capacity and cytotoxic activity of lymphocytes and neutrophil (PMN) mediated phagocytosis respectively. The observations suggest that SRBC down regulate malignant growth pattern of experimental brain tumors either by an immunologically enhanced killing of tumor cells and/or by directly inhibiting the tumor growth possibly via a stimulated cytokine network. Thus, a corpuscular antigen, can potentiate CMI response in experimentally induced brain tumor animal model, in which response induced in the periphery are able to mediate anti-tumor effects in the brain.     

Improved functional properties of the ovoinhibitor by conjugating with galactomannan

The chicken egg white ovoinhibitor, a multi-type proteinase inhibitor, was conjugated with galactomannan through the Maillard reaction in a controlled dry heating state at 60 degrees C and 65% relative humidity. The formation of an ovoinhibitor-galactomannan conjugate during dry heating was confirmed by SDS-PAGE. The resulting ovoinhibitor-galactomannan conjugate showed almost the same inhibitory activity toward trypsin, chymotrypsin and elastase as that of the untreated ovoinhibitor, while the conjugate showed stronger heat stability and better emulsifying properties than the untreated ovoinhibitor. These results suggest that the ovoinhibitor-galactomannan conjugate can be used as a protease inhibitor having heat stability and outstanding emulsifying properties for industrial application.     

Strategic compartmentalization of Toll-like receptor 4 in the mouse gut

Pattern recognition receptors (PRRs), which include the Toll-like receptors (TLRs), are involved in the innate immune response to infection. TLR4 is a model for the TLR family and is the main LPS receptor. We wanted to determine the expression of TLR4 and compare it with that of TLR2 and CD14 along the gastrointestinal mucosa of normal and colitic BALB/c mice. Colitis was induced with 2.5% dextran sodium sulfate (DSS). Mucosa from seven segments of the digestive tract (stomach, small intestine in three parts, and colon in three parts) was isolated by two different methods. Mucosal TLR4, CD14, TLR2, MyD88, and IL-1beta mRNA were semiquantified by Northern blotting. TLR4 protein was determined by Western blotting. TLR4/MD-2 complex and CD14 were evaluated by immunohistochemistry. PRR genes were constitutively expressed and were especially stronger in colon. TLR4 and CD14 mRNA were increased in the distal colon, but TLR2 mRNA was expressed more strongly in the proximal colon, and MyD88 had a uniform expression throughout the gut. Accordingly, TLR4 and CD14 protein levels were higher in the distal colon. TLR4/MD-2 and CD14 were localized at crypt bottom epithelial cells. TLR4/MD2, but not CD14, was found in mucosal mononuclear cells. Finally, DSS-induced inflammation was localized in the distal colon. All genes studied were up-regulated during DSS-induced inflammation, but the normal colon-stressed gut distribution was preserved. Our findings demonstrate that TLR4, CD14, and TLR2 are expressed in a compartmentalized manner in the mouse gut and provide novel information about the in vivo localization of PRRs.     

The Southwest of Scotland Structural Chromosomal Abnormalities Database: an assessment of its contribution to genetic counselling in affected families

AIM: To assess the effect of establishing a genetic database on the provision of genetic counselling to individuals and families with structural chromosomal abnormalities. METHOD: For the four year period 1997-2000, we compared all cytogenetics laboratory records with entries on the database to determine its completeness. We assessed the extent to which families had been followed up, compared these findings with a previous four year period (1977-1980) and sought to discover why some families were not followed up. RESULTS: Of 215 probands identified during 1997-2000, 19 (9%) were not recorded on the register. Approximately one third of families were followed up completely, one third were partially followed up and one third had had no follow-up, for a variety of reasons. In this last group, there was evidence that some had received inadequate or incorrect genetic advice. There was no evidence that the database improved follow-up in families with structural chromosome abnormalities. Over 20 years, there has been a downward trend in the proportion of cases referred to the genetic clinic. CONCLUSIONS: Our register can be used to monitor trends in clinical practice but has had no direct effect on the service provided to patients and their families.