Search

Hepatitis C virus is a genetically heterogeneous RNA virus that is a major cause of liver disease worldwide. Here, we show that, despite its extensive heterogeneity, the evolution of hepatitis C virus is primarily shaped by negative selection and that numerous coordinated substitutions in the polyprotein can be organized into a scale-free network whose degree of connections between sites follows a power-law distribution. This network shares all major properties with many complex biological and technological networks. The topological structure and hierarchical organization of this network suggest that a small number of amino acid sites exert extensive impact on hepatitis C virus evolution. Nonstructural proteins are enriched for negatively selected sites of high centrality, whereas structural proteins are enriched for positively selected sites located in the periphery of the network. The complex network of coordinated substitutions is an emergent property of genetic systems with implications for evolution, vaccine research, and drug development. In addition to such properties as polymorphism or strength of selection, the epistatic connectivity mapped in the network is important for typing individual sites, proteins, or entire genetic systems. The network topology may help devise molecular intervention strategies for disrupting viral functions or impeding compensatory changes for vaccine escape or drug resistance mutations. Also, it may be used to find new therapeutic targets, as suggested in this study for the NS4A protein, which plays an important role in the network.

Hepatitis C is a major public health problem in the United States and worldwide. Outbreaks of hepatitis C virus (HCV) infections associated with unsafe injection practices, drug diversion, and other exposures to blood are difficult to detect and investigate. Molecular analysis has been frequently used in the study of HCV outbreaks and transmission chains; helping identify a cluster of sequences as linked by transmission if their genetic distances are below a previously defined threshold.

Hepatitis C is a major public health problem in the United States and worldwide. Outbreaks of hepatitis C virus (HCV) infections are associated with unsafe injection practices, drug diversion, and other exposures to blood and are difficult to detect and investigate. Here, we developed and validated a simple approach for molecular detection of HCV transmissions in outbreak settings. We obtained sequences from the HCV hypervariable region 1 (HVR1), using end-point limiting-dilution (EPLD) technique, from 127 cases involved in 32 epidemiologically defined HCV outbreaks and 193 individuals with unrelated HCV strains. We compared several types of genetic distances and calculated a threshold, using minimal Hamming distances, that identifies transmission clusters in all tested outbreaks with 100% accuracy. The approach was also validated on sequences obtained using next-generation sequencing from HCV strains recovered from 239 individuals, and findings showed the same accuracy as that for EPLD. On average, the nucleotide diversity of the intrahost population was 6.2 times greater in the source case than in any incident case, allowing the correct detection of transmission direction in 8 outbreaks for which source cases were known. A simple and accurate distance-based approach developed here for detecting HCV transmissions streamlines molecular investigation of outbreaks, thus improving the public health capacity for rapid and effective control of hepatitis C.

Summary. The aim of the present study was to establish the serum levels of inhibins and their relationship with the degree of seminal alteration in infertile men. Thirty-six patients with varicocele (Va) and seven non-obstructive azoospermic men (Az) were included. The Va group was divided into two subgroups: Va I (sperm concentration: >20 × 106 ; n = 21) and Va II (sperm concentration

Summary Background : Eradication rates of Helicobacter pylori with standard triple therapy are disappointing, and studies from several countries confirm this poor performance. Aim : To assess the eradication rate of a new sequential treatment regimen compared with conventional triple therapy for the eradication of H. pylori infection. Methods : One thousand and forty-nine dyspeptic patients were studied prospectively. H. pylori-infected patients were randomized to receive 10-day sequential therapy [rabeprazole (40 mg daily) plus amoxicillin (1 g twice daily) for the first 5 days, followed by rabeprazole (20 mg), clarithromycin (500 mg) and tinidazole (500 mg) twice daily for the remaining 5 days] or standard 7-day treatment [rabeprazole (20 mg), clarithromycin (500 mg) and amoxicillin (1 g) twice daily]. H. pylori status was assessed by histology, rapid urease test and 13C-urea breath test at baseline and 6 weeks or more after completion of treatment. Results : Higher eradication rates were found with the sequential regimen compared to the standard regimen (intention-to-treat: 92% vs. 74%, P 90%), as was the rate of side-effects, which were mild. Conclusions : This 10-day sequential treatment regimen achieves high eradication rates in peptic ulcer disease and non-ulcer dyspepsia.

Globally, hepatitis C Virus (HCV) infection is responsible for a large proportion of persons with liver disease, including cancer. The infection is highly prevalent in sub-Saharan Africa. West Africa was identified as a geographic origin of two HCV genotypes. However, little is known about the genetic composition of HCV populations in many countries of the region. Using conventional and next-generation sequencing (NGS), we identified and genetically characterized 65 HCV strains circulating among HCV-positive blood donors in Kumasi, Ghana. Phylogenetic analysis using consensus sequences derived from 3 genomic regions of the HCV genome, 5'-untranslated region, hypervariable region 1 (HVR1) and NS5B gene, consistently classified the HCV variants (n = 65) into genotypes 1 (HCV-1, 15%) and genotype 2 (HCV-2, 85%). The Ghanaian and West African HCV-2 NS5B sequences were found completely intermixed in the phylogenetic tree, indicating a substantial genetic heterogeneity of HCV-2 in Ghana. Analysis of HVR1 sequences from intra-host HCV variants obtained by NGS showed that three donors were infected with >1 HCV strain, including infections with 2 genotypes. Two other donors share an HCV strain, indicating HCV transmission between them. The HCV-2 strain sampled from one donor was replaced with another HCV-2 strain after only 2 months of observation, indicating rapid strain switching. Bayesian analysis estimated that the HCV-2 strains in Ghana were expanding since the 16th century. The blood donors in Kumasi, Ghana, are infected with a very heterogeneous HCV population of HCV-1 and HCV-2, with HCV-2 being prevalent. The detection of three cases of co- or super-infections and transmission linkage between 2 cases suggests frequent opportunities for HCV exposure among the blood donors and is consistent with the reported high HCV prevalence. The conditions for effective HCV-2 transmission existed for ~ 3–4 centuries, indicating a long epidemic history of HCV-2 in Ghana.

Liver sex hormone-binding globulin (SHBG) biosynthesis is regulated by triiodothyronine (T3). This regulation is responsible for increased serum SHBG concentrations in hyperthyroid states. However, in hypothyroidism, normal SHBG levels are frequently found. To understand this we have characterized circulating SHBG isoforms according to their sialic acid content, which determines its half-life, in euthyroid and hypothyroid women. Six euthyroid (aged 56 +/- 8 years) and five hypothyroid women (51 +/- 13 years) were studied. Their body mass index (BMI) range was 20-25. Hypothyroidism diagnosis was based on clinical findings, elevated basal thyrotropin (TSH) and decreased T3 and thyroxine (T4) values. Total SHBG was measured by radioimmunoassay (RIA) and SHBG isoforms were isolated using preparative isoelectrofocusing. For comparisons, two-tailed t test was applied. No statistical difference was found between the total SHBG levels of hypothyroid and euthyroid postmenopausal women. Three groups of SHBG isoforms were isolated in the euthyroid group: S(I): pl: 5.0-5.2: 20% +/- 4%, S(II) : pl 5.2-5.4: 50% +/- 3% and S(III): pl 5.4-5.6: 29% +/- 4%. In hypothyroid patients, although the three groups of isoforms were isolated in the same pH range, S(I) and S(II) proportions were different (p0.001) when compared to normal women: S(I): 34% +/- 4%, S(II): 33% +/- 9.9% and S(III): 29% +/- 5.7%. These results show that hypothyroid patients have a higher proportion of more acidic SHBG isoforms. This variation may explain the normal levels of serum SHBG observed in hypothyroidism.

Background Up to 30% of acute viral hepatitis has no known etiology. To determine the disease etiology in patients with acute hepatitis of unknown etiology (HUE), serum specimens were obtained from 38 patients residing in the United Kingdom and Vietnam and from 26 healthy US blood donors. All specimens tested negative for known viral infections causing hepatitis, using commercially available serological and nucleic acid assays. Methods Specimens were processed by sequence-independent complementary DNA amplification and next-generation sequencing (NGS). Sufficient material for individual NGS libraries was obtained from 12 HUE cases and 26 blood donors; the remaining HUE cases were sequenced as a pool. Read mapping was done by targeted and de novo assembly. Results Sequences from hepatitis B virus (HBV) were detected in 7 individuals with HUE (58.3%) and the pooled library, and hepatitis E virus (HEV) was detected in 2 individuals with HUE (16.7%) and the pooled library. Both HEV-positive cases were coinfected with HBV. HBV sequences belonged to genotypes A, D, or G, and HEV sequences belonged to genotype 3. No known hepatotropic viruses were detected in the tested normal human sera. Conclusions NGS-based detection of HBV and HEV infections is more sensitive than using commercially available assays. HBV and HEV may be cryptically associated with HUE.

Motivation: Next-generation sequencing (NGS) allows for analyzing a large number of viral sequences from infected patients, providing an opportunity to implement large-scale molecular surveillance of viral diseases. However, despite improvements in technology, traditional protocols for NGS of large numbers of samples are still highly cost and labor intensive. One of the possible cost-effective alternatives is combinatorial pooling. Although a number of pooling strategies for consensus sequencing of DNA samples and detection of SNPs have been proposed, these strategies cannot be applied to sequencing of highly heterogeneous viral populations. Results: We developed a cost-effective and reliable protocol for sequencing of viral samples, that combines NGS using barcoding and combinatorial pooling and a computational framework including algorithms for optimal virus-specific pools design and deconvolution of individual samples from sequenced pools. Evaluation of the framework on experimental and simulated data for hepatitis C virus showed that it substantially reduces the sequencing costs and allows deconvolution of viral populations with a high accuracy. Availability and implementation: The source code and experimental data sets are available at http://alan.cs.gsu.edu/NGS/?q=content/pooling Contact: kki8@cdc.gov, yek0@cdc.gov Supplementary information: Supplementary data are available at Bioinformatics online.

Made available in DSpace on 2015-03-18T15:55:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-07-14Bitstream added on 2015-03-18T16:28:35Z : No. of bitstreams: 1 WOS000345682800004.pdf: 1403582 bytes, checksum: 52ae6747619aa45c91860ded91be58cb (MD5) Centers for Disease Control and Prevention Background: Next-generation sequencing (NGS) allows for sampling numerous viral variants from infected patients. This provides a novel opportunity to represent and study the mutational landscape of Hepatitis C Virus (HCV) within a single host.Results: Intra-host variants of the HCV E1/E2 region were extensively sampled from 58 chronically infected patients. After NGS error correction, the average number of reads and variants obtained from each sample were 3202 and 464, respectively. The distance between each pair of variants was calculated and networks were created for each patient, where each node is a variant and two nodes are connected by a link if the nucleotide distance between them is 1. The work focused on large components having > 5% of all reads, which in average account for 93.7% of all reads found in a patient. The distance between any two variants calculated over the component correlated strongly with nucleotide distances (r = 0.9499; p = 0.0001), a better correlation than the one obtained with Neighbour-Joining trees (r = 0.7624; p = 0.0001). In each patient, components were well separated, with the average distance between (6.53%) being 10 times greater than within each component (0.68%). The ratio of nonsynonymous to synonymous changes was calculated and some patients (6.9%) showed a mixture of networks under strong negative and positive selection. All components were robust to in silico stochastic sampling; even after randomly removing 85% of all reads, the largest connected component in the new subsample still involved 82.4% of remaining nodes. In vitro sampling showed that 93.02% of components present in the original sample were also found in experimental replicas, with 81.6% of reads found in both. When syringe-sharing transmission events were simulated, 91.2% of all simulated transmission events seeded all components present in the source.Conclusions: Most intra-host variants are organized into distinct single-mutation components that are: well separated from each other, represent genetic distances between viral variants, robust to sampling, reproducible and likely seeded during transmission events. Facilitated by NGS, large components offer a novel evolutionary framework for genetic analysis of intra-host viral populations and understanding transmission, immune escape and drug resistance. Ctr Dis Control & Prevent, Div Viral Hepatitis, Atlanta, GA 30333 USA UNESP Sao Paulo State Univ, Dept Biol, Lab Genom Studies, Sao Paulo, Brazil Harvard Univ, Beth Israel Deaconess Med Ctr, Sch Med, Liver Ctr,Div Gastroenterol, Cambridge, MA 02138 USA UNESP Sao Paulo State Univ, Dept Biol, Lab Genom Studies, Sao Paulo, Brazil

Hepatitis C is a major public health problem in the United States and worldwide. Outbreaks of hepatitis C virus (HCV) infections are associated with unsafe injection practices, drug diversion, and other exposures to blood products. HCV outbreaks are difficult to detect and investigate because HCV infections can remain asymptomatic in >70% of infected persons for years, even decades. During the 5-year period of 2008-2013 in the United States, 18 Hepatitis C outbreaks related to healthcare were reported to CDC. These outbreaks involved 223 associated cases and more than 90,550 at-risk persons were notified for screening. The prevailing method for molecular detection of viral transmissions involves: (i) sequencing a small heterogeneous region of the HCV genome isolated from each patient serum, (ii) creating a phylogenetic tree using the sequences, and (iii) identifying transmission clusters as subtrees containing sequences related above a certain threshold. This framework has also been used for other heterogeneous pathogens such as hepatitis B virus and human immunodeficiency virus. The present work identifies and solves two problems of molecular detection of transmissions during outbreak investigations using small genomic regions. The studied dataset included 1073 HCV genome sequences and 370 HBV genome sequences obtained from GenBank. The first problem is selection of a genomic region most applicable for outbreak detection. Commonly, the chosen region is simply most variable. Variability is usually defined by creating a whole genome multiple-sequence alignment and then calculating genetic heterogeneity using a sliding window of 300 to 500 nt (most common size of amplicons). The major problem with this approach is that it implicitly assumes that the region with the highest global genetic heterogeneity will also provide the highest discrimination among closely related variants. We study the discrimination for each region by calculating the ‘local genetic heterogeneity’, defined as the nucleotide diversity of the region over the 10 nearest neighbors (defined using the whole genome), and averaged over all sequences. It was found that the local heterogeneity allows for discriminating between regions of a similar global heterogeneity. For instance, among several regions of high global heterogeneity, only the E1/E2 region had a high local genetic heterogeneity. For HBV, we found that the most discriminating region was the TP domain of the polymerase gene. This region is more suitable for detection of transmissions during outbreaks than the commonly used S gene. Phylogenetic trees obtained using small genomic regions and entire genomes are frequently distinctly different from each other, which generates a problem in application of small regions to the detection of clusters of closely related genetic viral variants consistent with transmission of a single viral strain among patients. To resolve this problem we calculated correlation between matrices of genetic distances among HCV amplicon-size genomic regions and whole-genome sequences. For these experiments, we used the E1/E2 genomic region, which is commonly applied for molecular detection of genetic relatedness among HCV strains. Considering the task as an optimization problem, we conducted a search for weights for each nucleotide position in a small region that yield the highest possible correlation between both matrices. We solved this optimization problem using a Genetic Algorithm, finding a marked improvement of the correlation between matrices from r = 0.785 to = 0.905 in genotype 1a (15.3% improvement) and from r = 0.450 to 0.705 in genotype 1b (56.7% improvement). These new computational methods provide a measurable improvement of the selection and analysis of short genomic regions for assessing genetic relatedness among genetically close variants and can be applied to other heterogeneous pathogens. Keywords— Viral Hepatits; Genetic relatedness; Viral quasispecies; Genetic algorithms. U.S. Government work not protected by U.S. copyright.

Y chromosome STR haplotypes have been widely used in population studies to establish the origin of diverse populations.We analyzed Y chromosome STR haplotypes (8 loci) in 134 Caucasian-mestizo and 137 African-descent Colombian unrelated individuals to correlate the geographical origin with historical data as well as the genetic relationships and possible admixture patterns.One hundred samples of African descent and 137 Caucasian-mestizo samples analyzed for Y chromosome STR haplotypes by PCR followed by acrylamide electrophoresis.No evidence of population substructure was found for the African descent. Two point fifty nine per cent of the haplotypes were shared between the two groups with the possible existence of Caucasian gene flow towards Afro-descendants.The Caucasian-Mestizo Colombian population is grouped with other populations of the Iberian Peninsula and Europe, while the Afro-Colombian population is grouped with other African populations reported.

Background : Following standard triple therapy, up to 20% of patients require further Helicobacter pylori eradication treatment. Data regarding the efficacy of re-treatment in these patients are scarce. Aim : To evaluate the efficacy of a triple therapy after one or more consecutive treatment failures. Methods : A total of 51 patients with persistent H. pylori infection after at least one unsuccessful standard 1-week regimen were enrolled in the study. H. pylori infection at entry was assessed by rapid urease test and histology on biopsies from the antrum and the corpus. Patients were given a 2-week triple therapy, comprising ranitidine bismuth citrate 400 mg b.d., tetracycline 500 mg t.d.s., and tinidazole 500 mg b.d. Ranitidine bismuth citrate was given during meals, whilst tetracycline and tinidazole was given after meals. Bacterial eradication was assessed by endoscopy (36 patients) or 13C-urea breath test (15 patients) 4–6 weeks after therapy had ended. Results : All 51 patients completed the study and H. pylori eradication was achieved in 46, with an eradication rate of 90% (95% CI: 82–98). In detail, bacterial eradication was obtained in 96% of patients who had previously failed one course of clarithromycin–amoxicillin based triple therapy, in 88% patients who had failed a clarithromycin–tinidazole based triple therapy, in 83% patients who had failed both treatment schedules, and in the only patient who had failed three consecutive therapeutic attempts. Two patients took the therapy for 9 and 10 days instead of the full 14 day-course. No major side-effects were reported, whilst six (12%) patients complained of mild side-effects. Conclusion : This study demonstrates that this triple therapy regimen is effective for re-treatment of H. pylori infection.

The aim of the present study was to evaluate inhibin secretion in rats with autoimmune orchitis. As we have previously described, experimental autoimmune orchitis (EAO) induced in rats by active immunization with testis homogenate and adjuvants is characterized by an interstitial mononuclear cell infiltrate and sloughing of the germinal epithelium. At 120 days after the first immunization 60% of the rats exhibited a severe orchitis with large areas of aspermatogenic seminiferous tubules in which only spermatogonia and Sertoli cells with cytoplasmic vacuolization remained attached to the tubular wall. None of the untreated (N) or control (C) rats revealed pathological alterations. Sixty percent decrease in testis weight was observed in rats with EAO compared with N or C groups. A 3-fold increase in serum FSH levels was observed in rats with EAO compared with N or C groups (19·8 ± 3·7 vs 5·6 ± 0·3 and 5·9 ± 0·1 ng/ml respectively). A significant decrease in inhibin B levels was observed in rats with EAO when compared with N or C groups (40 ± 4·6 vs 207 ± 38·8 and 221·4 ± 28·6 pg/ml respectively). An inverse correlation between inhibin B and FSH serum levels and a direct correlation between inhibin B and testis weight were found. Strong expression of the inhibin α-subunit in Sertoli cells of untreated and control rats was observed; this subunit was undetectable or poorly detectable in rats with orchitis. Positive staining for the inhibin α-subunit was also observed in Leydig cells of all groups studied. In conclusion, using a model of autoimmune orchitis our results show that circulating inhibin B levels and inhibin α-subunit expression in Sertoli cell cytoplasm closely correlate with the degree of damage of the germinal epithelium., Instituto Multidisciplinario de Biología Celular, Facultad de Ciencias Exactas

Association and linkage studies have established the importance of the major histocompatibility complex (MHC) in the susceptibility for multiple sclerosis (MS). We carried out a case-control study to investigate the ancestral haplotype A30B18DR3 and MS in the Nuoro population of Sardinia, which is isolated and genetically distinct from other populations in the Mediterranean basin and characterized by genetic homogeneity, high level of inbreeding, low migration, high prevalence of MS, high frequency of the relevant haplotype, and high past malaria prevalence. Cases and controls were serologically typed for the currently recognized HLA-A, B, and DR antigens. We used a log-linear approach to fit a wide class of models. We tested our hypothesis comparing different models via a likelihood ratio test. We overcame the complication due to unknown gametic phase using expectation-maximization (EM) algorithm as the estimation method. We estimated confidence intervals for odds ratio by using a profile likelihood approach. We found that: (1) the ancestral haplotype A30B18DR3 was associated to MS after allowing for a possible stratification in cases and controls; (2) DR3 allele was conditional independent on disease status, given A30B18 haplotype; (3) there was a tendency for ORs for the high-risk haplotypes to be higher in the high malaria strata; however, this indication did not achieve statistical significance (P = 0.11).

Studies regarding the functions of the bovine papillomavirus (BPV) E5 oncoprotein in vivo are lacking and no E5- mediated mechanism underlying epithelial carcinogenesis is known. We have shown that BPV-2 DNA is present in the majority of naturally occurring urinary bladder tumours of cattle and that E5 is expressed in the cancer cells. Here we show that the interaction between the platelet-derived growth factor (PDGF) b receptor and BPV E5, described in vitro in cultured cells, takes place in vivo in bovine urinary bladder cancers. In these cancers, E5 and PDGFb receptor colocalize,as shown by confocal microscopy, and physically interact, as shown by coimmunoprecipitation. Furthermore,the PDGFb receptor associated withE5 is highly phosphorylated,suggesting the functional activation of the receptor upon E5 interaction.Our results demonstrate,for the first time, that E5–PDGFb receptor interaction occurs during the natural history of bovine urinary bladder tumours, suggesting an important role for E5 in carcinogenesis. Finally, the system provides a suitable animal model of papillomavirus-associated cancer to test therapeutic vaccination against E5. Successful bladder tumour regression would provide a valuable model for therapeutic vaccination against papillomavirus-associated tumours.

Hepatitis C virus (HCV) infection presents an important, but underappreciated public health problem in Africa. In Côte d’Ivoire, very little is known about the molecular dynamics of HCV infection. Plasma samples (n = 608) from pregnant women collected in 1995 from Côte d’Ivoire were analyzed in this study. Only 18 specimens (~3%) were found to be HCV PCR-positive. Phylogenetic analysis of the HCV NS5b sequences showed that the HCV variants belong to genotype 1 (HCV1) (n = 12, 67%) and genotype 2 (HCV2) (n = 6, 33%), with a maximum genetic diversity among HCV variants in each genotype being 20.7% and 24.0%, respectively. Although all HCV2 variants were genetically distant from each other, six HCV1 variants formed two tight sub-clusters belonging to HCV1a and HCV1b. Analysis of molecular variance (AMOVA) showed that the genetic structure of HCV isolates from West Africa with Côte d’Ivoire included were significantly different from Central African strains (P = 0.0001). Examination of intra-host viral populations using next-generation sequencing of the HCV HVR1 showed a significant variation in intra-host genetic diversity among infected individuals, with some strains composed of sub-populations as distant from each other as viral populations from different hosts. Collectively, the results indicate a complex HCV evolution in Côte d’Ivoire, similar to the rest of West Africa, and suggest a unique HCV epidemic history in the country.

Bovine papillomavirus type 4 (BPV-4) does not possess an E6 ORF. The E6 oncoprotein of human papillomavirus (HPV) binds and degrades the tumour suppressor protein p53, thus contributing to tumour progression. Since BPV-4 lacks E6, it is unknown how the virus evades the tumour suppressor properties of p53 in the induction of tumours of the gastrointestinal tract. Mutations in the p53 gene have been detected both in papillomas and carcinomas, suggesting that p53 dysfunction plays a part in these neoplasias. BPV-4 can transform primary foetal bovine cells (PalFs) in cooperation with an activated ras gene, but the transformed cells are neither immortal nor tumorigenic. Co-transfection with the HPV-16 E6 (16E6) ORF confers immortality but not tumorigenicity. To investigate the role of p53 in BPV-4 cell transformation in vitro, we transfected PalFs and p53-null mouse fibroblasts with BPV-4 DNA in combinations with ras, 16E6 ORF and mutant (V143A) p53 cDNA. Transfection of PalFs with BPV-4 DNA, ras and mutant p53 led to cell immortalization, indicating that 16E6 and mutant p53 are functionally equivalent in conferring immortality. However, co-transfection of PalFs with BPV-4 DNA, ras, and both mutant p53 cDNA and 16E6 ORF resulted in cells which were fully transformed to tumorigenicity. In p53-null mouse fibroblasts, BPV-4 DNA induced transformation by itself, but the transformed cells were incapable of suspension growth. The co-transfection of BPV-4 DNA with 16E6 ORF produced many more transformed colonies and the cells were capable of growing in suspension. In this system, therefore, 16E6 confers anchorage-independence to BPV-4-transformed cells in a p53-independent fashion.

Mc Ardles disease, also known as Type V glycogen storage disease, is a rare deficiency of the enzyme glycogen phosphorylase in muscle cells, inherited as an autosomal recessive trait. In the absence of this enzyme, muscles cannot break down glycogen during exercise, so in patients affected by McArdles disease even moderate physical activity produces cramps, pain and fatigue. Anaerobic activity leads to severe fixed contractures and rhabdomyolisis with myoglobinuria and raised serum creatine-kinase, which, in turn, can lead to acute renal failure. Disease onset is usually in early childhood, although diagnosis is often not made until the second or third decade.We present the case of a 68-year-old man who presented to the Emergency Room with fatigue, vertigo, diarrhea and oliguria. The patient underwent five daily hemodialysis sessions, diuresis reappeared and there was progressive recovery of renal function. The patient described episodes of fatigue and muscular pain occurring since childhood: the positive personal history, together with persistently raised CPK levels in the absence of any infective or toxic cause of myositis, led us to suspect the presence of this rare metabolic disease, which was subsequently confirmed by muscle biopsy.To date, there is no specific treatment for type V glycogenosis, although a diet rich in protein and saccarose, vitamin B6 supplementation and creatine administration are generally recommended. Moderate physical activity can help manage symptoms by improving exercise tolerance and blood supply to the muscles, ensuring provision of glucose and free fatty acids for the muscle fibers.

Summary form only given. Neutrality theory states that the majority of genotypic changes in evolution is selectively neutral, which has been confirmed in the analysis of neutral networks in genotype-phenotype models of RNA secondary structure and protein folding. These analyses have shown that collections of neutral genotypes, which are connected via single mutational steps, form extended networks that permeate large regions of genotype space. An interesting consequence is that a population does not move randomly over a neutral network but tends to concentrate in those regions of the network that are highly connected and, therefore, more robust to mutations. Here, we study experimentally obtained samples of intra-host HCV variants and show that the properties of their neutral networks show high mutational robustness. Intra-host sequences of the HCV E1/E2 region were extensively sampled from 16 chronically infected patients using a Next-Generation sequencing (NGS) technology. After error correction was performed, a network was created for each sample of sequences, where a node is a unique HCV sequence and two nodes are connected by an edge if their nucleotide distance is exactly one. Based on these networks, several neutrality measures were calculated. The average number of unique sequences obtained from each patient was 747.93, with an average of 6091.18 reads. Over all patients, the average frequency of the major is 0.2916. All sequences were used to generate one-step networks. There are some with a frequency higher than 5%, which were called big networks. In average a patient has 2.625 big networks, which account for 92.83% of all reads in a patient. Over all big networks, the average number of sequences and reads is 214.05 and 2157.78, respectively. For each sample, population neutrality was calculated as the spectral radius of the adjacency matrix. The average population neutrality over all samples was 5.6347 (ranging from 3.3685 to 9.5765 in all samples), which can be understood as the average number of point mutations that leave the phenotype unaltered. If the degree distribution is highly uniform over the network, network neutrality is equal to the average degree. In this set of samples the average network neutrality was 3.4110. When the population neutrality is greater than network neutrality, their difference precisely quantifies mutational robustness or the extent to which a population occupies the most connected areas of the neutral network. In this case, population neutrality was greater than network neutrality, yielding a mutational robustness of 0.6537 (ranging from 0.0964 to 1.6623 in all samples), and showing that under selection and mutation, the population evolved a mutational robustness that is 65.3% higher than if it were to spread randomly over the network. We took advantage of the capacity of NGS to produce a massive number of sequences, facilitating the accurate assessment of frequency of viral variants. If the population tends to concentrate in those regions of the network that are highly connected then the high centrality of a sequence should be associated with the high frequency in the population. Accordingly, we found that the correlation between the eigenvector centrality of the nodes in each network was highly and significantly associated with frequency, with an average Pearson correlation coefficient of 0.8311 (p = 0.0001). In conclusion, we have shown a strong mutational robustness of the one-step network of intra-host HCV variants, indicating that, even under conditions of similar fitness, the HCV population has a tendency to concentrate in areas of sequence space where mutations have a higher chance to be neutral and leave the phenotype unaltered.

The bovine papillomavirus type 4 (BPV4) long control region (LCR) contains three consensus binding sites, E2(1), E2(2), and E2(3) (ACCN6GGT), for the viral E2 transcription factor and a fourth degenerate site, dE2 (ATCN6GGT), which lies 3 bp upstream of E2(3). The E2(2) site was found to bind the cellular transcription factor PEBP2, and mutations at this site reduced basal promoter activity by as much as 60%, indicating an important role for PEBP2 in LCR function. Mutation of the E2(3) or dE2 site slightly decreased basal promoter activity, but the cellular proteins binding these sites have not yet been characterized. E2 protein was found to have considerable influence upon LCR promoter activity in primary bovine palate keratinocytes. Thus, when high levels of BPV1 E2 were present, almost complete repression of the BPV4 LCR was observed, whereas smaller amounts of BPV1 or BPV4 E2 led to transactivation. Mutational analysis indicated that E2(1) and dE2 mediated transactivation by E2, whereas E2(2) and E2(3) were responsible for repression by E2. In vitro complexes of binding sites E2(1) and E2(2) with E2 protein demonstrated much greater stability than complexes formed by the E2(3) and dE2 sites. These data suggest that the four E2 sites in the BPV4 LCR each perform different functions in the control of transcription and that competition between cellular transcription factors and viral E2 proteins is essential in regulating the level of viral gene expression during papilloma development.

The minimal enhancer in the long control region of human papillomavirus type 16 regulates cell type and constitutive expression from the promoter P97. This region contains at least four DNase I footprints (fp4e, fp5e, fp6e, and fp7e). We have shown that fp5e is crucial to enhancer function and have described an apparently novel factor (PEF-1) binding fp5e (S. Cuthill, G. J. Sibbet, and M. S. Campo, Mol. Carcinog. 8:9-104, 1993). Further analyses reveal that Oct-1 or an Oct-related factor binds fp5e at a site overlapping that of PEF-1. The binding of Oct-1 to fp5e has been demonstrated by electrophoretic mobility shift assays, by oligonucleotide competition studies, and by using an Oct-1-specific anti-POU serum. The location of the Oct-1 site has been confirmed by a panel of mutants across fp5e. Mutations that block PEF-1 binding to fp5e also block enhancer/promoter activity of the long control region, whereas mutations that block Oct-1 binding significantly increase enhancer/promoter activity. Thus, although both PEF-1 and Oct-1 interact with fp5e, they appear to regulate human papillomavirus expression in opposite ways.

Exposure to the flavonoid quercetin and transfection with bovine papillomavirus type 4 (BPV-4) DNA lead to oncogenic transformation of primary bovine cells. Here we show that the synergism between quercetin and BPV-4 (or its E7 oncogene) is stronger the shorter the interval between the two treatments. Quercetin immortalizes transformed cells, confers anchorage-independent growth and induces tumorigenicity in cells transfected only with the E7 oncogene.

Detection of Trichuris trichiura during colonoscopic examination is an unusual finding, at least in developed countries. We report a case of a coincidental endoscopic diagnosis of whipworm infestation performed in a patient referred to our open-access endoscopy even before a faecal examination for ova and/or parasites had been performed. Review of literature on colonoscopic diagnosis of T. trichuria is provided.

Hepatitis C virus (HCV) is a major cause of liver disease world-wide. Current interferon and ribavirin (IFN/RBV) therapy is effective in 50%-60% of patients. HCV exists in infected patients as a large viral population of intra-host variants (quasispecies), which may be differentially resistant to interferon treatment. We present a method for measuring differential interferon resistance of HCV quasispecies based on mathematical modeling and analysis of HCV population dynamics during the first hours of interferon therapy. The mathematical models showed that individual intra-host HCV variants have a wide range of resistance to IFN treatment in each patient. Analysis of differential IFN resistance among intra-host HCV variants allows for accurate prediction of response to IFN therapy. The models strongly suggest that resistance to interferon may vary broadly among closely related variants in infected hosts and therapy outcome may be defined by a single or a few variants irrespective of their frequency in the intra-host HCV population before treatment.

Hepatitis C Virus sequence studies mainly focus on the viral amplicon containing the Hypervariable region 1 (HVR1) to obtain a sample of sequences from which several population genetics parameters can be calculated. Recent advances in sequencing methods allow for analyzing an unprecedented number of viral variants from infected patients and present a novel opportunity for understanding viral evolution, drug resistance and immune escape. In the present paper, we compared three recent technologies for amplicon analysis: (i) Next-Generation Sequencing; (ii) Clonal sequencing using End-point Limiting-dilution for isolation of individual sequence variants followed by Real-Time PCR and sequencing; and (iii) Mass spectrometry of base-specific cleavage reactions of a target sequence. These three technologies were used to assess intra-host diversity and inter-host genetic relatedness in HVR1 amplicons obtained from 38 patients (subgenotypes 1a and 1b). Assessments of intra-host diversity varied greatly between sequence-based and mass-spectrometry-based data. However, assessments of inter-host variability by all three technologies were equally accurate in identification of genetic relatedness among viral strains. These results support the application of all three technologies for molecular epidemiology and population genetics studies. Mass spectrometry is especially promising given its high throughput, low cost and comparable results with sequence-based methods.

The detection of compensatory mutations that abrogate negative fitness effects of drug-resistance and vaccine-escape mutations indicates the important role of epistatic connectivity in evolution of viruses, especially under the strong selection pressures. Mapping of epistatic connectivity in the form of coordinated substitutions should help to characterize molecular mechanisms shaping viral evolution and provides a tool for the development of novel anti-viral drugs and vaccines. We analyzed coordinated variation among amino acid sites in 370 the hepatitis B virus (HBV) polymerase sequences using Bayesian networks. Among the HBV polymerase domains the spacer domain separating terminal protein from the reverse-transcriptase domain, showed the highest network centrality. Coordinated substitutions preserve the hydrophobicity and charge of Spacer. Maximum likelihood estimates of codon selection showed that Spacer contains the highest number of positively selected sites. Identification of 67% of the domain lacking an ordered structure suggests that Spacer belongs to the class of intrinsically disordered domains and proteins whose crucial functional role in the regulation of transcription, translation and cellular signal transduction has only recently been recognized. Spacer plays a central role in the epistatic network associating substitutions across the HBV genome, including those conferring viral virulence, drug resistance and vaccine escape. The data suggest that Spacer is extensively involved in coordination of HBV evolution.

Detection of genotypes and drug resistance mutations are important molecular tools assisting in clinical management of patients with chronic hepatitis B and C. Together with methods for assessment of genetic heterogeneity and relatedness of viral strains, they form the foundation of molecular surveillance. Currently, all these methods are based mainly on DNA sequencing followed by phylogenetic analysis. Mass spectrometry (MS) emerged recently as a rapid, cost-effective, reproducible and accurate alternative approach. MS-based molecular assays are highly amenable to automation and provide a suitable platform for routine application to the surveillance of HBV and HCV infections.

Treatment with lamivudine of patients infected with hepatitis B virus (HBV) results in a high rate of drug resistance, which is primarily associated with the rtM204I/V substitution in the HBV reverse transcriptase domain. Here we show that the rtM204I/V substitution, although essential, is insufficient for establishing resistance against lamivudine. The analysis of 639 HBV whole-genome sequences obtained from 11 patients shows that rtM204I/V is independently acquired by more than one intra-host HBV variant, indicating the convergent nature of lamivudine resistance. The differential capacity of HBV variants to develop drug resistance suggests that fitness effects of drug-resistance mutations depend on the genetic structure of the HBV genome. An analysis of Bayesian networks that connect rtM204I/V to many sites of HBV proteins confirms that lamivudine resistance is a complex trait encoded by the entire HBV genome rather than by a single mutation. These findings have implications for public health and offer a more general framework for understanding drug resistance., Lamivudine treatment of hepatitis B is associated with drug-resistance mutations in the virus’ DNA polymerase. In this study, 11 patients with drug resistance are investigated and the primary mutation in the DNA polymerase shown to be essential but not sufficient for establishing drug resistance.

We investigated the molecular epidemiology and population dynamics of HCV infection among indigenes of two semi-isolated communities in North-Central Nigeria. Despite remoteness and isolation, ~15 % of the population had serological or molecular markers of hepatitis C virus (HCV) infection. Phylogenetic analysis of the NS5b sequences obtained from 60 HCV-infected residents showed that HCV variants belonged to genotype 1 (n = 51; 85 %) and genotype 2 (n = 9; 15 %). All sequences were unique and intermixed in the phylogenetic tree with HCV sequences from people infected from other West African countries. The high-throughput 454 pyrosequencing of the HCV hypervariable region 1 and an empirical threshold error correction algorithm were used to evaluate intra-host heterogeneity of HCV strains of genotype 1 (n = 43) and genotype 2 (n = 6) from residents of the communities. Analysis revealed a rare detectable intermixing of HCV intra-host variants among residents. Identification of genetically close HCV variants among all known groups of relatives suggests a common intra-familial HCV transmission in the communities. Applying Bayesian coalescent analysis to the NS5b sequences, the most recent common ancestors for genotype 1 and 2 variants were estimated to have existed 675 and 286 years ago, respectively. Bayesian skyline plots suggest that HCV lineages of both genotypes identified in the Nigerian communities experienced epidemic growth for 200–300 years until the mid-20th century. The data suggest a massive introduction of numerous HCV variants to the communities during the 20th century in the background of a dynamic evolutionary history of the hepatitis C epidemic in Nigeria over the past three centuries.

Vaccine development against hepatitis C virus (HCV) is hindered by poor understanding of factors defining cross-immunoreactivity among heterogeneous epitopes. Using synthetic peptides and mouse immunization as a model, we conducted a quantitative analysis of cross-immunoreactivity among variants of the HCV hypervariable region 1 (HVR1). Analysis of 26,883 immunological reactions among pairs of peptides showed that the distribution of cross-immunoreactivity among HVR1 variants was skewed, with antibodies against a few variants reacting with all tested peptides. The HVR1 cross-immunoreactivity was accurately modeled based on amino acid sequence alone. The tested peptides were mapped in the HVR1 sequence space, which was visualized as a network of 11,319 sequences. The HVR1 variants with a greater network centrality showed a broader cross-immunoreactivity. The entire sequence space is explored by each HCV genotype and subtype. These findings indicate that HVR1 antigenic diversity is extensively convergent and effectively limited, suggesting significant implications for vaccine development.

Recent advances in sequencing methods allow the analysis of an unprecedented number of viral variants from infected patients and present a novel opportunity for understanding viral evolution, drug resistance and immune escape. In the present paper, we compared three technologies for amplicon analysis: (i) Next Generation Sequencing; (ii) Clonal sequencing using End-point Limiting-dilution for isolation of individual sequence variants followed by Real-Time PCR and sequencing; and (iii) Mass spectrometry of base-specific cleavage reactions of a target sequence. Hypervariable region 1 of hepatitis C virus was analyzed using these three technologies to assess diversity and genetic relatedness of intra-host viral populations in specimens obtained from 38 patients. Estimates of population heterogeneity varied among technologies. However, all three technologies were equally accurate in identification of genetic relatedness among viral strains, supporting their application in molecular epidemiology for tracking viral variants and detecting transmission events.

Differentiation between acute and chronic HCV infections is clinically important given that early treatment of infected patients leads to high rates of sustained virological response. Analysis of HVR1 sequences (n=2179) from samples obtained from patients with acute (n=49) and chronic (n=102) HCV infections showed that intra-host HVR1 diversity was 1.8 times higher in patients with chronic than acute infection. Analysis of molecular variance showed significant differences between sequences from acute and chronic patients. We found statistically significant differences in polarity, volume and hydrophobicity of amino acids at 10 HVR1 positions. A classification model constructed using the 10 positions in HVR1 distinguished between acute and chronic cases with accuracy of 88% in cross-validation experiments. The results indicate that progression from acute to chronic stage of HCV infection is accompanied by characteristic changes in amino acid composition of HVR1, suggesting a substantial regularity of the intra-host HVR1 evolution.

We analyzed coordinated variation among amino acid sites in 370 HBV polymerase sequences using Bayesian networks. Among the HBV polymerase domains the spacer domain separating terminal protein from the reverse-transcriptase domain, showed the highest network centrality. Coordinated substitutions preserve the hydrophobicity and charge of Spacer. Maximum likelihood estimates of codon selection showed that Spacer contains the highest number of positively selected sites. Identification of 67% of the domain lacking an ordered structure suggests that Spacer belongs to the class of intrinsically unstructured domains and proteins whose crucial functional role in the regulation of transcription, translation and cellular signal transduction has only recently been recognized. Spacer plays a central role in the epistatic network associating substitutions across the HBV genome, including those conferring viral virulence, drug resistance and vaccine escape.

Hepatitis C virus (HCV) is a major cause of liver disease worldwide. Current interferon and ribavirin (IFN/RBV) therapy is effective in 50%–60% of patients. HCV exists in infected patients as a large viral population of intra-host variants (quasispecies), which form a certain topological structure (sequence space) and may be differentially resistant to interferon treatment. We present a method for measuring differential interferon resistance of HCV quasispecies based on mathematical modeling and analysis of HCV population dynamics during the first hours of interferon therapy. The mathematical models showed that individual intra-host HCV variants have a wide range of resistance to IFN treatment in each patient. Analysis of differential IFN resistance among intra-host HCV variants allows to accurately predict outcome of IFN therapy. The models strongly suggest that resistance to interferon may vary broadly among closely related variants in infected hosts and therapy outcome may be defined by a single or a few variants irrespective of their frequency in the intra-host HCV population before treatment.

The intrahost evolution of hepatitis C virus (HCV) holds keys to understanding mechanisms responsible for the establishment of chronic infections and to development of a vaccine and therapeutics. In this study, intrahost variants of two variable HCV genomic regions, HVR1 and NS5A, were sequenced from four treatment-naïve chronically infected patients who were followed up from the acute stage of infection for 9 to 18 years. Median-joining network analysis indicated that the majority of the HCV intrahost variants were observed only at certain time points, but some variants were detectable at more than one time point. In all patients, these variants were found organized into communities or subpopulations. We hypothesize that HCV intrahost evolution is defined by two processes: incremental changes within communities through random mutation and alternations between coexisting communities. The HCV population was observed to incrementally evolve within a single community during approximately the first 3 years of infection, followed by dispersion into several subpopulations. Two patients demonstrated this pattern of dispersion for the rest of the observation period, while HCV variants in the other two patients converged into another single subpopulation after ∼9 to 12 years of dispersion. The final subpopulation in these two patients was under purifying selection. Intrahost HCV evolution in all four patients was characterized by a consistent increase in negative selection over time, suggesting the increasing HCV adaptation to the host late in infection. The data suggest specific staging of HCV intrahost evolution.

BACKGROUND: In Nigeria, hepatitis B virus (HBV) infection has reached hyperendemic levels and its nature and origin have been described as a puzzle. In this study, we investigated the molecular epidemiology and epidemic history of HBV infection in two semi-isolated rural communities in North/Central Nigeria. It was expected that only a few, if any, HBV strains could have been introduced and effectively transmitted among these residents, reflecting limited contacts of these communities with the general population in the country. METHODS AND FINDINGS: Despite remoteness and isolation, approximately 11% of the entire population in these communities was HBV-DNA seropositive. Analyses of the S-gene sequences obtained from 55 HBV-seropositive individuals showed the circulation of 37 distinct HBV variants. These HBV isolates belong predominantly to genotype E (HBV/E) (n=53, 96.4%), with only 2 classified as sub-genotype A3 (HBV/A3). Phylogenetic analysis showed extensive intermixing between HBV/E variants identified in these communities and different countries in Africa. Quasispecies analysis of 22 HBV/E strains using end-point limiting-dilution real-time PCR, sequencing and median joining networks showed extensive intra-host heterogeneity and inter-host variant sharing. To investigate events that resulted in such remarkable HBV/E diversity, HBV full-size genome sequences were obtained from 47 HBV/E infected persons and P gene was subjected to Bayesian coalescent analysis. The time to the most recent common ancestor (tMRCA) for these HBV/E variants was estimated to be year 1952 (95% highest posterior density (95% HPD): 1927-1970). Using additional HBV/E sequences from other African countries, the tMRCA was estimated to be year 1948 (95% HPD: 1924-1966), indicating that HBV/E in these remote communities has a similar time of origin with multiple HBV/E variants broadly circulating in West/Central Africa. Phylogenetic analysis and statistical neutrality tests suggested rapid HBV/E population expansion. Additionally, skyline plot analysis showed an increase in the size of the HBV/E-infected population over the last approximately 30-40 years. CONCLUSIONS: Our data suggest a massive introduction and relatively recent HBV/E expansion in the human population in Africa. Collectively, these data show a significant shift in the HBV/E epidemic dynamics in Africa over the last century.

A combined extraction system for the selective and quantitative determination of the monoamines noradrenaline, adrenaline, dopamine and L-DOPA from the flowers and leaves of Crataegus oxyacantha L. has been developed. The method is based on a preliminary isolation of monoamines by successive petroleum ether and chloroform clean-up, absorption on alumina and passage through Sep-Pak C18 cartridges, followed by measurement of the catecholamines by high pressure liquid chromatography, with electrochemical detection, on a reversed phase column. The described method appears sufficiently simple, sensitive, precise and accurate for routine use and holds great promise for pharmacognostic studies.

The neuromasts of the lateral line system are regarded as a model to study the mechanisms of hearing, deafness, and ototoxicity. The neurotrophins (NTs), especially brain-derived neurotrophic factor (BDNF), and its signaling receptor TrkB are involved in the development and maintenance of neuromasts. To know the period in which the BDNF/TrkB complex has more effects in the neuromast biology, the age-related changes were studied. Normal zebrafish from 10 to 180 days post-fertilization (dpf), as well as transgenic ET4 zebrafish 10 and 20 dpf, was analyzed using qRT-PCR, western blot, and immunohistochemistry. BDNF and TrkB mRNAs followed a parallel course, peaking at 20 dpf, and thereafter progressively decreased. Specific immunoreactivity for BDNF and TrkB was found co-localized in all hairy cells of neuromasts in 20 and 30 dpf; then, the number of immunoreactive cells decreased, and by 180 dpf BDNF remains restricted to a subpopulation of hairy cells, and TrkB to a few number of sensory and non-sensory cells. At all ages examined, TrkB immunoreactivity was detected in sensory ganglia innervating the neuromasts. The present results demonstrate that there is a parallel time-related decline in the expression of BDNF and TrkB in zebrafish. Also, the patterns of cell expression suggest that autocrine/paracrine mechanisms for this NT system might occur within the neuromasts. Because TrkB in lateral line ganglia did not vary with age, their neurons are potentially capable to respond to BDNF during the entire lifespan of zebrafish.

Reactive oxygen species (ROC) are the main causes of carbon tetrachloride (CCl4)-induced acute liver injury. Chondroitin-4-sulphate (C4S) is known to inhibit lipid peroxidation through antioxidant mechanisms. Activation of nuclear factor (NF)-kappaB and caspases may strongly intensify inflammation and cell damage, in addition to that directly exerted by ROS. We investigated whether treatment with C4S, besides exerting antioxidant activity, was able to modulate NF-kappaB and apoptosis activation in CCl4-induced liver injury in mice.Acute hepatitis was induced in mice by an i.p. injection of CCl(4). Varying doses of C4S were administered i.p. 1 h before, 6 and 12 h after CCl4 injection. 24 h after CCl4 injection, the mice were killed for biochemical and histological analysis.CCl4 injection produced: marked elevation of alanine aminotransferase and aspartate aminotransferase; hepatic membrane lipid peroxidation, assayed by 8-isoprostane levels; and depletion of reduced glutathione and superoxide dismutase. CCl4 also decreased NF-kappaB translocation and IkBalpha, and increased gene expression of mRNA and protein of metalloproteases (MMP)-2 and -9, and of pro- and cleaved forms of caspases-3 and -7. There was also increased liver polymorphonuclear infiltration, evaluated by elastase assay, and hepatic cell disruption.C4S treatment inhibited lipid peroxidation; blocked NF-kappaB activation and IkBalpha protein loss; decreased mRNA and proteins for MMPs and caspases; restored endogenous antioxidants; limited hepatic polymorphonuclear accumulation and tissue damage.As antioxidants may inhibit NF-kappaB and caspase activation, we hypothesize that treatment with C4S was able to inhibit NF-kappaB and apoptosis activation in hepatic injury.

The aim of this study was to compare the effect of cadexomer on reducing wound surface area of leg ulcers compared to that obtained in a group patients whose ulcers were treated by compression therapy.For each ulcer group, wound surface area was calculated at day 0 and after 28 days of treatment: this allowed to calculate the average wound surface area reduction, the percent reduction in wound size, as well as the weekly wound size reduction index.In the cadexomer-treated ulcers the total wound area reduction was 9.67 cm(2)/week, with a weekly wound size reduction index per patient of 0.96 cm(2); in the controls (compression therapy-treated patients) the total wound area reduction was 6.11 cm(2)/week, with a weekly reduction index per patient of 0.61 cm(2). At the end of treatment, in the group of patients whose ulcers were treated with cadexomer ointment the average wound size reduction was 43%, whereas in the control-treated patient group the average wound size reduction was 28%.These data suggest that cadexomer can play an important role in the healing of chronic leg ulcers.