Hepatitis C virus (HCV) is the second viral agent that causes the majority of chronic hepatic infections worldwide, following Hepatitis B virus (HBV) infection. HCV infection comprises several steps, from the attachment to the receptors to the delivery of the viral genetic material and replication inside the cells. Tetraspanin CD81 is a key entry factor for HCV as it accompanies the virus during attachment and internalization through clathrin-mediated endocytosis. HCV-CD81 binding takes place through the viral glycoprotein E2. We performed full-atom molecular dynamics simulations reproducing the pH conditions that occur during the viral attachment to the hepatocytes (pH 7.4) and internalization (pH 6.2-4.6). We observed that changing the pH from 7.4 to 6.2 triggers a large conformational change in the binding orientation between E2core (E2core corresponds to residues 412-645 of the viral glycoprotein E2) and CD81LEL (CD81LEL corresponds to residues 112-204 of CD81) that occurs even more rapidly at low pH 4.6. This pH-induced switching mechanism has never been observed before and could allow the virus particles to sense the right moment during the maturation of the endosome to start fusion.

CD81 is a tetraspanin receptor that clusters into microdomains to mediate cell signalling processes. CD81 is also one of the four primary cellular receptors of the Hepatitis C virus (HCV). Previous structural studies on the α-helical CD81 large-extracellular-loop domain (CD81 LEL ) have shown that it can adopt different conformations (from closed to open), likely depending on the environmental conditions. This conformational plasticity has been implicated in the endosomal fusion of HCV upon entry. However, the precise mechanism governing the CD81 LEL plasticity has remained elusive so far. Here, by combining molecular dynamics simulations and circular dichroism experiments on wt-CD81 LEL and two mutants at different endosomal pH conditions, pH 5.5 and pH 4.6, we show that the modulation of the solvation shell governs the plasticity of CD81 LEL . The primarily implicated residues are D139 and E188, respectively, located near a loop preceded by a helix. At acidic conditions, their interaction with water is reduced, causing a re-ordering of the water molecules, and thus triggering the dynamics of CD81 LEL . However, mutations E188Q and D139N retain the solvation shell and restrict the conformational space that the head subdomain can explore. We propose that residues E188 and D139 control the solvent-induced allosteric transition of the CD81 LEL domain. This mechanism might play a role in other cellular receptors that function along the endosomal pathway. Popular Summary Understanding the cellular mechanisms that are exploited by viruses to infect their host is key for the development of therapeutics. Here, in the context of Hepatitis C Virus infection we report the mechanism that governs the plasticity of the extra-cellular domain of tetraspanin CD81, one of the major cellular receptors of this virus. The mechanism proposed here is a novel form of solvent-induced allosteric transition in proteins mediated by two antenna residues located in the head subdomain of CD81. We propose that it could serve as a pH sensing strategy to time the endosomal pathway and trigger a signal at the right time for HCV fusion.

Non-homologous end-joining (NHEJ) is the preferred mechanism used by hematopoietic stem cells (HSCs) to repair double-stranded DNA breaks and is particularly increased in cells deficient in the Fanconi anemia (FA) pathway. Here, we show feasible correction of compromised functional phenotypes in hematopoietic cells from multiple FA complementation groups, including FA-A, FA-C, FA-D1, and FA-D2. NHEJ-mediated repair of targeted CRISPR-Cas9-induced DNA breaks generated compensatory insertions and deletions that restore the coding frame of the mutated gene. NHEJ-mediated editing efficacy was initially verified in FA lymphoblastic cell lines and then in primary FA patient-derived CD34(+) cells, which showed marked proliferative advantage and phenotypic correction both in vitro and after transplantation. Importantly, and in contrast to homologous directed repair, NHEJ efficiently targeted primitive human HSCs, indicating that NHEJ editing approaches may constitute a sound alternative for editing self-renewing human HSCs and consequently for treatment of FA and other monogenic diseases affecting the hematopoietic system.

21171 Background: Background: Axillary lymph node metastasis is the most significant marker for the pathologic staging of breast cancer. However a proportion of lymph node-negative breast cancer will develop metastatic disease. Therefore, molecular markers of invasion in these patients are needed Methods: We selected 10 primary breast cancer cases, 5 lymph node-negative (T1N0) and 4 lymph node-positive (T1N1). In these cases we searched for gene expression of 8 genes (bbc3, cegp1, fgf18, flt1, cffm4, gstm3, hec, tgfb3) selected from previous studies as a good candidates for metastasis prediction. A quantitative Real-Time PCR was performed using beta-2- microglobulin gene expression to normalized gene expression of each gene. The expression average of beta-2-microglobulin was 303,291 among T1N0 and 342,533 among T1N1 cases (ratio 0.88). The expression average of these 8 genes was 2.11 and 0.002 at T1N0 and T1N1 respectively (ratio 1,055). At least 3 genes were significantly down-regulated in T1N1 (bbc3, flt1, gstm3) in comparison with T1N1 breast carcinomas Results: Computational analysis reveals that these 3 genes (bbc3, flt1, gstm3) contain CpG islands in their promoter regions. Although preliminary data, we found group of genes that are down-regulated in T1N1 tumor Conclusions: The finding that these genes are epigenetically regulated, suggest that could be a good candidates for metastasis prediction, by methylation approach (MethyLight System). Since this down-regulation occurs in primary tumor, the analysis of these genes might be useful to predict metastasis in lymph node-negative breast cancer. No significant financial relationships to disclose.

The t(1;5)(q23;q33) is a rare genetic anomaly that was reported previously in two infants with a myeloproliferative disorder and eosinophilia and in one adult patient with acute nonlymphocytic leukemia (ANLL). A 13-year-old boy with high-risk early pre-B acute lymphoblastic leukemia (ALL) who presented to our institution carried the t(1;5)(q23;q33). He had an initial blast count of 230 X 10(9)/L and responded poorly to prednisone. Complete remission (CR) was achieved, and he had a bone marrow (BM) relapse 3 months after despite intensive consolidation therapy. He underwent allogeneic BM transplantation (BMT) from a human leukocyte antigen (HLA)-identical siblings in early relapse with total body irradiation (TBI) and cyclophosphamide conditioning. He had a short second CR with a central nervous system (CNS) relapse on day + 106 after BMT. Two of the previously reported patients also did not respond to chemotherapy. The t(1;5)(q23;q33) appears to be a rare lineage nonspecific anomaly related to hematologic malignancies that are resistant to current therapy.

We have analyzed the sequence of 40 VDJ rearrangements of the immunoglobulin heavy chain gene locus on 32 unselected children from Chile with precursor B cell ALL at diagnosis. Rearrangements were derived by PCR with VH gene family-specific primers and sequenced directly. The number of VDJ rearrangements, and the pattern of VH, DH and JH gene usage was identical to the one reported by groups from developed countries. CDR3 regions represented an unbiased repertoire; VH to JH joinings were in frame in 36% of cases. Absent N nucleotides in the DJ border, suggestive of fetal origin of ALL, were seen in 9/40 rearrangements but they did not correlate with younger age. More than one rearrangement was sequenced in six patients, representing independent events with no signs of clonal evolution. One patient was analyzed at first bone marrow relapse showing persistence of one rearrangement and evolution of a second one which conserved the DJ border. The subset of B cell precursors which suffer malignant transformation to ALL appear to be common in different parts of the world.

We describe a case of neonatal mixed lineage leukaemia which presented with a dominant B progenitor lymphoblast population plus a minor monocytic component. Treatment of the patient with corticosteroid and Ara-C resulted in loss of lymphoblasts and a rapid (within 7 days) increase and dominance of the monocytic component. The common clonal origin of the two cell types was evident from the identical rearrangement in the MLL gene and a shared rearrangement of one IGH allele. In common with other neonatal or infant ALL with MLL gene rearrangements, this leukaemia may have originated in a common B-monocytic lineage stem cell during foetal haemopoiesis. The observations further suggest that the therapeutic impact of the MLL gene rearrangement is to some extent dependent on the cellular context in which it is expressed.

The aim of this study was to perform a cytogenetic and molecular study in patients with chronic myelogenous leukemia and to seek a possible relation between bcr gene break points and the clinical evolution of the disease. The cytogenetic study allowed to establish the presence of Ph chromosome and the molecular study localized the break point in bcr region of chromosome 22 using the Southern technique, hybridizing with bcr fragment derived probes bcr1 and bcr2. Forty eight patients were studied, 27 male (aged 46.5 years) and 21 female (aged 56). Forty seven patients were Ph +. A rearrangement in 3' bcr region was found in 25 patients and in 5' region in 23. During the follow up period 20 patients developed a blast crisis or accelerated phase. In 11 of these the rearrangement was in region'3 and their chronic phase lasted a mean of 33.1 months; in 9 the rearrangement was in region 5' and their chronic phase lasted 44.1 months. There were no differences in event-free survival between those with rearrangement in region 3' or 5', however these was a tendency towards a longer chronic phase duration in those with 5' breaks. The lack of correlation between the location of break points and the evolution of the disease may be due to a selection of patients with a better evolution and the exclusion of those with a rapid progression to blast crisis or accelerated phase.

The aim of this study was to perform a cytogenetic and molecular study in patients with chronic myelogenous leukemia and to seek a possible relation beetween bcr gene break points and the clinical evolution of the disease. The cytogenetic study allowed to establish the presence of Ph chromosome and the molecular study localized the break point in bcr region of chromosome 22 using the Southern tecnique, hybridizing with bcr fragment derived probes bcr1 and bcr2. Forty eight patients were studied, 27 male (aged 46.5 years) and 21 female (aged 56). Forty seven patients were Ph +. A rearrangement in 3' bcr region was found in 25 patients and in 5' region in 23. During the follow up period 20 patients developed a blast crisis or accelerated phase. In 11 of these the rearrangement was in region'3 and their chronic phase lasted a mean of 33.1 months; in 9 the rearrangement was in region 5' and their chronic phase lasted 44.1 months. There were no differences in event - free survival between those with rearrangement in region 3' or 5', however these was a tendency towards a longer chronic phase duration in those with 5' breaks. The lack of correlation between the location of break points and the evolution of the disease may be due to a selection of patients with a better evolution and the exclusion of those with a rapid progession to blast crisis or accelerated phase.

We have previously shown that the endemic (African) and sporadic (North American) forms of Burkitt's lymphoma (BL) differ at a molecular level. We have now extended our studies to the molecular epidemiology of BL in South America, specifically to two climatic regions: temperate (Argentina and Chile) and tropical (Brazil). We have examined the patterns of chromosomal breakpoint locations in 39 tumors with respect to geography and Epstein-Barr virus (EBV) association. The result of these analyses provide further support for the existence of pathogenetically distinct subtypes of BL in different world regions. The majority of breakpoints on chromosome 8 in South American BL (41%) occurred in the immediate flanking region of c-myc, ie, further 5′ of the “typical” sporadic breakpoints, in the first exon/intron region, and further 3′ of the “typical” endemic breakpoints, which are usually distant from c-myc. However, the distribution of breakpoints on chromosome 14 in tumors from the temperate and tropical regions of South America is similar to that observed in sporadic and endemic tumors. Interestingly, only one tumor with an unrearranged c-myc gene joined to the S mu region of chromosome 14 was observed. This combination was also rarely observed in our earlier series and presumably is either less readily generated by the mechanism that mediates 8;14 translocation or requires other, infrequent genetic changes to provide the necessary selective advantage for lymphomagenesis. The frequency of EBV association in South American BL (51%) is also intermediate with respect to tumors from the United States (30%) and Africa (100%). No correlation with the breakpoint location on chromosome 8 was discernable. Surprisingly, only 54% of tumors with breakpoint outside c-myc were EBV positive. This is in contrast to endemic tumors and suggests that any pathogenetic contribution of EBV is not dependent on breakpoint location, but is more likely to complement additional pathogenetic elements that differ in different world regions.