BACKGROUND: The hepatitis B e antigen (HBeAg)-negative infection Phase 3 is characterized by no or minimal signs of hepatic inflammation and the absence of hepatic fibrosis. However, underlying molecular mechanisms leading to this benign phenotype are poorly understood.
METHODS: Genotype A, B and D HBeAg-negative patient isolates with precore mutation G1896A from Phase 3 were analysed in comparison with respective HBeAg-positive rescue mutant and HBeAg-positive wild-type reference genomes regarding differences in viral replication, morphogenesis, infectivity and impact on NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE)-dependent gene expression and cellular kinome.
RESULTS: In comparison with reference genomes, the patient isolates are characterized by a lower intra- and extracellular hepatitis B surface antigen (HBsAg)-amount, and HBsAg-retention in the endoplasmic reticulum. Rescue of HBeAg expression increased HBsAg-amount but not its release. Expression of the isolated genomes is associated with a higher Nrf2/ARE-dependent gene expression as compared to reference genomes independent of HBeAg expression. Kinome analyses revealed a decreased activity of receptors involved in regulation of proliferative pathways for all patient isolates compared to the reference genomes. No specific conserved mutations could be found between all genomes from Phase 3.
CONCLUSIONS: HBeAg-negative genomes from Phase 3 exhibit distinct molecular characteristics leading to lower HBsAg synthesis and release, enhanced oxidative stress protection and decreased activity of key kinases, triggering an antiproliferative stage, which might contribute to the lower probability of hepatocellular carcinoma. The observed differences cannot be associated with loss of HBeAg or specific mutations common to all analysed isolates, indicating the phenotype of Phase 3 derived genomes to be the result of a multifactorial process likely reflecting a conserved natural selection process.

Reverse genetic approaches are common tools in genomics for elucidating gene functions, involving techniques such as gene deletion followed by screening for aberrant phenotypes. If the generation of gene deletion mutants fails, the question arises whether the failure stems from technical issues or because the gene of interest (GOI) is essential, meaning that the deletion causes lethality. In this report, we introduce a novel method for assessing gene essentiality using the phytopathogenic ascomycete Magnaporthe oryzae. The method is based on the observation that telomere vectors are lost in transformants during cultivation without selection pressure. We tested the hypothesis that essential genes can be identified in deletion mutants co-transformed with a telomere vector. The M. oryzae gene MoPKC, described in literature as essential, was chosen as GOI. Using CRISPR/Cas9 technology transformants with deleted GOI were generated and backed up by a telomere vector carrying a copy of the GOI and conferring fenhexamid resistance. Transformants in which the GOI deletion in the genome was not successful lost the telomere vector on media without fenhexamid. In contrast, transformants with confirmed GOI deletion retained the telomere vector even in absence of fenhexamid selection. In the latter case, the maintenance of the telomere indicates that the GOI is essential for the surveillance of the fungi, as it would have been lost otherwise. The method presented here allows to test for essentiality of genes when no mutants can be obtained from gene deletion approaches, thereby expanding the toolbox for studying gene function in ascomycetes.

UNASSIGNED: This study described the epidemiological, clinical, and survival profiles of patients with gastrointestinal stromal tumor (GIST) in North Africa and the Middle East (AfME).
UNASSIGNED: This regional, multicenter, observational, retrospective study collected 11-year data on demographics, medical history, disease characteristics, current treatment approaches of GIST, the safety of the most common tyrosine kinase inhibitors (TKIs), second cancers, and survival status.
UNASSIGNED: Data of 201 eligible patients were analyzed: mean age was 56.9 ± 12.6 years; 111 (55.2%) patients were men, 21 (10.4%) patients had previous personal malignancy. The most common clinical presentation of GIST was dysphagia [92 (45.8%) patients]. The stomach was the most common primary site in 120 (60.7%) patients, 171 (85.1%) patients had localized disease at diagnosis. 198 (98.5%) GIST cases were CD117/CD34-positive. Imatinib was used in the neoadjuvant (18/21 patients), adjuvant (85/89 patients), and first-line metastatic treatment (28/33 patients) settings. The most common non-hematological toxicity associated with TKIs was vomiting in 32/85 (37.6%) patients. Overall, 100 (49.8%) patients (95%CI: 42.8-56.7%) were alive and disease-free while 30 (14.9%) patients were alive with active disease.
UNASSIGNED: Presentation of GIST in our AfME population is consistent with global reports, being more frequent in patients >50 years old and having the stomach as the most common primary site. Unlike what is usually reported, though, we did have more patients with lymphatic spread of the disease. Despite the global trend and advances in the treatment of GIST according to molecular profile, this is still far to happen in our population given the lack of access to molecular profiles and the high associated cost.

Zika virus is responsible for causing Zika infections and was declared as a public health emergency of international concern in February 2016. The Zika virus NS3-helicase is a viable drug target for the design of inhibitors due to its essential role in the replication of viral genome. The viral RNA is unwound by the NS3-helicase in order to enable the reproduction of viral genome by the NS5 protein. Zika virus infections in humans are being reported for the last 15 years. We have therefore carried out amino acid mutational analyses of NS3-helicase. NS3-helicase has two crucial binding sites: the ATP and RNA binding sites. The cofactor-ATP based pharmacophore was generated for virtual screening of ZINC database using Pharmit server, that is followed by molecular docking and molecular dynamics simulations of potential hits as probable Zika virus NS3-helicase inhibitors at the cofactor binding site. The drug-like properties of the molecules were analysed and, DFT calculations were performed on the five best molecules to reveal their stability in solvent phase compared to gas phase, the HOMO and LUMO and electrostatic potential maps to analyze the electronic and geometric characteristics. These are significant findings towards the discovery of new inhibitors of Zika virus NS3-helicase, a promising drug target to treat the Zika virus infection.Communicated by Ramaswamy H. Sarma.

Four cellular retinol-binding protein (CRBP) types (CRBP1,2,3,4) are encoded in the human genome. Here, we report on X-ray analyses of human apo- and holo-CRBP1, showing nearly identical structures, at variance with the results of a recent study on the same proteins containing a His-Tag, which appears to be responsible for a destabilizing effect on the apoprotein. The analysis of crystallographic B-factors for our structures indicates that the putative portal region, in particular α-helix-II, along with Arg58 and the E-F loop, is the most flexible part of both apo- and holoprotein, consistent with its role in ligand uptake and release. Fluorometric titrations of wild type and mutant forms of apo-CRBP1, coupled with X-ray analyses, provided insight into structural and molecular determinants for the interaction of retinol with CRBP1. An approximately stoichiometric binding of retinol to wild type apo-CRBP1 (Kd∼4.5nM), significantly lower binding affinity for both mutants Q108L (Kd∼65nM) and K40L (Kd∼70nM) and very low binding affinity for the double mutant Q108L/K40L (Kd∼250nM) were determined, respectively. Overall, our data indicate that the extensive apolar interactions between the ligand and hydrophobic residues lining the retinol binding cavity are sufficient to keep it in its position bound to CRBP1. However, polar interactions of the retinol hydroxyl end group with Gln108 and Lys40 play a key role to induce a high binding affinity and specificity for the interaction.