Roxana M. Coman

Drug-associated changes in amino acid residues in Gag p2, p7NC, and p6Gag/p6Pol in human immunodeficiency virus type 1 (HIV-1) display a dominant effect on replicative fitness and drug response

Regions of HIV-1 gag between p2 and p6(Gag)/p6(Pol), in addition to protease (PR), develop genetic diversity in HIV-1 infected individuals who fail to suppress virus replication by combination protease inhibitor (PI) therapy. To elucidate functional consequences for viral replication and PI susceptibility by changes in Gag that evolve in vivo during PI therapy, a panel of recombinant viruses was constructed. Residues in Gag p2/p7(NC) cleavage site and p7(NC), combined with residues in the flap of PR, defined novel fitness determinants that restored replicative capacity to the posttherapy virus. Multiple determinants in Gag have a dominant effect on PR phenotype and increase susceptibility to inhibitors of drug-resistant or drug-sensitive PR genes. Gag determinants of drug sensitivity and replication alter the fitness landscape of the virus, and viral replicative capacity can be independent of drug sensitivity. The functional linkage between Gag and PR provides targets for novel therapeutics to inhibit drug-resistant viruses.

Training in endoscopic submucosal dissection

Endoscopic submucosal dissection (ESD) represents an important advancement in the therapy of early neoplastic gastrointestinal lesions by providing higher en-bloc curative resection rate with lower recurrence compared to endoscopic mucosal resection (EMR) and by sparing the involved organ and protecting patients quality of life. Despite these advantages ESD is associated with long procedure times and a higher rate of complications, making ESD a challenging procedure which requires advanced endoscopic skills. Thus, there has been a recognized need for structured training system for ESD to enhance trainee experience and, to reduce the risks of complications and inadequate treatment. ESD has a very flat learning curve. However, we do not have uniformly accepted benchmarks for competency. Nevertheless, it appears that, in Japan, more than 30 supervised gastric ESD procedures are required to achieve technical proficiency and minimize complications. A number of training algorithms have been proposed in Japan with the aim to standardize ESD training. These algorithms cannot be directly applied in the West due to substantial differences including the availability of highly qualified mentors, the type of pathology seen, choice of devices, and trainees background. We propose a training algorithm for Western physicians which integrates both hands-on training courses, animal model work as well as visits to expert centers. No specific preceptor training programs have been yet developed but there is a consensus that these programs are important for permeation of ESD worldwide.

Structure of the unbound form of HIV-1 subtype A protease: comparison with unbound forms of proteases from other HIV subtypes.

The crystal structure of the unbound form of HIV-1 subtype A protease (PR) has been determined to 1.7 A resolution and refined as a homodimer in the hexagonal space group P6(1) to an R(cryst) of 20.5%. The structure is similar in overall shape and fold to the previously determined subtype B, C and F PRs. The major differences lie in the conformation of the flap region. The flaps in the crystal structures of the unbound subtype B and C PRs, which were crystallized in tetragonal space groups, are either semi-open or wide open. In the present structure of subtype A PR the flaps are found in the closed position, a conformation that would be more anticipated in the structure of HIV protease complexed with an inhibitor. The amino-acid differences between the subtypes and their respective crystal space groups are discussed in terms of the differences in the flap conformations.

High‐resolution structure of unbound human immunodeficiency virus 1 subtype C protease: implications of flap dynamics and drug resistance

The X-ray crystal structure of the unbound state of human immunodeficiency virus 1 (HIV-1) subtype C protease (C PR) has been determined to 1.20 angstroms resolution in the tetragonal space group P4(1)2(1)2, with one monomer per asymmetric unit and unit-cell parameters a = 46.7, c = 100.8 angstroms, allowing full anisotropic least-squares refinement. The refined model has a conventional R factor of 14.1% for all reflections and estimated standard deviations in bond lengths and angles for all main-chain non-H atoms of 0.014 angstroms and 0.030 degrees , respectively. The structure is compared with three unbound subtype B proteases (B PRs) to identify structural changes arising from the naturally occurring polymorphisms and delineate their implications in antiretroviral drug resistance/susceptibility. The unbound C PR exhibits a larger distance between the tips of the flaps, a downward displacement of the 36-41 loop and an increased thermal stability of the 10s loop when compared with the B PR structures. The C PR structure presents the highest resolution of the unbound state of a non-subtype-B PR and adds to the understanding of flap dynamics and drug resistance.

Training for complex endoscopic procedures: how to incorporate endoscopic submucosal dissection skills in the West?

Endoscopic submucosal dissection (ESD) has been employed for removal of early neoplastic gastrointestinal lesions and has well-documented higher en bloc and curative resection rates compared with endoscopic mucosal resection. Based on these advantages, ESD has gained widespread use in Asia but unfortunately not in the West. The main obstacles remain the very flat learning curve and lack of training resources. In Asia, ESD skills are acquired in the time-honored mentor/apprentice model over a period of few years. This algorithm cannot be directly applied in the West due to substantial differences. Can we train Western endoscopists in ESD in a model that bridges the gap between the traditional approach that requires years to gain proficiency and the weekend crash course approach that does not do justice to our patients? We propose a training algorithm that would guide the ESD training of Western endoscopists in a way to reliably deliver high-quality end product.

Role of observation of live cases done by Japanese experts in the acquisition of ESD skills by a western endoscopist.

AIM To evaluate the role of observation of experts performing endoscopic submucosal dissection (ESD) in the acquisition of ESD skills. METHODS This prospective study is documenting the learning curve of one Western endoscopist. The study consisted of three periods. In the first period (pre-observation), the trainee performed ESDs in animal models in his home institution in the United States. The second period (observation) consisted of visit to Japan and observation of live ESD cases done by experts. The observation of cases occurred over a 5-wk period. During the third period (post-observation), the trainee performed ESD in animal models in a similar fashion as in the first period. Three animal models were used: live 40-50 kg Yorkshire pig, explanted pig stomach model, and explanted pig rectum model. The outcomes from the ESDs done in the animal models before and after observation of live human cases (main study intervention) were compared. Statistical analysis of the data included: Fishers exact test to compare distributions of a categorical variable, Wilcoxon rank sum test to compare distributions of a continuous variable between the two groups (pre-observation and post-observation), and Kruskal-Wallis test to evaluate the impact of lesion location and type of model (ex-vivo vs live pig) on lesion removal time. RESULTS The trainee performed 38 ESDs in animal model (29 pre-observation/9 post-observation). The removal times post-observation were significantly shorter than those pre-observation (32.7 ± 15.0 min vs 63.5 ± 9.8 min, P < 0.001). To minimize the impact of improving physician skill, the 9 lesions post-observation were compared to the last 9 lesions pre-observation and the removal times remained significantly shorter (32.7 ± 15.0 min vs 61.0 ± 7.4 min, P = 0.0011). Regression analysis showed that ESD observation significantly reduced removal time when controlling for the sequence of lesion removal (P = 0.025). Furthermore, it was also noted a trend towards decrease in failure to remove lesions and decrease in complications after the period of observation. This study did not find a significant difference in the time needed to remove lesions in different animal models. This finding could have important implications in designing training programs due to the substantial difference in cost between live animal and explanted organ models. The main limitation of this study is that it reflects the experience of a single endoscopist. CONCLUSION Observation of experts performing ESD over short period of time can significantly contribute to the acquisition of ESD skills.

Prospective evaluation of the clinical utility of endoscopic submucosal dissection (ESD) in patients with Barrett’s esophagus: a Western center experience

Background and study aims: Endoscopic submucosal dissection (ESD) carries significant advantages over endoscopic mucosal resection. As such, ESD is an established therapy for esophageal squamous cell carcinoma but there are only limited data on ESD as therapy for Barrett’s esophagus (BE). Thus, we prospectively evaluated the outcomes of ESD in patients with BE with high-grade dysplasia (HGD) and early esophageal adenocarcinoma (EAC) performed in a Western center. Patients and methods: This is a prospective cohort study. Indications for ESD included: (1) early EAC defined as lesions with intramucosal cancer or superficial submucosal invasion; (2) early EAC with positive lateral margin after EMR; and (3) nodularity with HGD that could not be removed en-bloc with EMR Results: From October 2013 to July 2015, 36 consecutive patients (median age 69, 32 males) underwent ESD at our center. Median procedure time was 88 minutes, with median maximal diameter of resected specimens of 49 mm. En-bloc, R0, and curative resection rates were 100 %, 81 %, and 69 %, respectively. Intramucosal EAC was found in 13 patients (36 %), and submucosal invasion in 13 patients (36 %). In 59 % of the cases, there was discrepancy in the pre- and post-ESD histopathologic diagnosis. Adverse events occurred in 8 patients (22 %), including one episode of bleeding treated with endoscopy and seven esophageal strictures, which were successfully managed with dilations. Conclusions: ESD for BE with HGD/early EAC is feasible and safe with resulting very high en-bloc and R0 resection rates. ESD provided for more accurate pathologic evaluation and significant discrepancy between the pre- and post-ESD histopathological diagnosis was noted.

Influence of host immunoregulatory genes, ER stress and gut microbiota on the shared pathogenesis of inflammatory bowel disease and Type 1 diabetes.

Inflammatory bowel disease (IBD) with its two distinct entities, Crohns disease and ulcerative colitis, and Type 1 diabetes mellitus (T1D) are autoimmune diseases. The prevalence of these diseases continues to rapidly rise in the industrialized world. Despite the identification of several genetic loci that are associated with both IBD and T1D, thus far, there is a paucity of epidemiological data to support a clinical overlap. In an effort to better understand the underlying pathogenic mechanisms of both IBD and T1D, this review summarizes the literature about these related autoimmune diseases, describes the most recent advances in their etiopathogenesis and emphasizes the genetic and nongenetic factors that exercise a differential influence. Genome-wide association studies have identified genetic loci with a role in immune response regulation that are linked to both IBD (particularly Crohns disease) and T1D. Some of these genetic loci (e.g., IL-18RAP) have a divergent role, conferring risk for one disease and protection for the other. Recent evidence highlights an important role of gut microbiota and cellular responses (e.g., endoplasmic reticulum stress) in the pathogenesis of both IBD and T1D.

Genetic determinants in HIV-1 Gag and Env V3 are related to viral response to combination antiretroviral therapy with a protease inhibitor

Objective:To identify novel viral determinants in HIV-1 protease, Gag, and envelope V3 that relate to outcomes to initial protease inhibitor-based antiretroviral therapy. Design:A longitudinal cohort study of protease inhibitor-naive, HIV-infected individuals was designed to identify genetic variables in viral Gag and envelope sequences associated with response to antiretroviral therapy. Methods:Genetic and statistical models, including amino acid profiles, phylogenetic analyses, receiver operating characteristic analyses, and covariation analyses, were used to evaluate viral sequences and clinical variables from individuals who developed immune reconstitution with or without suppression of viral replication. Results:Pretherapy chemokine (C–X–C motif) receptor 4-using V3 regions had significant associations with viral failure (P = 0.04). Amino acid residues in protease covaried with Gag residues, particularly in p7NC, independent of cleavage sites. Pretherapy V3 charge combined with p6Pol and p2/p7NC cleavage site genotypes produced the best three-variable model to predict viral suppression in 88% of individuals. Combinations of baseline CD4 cell percentage with genetic determinants in Gag–protease predicted viral fitness in 100% of individuals who failed to suppress viral replication. Conclusion:Baseline genetic determinants in Gag p6Pol and p2/p7NC, as well as envelope, provide novel combinations of biomarkers for predicting emergence of viral resistance to initial therapy regimens.

Expression, purification and preliminary X-ray crystallographic studies of the human immunodeficiency virus 1 subtype C protease

Crystals of the human immunodeficiency virus 1 (HIV-1) subtype C protease (PR) complexed with the clinically used inhibitors indinavir (IDV) and nelfinavir (NFV) have been grown in the monoclinic space group P2(1), with mean unit-cell parameters a = 46.7 (+/-0.1), b = 59.8 (+/-0.3), c = 87.0 (+/-0.4) A, beta = 95.2 (+/-0.5) degrees. The crystals of both complexes have been shown to diffract X-rays to 2.3 A resolution. The diffraction data for the subtype C PR complexes with IDV and NFV were subsequently processed and reduced, with overall R(sym) values of 8.4 and 11.4%, respectively. Based on the unit-cell volumes, molecular-replacement results and packing considerations, there are two protease homodimers per crystallographic asymmetric unit in each of the complexes. The data were initially phased using a model based on the crystal structure of HIV-1 subtype B PR; the structures have been determined and further refinement and analysis are in progress. These structures and subsequent studies with other inhibitors will greatly aid in correlating the amino-acid variation between the different HIV PRs and understanding their differential sensitivity and resistance to current drug therapy.