Research toward the development of a protocol to electrotransform Bacillus megaterium through exploration of parameters and variables that have been used to successfully electrotransform other gram-positive bacteria

Includes bibliographical references.This project was proposed to develop a protocol to electrotransform Bacillus megaterium (B. megaterium) through investigation of techniques that have been used to electrotransform other species of gram-positive bacteria. Electrotransformation is a process by which exogenous DNA can be introduced into a recipient cell through the application of an electric pulse. It was decided that successful protocols be studied and applied as closely as possible to B. megaterium. Specific areas of inquiry included; finding the machine settings on the apparatus needed to achieve electric pulse times that have been found to be optimum in successful protocols, constructing a table of applied voltage vs incidence of cell death in the range of applied voltages used in other protocols, and investigating the effect of adding various substances, such as glycine and polyethelene glycol, to the growth medium or the electrotransformation buffer to assess whether damage to, or reconfiguration of the cell wall and capsule could be induced. It was found that similar, but not identical, voltages and pulse times as those utilized in the protocols studied could be achieved using the available apparatus. Surprisingly, applying similar pulse times and voltages to B. megaterium did not result in any significant or even measurable level of cell death -- a condition thought to be critical to successful electrotransformation. The most promising procedure proved to be the addition of glycine to the growth medium. Significant morphological changes were observed after growing a culture in varying concentrations of glycine to a stage that showed a 75-90% reduction in optical density when compared to a control grown without glycine. In addition, when an electric pulse was applied to these cells they appeared to have been damaged. In spite of this progress, when the procedure was followed using plasmid DNA as the donor, it yielded no transformants. There is some question as to the suitability of the plasmid that was used,(pHV 33) as an electrotransformation vector, adding complexity to the problem. In addition to the work described above, two plasmid DNA extractions using cesium chloride gradients were performed and another plasmid, the 8.1kb plasmid of B. megaterium was isolated using electroelution.B.S. (Bachelor of Science

A new small molecule inhibitor of soluble guanylate cyclase

Soluble guanylate cyclase (sGC) is a haem containing enzyme that regulates cardiovascular homeostasis and multiple mechanisms in the central and peripheral nervous system. Commonly used inhibitors of sGC activity act through oxidation of the haem moiety, however they also bind haemoglobin and this limits their bioavailability for in vivo studies. We have discovered a new class of small molecule inhibitors of sGC and have characterised a compound designated D12 (compound 10) which binds to the catalytic domain of the enzyme with a KD of 11 μM in a SPR assay

Biomarkers and overall survival in patients with advanced hepatocellular carcinoma treated with TGF-βRI inhibitor galunisertib.

BackgroundTransforming growth factor beta (TGF-β) signalling is involved in the development of hepatocellular carcinoma (HCC). We followed changes in biomarkers during treatment of patients with HCC with the TGF-βRI/ALK5 inhibitor galunisertib.MethodsThis phase 2 study (NCT01246986) enrolled second-line patients with advanced HCC into one of two cohorts of baseline serum alpha-fetoprotein (AFP): Part A (AFP ≥1.5x ULN) or Part B (AFP <1.5x ULN). Baseline and postbaseline levels of AFP, TGF-β1, E-cadherin, selected miRNAs, and other plasma proteins were monitored.ResultsThe study enrolled 149 patients (Part A, 109; Part B, 40). Median OS was 7.3 months in Part A and 16.8 months in Part B. Baseline AFP, TGF-β1, E-cadherin, and an additional 16 plasma proteins (such as M-CSF, IL-6, ErbB3, ANG-2, neuropilin-1, MIP-3 alpha, KIM-1, uPA, IL-8, TIMP-1, ICAM-1, Apo A-1, CA-125, osteopontin, tetranectin, and IGFBP-1) were found to correlate with OS. In addition, a range of miRs were found to be associated with OS. In AFP responders (21% of patients in Part A with decrease of >20% from baseline) versus non-responders, median OS was 21.5 months versus 6.8 months (p = 0.0015). In TGF-β1 responders (51% of all patients) versus non-responders, median OS was 11.2 months versus 5.3 months (p = 0.0036).ConclusionsConsistent with previous findings, both baseline levels and changes from baseline of circulating AFP and TGF-β1 function as prognostic indicators of survival. Future trials are needed to confirm and extend these results

Design, synthesis, and evaluation of peptide-imidazo[1,2-a]pyrazine bioconjugates as potential bivalent inhibitors of the VirB11 ATPase HP0525

Helicobacter pylori (H. pylori) infections have been implicated in the development of gastric ulcers and various cancers: however, the success of current therapies is compromised by rising antibiotic resistance. The virulence and pathogenicity of H. pylori is mediated by the type IV secretion system (T4SS), a multiprotein macromolecular nanomachine that transfers toxic bacterial factors and plasmid DNA between bacterial cells, thus contributing to the spread of antibiotic resistance. A key component of the T4SS is the VirB11 ATPase HP0525, which is a hexameric protein assembly. We have previously reported the design and synthesis of a series of novel 8-amino imidazo[1,2-a]pyrazine derivatives as inhibitors of HP0525. In order to improve their selectivity, and potentially develop these compounds as tools for probing the assembly of the HP0525 hexamer, we have explored the design and synthesis of potential bivalent inhibitors. We used the structural details of the subunit-subunit interactions within the HP0525 hexamer to design peptide recognition moieties of the subunit interface. Different methods (cross metathesis, click chemistry, and cysteine-malemide) for bioconjugation to selected 8-amino imidazo[1,2-a]pyrazines were explored, as well as peptides spanning larger or smaller regions of the interface. The IC50 values of the resulting linker-8-amino imidazo[1,2-a]pyrazine derivatives, and the bivalent inhibitors, were related to docking studies with the HP0525 crystal structure and to molecular dynamics simulations of the peptide recognition moieties

Evolutionary Analysis of the LAFL Genes Involved in the Land Plant Seed Maturation Program

Seeds are one of the most significant innovations in the land plant lineage, critical to the diversification and adaptation of plants to terrestrial environments. From perspective of seed evo-devo, the most crucial developmental stage in this innovation is seed maturation, which includes accumulation of storage reserves, acquisition of desiccation tolerance, and induction of dormancy. Based on previous studies of seed development in the model plant Arabidopsis thaliana, seed maturation is mainly controlled by the LAFL regulatory network, which includes LEAFY COTYLEDON1 (LEC1) and LEC1-LIKE (L1L) of the NF-YB gene family, and ABSCISIC ACID INSENSITIVE3 (ABI3), FUSCA3 (FUS3), and LEC2 (LEAFY COTYLEDON2) of the B3-AFL gene family. In the present study, molecular evolution of these LAFL genes was analyzed, using representative species from across the major plant lineages. Additionally, to elucidate the molecular mechanisms of the seed maturation program, co-expression pattern analyses of LAFL genes were conducted across vascular plants. The results show that the origin of AFL gene family dates back to a common ancestor of bryophytes and vascular plants, while LEC1-type genes are only found in vascular plants. LAFL genes of vascular plants likely specify their co-expression in two different developmental phrases, spore and seed maturation, respectively, and expression patterns vary slightly across the major vascular plants lineages. All the information presented in this study will provide insights into the origin and diversification of seed plants.National Natural Science Foundation of China (NSFC) [91231105]SCI(E)ARTICLE

Combination interventions to prevent HCV transmission among people who inject drugs: modelling the impact of antiviral treatment, needle and syringe programs, and opiate substitution therapy

BackgroundInterventions such as opiate substitution therapy (OST) and high-coverage needle and syringe programs (HCNSP) cannot substantially reduce hepatitis C virus (HCV) prevalence among people who inject drugs (PWID). HCV antiviral treatment may prevent onward transmission. We project the impact of combining OST, HCNSP, and antiviral treatment on HCV prevalence/incidence among PWID.MethodsAn HCV transmission model among PWID was used to project the combinations of OST, HCNSP, and antiviral treatment required to achieve different prevalence and incidence reductions within 10 years for 3 chronic prevalence scenarios and the impact of HCV treatment if only delivered through OST programs. Multivariate and univariate sensitivity analyses were performed.ResultsLarge reductions (>45%) in HCV chronic prevalence over 10 years require HCV antiviral treatment. Scaling up OST and HCNSP substantially reduces the treatment rate required to achieve specific HCV prevalence reductions. If OST and HCNSP coverage were increased to 40% each (no coverage at baseline), then annually treating 10, 23, or 42 per 1000 PWID over 10 years would halve prevalence for 20%, 40%, or 60% baseline chronic HCV prevalences, respectively. Approximately 30% fewer treatments are necessary with new direct-acting antivirals. If coverage of OST and HCNSP is 50% at baseline, similar prevalence reductions require higher treatment rates for the same OST and HCNSP coverage.ConclusionsCombining antiviral treatment with OST with HCNSP is critical for achieving substantial reductions (>50%) in HCV chronic prevalence over 10 years. Empirical studies are required on how best to scale up antiviral treatment and combine treatment with other interventions

Insights into the molecular triggers of parosmia based on gas chromatography olfactometry

Background Parosmia is a debilitating condition in which familiar smells become distorted and disgusting, with consequences for diet and mental health. It is a feature of post-infectious olfactory loss, particularly resulting from COVID-19. There is currently little understanding of its pathophysiology, and the prevailing hypothesis for the underlying mechanism is aberrant growth of regenerating olfactory sensory neurons after damage. Methods We use gas-chromatograph olfactometry to individually present components of a complex olfactory mixture as a rapid screening tool for assessment of both quantitative and qualitative olfactory dysfunction in those with and without parosmia. This allows them to report the associated sensory effects and to identify those molecules which are altered or parosmic in nature. Results Here we show 15 different molecular triggers of this symptom. These trigger molecules are common to many in the parosmic volunteer group and share certain characteristics such as extremely low olfactory threshold and common molecular structure Conclusions We posit that specific highly odour-active molecules are the cause of the parosmic symptom in most cases and initiate the sense of disgust, suggesting that parosmia is, at least in part, a receptor-level phenomenon

Mapping ecologically sensitive, significant and salient areas of Western Ghats: proposed protocols and methodology

The Western Ghats Ecology Expert Panel (WGEEP) of the Ministry of Environment and Forests, Government of India (GOI) has been asked to identify ecologically sensitive areas (ESAs) along the Western Ghats, and to suggest how to manage them. The concept of ESAs has been extensively discussed in the literature. Several ESAs have been set up in India over the last 22 years under the Environment Protection Act, 1986, and a GOI committee under the chairmanship of Pranob Sen has proposed certain criteria for identification of ESAs. However, WGEEP noted that we still lack a global consensus either on the criteria to define ESAs or on a workable methodology to identify them. Furthermore, there are no clear guidelines on the management regime that should prevail in ESAs, and the Pranob Sen Committee has not addressed this issue at all. Hence, WGEEP decided to undertake an exercise of defining ESAs and developing a workable methodology to assign levels of ecological significance/sensitivity as a first step towards putting ESAs on the map of the Western Ghats. This article provides a report on the outcome of a series of discussions and consultations held by WGEEP to build a consensus on defining and mapping ESAs. It hopes to provoke discussion and feedback from a wider section of experts, with the aim of finalizing a generic methodology for mapping ESAs in other ecologically sign ificant, biodiversity-rich areas within and outside the country. We hope to shortly prepare a companion paper that will address the equally vital management issues

The scientific basis of combination therapy for chronic hepatitis B functional cure

Functional cure of chronic hepatitis B (CHB) — or hepatitis B surface antigen (HBsAg) loss after 24 weeks off therapy — is now the goal of treatment, but is rarely achieved with current therapy. Understanding the hepatitis B virus (HBV) life cycle and immunological defects that lead to persistence can identify targets for novel therapy. Broadly, treatments fall into three categories: those that reduce viral replication, those that reduce antigen load and immunotherapies. Profound viral suppression alone does not achieve quantitative (q)HBsAg reduction or HBsAg loss. Combining nucleos(t)ide analogues and immunotherapy reduces qHBsAg levels and induces HBsAg loss in some patients, particularly those with low baseline qHBsAg levels. Even agents that are specifically designed to reduce viral antigen load might not be able to achieve sustained HBsAg loss when used alone. Thus, rationale exists for the use of combinations of all three therapy types. Monitoring during therapy is important not just to predict HBsAg loss but also to understand mechanisms of HBsAg loss using viral and immunological biomarkers, and in selected cases intrahepatic sampling. We consider various paths to functional cure of CHB and the need to individualize treatment of this heterogeneous infection until a therapeutic avenue for all patients with CHB is available

Efficacy of Sofosbuvir, Velpatasvir, and GS-9857 in Patients With Hepatitis C Virus Genotype 2, 3, 4, or 6 Infections in an Open-Label, Phase 2 Trial

© 2016 AGA Institute Background & Aims Studies are needed to determine the optimal regimen for patients with chronic hepatitis C virus (HCV) genotype 2, 3, 4, or 6 infections whose prior course of antiviral therapy has failed, and the feasibility of shortening treatment duration. We performed a phase 2 study to determine the efficacy and safety of the combination of the nucleotide polymerase inhibitor sofosbuvir, the NS5A inhibitor velpatasvir, and the NS3/4A protease inhibitor GS-9857 in these patients. Methods We performed a multicenter, open-label trial at 32 sites in the United States and 2 sites in New Zealand from March 3, 2015 to April 27, 2015. Our study included 128 treatment-naïve and treatment-experienced patients (1 with HCV genotype 1b; 33 with HCV genotype 2; 74 with HCV genotype 3; 17 with genotype HCV 4; and 3 with HCV genotype 6), with or without compensated cirrhosis. All patients received sofosbuvir-velpatasvir (400 mg/100 mg fixed-dose combination tablet) and GS-9857 (100 mg) once daily for 6–12 weeks. The primary end point was sustained virologic response 12 weeks after treatment (SVR12). Results After 6 weeks of treatment, SVR12s were achieved by 88% of treatment-naïve patients without cirrhosis (29 of 33; 95% confidence interval, 72%–97%). After 8 weeks of treatment, SVR12s were achieved by 93% of treatment-naïve patients with cirrhosis (28 of 30; 95% CI, 78%–99%). After 12 weeks of treatment, SVR12s were achieved by all treatment-experienced patients without cirrhosis (36 of 36; 95% CI, 90%–100%) and 97% of treatment-experienced patients with cirrhosis (28 of 29; 95% CI, 82%–100%). The most common adverse events were headache, diarrhea, fatigue, and nausea. Three patients (1%) discontinued treatment due to adverse events. Conclusions In a phase 2 open-label trial, we found sofosbuvir-velpatasvir plus GS-9857 (8 weeks in treatment-naïve patients or 12 weeks in treatment-experienced patients) to be safe and effective for patients with HCV genotype 2, 3, 4, or 6 infections, with or without compensated cirrhosis. ClinicalTrials.gov ID: NCT02378961