Multistability maintains redox homeostasis in human cells

Cells metabolize nutrients through a complex metabolic and signaling network that governs redox homeostasis. At the core of this, redox regulatory network is a mutually inhibitory relationship between reduced glutathione and reactive oxygen species (ROS)—two opposing metabolites that are linked to upstream nutrient metabolic pathways (glucose, cysteine, and glutamine) and downstream feedback loops of signaling pathways (calcium and NADPH oxidase). We developed a nutrient‐redox model of human cells to understand system‐level properties of this network. Combining in silico modeling and ROS measurements in individual cells, we show that ROS dynamics follow a switch‐like, all‐or‐none response upon glucose deprivation at a threshold that is approximately two orders of magnitude lower than its physiological concentration. We also confirm that this ROS switch can be irreversible and exhibits hysteresis, a hallmark of bistability. Our findings evidence that bistability modulates redox homeostasis in human cells and provide a general framework for quantitative investigations of redox regulation in humans.     

Characterization of photodamage to escherichia coli in optical traps

Optical tweezers (infrared laser-based optical traps) have emerged as a powerful tool in molecular and cell biology. However, their usefulness has been limited, particularly in vivo, by the potential for damage to specimens resulting from the trapping laser. Relatively little is known about the origin of this phenomenon. Here we employed a wavelength-tunable optical trap in which the microscope objective transmission was fully characterized throughout the near infrared, in conjunction with a sensitive, rotating bacterial cell assay. Single cells of Escherichia coli were tethered to a glass coverslip by means of a single flagellum: such cells rotate at rates proportional to their transmembrane proton potential (. J. Mol. Biol. 138:541-561). Monitoring the rotation rates of cells subjected to laser illumination permits a rapid and quantitative measure of their metabolic state. Employing this assay, we characterized photodamage throughout the near-infrared region favored for optical trapping (790-1064 nm). The action spectrum for photodamage exhibits minima at 830 and 970 nm, and maxima at 870 and 930 nm. Damage was reduced to background levels under anaerobic conditions, implicating oxygen in the photodamage pathway. The intensity dependence for photodamage was linear, supporting a single-photon process. These findings may help guide the selection of lasers and experimental protocols best suited for optical trapping work.     

Hepatitis C (HCV) therapy for HCV mono-infected and HIV-HCV co-infected individuals living in Nepal

BackgroundDespite direct-acting antivirals (DAA), aims to “eradicate” viral hepatitis by 2030 remain unlikely. In Nepal, an expert consortium was established to treat HCV through Nepal earthquakes aftermath offering a model for HCV treatment expansion in a resource-poor setting.Methodology/Principal findingsIn 2015, we established a network of hepatologists, laboratory experts, and community-based leaders at 6 Opioid Substitution Treatment (OST) sites from 4 cities in Nepal screening 838 patients for a treatment cohort of 600 individuals with HCV infection and past or current drug use. During phase 1, patients were treated with interferon-based regimens (n = 46). During phase 2, 135 patients with optimal predictors (HIV controlled, without cirrhosis, low baseline HCV viral load) were treated with DAA-based regimens. During phase 3, IFN-free DAA treatment was expanded, regardless of HCV disease severity, HIV viremia or drug use. Sustained virologic response (SVR) was assessed at 12 weeks.Median age was 37 years and 95.5% were males. HCV genotype was 3 (53.2%) or 1a (40.7%) and 32% had cirrhosis; 42.5% were HIV-HCV coinfected. The intention-to-treat (ITT) SVR rates in phase 2 and 3 were 97% and 81%, respectively. The overall per-protocol and ITT SVR rates were 97% and 85%, respectively. By multivariable analysis, treatment at the Kathmandu site was protective and substance use, treatment during phase 3 were associated with failure to achieve SVR.Conclusions/SignificanceVery high SVR rates may be achieved in a difficult-to-treat, low-income population whatever the patient’s profile and disease severity. The excellent treatment outcomes observed in this real-life community study should prompt further HCV treatment initiatives in Nepal.     

Meeting report: SMART timing—principles of single molecule techniques course at the University of Michigan 2014

Four days after the announcement of the 2014 Nobel Prize in Chemistry for “the development of super‐resolved fluorescence microscopy” based on single molecule detection, the Single Molecule Analysis in Real‐Time (SMART) Center at the University of Michigan hosted a “Principles of Single Molecule Techniques 2014” course. Through a combination of plenary lectures and an Open House at the SMART Center, the course took a snapshot of a technology with an especially broad and rapidly expanding range of applications in the biomedical and materials sciences. Highlighting the continued rapid emergence of technical and scientific advances, the course underscored just how brightly the future of the single molecule field shines. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 296–302, 2015.     

Ranking and compacting binding segments of protein families using aligned pattern clusters

Background

Discovering sequence patterns with variation can unveil functions of a protein family that are important for drug discovery. Exploring protein families using existing methods such as multiple sequence alignment is computationally expensive, thus pattern search, called motif finding in Bioinformatics, is used. However, at present, combinatorial algorithms result in large sets of solutions, and probabilistic models require a richer representation of the amino acid associations. To overcome these shortcomings, we present a method for ranking and compacting these solutions in a new representation referred to as Aligned Pattern Clusters (APCs). To tackle the problem of a large solution set, our method reveals a reduced set of candidate solutions without losing any information. To address the problem of representation, our method captures the amino acid associations and conservations of the aligned patterns. Our algorithm renders a set of APCs in which a set of patterns is discovered, pruned, aligned, and synthesized from the input sequences of a protein family.

Results

Our algorithm identifies the binding or other functional segments and their embedded residues which are important drug targets from the cytochrome c and the ubiquitin protein families taken from Unitprot. The results are independently confirmed by pFam's multiple sequence alignment. For cytochrome c protein the number of resulting patterns with variations are reduced by 76.62% from the number of original patterns without variations. Furthermore, all of the top four candidate APCs correspond to the binding segments with one of each of their conserved amino acid as the binding residue. The discovered proximal APCs agree with pFam and PROSITE results. Surprisingly, the distal binding site discovered by our algorithm is not discovered by pFam nor PROSITE, but confirmed by the three-dimensional cytochrome c structure. When applied to the ubiquitin protein family, our results agree with pFam and reveals six of the seven Lysine binding residues as conserved aligned columns with entropy redundancy measure of 1.0.

Conclusion

The discovery, ranking, reduction, and representation of a set of patterns is important to avert time-consuming and expensive simulations and experimentations during proteomic study and drug discovery.

     

Inhibitory effects of biochanin A on titanium particle‐induced osteoclast activation and inflammatory bone resorption via NF‐κB and MAPK pathways

Revision operations have become a new issue after successful artificial joint replacements, and periprosthetic osteolysis leading to prosthetic loosening is the main cause of why the overactivation of osteoclasts (OCs) plays an important role. The effect of biochanin A (BCA) has been examined in osteoporosis, but no study on the role of BCA in prosthetic loosening osteolysis has been conducted yet. In this study, we utilised enzyme‐linked immunosorbent assay, computed tomography imaging, and histological analysis. Results showed that BCA downregulated the secretion levels of tumor necrosis factor‐α, interleukin‐1α (IL‐1α), and IL‐1β to suppress inflammatory responses. The secretion levels of receptor‐activated nuclear factor‐κB ligand, CTX‐1, and osteoclast‐associated receptor as well as Ti‐induced osteolysis were also reduced. BCA effectively inhibited osteoclastogenesis and suppressed hydroxyapatite resorption by downregulating OC‐related genes in vitro. Analysis of mechanisms indicated that BCA inhibited the signalling pathways of mitogen‐activated protein kinase (P38, extracellular signal‐regulated kinase, and c‐JUN N‐terminal kinase) and nuclear factor‐κB (inhibitor κB‐α and P65), thereby downregulating the expression of nuclear factor of activated T cell 1 and c‐Fos. In conclusion, BCA may be an alternative choice for the prevention of prosthetic loosening caused by OCs.     

Atypical B cells are part of an alternative lineage of B cells that participates in responses to vaccination and infection in humans

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PfSPZ-CVac efficacy against malaria increases from 0% to 75% when administered in the absence of erythrocyte stage parasitemia: A randomized,placebo-controlled trial with controlled human malaria infection

PfSPZ-CVac combines ‘PfSPZ Challenge’, which consists of infectious Plasmodium falciparum sporozoites (PfSPZ), with concurrent antimalarial chemoprophylaxis. In a previously-published PfSPZ-CVac study, three doses of 5.12x10⁴ PfSPZ-CVac given 28 days apart had 100% vaccine efficacy (VE) against controlled human malaria infection (CHMI) 10 weeks after the last immunization, while the same dose given as three injections five days apart had 63% VE. Here, we conducted a dose escalation trial of similarly condensed schedules. Of the groups proceeding to CHMI, the first study group received three direct venous inoculations (DVIs) of a dose of 5.12x10⁴ PfSPZ-CVac seven days apart and the next full dose group received three DVIs of a higher dose of 1.024x10⁵ PfSPZ-CVac five days apart. CHMI (3.2x10³ PfSPZ Challenge) was performed by DVI 10 weeks after the last vaccination. In both CHMI groups, transient parasitemia occurred starting seven days after each vaccination. For the seven-day interval group, the second and third vaccinations were therefore administered coincident with parasitemia from the prior vaccination. Parasitemia was associated with systemic symptoms which were severe in 25% of subjects. VE in the seven-day group was 0% (7/7 infected) and in the higher-dose, five-day group was 75% (2/8 infected). Thus, the same dose of PfSPZ-CVac previously associated with 63% VE when given on a five-day schedule in the prior study had zero VE here when given on a seven-day schedule, while a double dose given on a five-day schedule here achieved 75% VE. The relative contributions of the five-day schedule and/or the higher dose to improved VE warrant further investigation. It is notable that administration of PfSPZ-CVac on a schedule where vaccine administration coincided with blood-stage parasitemia was associated with an absence of sterile protective immunity.Clinical trials registration: {"type":"clinical-trial","attrs":{"text":"NCT02773979","term_id":"NCT02773979"}}NCT02773979.