SP3-Enabled Rapid and High Coverage Chemoproteomic Identification of Cell-State–Dependent Redox-Sensitive Cysteines

Proteinaceous cysteine residues act as privileged sensors of oxidative stress. As reactive oxygen and nitrogen species have been implicated in numerous pathophysiological processes, deciphering which cysteines are sensitive to oxidative modification and the specific nature of these modifications is essential to understanding protein and cellular function in health and disease. While established mass spectrometry-based proteomic platforms have improved our understanding of the redox proteome, the widespread adoption of these methods is often hindered by complex sample preparation workflows, prohibitive cost of isotopic labeling reagents, and requirements for custom data analysis workflows. Here, we present the SP3-Rox redox proteomics method that combines tailored low cost isotopically labeled capture reagents with SP3 sample cleanup to achieve high throughput and high coverage proteome-wide identification of redox-sensitive cysteines. By implementing a customized workflow in the free FragPipe computational pipeline, we achieve accurate MS1-based quantitation, including for peptides containing multiple cysteine residues. Application of the SP3-Rox method to cellular proteomes identified cysteines sensitive to the oxidative stressor GSNO and cysteine oxidation state changes that occur during T cell activation.     

Immunological effect of Moringa Oleifera leaf extract on vaccinated and non-vaccinated Hubbard chickens experimentally infected with Newcastle virus

In veterinary medicine plant based medicine is achieving a huge importance worldwide. This research was subjected to rectify the hydrophilic Moringa Oleifera alcoholic leaves extract could improve the immune system in vaccinated and non-vaccinated broiler Hubbard chickens experimentally exposed to Newcastle disease (ND) virus. Seventy five chicks with age one day old were splitted randomly into five groups equally in distribution with fifteen chick in each group. Group I was untreated unvaccinated (control negative group) while group IV was infected group with NDV (control positive group). The experimental Groups II and V were given daily oral treatment of hydrophilic alcoholic leaves extract of M. oleifera at 200 mg/kg body weight until day 21 of age while groups III and V were ND vaccinated with La Sota strain of ND vaccines. The four groups (II, III, IV, V) were infected with ND virus velogenic strain (VNDV) on day 21. Following to infection, Monitoring of birds were done daily for clinical signs, postmortem examination, morbidity and mortality. Cellular, humeral immune response and phagocytic activity were evaluated and the data were statistically analyzed using (SPSS). Total and differential cell numbers as well as Haemagglutination inhibition (HI) titre increased in the extract treated and vaccinated group which give total protection against NDV much more than treated and unvaccinated group. As a result it could be recommended to use M. Olifera extract from the first day of rearing in Hubbard chicken with ND vaccination program as a prophylactic treatment in protection of birds against ND infection.     

Anti-salmonella properties of kefir yeast isolates: An in vitro screening for potential infection control

The rise of antibiotic resistance has increased the need for alternative ways of preventing and treating enteropathogenic bacterial infection. Various probiotic bacteria have been used in animal and human. However, Saccharomyces boulardii is the only yeast currently used in humans as probiotic. There is scarce research conducted on yeast species commonly found in kefir despite its claimed potential preventative and curative effects. This work focused on adhesion properties, and antibacterial metabolites produced by Kluyveromyces lactis and Saccharomyces unisporus isolated from traditional kefir grains compared to Saccharomyces boulardii strains. Adhesion and sedimentation assay, slide agglutination, microscopy and turbidimetry assay were used to analyze adhesion of Salmonella Arizonae and Salmonella Typhimurium onto yeast cells. Salmonella growth inhibition due to the antimicrobial metabolites produced by yeasts in killer toxin medium was analyzed by slab on the lawn, turbidimetry, tube dilution and solid agar plating assays. Alcohol and antimicrobial proteins production by yeasts in killer toxin medium were analyzed using gas chromatography and shotgun proteomics, respectively. Salmonella adhered onto viable and non-viable yeast isolates cell wall. Adhesion was visualized using scanning electron microscope. Yeasts-fermented killer toxin medium showed Salmonella growth inhibition. The highest alcohol concentration detected was 1.55%, and proteins with known antimicrobial properties including cathelicidin, xanthine dehydrogenase, mucin-1, lactadherin, lactoperoxidase, serum amyloid A protein and lactotransferrin were detected in yeasts fermented killer medium. These proteins are suggested to be responsible for the observed growth inhibition effect of yeasts-fermented killer toxin medium. Kluyveromyces lactis and Saccharomyces unisporus have anti-salmonella effect comparable to Saccharomyces boulardii strains, and therefore have potential to control Salmonella infection.     

Biochemical and structural insights into intramembrane metalloprotease mechanisms

Intramembrane metalloproteases are nearly ubiquitous in living organisms and they function in diverse processes ranging from cholesterol homeostasis and the unfolded protein response in humans to sporulation, stress responses, and virulence of bacteria. Understanding how these enzymes function in membranes is a challenge of fundamental interest with potential applications if modulators can be devised. Progress is described toward a mechanistic understanding, based primarily on molecular genetic and biochemical studies of human S2P and bacterial SpoIVFB and RseP, and on the structure of the membrane domain of an archaeal enzyme. Conserved features of the enzymes appear to include transmembrane helices and loops around the active site zinc ion, which may be near the membrane surface. Extramembrane domains such as PDZ (PSD-95, DLG, ZO-1) or CBS (cystathionine-β-synthase) domains govern substrate access to the active site, but several different mechanisms of access and cleavage site selection can be envisioned, which might differ depending on the substrate and the enzyme. More work is needed to distinguish between these mechanisms, both for enzymes that have been relatively well-studied, and for enzymes lacking PDZ and CBS domains, which have not been studied. This article is part of a Special Issue entitled: Intramembrane Proteases.     

Evaluation of Superoxide Scavenging Activity of OPC-14117 By Electron Spin Resonance Technique

OPC-14117 is a potent drug which has both brain function activating effect and protective effect against cerebral ischemia. Occurrences of these effects might be expected due to superoxide dismutase-like activity of OPC-14117. The present study has been conducted to evaluate the active oxygen scavenging activity of OPC-14117 and to explain the mechanisms of its pharmacological activities. The reaction of OPC-14117 and superoxide anion. generated in potassium superoxide, was examined by electron spin resonance technique at both liquid nitrogen (77 K) and room (22°C) temperatures. OPC-14117 showed a higher superoxide scavenging activity than that of α-tocopherol in an aprotic solvent system. The active moiety of OPC-14117 to provide the scavenging effect was found due to the phenolic hydroxyl group of its indan skeleton.     

Point mutations in firefly luciferase C-domain demonstrate its significance in green color of bioluminescence

Firefly luciferase is a two-domain enzyme that catalyzes the bioluminescent reaction of firefly luciferin oxidation. Color of the emitted light depends on the structure of the enzyme, yet the exact color-tuning mechanism remains unknown by now, and the role of the C-domain in it is rarely discussed, because a very few color-shifting mutations in the C-domain were described. Recently we reported a strong red-shifting mutation E457K in the C-domain; the bioluminescence spectra of this enzyme were independent of temperature or pH. In the present study we investigated the role of the residue E457 in the enzyme using the Luciola mingrelica luciferase with a thermostabilized N-domain as a parent enzyme for site-directed mutagenesis. We obtained a set of mutants and studied their catalytic properties, thermal stability and bioluminescence spectra. Experimental spectra were represented as a sum of two components (bioluminescence spectra of putative “red” and “green” emitters); λ_max of these components were constant for all the mutants, but the ratio of these emitters was defined by temperature and mutations in the C-domain. We suggest that each emitter is stabilized by a specific conformation of the active site; thus, enzymes with two forms of the active site coexist in the reactive media. The rigid structure of the C-domain is crucial for maintaining the conformation corresponding to the “green” emitter. We presume that the emitters are the keto- and enol forms of oxyluciferin.     

Taxonomic notes on the genus Eupoa ?abka, 1985 (Arachnida,Araneae, Salticidae)

The south-east Asian genus Eupoa is redescribed and diagnosed. Seven new species are diagnosed, described and illustrated: E. daklak sp. n. (♀) from Viet-Nam; E. lehtineni sp. n. (♂♀) from India, Thailand and Viet-Nam; E. lobli sp. n. (♂) from Malaysia; E. pappi sp. n. (♂) from Thailand; E. pulchella sp. n.(♂) from Thailand; E. schwendingeri sp. n. (♂♀) from Thailand; and E. thailandica sp. n. (♂♀) from Thailand. Eupoa prima Żabka, 1985 and E. yunnanensis Peng & Kim, 1997 are redescribed and illustrated on the basis of type and/or newly collected materials. The female of E. yunnanensis Peng & Kim, 1997 is found and described for the first time.     

Changing seasonality and phenological responses of free-living male arctic ground squirrels: the importance of sex

Many studies have addressed the effects of climate change on species as a whole; however, few have examined the possibility of sex-specific differences. To understand better the impact that changing patterns of snow-cover have on an important resident Arctic mammal, we investigated the long-term (13 years) phenology of hibernating male arctic ground squirrels living at two nearby sites in northern Alaska that experience significantly different snow-cover regimes. Previously, we demonstrated that snow-cover influences the timing of phenological events in females. Our results here suggest that the end of heterothermy in males is influenced by soil temperature and an endogenous circannual clock, but timing of male emergence from hibernation is influenced by the timing of female emergence. Males at both sites, Atigun and Toolik, end heterothermy on the same date in spring, but remain in their burrows while undergoing reproductive maturation. However, at Atigun, where snowmelt and female emergence occur relatively early, males emerge 8 days earlier than those at Toolik, maintaining a 12-day period between male and female emergence found at each site, but reducing the pre-emergence euthermic period that is critical for reproductive maturation. This sensitivity in timing of male emergence to female emergence will need to be matched by phase shifts in the circannual clock and responsiveness to environmental factors that time the end of heterothermy, if synchrony in reproductive readiness between the sexes is to be preserved in a rapidly changing climate.