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Antigen processing in the class II MHC pathway depends on conventional proteolytic enzymes, potentially acting on antigens in native-like conformational states. CD4+ epitope dominance arises from a competition between antigen folding, proteolysis, and MHCII binding. Protease-sensitive sites, linear antibody epitopes, and CD4+ T-cell epitopes were mapped in the plague vaccine candidate F1-V to evaluate the various contributions to CD4+ epitope dominance. Using X-ray crystal structures, antigen processing likelihood (APL) predicts CD4+ epitopes with significant accuracy without considering peptide-MHCII binding affinity. The profiles of conformational flexibility derived from the X-ray crystal structures of the F1-V proteins, Caf1 and LcrV, were similar to the biochemical profiles of linear antibody epitope reactivity and protease-sensitivity, suggesting that the role of structure in proteolysis was captured by the analysis of the crystal structures. The patterns of CD4+ T-cell epitope dominance in C57BL/6, CBA, and BALB/c mice were compared to epitope predictions based on APL, peptide binding to MHCII proteins, or both. For a sample of 13 diverse antigens larger than 200 residues, accuracy of epitope prediction by the combination of APL and I-Ab-MHCII-peptide affinity approached 40%. When MHCII allele specificity is also diverse, such as in human immunity, prediction of dominant epitopes by APL alone approached 40%. Since dominant CD4+ epitopes tend to occur in conformationally stable antigen domains, crystal structures typically are available for analysis by APL; and thus, the requirement for a crystal structure is not a severe limitation.

Age-related impairments in cognitive function occur in multiple animal species including humans and nonhuman primates. Humans and rhesus monkeys exhibit a similar pattern of cognitive decline beginning in middle age, particularly within the domain of executive function. The prefrontal cortex is the brain region most closely associated with mediating executive function. Previous studies in rhesus monkeys have demonstrated that normal aging leads to an increase in myelin degradation in the prefrontal regions that correlates with cognitive decline. This myelin deterioration is thought to result, at least in part, from the age-related emergence of chronic low levels of inflammation. One therapeutic that may arrest the deleterious effects of neuroinflammation is curcumin (CUR), the primary component of the spice turmeric. CUR has been shown to be a potent anti-inflammatory and antioxidant and improves performance on tasks for working memory and motor function. In the present study, middle-aged monkeys (12-21 years old) were given daily dietary supplementation of 500 mg of curcumin or vehicle over a period of 3-4 years. Here, we present data from a series of both object and spatial reversal tasks. Compared to vehicle, the CUR group showed enhanced performance on object, but not spatial reversal learning. These findings suggest that curcumin may improve specific aspects of executive function. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Kinases are signaling proteins that are involved in many different cellular processes. There are over 500 different kinases to accomplish these various tasks. Abnormal kinase signaling can lead to severe disease outcomes. Studying these disruptions have led to fundamental discoveries about human and cellular biology. Chapter 1 introduces a number of cases in both cancer and infectious disease where kinase signaling is disrupted. The subsequent studies used to elucidate why these disruptions occur have helped advance our current knowledge of human biology. In Chapter 2 I describe about some of our studies with eukaryotic-like ser/thr protein kinases used as effector proteins by the bacteria Legionella pneumophila, the causative agent of Legionnaires’ disease. This chapter explores in vitro and in vivo assays to characterize four of the known and conserved effector kinases from Legionella. Chapter 3 takes a comprehensive look at Legionella kinase 4 (LegK4), one of the effector kinases that showed an interesting Golgi fragmentation phenotype. We discovered that LegK4 targets host Hsp70, reduced the chaperone’s refolding activity, and subsequently reduced global translation of the host cell. Further work showed that phosphorylation of Hsp70 by LegK4 increases the amount of the chaperone present on highly translating polysomes. Chapter 4 reviews the available kinase substrate identification techniques, and explain how we used one of them to explore substrates of Protein Kinase A (PKA) in cell lines of small cell lung cancer. While many substrates were found, a general theme emerged where a number of the direct targets of PKA were involved in cell cycle and cell proliferation. Follow-up experiments performed by our collaborators showed that these results align well with a global phosphoproteomic analysis carried out for PKA in a small cell lung cancer setting. All of these examples show kinase signaling that helped to inform us about new human biology.

Organisms throughout the natural world have evolved different methods for biosynthesis of natural products, utilized in their native environs for defense, structure and signaling. One such method is the employment of modular polyketide synthase and non-ribosomal peptide synthetase enzymes to produce molecules from acetate and amino acid building blocks. The combinatorial capacity of these modules, combined with associated tailoring enzymes and unique protein-protein interaction strategies, has enabled a wide diversity of chemical structures and scaffolds. Many secondary metabolites or semi-synthetic derivatives have strong affinity for enzymatic targets and are used therapeutically to treat certain diseases. Photosynthetic marine filamentous cyanobacteria are prominent sources of bioactive natural products and present unique enzymatic methods for generating new chemical structures. This thesis describes the use of chemical biology techniques, including biochemistry, genomics, stable-isotope labeled feeding studies, NMR and mass spectrometry-based isolation and structure elucidation to explore and understand these novel methods for generating chemical structure diversity in cyanobacteria. Following an introduction and background in chapter one, chapter two describes new strategies for biosynthesis in the production of type A malyngamides in the species Okeania hirsuta sp. PAB10Feb10-1 and sp. PAP21Jun06-1, including neofunctionalization of a lipoic acid synthesis enzyme and ketoreductase domain inactivation. Chapter three of this thesis describes an unprecedented combinatorial approach to natural products biosynthesis in Moorea producens sp. ASI16Jul14-2: identical protein-protein interaction motifs guide a polyketide synthase-generated fatty acid tail to disparate downstream NRPS modules in the same biosynthetic gene cluster, representing a new mechanism for generating combinatorial chemical diversity in the productions of vatiamides A-F. Chapter four of this thesis highlights investigations toward understanding the biosynthesis of the t-butyl group in apratoxin A, a cyclic lipopeptide derived from cyanobacterium Moorea bouillonii PNG5-198. Lastly, chapter five presents a comprehensive overview of the findings described here, and discusses future avenues for research in microbial biosynthesis and natural products drug discovery as a whole.

This study was to characterise the undigested nutrients present along the gastrointestinal tract of birds offered common wheat- or maize-based diets, with the goal of optimising utilisation of enzymes to enhance digestive efficiency. Wheat- and maize-based diets were offered to 240 mixed-sex broilers (10 birds/pen; n = 12) from 1 to 35 d post–hatch. Digestibility of dry matter, starch, crude protein and non-starch polysaccharides (NSP) were measured in the crop, gizzard, duodenum, jejunum, ileum, caeca and excreta at d 12 and 35 post–hatch. Analysis of nutrient levels in the excreta presented that more than 30% of nutrients provided in the feed was wasted, irrespective of wheat or maize diet type. On average, 92 g/kg crude protein, 92 g/kg insoluble NSP and 14 g/kg oligosaccharides were not utilised by birds at d 12 post–hatch. The quantity of water-insoluble NSP in the small intestine at d 12 was lower in birds offered the wheat-based diet compared to those fed the maize-based diet (P

A study was conducted to evaluate the effects of supplemental protease and cereal grain type on nutrient digestibility (jejunum and ileum) and performance of broilers offered diets with reduced amino acid concentrations and supplemental xylanase and phytase. A total of 624 male broiler chicks (Ross 308) were randomly distributed into 48 floor pens (13 chicks/pen; 0.07 m2/bird) and offered one of six dietary treatments with eight replicates per treatment. Dietary treatments were either maize- or wheat-based with a positive control (PC) reference diet, a negative control diet without protease (NC; 60 g/kg lower amino acid density than PC), and an NC diet with protease. The reduction in amino acid density affected (P

Recent work has shown that heterojunction photoelectrodes can achieve synergistically higher water splitting activity than their parent materials. To optimize the performance in such layered systems, it is necessary to develop new methods capable of assessing heterojunction phase space. Herein, we explore WO3/TiO2 heterojunction phase space as a model system. Using chemical vapor deposition, 71 unique photoanodes were grown (15 single-layer; 56 heterojunctions). The materials were physically characterized using X-ray diffraction, Raman spectroscopy, scanning electron microscopy, energy-dispersive X-ray spectroscopy analysis, and ultraviolet–visible transmission spectroscopy. Various key performance indicators were measured. Within this WO3/TiO2 heterojunction phase space, the onset potentials ranged from ∼0.45 to ∼0.81 VRHE; the incident-photon-to-current efficiencies at 350, 375, and 400 nm ranged from ∼0.6 to ∼50.9, ∼0.1 to ∼30.0, and ∼0 to ∼15.6%, respectively; and the theoretical solar photocurrents ranged from ∼0.01 to ∼0.94 mA cm–2. Contour plots allowed us to identify regions of heterojunction phase space with high activity and establish trends. We identified an electronic barrier to charge transfer between the heterojunction layers that required a sufficiently high applied potential (≥1.0 VRHE) to be surpassed for synergetic improvements in activity to be observed. We recommend that the methods developed herein, for assessing the performance of sample libraries of heterojunction photoelectrodes, be used alongside combinatorial synthesis methods and high-throughput photoelectrochemical measurements to optimize promising heterojunction systems more rigorously and rapidly.

Inorganic trace minerals may exacerbate lipid peroxidation, thereby impacting lipid metabolism. This study aimed to compare the effects of inorganic and coated trace minerals in diets with different fat sources, on the performance, slaughter characteristics, and antioxidant status of broiler chickens. A total of 576 21-day-old Abor Acres broiler birds were randomly divided into four dietary treatment groups in a 2 (non-coated and coated trace minerals)×2 (soybean oil and lard) factorial design. Each treatment was replicated 12 times (12 birds per replicate). The results showed that coated minerals significantly improved the average daily gain (ADG) in weight and the feed conversion ratio (P

BACKGROUND Patients' self-reports suggest that acute alcohol consumption may trigger a discrete atrial fibrillation (AF) event. OBJECTIVE To objectively ascertain whether alcohol consumption heightens risk for an AF episode. DESIGN A prospective, case-crossover analysis. SETTING Ambulatory persons in their natural environments. PARTICIPANTS Consenting patients with paroxysmal AF. MEASUREMENTS Participants were fitted with a continuous electrocardiogram (ECG) monitor and an ankle-worn transdermal ethanol sensor for 4 weeks. Real-time documentation of each alcoholic drink consumed was self-recorded using a button on the ECG recording device. Fingerstick blood tests for phosphatidylethanol (PEth) were used to corroborate ascertainments of drinking events. RESULTS Of 100 participants (mean age, 64 years [SD, 15]; 79% male; 85% White), 56 had at least 1 episode of AF. Results of PEth testing correlated with the number of real-time recorded drinks and with events detected by the transdermal alcohol sensor. An AF episode was associated with 2-fold higher odds of 1 alcoholic drink (odds ratio [OR], 2.02 [95% CI, 1.38 to 3.17]) and greater than 3-fold higher odds of at least 2 drinks (OR, 3.58 [CI, 1.63 to 7.89]) in the preceding 4 hours. Episodes of AF were also associated with higher odds of peak blood alcohol concentration (OR, 1.38 [CI, 1.04 to 1.83] per 0.1% increase in blood alcohol concentration) and the total area under the curve of alcohol exposure (OR, 1.14 [CI, 1.06 to 1.22] per 4.7% increase in alcohol exposure) inferred from the transdermal ethanol sensor in the preceding 12 hours. LIMITATION Confounding by other time-varying exposures that may accompany alcohol consumption cannot be excluded, and the findings from the current study of patients with AF consuming alcohol may not apply to the general population. CONCLUSION Individual AF episodes were associated with higher odds of recent alcohol consumption, providing objective evidence that a modifiable behavior may influence the probability that a discrete AF event will occur. PRIMARY FUNDING SOURCE National Institute on Alcohol Abuse and Alcoholism.

Locus for Enterocyte Effacement (LEE)-positive Shiga-toxigenic Escherichia coli (STEC) contributes to many global foodborne diseases, with infection characterized by severe gastrointestinal symptoms, including bloody diarrhea. The incidence of LEE-negative STEC-mediated disease is also increasing globally. Subtilase cytotoxin (SubAB) is released by some LEE-negative STEC strains. It cleaves BiP, which is a chaperone protein located in the endoplasmic reticulum (ER), thereby causing apoptosis induced by ER stress. To date, the apoptotic signaling pathway mediated by SubAB has not been identified. In the current study, RNA-seq analysis showed that SubAB significantly induced the expression of Kelch domain containing 7B (KLHDC7B). We explored the role of KLHDC7B in the SubAB-induced apoptotic pathway. SubAB-induced KLHDC7B mRNA expression was increased after 12 h of incubation of toxin with HeLa cells. KLHDC7B expression was downregulated by knockdown of PKR-like endoplasmic reticulum kinase (PERK), CEBP homologous protein (CHOP), activating transcription factor 4 (ATF4), and CEBP β (CEBPB). KLHDC7B knockdown suppressed SubAB-stimulated CHOP expression, poly(ADP-ribose) polymerase (PARP) cleavage, and cytotoxicity. The over-expressed KLHDC7B was localized to the nucleus and cytosolic fractions. Next, we used RNA-seq to analyze the effect of KLHDC7B knockdown on apoptosis induced by SubAB, and found that the gene encoding for the pro-apoptotic Bcl-2 family protein, Harakiri (HRK), was upregulated in SubAB-treated control cells. However, this effect was not observed in SubAB-treated KLHDC7B-knockdown cells. Therefore, we identified the pathway through which SubAB-induced KLHDC7B regulates HRK expression, which is essential for apoptosis in toxin-mediated ER stress.

Objective: MR (mineralocorticoid receptor) activation associates with increased risk of cardiovascular ischemia while MR inhibition reduces cardiovascular-related mortality and plaque inflammation in mouse atherosclerosis. MR in myeloid cells (My-MR) promotes inflammatory cell infiltration into injured tissues and atherosclerotic plaque inflammation by unclear mechanisms. Here, we examined the role of My-MR in leukocyte trafficking and the impact of sex. Approach and Results: We confirm in vivo that My-MR deletion (My-MR-KO) in ApoE-KO mice decreased plaque size. Flow cytometry revealed fewer plaque macrophages with My-MR-KO. By intravital microscopy, My-MR-KO significantly attenuated monocyte slow-rolling and adhesion to mesenteric vessels and decreased peritoneal infiltration of myeloid cells in response to inflammatory stimuli in male but not female mice. My-MR-KO mice had significantly less PSGL1 (P-selectin glycoprotein ligand 1) mRNA in peritoneal macrophages and surface PSGL1 protein on circulating monocytes in males. In vitro, MR activation with aldosterone significantly increased PSGL1 mRNA only in monocytes from MR-intact males. Similarly, aldosterone induced, and MR antagonist spironolactone inhibited, PSGL1 expression in human U937 monocytes. Mechanistically, aldosterone stimulated MR binding to a predicted MR response element in intron-1 of the PSGL1 gene by ChIP-qPCR. Reporter assays demonstrated that this PSGL1 MR response element is necessary and sufficient for aldosterone-activated, MR-dependent transcriptional activity. Conclusions: These data identify PSGL1 as a My-MR target gene that drives leukocyte trafficking to enhance atherosclerotic plaque inflammation. These novel and sexually dimorphic findings provide insight into increased ischemia risk with MR activation, cardiovascular protection in women, and the role of MR in atherosclerosis and tissue inflammation. Graphic Abstract: A graphic abstract is available for this article.

Purpose Veliparib (V), an oral poly(ADP-ribose) polymerase (PARP) inhibitor, potentiates effects of alkylating agents and topoisomerase inhibitors in preclinical tumor models. We conducted a phase I trial of V with iv cyclophosphamide (C) and V plus iv doxorubicin (A) and C. Methods Objectives were to establish the maximum tolerated dose (MTD) of the combinations, characterize V pharmacokinetics (PK) in the presence and absence of C, measure PAR in peripheral blood mononuclear cells (PBMCs) and γH2AX in circulating tumor cells (CTCs). In Group 1, dose escalations of V from 10 to 50 mg every 12 h Days 1-4 plus C 450 to 750 mg/m2 Day 3 in 21-day cycles were evaluated. In Group 2, V doses ranged from 50 to 150 mg every 12 h Days 1-4 with AC (60/600 mg/m2) Day 3 in 21-day cycles. In Group 3, patients received AC Day 1 plus V Days 1-7, and in Group 4, AC Day 1 plus V Days 1-14 was given in 21-day cycles to evaluate effects on γH2AX foci. Results Eighty patients were enrolled. MTD was not reached for V and C. MTD for V and AC was V 100 mg every 12 h Days 1-4 with AC (60/600 mg/m2) Day 3 every 21 days. V PK appears to be dose-dependent and has no effect on the PK of C. Overall, neutropenia and anemia were the most common adverse events. Objective response in V and AC treated groups was 22% (11/49). Overall clinical benefit rate was 31% (25/80). PAR decreased in PBMCs. Percentage of γH2AX-positive CTCs increased after treatment with V and AC. Conclusion V and AC can be safely combined. Activity was observed in patients with metastatic breast cancer.

We theoretically investigate and optimize the performance of four-wave mixing (FWM) in microring resonators (MRRs) integrated with two-dimensional (2D) layered graphene oxide (GO) films. Owing to the interaction between the MRRs and the highly nonlinear GO films as well as to the resonant enhancement effect, the FWM efficiency in GO-coated MRRs can be significantly improved. Based on previous experiments, we perform detailed analysis for the influence of the GO film parameters and MRR coupling strength on the FWM conversion efficiency (CE) of the hybrid MRRs. By optimizing the device parameters to balance the trade-off between the Kerr nonlinearity and loss, we achieve a high CE enhancement of ∼18.6 dB relative to the uncoated MRR, which is ∼8.3 dB higher than previous experimental results. The influence of photo-thermal changes in the GO films as well as variations in the MRR parameters such as the ring radius and waveguide dispersion on the FWM performance is also discussed. These results highlight the significantly improved FWM performance that can be achieved in MRRs incorporating GO films and provide a guide for optimizing their FWM performance.

According to the 1994 IUBMB-IUPAC Joint Commission on Biochemical Nomenclature (JCBN) on chemical and biochemical reactions, two categories of thermodynamics, based on different concepts and different formalisms, are established: (i) chemical thermodynamics, which employ conventional thermodynamic potentials to deal with chemical reactions [1], [2], [3]; and (ii) biochemical thermodynamics, which employ transformed thermodynamic quantities to deal with biochemical reactions based on the formalism proposed by Alberty [4], [5], [6], [7]. We showed that the two worlds of chemical and biochemical thermodynamics, which so far have been treated separately, can be reunified within the same thermodynamic framework. The thermodynamics of chemical reactions, in which all species are explicitly considered with their atoms and charge balanced, are compared with the transformed thermodynamics generally used to treat biochemical reactions where atoms and charges are not balanced. The transformed thermodynamic quantities suggested by Alberty are obtained by a mathematical transformation of the usual thermodynamic quantities. The present analysis demonstrates that the transformed values for Δr G′0 and Δr H′0 can be obtained directly, without performing any transformation, by simply writing the chemical reactions with all the pseudoisomers explicitly included and the elements and charges balanced. The appropriate procedures for computing the stoichiometric coefficients for the pseudoisomers are fully explained by means of an example calculation for the biochemical ATP hydrolysis reaction. It is concluded that the analysis reunifies the “two separate worlds” of conventional thermodynamics and transformed thermodynamics.

This experiment evaluated the effects of supplemental protease in maize-based diets formulated with reduced density of digestible amino acids (dAA) on net energy (NE) utilisation and nutrient digestibility in broilers. A total of 312, one-day-old, male broilers (Ross 308) were randomly distributed into 24 floor pens and fed one of three treatments, with eight pen replicates per treatment throughout the starter (1 to 14 d of age) and grower (15 to 28 d of age) phases. Dietary treatments consisted of a positive control (PC) reference diet, a negative control diet (NC; dAA density 60 g/kg less than PC diet), and a NC diet with supplemental protease (200 mg/kg). All diets contained supplemental phytase (200 mg/kg) and xylanase (200 mg/kg). The reduction in dAA density between the PC and NC did not affect (P>0.05) NE, but protease supplementation in the NC diet increased (P0.05) affect nitrogen, starch, or DE in the jejunum or ileum. Likewise, no differences (P>0.05) in jejunal (nitrogen, starch, and DE) and ileal (starch and DE) digestibility values were observed between those offered the NC diets without or with protease, but a small difference (P

Background: Cervical cancer is common in women in less developed regions of the world. The plant biomolecules can be employed for synergistic activity with chemo- and radiotherapy. This combinations might result in reduced toxicity and increased efficacy of the treatment regimen. Objectives: The anti-HeLa cells activity of the acetone extracts of S. plumosum, T. cilliata and S. pinnata was assessed using different parameters. Methods: Secondary metabolite detection and antioxidant activity quantification were determined using the DPPH and ferric iron reducing assays. HeLa cell growth inhibition and mechanistics were assessed by employing MTT and Annexin-V flous assays. Results: Observations revealed the presence of phenolic, flavonoids, tannins steroids and coumarins in all the plants ex- tracts. High amount of total phenolic and flavonoid content were detected in S. plumosum and T. cilliata. S. plumosum extract had the best DPPH scavenging activity and ferric reducing powers. Conclusion: Observable concentration dependent cell proliferation inhibition by test materials was exhibited. The leaf ex- tracts from T. cilliata, S. plumosum and S. pinnata contain compounds of various polarities with free-radical, antioxidant and anti-cancerous activities that may play a beneficial role in treatment. Keywords: Medicinal plants; anticancer activity; antioxidant activity.

The Kerr nonlinear optical performance of silicon nanowire waveguides integrated with 2D layered graphene oxide (GO) films is theoretically studied and optimized based on experimentally measured linear and nonlinear optical parameters of the GO films. The strong mode overlap between the silicon nanowires and highly nonlinear GO films yields a significantly enhanced Kerr nonlinearity for the hybrid waveguides. A detailed analysis for the influence of waveguide geometry and GO film thickness on the propagation loss, nonlinear parameter, and nonlinear figure of merit (FOM) is performed. The results show that the effective nonlinear parameter and nonlinear FOM can be increased by up to 52 and 79 times relative to bare silicon nanowires, respectively. Self-phase modulation (SPM)-induced spectral broadening of optical pulses is used as a benchmark to evaluate the nonlinear performance, examining the tradeoff between enhancing Kerr nonlinearity and minimizing loss. By optimizing the device parameters to balance this, a high spectral broadening factor of 27.8 can be achieved, more than 6 times that achieved in previous experiments. Finally, the influence of pulse chirp, material anisotropy, and the interplay between saturable absorption and SPM is also discussed, together with the comparison between the spectral broadening after going through GO-coated and graphene-coated silicon waveguides. These results provide useful guidance for optimizing the Kerr nonlinear optical performance of silicon waveguides integrated with 2D layered GO films.

Caffeine is widely accepted as an endurance-performance enhancing supplement. Most scientific research studies use doses of 3–6 mg/kg of caffeine 60 min prior to exercise based on pharmacokinetics. It is not well understood whether endurance athletes employ similar supplementation strategies in practice. The purpose of this study was to investigate caffeine supplementation protocols among endurance athletes. A survey conducted on Qualtrics returned responses regarding caffeine supplementation from 254 endurance athletes (f = 134, m =120; age = 39.4 ± 13.9 y; pro = 11, current collegiate athlete = 37, recreational = 206; running = 98, triathlon = 83, cycling = 54, other = 19; training days per week = 5.4 ± 1.3). Most participants reported habitual caffeine consumption (85.0%; 41.2% multiple times daily). However, only 24.0% used caffeine supplements. A greater proportion of men (31.7%) used caffeine supplements compared with women (17.2%; p = 0.007). Caffeine use was also more prevalent among professional (45.5%) and recreational athletes (25.1%) than in collegiate athletes (9.4%). Type of sport (p = 0.641), household income (p = 0.263), education (p = 0.570) or working with a coach (p = 0.612) did not have an impact on caffeine supplementation prevalence. Of those reporting specific timing of caffeine supplementation, 49.1% and 34.9% reported consuming caffeine within 30 min of training and races respectively; 38.6 and 36.5% used caffeine 30–60 min before training and races. Recreational athletes reported consuming smaller amounts of caffeine before training (1.6 ± 1.0 mg/kg) and races (2.0 ± 1.2 mg/kg) compared with collegiate (TRG: 2.1 ± 1.2 mg/kg; RACE: 3.6 ± 0.2 mg/kg) and professional (TRG: 2.4 ± 1.1 mg/kg; RACE: 3.5 ± 0.6 mg/kg) athletes. Overall, participants reported minor to moderate perceived effectiveness of caffeine supplementation (2.31 ± 0.9 on a four-point Likert-type scale) with greatest effectiveness during longer sessions (2.8 ± 1.1). It appears that recreational athletes use lower caffeine amounts than what has been established as ergogenic in laboratory protocols; further, they consume caffeine closer to exercise compared with typical research protocols. Thus, better education of recreational athletes and additional research into alternative supplementation strategies are warranted.

E1 and E2 (E1E2), the fusion proteins of Hepatitis C Virus (HCV), are unlike that of any other virus yet described, and the detailed molecular mechanisms of HCV entry/fusion remain unknown. Hypervariable region-1 (HVR-1) of E2 is a putative intrinsically disordered protein tail. Here, we demonstrate that HVR-1 has an autoinhibitory function that suppresses the activity of E1E2 on free virions; this is dependent on its conformational entropy. Thus, HVR-1 is akin to a safety catch that prevents premature triggering of E1E2 activity. Crucially, this mechanism is turned off by host receptor interactions at the cell surface to allow entry. Mutations that reduce conformational entropy in HVR-1, or genetic deletion of HVR-1, turn off the safety catch to generate hyper-reactive HCV that exhibits enhanced virus entry but is thermally unstable and acutely sensitive to neutralising antibodies. Therefore, the HVR-1 safety catch controls the efficiency of virus entry and maintains resistance to neutralising antibodies. This discovery provides an explanation for the ability of HCV to persist in the face of continual immune assault and represents a novel regulatory mechanism that is likely to be found in other viral fusion machinery.

Synthetic materials designed for improved biomimicry of the extracellular matrix must contain fibrous, bioactive, and mechanical cues. Self-assembly of low molecular weight gelator (LMWG) peptides Fmoc-DIKVAV (Fmoc-aspartic acid-isoleucine-lysine-valine-alanine-valine) and Fmoc-FRGDF (Fmoc-phenylalanine-arginine-glycine-aspartic acid-phenylalanine) creates fibrous and bioactive hydrogels. Polysaccharides such as agarose are biocompatible, degradable, and non-toxic. Agarose and these Fmoc-peptides have both demonstrated efficacy in vitro and in vivo. These materials have complementary properties; agarose has known mechanics in the physiological range but is inert and would benefit from bioactive and topographical cues found in the fibrous, protein-rich extracellular matrix. Fmoc-DIKVAV and Fmoc-FRGDF are synthetic self-assembling peptides that present bioactive cues "IKVAV" and "RGD" designed from the ECM proteins laminin and fibronectin. The work presented here demonstrates that the addition of agarose to Fmoc-DIKVAV and Fmoc-FRGDF results in physical characteristics that are dependent on agarose concentration. The networks are peptide-dominated at low agarose concentrations, and agarose-dominated at high agarose concentrations, resulting in distinct changes in structural morphology. Interestingly, at mid-range agarose concentration, a hybrid network is formed with structural similarities to both peptide and agarose systems, demonstrating reinforced mechanical properties. Bioactive-LMWG polysaccharide hydrogels demonstrate controllable microenvironmental properties, providing the ability for tissue-specific biomaterial design for tissue engineering and 3D cell culture.

Chicken ovalbumin (cOVA) has been studied for decades primarily due to the robust genetic and molecular resources that are available for experimental investigations. cOVA is a member of the serpin superfamily of proteins that function as protease inhibitors, although cOVA does not exhibit this activity. As a serpin, cOVA possesses a protease-sensitive reactive center loop that lies adjacent to the OVA 323–339 CD4+ T-cell epitope. We took advantage of the previously described single-substitution variant, OVA R339T, which can undergo the dramatic structural transition observed in serpins, to study how changes in loop size and protein stability influence the processing and presentation of the OVA 323–339 epitope. We observed that the OVA R339T loop insertion increases the stability and protease resistance, resulting in the reduced presentation of the OVA 323–339 epitope in vitro. These findings have implications for the design of more effective vaccines for the treatment of infectious diseases and cancer as well as the development of more robust CD4+ T-cell epitope prediction tools.

We theoretically investigate and optimize four-wave mixing (FWM) in silicon nitride (SiN) waveguides integrated with two-dimensional (2D) layered graphene oxide (GO) films. Based on extensive previous measurements of the material parameters of the GO films, we perform detailed analysis for the influence of device parameters including waveguide geometry, GO film thickness, length, and coating position on the FWM conversion efficiency (CE) and conversion bandwidth (CB). The influence of dispersion and photo-thermal changes in the GO films is also discussed. Owing to the strong mode overlap between the SiN waveguides and the highly nonlinear GO films, FWM in the hybrid waveguides can be significantly enhanced. We obtain good agreement with previous experimental results and show that by optimizing the device parameters to balance the trade-off between Kerr nonlinearity and loss, the FWM CE can be improved by as much as ~20.7 dB and the FWM CB can be increased by ~4.4 folds, relative to the uncoated waveguides. These results highlight the significantly enhanced FWM performance that can be achieved in SiN waveguides by integrating 2D layered GO films.

Detailed knowledge of the ultrastructure of intracellular compartments is a prerequisite for our understanding of how cells function. In cardiac muscle cells, close apposition of transverse (t)-tubule (TT) and sarcoplasmic reticulum (SR) membranes supports stable high-gain excitation-contraction coupling. Here, the fine structure of this key intracellular element is examined in rabbit and mouse ventricular cardiomyocytes, using ultra-rapid high-pressure freezing (HPF, omitting aldehyde fixation) and electron microscopy. 3D electron tomograms were used to quantify the dimensions of TT, terminal cisternae of the SR, and the space between SR and TT membranes (dyadic cleft). In comparison to conventional aldehyde-based chemical sample fixation, HPF-preserved samples of both species show considerably more voluminous SR terminal cisternae, both in absolute dimensions and in terms of junctional SR to TT volume ratio. In rabbit cardiomyocytes, the average dyadic cleft surface area of HPF and chemically fixed myocytes did not differ, but cleft volume was significantly smaller in HPF samples than in conventionally fixed tissue; in murine cardiomyocytes, the dyadic cleft surface area was higher in HPF samples with no difference in cleft volume. In both species, the apposition of the TT and SR membranes in the dyad was more likely to be closer than 10 nm in HPF samples compared to CFD, presumably resulting from avoidance of sample shrinkage associated with conventional fixation techniques. Overall, we provide a note of caution regarding quantitative interpretation of chemically-fixed ultrastructures, and offer novel insight into cardiac TT and SR ultrastructure with relevance for our understanding of cardiac physiology., Graphical abstract Unlabelled Image, Highlights • The cardiac dyadic cleft, where T-tubules and sarcoplasmic reticulum are closely opposed, is key to cardiac excitation-contraction coupling. • Most knowledge about the ultrastructure of the dyad comes from electron microscopic studies, traditionally involving chemical fixation. • By using ultra-fast vitrification, we demonstrate that the dyadic cleft has dramatic qualitative and quantitative differences in morphology.

Background: ANDHI was done to assess the efficacy of benralizumab, including onset of effect and impact on health-related quality of life (HRQOL), exacerbation rate, lung function, and nasal polyposis symptoms. Methods: This phase 3b, randomised, double-blind, parallel-group, placebo-controlled ANDHI study was completed in adults (aged 18–75 years) with severe eosinophilic asthma with at least 2 exacerbations in the previous year, despite high-dose inhaled corticosteroid plus additional controllers, screening blood eosinophil counts of at least 150 cells per μL, and an Asthma Control Questionnaire 6 (ACQ-6) score of 1·5 or more. Patients who met eligibility criteria were randomly assigned (2:1; stratified by previous exacerbation count [two, or three or more], maintenance oral corticosteroid use, and region), using an integrated web-based response system, to receive benralizumab at 30 mg every 8 weeks (first three doses given 4 weeks apart) or matched placebo for 24 weeks. Primary efficacy measure was annualised asthma exacerbation rate, with rate ratio (RR) calculated over the approximate 24-week follow-up. Secondary efficacy measures included change from baseline to end of treatment (week 24) in St George's Respiratory Questionnaire (SGRQ) total score (key secondary endpoint), FEV1, peak expiratory flow (PEF), ACQ-6, Predominant Symptom and Impairment Assessment (PSIA), Clinician Global Impression of Change (CGI-C), Patient Global Impression of Change (PGI-C), and Sino-Nasal Outcome Test-22 (SNOT-22). All efficacy analyses, except for SNOT-22, were summarised and analysed using the full analysis set on an intention-to-treat population (all randomly assigned patients receiving investigational product, regardless of protocol adherence or continued participation in the study). SNOT-22 was summarised for the subgroup of patients with physician-diagnosed nasal polyposis with informed consent. This study is registered with ClinicalTrials.gov, NCT03170271. Findings: Between July 7, 2017, and Sept 25, 2019, 656 patients received benralizumab (n=427) or placebo (n=229). Baseline characteristics were consistent with severe eosinophilic asthma. Benralizumab significantly reduced exacerbation risk by 49% compared with placebo (RR estimate 0·51, 95% CI 0·39–0·65; p5%) were nasopharyngitis (30 [7%]), headache (37 [9%]), sinusitis (28 [7%]), bronchitis (22 [5%]), and pyrexia (26 [6%]). Fewer serious adverse events were reported for benralizumab (23 [5%]) versus placebo (25 [11%]), and the only common serious adverse event (experienced by >1% of patients) was worsening of asthma, which was reported for nine (2%) patients in the benralizumab group and nine (4%) patients in the placebo group. Interpretation: Our results extend the efficacy profile of benralizumab for patients with severe eosinophilic asthma, showing early clinical benefits in patient-reported outcomes, HRQOL, lung function, and nasal polyposis symptoms. Funding: AstraZeneca.