Receptor binding and immunogenic properties of the receptor binding domain of influenza D virus hemagglutinin-esterase-fusion protein expressed from Escherichia coli.

The hemagglutinin-esterase-fusion (HEF) protein binds 9-O-acetylated sialic acids-containing glycans on the cell surface and drives influenza D virus (IDV) entry. The HEF is a primary determinant of the exceptional thermal and acid stability observed in IDV infection biology. Here, we expressed and purified the receptor binding domain (RBD) of the IDV HEF protein in Escherichia coli and characterized its receptor binding and antigenic properties. The data from these experiments indicate that (i) the RBD can bind with specificity to turkey red blood cells (RBC), and its binding can be specifically inhibited by IDV antibody; (ii) the RBD efficiently binds to the cell surface of MDCK cells expressing the receptor of IDV; and (iii) anti-RBD antibodies are capable of blocking RBD attachment to MDCK cells as well as of inhibiting the virus from agglutinating RBCs. These observations support the utility of this RBD in future receptor and entry studies of IDV.

Statistical considerations and database limitations in NMR-based metabolic profiling studies.

INTRODUCTION: Interpretation and analysis of NMR-based metabolic profiling studies is limited by substantially incomplete commercial and academic databases. Statistical significance tests, including p-values, VIP scores, AUC values and FC values, can be largely inconsistent. Data normalization prior to statistical analysis can cause erroneous outcomes. OBJECTIVES: The objectives were (1) to quantitatively assess consistency among p-values, VIP scores, AUC values and FC values in representative NMR-based metabolic profiling datasets, (2) to assess how data normalization can impact statistical significance outcomes, (3) to determine resonance peak assignment completion potential using commonly used databases and (4) to analyze intersection and uniqueness of metabolite space in these databases. METHODS: P-values, VIP scores, AUC values and FC values, and their dependence on data normalization, were determined in orthotopic mouse model of pancreatic cancer and two human pancreatic cancer cell lines. Completeness of resonance assignments were evaluated using Chenomx, the human metabolite database (HMDB) and the COLMAR database. The intersection and uniqueness of the databases was quantified. RESULTS: P-values and AUC values were strongly correlated compared to VIP or FC values. Distributions of statistically significant bins depended strongly on whether or not datasets were normalized. 40-45% of peaks had either no or ambiguous database matches. 9-22% of metabolites were unique to each database. CONCLUSIONS: Lack of consistency in statistical analyses of metabolomics data can lead to misleading or inconsistent interpretation. Data normalization can have large effects on statistical analysis and should be justified. About 40% of peak assignments remain ambiguous or impossible with current databases. 1D and 2D databases should be made consistent to maximize metabolite assignment confidence and validation.

Safety and efficacy of autologous cell vaccines in solid tumors: a systematic review and meta-analysis of randomized control trials.

We conducted a systematic review and meta-analysis of randomized control trials to formally assess the safety and efficacy of autologous whole cell vaccines as immunotherapies for solid tumors. Our primary safety outcome was number, and grade of adverse events. Our primary efficacy outcome was clinical responses. Secondary outcomes included survival metrics and correlative immune assays. We searched MEDLINE, Embase, and the Cochrane Central Register of Controlled Trials for studies published between 1946 and August 2020 using any autologous whole cell product in the treatment of any solid tumor. The Cochrane Randomized Controlled Trial risk of bias tool was used to assess risk of bias. Eighteen manuscripts were identified with a total of 714 patients enrolled in control and 808 in vaccine arms. In 698 patients receiving at least one dose of vaccine, treatment was well tolerated with a total of 5 grade III or higher adverse events. Clinical response was reported in a minority (n = 2, 14%) of studies. Autologous cell vaccines were associated with improved overall (HR 1.28, 95% CI 1.01-1.63) and disease-free survival (HR 1.33, 95% CI 1.05-1.67) over thirteen and ten trials respectively. Where reported, immune assays correlated well with clinical outcomes. Our results suggest that autologous whole cell vaccination is safe and efficacious in increasing survival in patients undergoing treatment for solid tumors.Registration: PROSPERO CRD42019140187.

Preventing Surgery-Induced NK Cell Dysfunction Using Anti-TGF-ß Immunotherapeutics.

Natural Killer (NK) cell cytotoxicity and interferon-gamma (IFNγ) production are profoundly suppressed postoperatively. This dysfunction is associated with increased morbidity and cancer recurrence. NK activity depends on the integration of activating and inhibitory signals, which may be modulated by transforming growth factor-beta (TGF-ß). We hypothesized that impaired postoperative NK cell IFNγ production is due to altered signaling pathways caused by postoperative TGF-ß. NK cell receptor expression, downstream phosphorylated targets, and IFNγ production were assessed using peripheral blood mononuclear cells (PBMCs) from patients undergoing cancer surgery. Healthy NK cells were incubated in the presence of healthy/baseline/postoperative day (POD) 1 plasma and in the presence/absence of a TGF-ß-blocking monoclonal antibody (mAb) or the small molecule inhibitor (smi) SB525334. Single-cell RNA sequencing (scRNA-seq) was performed on PBMCs from six patients with colorectal cancer having surgery at baseline/on POD1. Intracellular IFNγ, activating receptors (CD132, CD212, NKG2D, DNAM-1), and downstream target (STAT5, STAT4, p38 MAPK, S6) phosphorylation were significantly reduced on POD1. Furthermore, this dysfunction was phenocopied in healthy NK cells through incubation with rTGF-ß1 or POD1 plasma and was prevented by the addition of anti-TGF-ß immunotherapeutics (anti-TGF-ß mAb or TGF-ßR smi). Targeted gene analysis revealed significant decreases in S6 and FKBP12, an increase in Shp-2, and a reduction in NK metabolism-associated transcripts on POD1. pSmad2/3 was increased and pS6 was reduced in response to rTGF-ß1 on POD1, changes that were prevented by anti-TGF-ß immunotherapeutics. Together, these results suggest that both canonical and mTOR pathways downstream of TGF-ß mediate phenotypic changes that result in postoperative NK cell dysfunction.

A Translational Randomized Trial of Perioperative Arginine Immunonutrition on Natural Killer Cell Function in Colorectal Cancer Surgery Patients.

BACKGROUND: Surgery results in severe impairment of natural killer (NK) cell cytotoxicity (NKC) and activity (NKA, cytokine secretion), and a dramatic drop in arginine levels. Postoperative immunosuppression is associated with increased complications and recurrence. Perioperative arginine is reported to reduce postoperative complications. Because arginine modulates NK cell function, this study aimed to determine whether perioperative consumption of arginine-enriched supplements (AES) can improve NK cell function in colorectal cancer (CRC) surgery patients. METHODS: This study randomized 24 CRC patients to receive the AES or isocaloric/isonitrogenous control supplement three times a day for five days before and after surgery. The AES contained 4.2 g of arginine per dose (12.6 g/day). The primary objective was to determine whether AES improved NKC by 50 % compared with the control group after surgery. RESULTS: On surgery day (SD) 1, NKC was significantly reduced postoperatively in the control group by 50 % (interquartile range [IQR], 36-55 %; p = 0.02) but not in the AES group (25 % reduction; IQR, 28-75 %; p = 0.3). Furthermore, AES had no benefit in terms of NKA or NK cell number. Compliance was much greater preoperatively (>91 %) than postoperatively (<46 %). However, despite excellent preoperative compliance, arginine was rapidly cleared from the blood within 4 h after consumption and therefore, did not prevent the postoperative drop in arginine. CONCLUSIONS: Oral consumption of arginine immunonutrition resulted in a modest improvement in NKC after surgery but was unable to prevent postoperative arginine depletion or the suppression of NKA (ClinicalTrials.gov NCT02987296).

Perioperative arginine prevents metastases by accelerating natural killer cell recovery after surgery.

Profound natural killer (NK) cell suppression after cancer surgery is a main driver of metastases and recurrence, for which there is no clinically approved intervention available. Surgical stress is known to cause systemic postoperative changes that negatively modulate NK cell function including the expansion of surgery-induced myeloid-derived suppressor cells (Sx-MDSCs) and a marked reduction in arginine bioavailability. In this study, we determine that Sx-MDSCs regulate systemic arginine levels in the postoperative period and that restoring arginine imbalance after surgery by dietary intake alone was sufficient to significantly reduce surgery-induced metastases in our preclinical murine models. Importantly, the effects of perioperative arginine were dependent upon NK cells. Although perioperative arginine did not prevent immediate NK cell immunoparalysis after surgery, it did accelerate their return to preoperative cytotoxicity, interferon gamma secretion, and activating receptor expression. Finally, in a cohort of patients with colorectal cancer, postoperative arginine levels were shown to correlate with their Sx-MDSC levels. Therefore, this study lends further support for the use of perioperative arginine supplementation by improving NK cell recovery after surgery.

Current challenges in the manufacture of clinical-grade autologous whole cell vaccines for hematological malignancies.

Autologous whole cell vaccines use a patient's own tumor cells as a source of antigen to elicit an anti-tumor immune response in vivo. Recently, the authors conducted a systematic review of clinical trials employing these products in hematological cancers that showed a favorable safety profile and trend toward efficacy. However, it was noted that manufacturing challenges limit both the efficacy and clinical implementation of these vaccine products. In the current literature review, the authors sought to define the issues surrounding the manufacture of autologous whole cell products for hematological cancers. The authors describe key factors, including the acquisition, culture, cryopreservation and transduction of malignant cells, that require optimization for further advancement of the field. Furthermore, the authors provide a summary of pre-clinical work that informs how the identified challenges may be overcome. The authors also highlight areas in which future basic research would be of benefit to the field. The goal of this review is to provide a roadmap for investigators seeking to advance the field of autologous cell vaccines as it applies to hematological malignancies.

Safety and efficacy of autologous whole cell vaccines in hematologic malignancies: A systematic review and meta-analysis.

Autologous cell vaccines use a patient's tumor cells to stimulate a broad antitumor response in vivo. This approach shows promise for treating hematologic cancers in early phase clinical trials, but overall safety and efficacy remain poorly described. We conducted a systematic review assessing the use of autologous cell vaccination in treating hematologic cancers. Primary outcomes of interest were safety and clinical response, with secondary outcomes including survival, relapse rate, correlative immune assays and health-quality related metrics. We performed a search of MEDLINE, Embase and the Cochrane Register of Controlled Trials including any interventional trial employing an autologous, whole cell product in any hematologic malignancy. Risk of bias was assessed using a modified Institute of Health Economics tool. Across 20 single arm studies, only 341 of 592 enrolled participants received one or more vaccinations. Primary reasons for not receiving vaccination included rapid disease progression/death and manufacturing challenges. Overall, few high-grade adverse events were observed. One death was reported and attributed to a GM-CSF producing allogeneic cell line co-administered with the autologous vaccine. Of 58 evaluable patients, the complete response rate was 21.0% [95% CI, 10.4%-37.8%)] and overall response rate was 35.8% (95% CI, 24.4%-49.0%). Of 97 evaluable patients for survival, the 5-years overall survival rate was 64.9% (95% CI, 52.6%-77.2%) and disease-free survival was 59.7% (95% CI, 47.7%-71.7%). We conclude that, in hematologic malignancies, based on limited available data, autologous cell vaccines are safe and display a trend towards efficacy but that challenges exist in vaccine manufacture and administration.

Current Understanding of the Structure and Function of Pentapeptide Repeat Proteins.

The pentapeptide repeat protein (PRP) superfamily, identified in 1998, has grown to nearly 39,000 sequences from over 3300 species. PRPs, recognized as having at least eight contiguous pentapeptide repeats (PRs) of a consensus pentapeptide sequence, adopt a remarkable structure, namely, a right-handed quadrilateral ß-helix with four consecutive PRs forming a single ß-helix coil. Adjacent coils join together to form a ß-helix "tower" stabilized by ß-ladders on the tower faces and type I, type II, or type IV ß-turns facilitating an approximately -90° redirection of the polypeptide chain joining one coil face to the next. PRPs have been found in all branches of life, but they are predominantly found in cyanobacteria. Cyanobacteria have existed on earth for more than two billion years and are thought to be responsible for oxygenation of the earth's atmosphere. Filamentous cyanobacteria such as Nostoc sp. strain PCC 7120 may also represent the oldest and simplest multicellular organisms known to undergo cell differentiation on earth. Knowledge of the biochemical function of these PRPs is essential to understanding how ancient cyanobacteria achieved functions critical to early development of life on earth. PRPs are predicted to exist in all cyanobacteria compartments including thylakoid and cell-wall membranes, cytoplasm, and thylakoid periplasmic space. Despite their intriguing structure and importance to understanding ancient cyanobacteria, the biochemical functions of PRPs in cyanobacteria remain almost completely unknown. The precise biochemical function of only a handful of PRPs is currently known from any organisms, and three-dimensional structures of only sixteen PRPs or PRP-containing multidomain proteins from any organism have been reported. In this review, the current knowledge of the structures and functions of PRPs is presented and discussed.

Adjuvant melatonin for the prevention of recurrence and mortality following lung cancer resection (AMPLCaRe): A randomized placebo controlled clinical trial.

BACKGROUND: Despite curative intent resection in patients with non-small cell lung cancer (NSCLC), recurrence leading to mortality remains too common. Melatonin has shown promise for the treatment of patients with lung cancer; however, its effect following cancer resection has not been studied. We evaluated if melatonin taken after complete resection reduces lung cancer recurrence and mortality, or impacts quality of life (QOL), symptomatology or immune function. METHODS: Participants received melatonin (20 mg) or placebo nightly for one year following surgical resection of primary NSCLC. The primary outcome was two-year disease-free survival (DFS). Secondary outcomes included five-year DFS, adverse events, QOL, fatigue, sleep, depression, anxiety, pain, and biomarkers assessing for immune function/inflammation. This study is registered at https://clinicaltrials.gov NCT00668707. FINDINGS: 709 patients across eight centres were randomized to melatonin (n = 356) versus placebo (n = 353). At two years, melatonin showed a relative risk of 1·01 (95% CI 0·83-1·22), p = 0·94 for DFS. At five years, melatonin showed a hazard ratio of 0·97 (95% CI 0·86-1·09), p = 0·84 for DFS. When stratified by cancer stage (I/II and III/IV), a hazard reduction of 25% (HR 0·75, 95% CI 0·61-0·92, p = 0·005) in five-year DFS was seen for participants in the treatment arm with advanced cancer (stage III/IV). No meaningful differences were seen in any other outcomes. INTERPRETATION: Adjuvant melatonin following resection of NSCLC does not affect DFS for patients with resected early stage NSCLC, yet may increase DFS in patients with late stage disease. Further study is needed to confirm this positive result. No beneficial effects were seen in QOL, symptoms, or immune function. FUNDING: This study was funded by the Lotte and John Hecht Memorial Foundation and the Gateway for Cancer Research Foundation.

A Method of Assessment of Human Natural Killer Cell Phenotype and Function in Whole Blood.

The majority of data on human Natural Killer (NK) cell phenotype and function has been generated using cryopreserved peripheral blood mononuclear cells (PBMCs). However, cryopreservation can have adverse effects on PBMCs. In contrast, investigating immune cells in whole blood can reduce the time, volume of blood required, and potential artefacts associated with manipulation of the cells. Whole blood collected from healthy donors and cancer patients was processed by three separate protocols that can be used independently or in parallel to assess extracellular receptors, intracellular signaling protein phosphorylation, and intracellular and extracellular cytokine production in human NK cells. To assess extracellular receptor expression, 200 µL of whole blood was incubated with an extracellular staining (ECS) mix and cells were subsequently fixed and RBCs lysed prior to analysis. The phosphorylation status of signaling proteins was assessed in 500 µL of whole blood following co-incubation with interleukin (IL)-2/12 and an ECS mix for 20 min prior to cell fixation, RBC lysis, and subsequent permeabilization for staining with an intracellular staining (ICS) mix. Cytokine production (IFNγ) was similarly assessed by incubating 1 mL of whole blood with PMA-ionomycin or IL-2/12 prior to incubation with ECS and subsequent ICS antibodies. In addition, plasma was collected from stimulated samples prior to ECS for quantification of secreted IFNγ by ELISA. Results were consistent, despite inherent inter-patient variability. Although we did not investigate an exhaustive list of targets, this approach enabled quantification of representative ECS surface markers including activating (NKG2D and DNAM-1) and inhibitory (NKG2A, PD-1, TIGIT, and TIM-3) receptors, cytokine receptors (CD25, CD122, CD132, and CD212) and ICS markers associated with NK cell activation following stimulation, including signaling protein phosphorylation (p-STAT4, p-STAT5, p-p38 MAPK, p-S6) and IFNγ in both healthy donors and cancer patients. In addition, we compared extracellular receptor expression using whole blood vs. cryopreserved PBMCs and observed a significant difference in the expression of almost all receptors. The methods presented permit a relatively rapid parallel assessment of immune cell receptor expression, signaling protein activity, and cytokine production in a minimal volume of whole blood from both healthy donors and cancer patients.

Safety and efficacy of autologous tumour cell vaccines as a cancer therapeutic to treat solid tumours and haematological malignancies: a meta-analysis protocol for two systematic reviews.

INTRODUCTION: Autologous cancer cell vaccines are promising personalised immunotherapeutic options for solid and haematological malignancies that uses the patient's own cells to arm an immune response. Evidence suggests that among patients receiving these vaccines, those who mount an immune response against their own tumour cells have better prognosis, and a myriad of preclinical studies have demonstrated the same. Recently, two autologous cell vaccines Vigil and OncoVAX have made it to phase III clinical trials. Here, we outline a protocol to be used for two separate systematic reviews using a parallel approach for inclusion criteria, data extraction and analysis for autologous cell vaccines in (1) solid and (2) haematological malignancies. We aim to review evidence from controlled and uncontrolled interventional studies of autologous cell vaccines administered to patients with cancer to determine their historical efficacy (with or without associated adjuvants or modifications) with clinical response rates and safety outcomes being of particular importance. METHODS AND ANALYSIS: We will search MEDLINE (OVID interface, including In-Process and Epub Ahead of Print), Embase (OVID interface) and the Cochrane Central Register of Controlled Trials (Wiley interface) for articles published from 1947 until 30 July 2018 (date search was performed). Studies will be screened first by title and abstract, then by full-text in duplicate. Interventional trials that report the use of an autologous cell vaccine to patients with cancer of any age will be included. The primary outcomes of interest in this review are clinical response (complete or overall/objective response) and safety outcomes (adverse events). Secondary outcomes include immune response, disease-free survival and overall survival. The risk of bias within studies will be assessed using the appropriate Cochrane Risk of Bias tool. If appropriate, a random effects meta-analysis will be performed to synthesise the data and report summary estimates of effect. Statistical heterogeneity will be assessed using the I2 statistic. ETHICS AND DISSEMINATION: Ethics approval is not required for this systematic review protocol as the review will solely use published literature. Results will be submitted to peer-reviewed journals for publication and presented to relevant stakeholders and scientific meetings. PROSPERO REGISTRATION NUMBER: CRD42019140187.

Structural dynamics of pentapeptide repeat proteins.

Pentapeptide repeat proteins (PRPs) represent a large superfamily with more than 38 000 sequences in nearly 3500 species, the majority belonging to cyanobacteria but represented among all branches of life. PRPs contain at least eight consecutive pentapeptide repeats with the consensus (A/C/S/V/T/L/I)(D/N/S/K/E/I/R)(L/F)(S/T/R/E/Q/K/V/D)(G/D/E/N/R/Q/K). PRPs fold into right-handed quadrilateral ß helices, also known as repeat-five-residue (Rfr)-folds, with four consecutive pentapeptide repeats comprising a single coil, the ~90° change in polypeptide direction in square-shaped coils achieved by type I, II and IV ß turns, and hydrogen bonds between coils establishing ß ladders on each Rfr-fold face. PRPs are broadly categorized into group 1 and 2 involved in antibiotic resistance and group 3 currently having unknown functions. Motivated by their intriguing structures, we are investigating PRP biophysical characteristics, including Rfr-fold thermal stability, ß turn and ß ladder hydrogen bond amide exchange rates and backbone dynamics. Here, we present analysis of 20 ns molecular dynamics (MD) simulations and all atom normal mode analysis (aaNMA) calculations for four group 1 and group 2 and four group 3 PRPs whose structures have been determined by X-ray crystallography. The MD cross-correlation matrices and aaNMA indicated strong correlated motion between adjacent coils and weak coupled motion between coils separated by one or more intervening coils. Slow anticorrelated motions were detected between adjacent coils in aaNMA modes that we hypothesize are requisite to access exchange-competent states necessary to permit solvent exchange of amide hydrogens involved in ß-ladder and ß-turns hydrogen bonds, which can have lifetimes on the order of months.

Molecular basis of P[II] major human rotavirus VP8* domain recognition of histo-blood group antigens.

Initial cell attachment of rotavirus (RV) to specific cell surface glycan receptors, which is the essential first step in RV infection, is mediated by the VP8* domain of the spike protein VP4. Recently, human histo-blood group antigens (HBGAs) have been identified as receptors or attachment factors for human RV strains. RV strains in the P[4] and P[8] genotypes of the P[II] genogroup share common recognition of the Lewis b (Leb) and H type 1 antigens, however, the molecular basis of receptor recognition by the major human P[8] RVs remains unknown due to lack of experimental structural information. Here, we used nuclear magnetic resonance (NMR) spectroscopy-based titration experiments and NMR-derived high ambiguity driven docking (HADDOCK) methods to elucidate the molecular basis for P[8] VP8* recognition of the Leb (LNDFH I) and type 1 HBGAs. We also used X-ray crystallography to determine the molecular details underlying P[6] recognition of H type 1 HBGAs. Unlike P[6]/P[19] VP8*s that recognize H type 1 HBGAs in a binding surface composed of an α-helix and a ß-sheet, referred as the "ßα binding site", the P[8] and P[4] VP8*s bind Leb HBGAs in a previously undescribed pocket formed by the edges of two ß-sheets, referred to as the "ßß binding site". Importantly, the P[8] and P[4] VP8*s retain binding capability to non-Leb type 1 HBGAs using the ßα binding site. The presence of two distinct binding sites for Leb and non-Leb HBGA glycans in the P[8] and P[4] VP8* domains suggests host-pathogen co-evolution under structural and functional adaptation of RV pathogens to host glycan polymorphisms. Assessment and understanding of the precise impact of this co-evolutionary process in determining RV host ranges and cross-species RV transmission should facilitate improved RV vaccine development and prediction of future RV strain emergence and epidemics.

Influence of Drying Method on NMR-Based Metabolic Profiling of Human Cell Lines.

Metabolic profiling of cell line and tissue extracts involves sample processing that includes a drying step prior to re-dissolving the cell or tissue extracts in a buffer for analysis by GC/LC-MS or NMR. Two of the most commonly used drying techniques are centrifugal evaporation under vacuum (SpeedVac) and lyophilization. Here, NMR spectroscopy was used to determine how the metabolic profiles of hydrophilic extracts of three human pancreatic cancer cell lines, MiaPaCa-2, Panc-1 and AsPC-1, were influenced by the choice of drying technique. In each of the three cell lines, 40-50 metabolites were identified as having statistically significant differences in abundance in redissolved extract samples depending on the drying technique used during sample preparation. In addition to these differences, some metabolites were only present in the lyophilized samples, for example, n-methyl-α-aminoisobutyric acid, n-methylnicotimamide, sarcosine and 3-hydroxyisovaleric acid, whereas some metabolites were only present in SpeedVac dried samples, for example, trimethylamine. This research demonstrates that the choice of drying technique used during the preparation of samples of human cell lines or tissue extracts can significantly influence the observed metabolome, making it important to carefully consider the selection of a drying method prior to preparation of such samples for metabolic profiling.

Solution NMR structure and ligand identification of human Gas7 SH3 domain reveal a typical SH3 fold but a non-canonical ligand-binding mode.

Growth arrest specific 7 (Gas7) protein is a cytoskeleton regulator playing a crucial role in neural cell development and function, and has been implicated in Alzheimer disease, schizophrenia and cancers. In human, three Gas7 isoforms can be expressed from a single Gas7 gene, while only the longest isoform, hGas7c, possesses an SH3 domain at the N-terminus. To date, the structure and function of hGas7 SH3 domain are still unclear. Here, we reported the solution NMR structure of hGas7 SH3 domain (hGas7-SH3), which displays a typical SH3 ß-barrel fold comprising five ß-strands and one 310-helix. Structural and sequence comparison showed that hGas7-SH3 shares high similarity with Abl SH3 domain, which binds to a high-affinity proline-rich peptide P41 in a canonical SH3-ligand binding mode through two hydrophobic pockets and a specificity site in the RT-loop. However, unlike Abl-SH3, only six residues in the RT-loop and two residues adjacent to but not in the two hydrophobic pockets of hGas7-SH3 showed significant chemical shift perturbations in NMR titrations, suggesting a low affinity and a non-canonical binding mode of hGas7-SH3 for P41. Furthermore, four peptides selected from phage-displayed libraries also bound weakly to hGas7-SH3, and the binding region of hGas7-SH3 was mainly located in the RT-loop as well. The ligand identifications through structural similarity searching and peptide library screening in this study imply that although hGas7-SH3 adopts a typical SH3 fold, it probably possesses distinctive ligand-binding specificity.

Evaluation of the Antibacterial Activity of 75 Mushrooms Collected in the Vicinity of Oxford, Ohio (USA).

Antibiotic-resistant bacteria are an increasing and serious health concern worldwide, and multidrug-resistant pathogens are increasingly emerging among patients across the United States. Researchers are exploring sources of traditional medicines, including mushrooms, to find new antibiotic compounds. In this study, the antibiotic activities of 75 mushrooms collected in the area surrounding Oxford, Ohio (USA), were assayed for antibiotic activity against 6 bacterial strains (Pseudomonas aeruginosa reference strains PAO1 and PA14, P. fluorescens, Bacillus subtilis, Staphylococcus epidermidis, and Micrococcus luteus). Mushroom samples were identified by using DNA ribotyping. We used methanol and water extracts of mushrooms in agar diffusion assays to screen for antibiotic activity toward each bacterial strain. A total of 25 mushroom species had antibacterial activity against at least 1 bacterium. Water extracts of Polyporus squamosus, Ganoderma applanatum, Lentinellus subaustralis, Laetiporus sulphureus, G. lucidum, and Trametes versicolor exhibited strong antibiotic activity against all bacterial strains tested. Water and methanol extracts from 25 mushroom species had significant activity against most of the bacteria tested. A minimum inhibitory concentration (MIC) against S. epidermidis was determined for all samples that exhibited antibiotic activity in the disk assay. The G. lucidum and L. sulphureus extracts displayed the strongest inhibition, with an MIC of 0.1 mg/mL.

Type I beta turns make a new twist in pentapeptide repeat proteins: Crystal structure of Alr5209 from Nostoc sp. PCC 7120 determined at 1.7 angström resolution.

Pentapeptide repeat proteins (PRPs) are found abundantly in cyanobacteria, numbering in the dozens in some genomes, e.g. in Nostoc sp. PCC 7120. PRPs, comprised of a repeating consensus sequence of five amino acids, adopt a distinctive right-handed quadrilateral ß-helical structure, also referred to as a repeat five residue (Rfr) fold, made up of stacks of coils formed by four consecutive pentapeptide repeats. The right-handed quadrilateral ß-helical PRP structure is constructed by repeating ß turns at each of four corners in a given coil, each causing a 90° change in direction of the polypeptide chain. Until now, all PRP structures have consisted either of type II and IV ß turns or exclusively of type II ß turns. Here, we report the first structure of a PRP comprised of type I and II ß turns, Alr5209 from Nostoc sp. PCC 7120. The alr5209 gene encodes 129 amino acids containing 16 tandem pentapeptide repeats. The Alr5209 structure was analyzed in comparison to all other PRPs to determine how type I ß turns can be accommodated in Rfr folds and the consequences of type I ß turns on the right-handed quadrilateral ß-helical structure. Given that Alr5209 represents the first PRP structure containing type I ß turns, the PRP consensus sequence was reevaluated and updated. Despite a growing number of PRP structural investigations, their function remains largely unknown. Genome analysis indicated that alr5209 resides in a five-gene operon (alr5208-alr5212) with Alr5211 annotated to be a NADH dehydrogenase indicating Alr5209 may be involved in oxidative phosphorylation.

The Potential for Cancer Immunotherapy in Targeting Surgery-Induced Natural Killer Cell Dysfunction.

Natural Killer (NK) cells are granular lymphocytes of the innate immune system that are able to recognize and kill tumor cells without undergoing clonal selection. Discovered over 40 years ago, they have since been recognized to possess both cytotoxic and cytokine-producing effector functions. Following trauma, NK cells are suppressed and their effector functions are impaired. This is especially important for cancer patients undergoing the removal of solid tumors, as surgery has shown to contribute to the development of metastasis and cancer recurrence postoperatively. We have recently shown that NK cells are critical mediators in the formation of metastasis after surgery. While research into the mechanism(s) responsible for NK cell dysfunction is ongoing, knowledge of these mechanisms will pave the way for perioperative therapeutics with the potential to improve cancer outcomes by reversing NK cell dysfunction. This review will discuss mechanisms of suppression in the postoperative environment, including hypercoagulability, suppressive soluble factors, the expansion of suppressive cell populations, and how this affects NK cell biology, including modulation of cell surface receptors, the potential for anergy, and immunosuppressive NK cell functions. This review will also outline potential immunotherapies to reverse postoperative NK dysfunction, with the goal of preventing surgery-induced metastasis.