Cancer surgery can promote tumour metastases and worsen prognosis, however, the effect of perioperative complications on metastatic disease remains unclear. In this study we sought to evaluate the effect of common perioperative complications including perioperative blood loss, hypothermia, and sepsis on tumour metastases in a murine model.

We have determined the solution NMR structure of the intermembrane space domain (IMSD) of the human mitochondrial ATPase associated with various activities (AAA) protease known as AFG3-like protein 2 (AFG3L2). Our structural analysis and molecular dynamics results indicate that the IMSD is peripherally bound to the membrane surface. This is a modification to the location of the six IMSDs in a model of the full length yeast hexaoligomeric homolog of AFG3L2 determined at low resolution by electron cryomicroscopy [1]. The predicted protein-protein interaction surface, located on the side furthest from the membrane, may mediate binding to substrates as well as prohibitins.

Genetic and functional studies showed that some components of the Acinetobacter baumannii ATCC 17978 A1S_0112-A1S_0119 gene cluster are critical for biofilm biogenesis and surface motility. Recently, our group has shown that the A1S_0114 gene was involved in biofilm formation, a process related with pathogenesis. Confirming our previous results, microscopy images revealed that the ATCC 17978 Δ0114 derivative lacking this gene was unable to form a mature biofilm structure. Therefore, other bacterial phenotypes were analyzed to determine the role of this gene in the pathogenicity of A. baumannii ATCC 17978. The interaction of the ATCC 17978 parental strain and the Δ0114 mutant with A549 human alveolar epithelial cells was quantified revealing that the A1S_0114 gene was necessary for proper attachment to A549 cells. This dependency correlates with the negative effect of the A1S_0114 deletion on the expression of genes coding for surface proteins and pili-assembly systems, which are known to play a role in adhesion. Three different experimental animal models, including vertebrate and invertebrate hosts, confirmed the role of the A1S_0114 gene in virulence. All of the experimental infection assays indicated that the virulence of the ATCC 17978 was significantly reduced when this gene was inactivated. Finally, we discovered that the A1S_0114 gene was involved in the production of a small lipopeptide-like compound herein referred to as acinetin 505 (Ac-505). Ac-505 was isolated from ATCC 17978 spent media and its chemical structure was interpreted by mass spectrometry. Overall, our observations provide novel information on the role of the A1S_0114 gene in A. baumannii's pathobiology and lay the foundation for future work to determine the mechanisms by which Ac-505, or possibly an Ac-505 precursor, could execute critical functions as a secondary metabolite.

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.

Recent advances in molecular modeling of protein structures are changing the field of structural biology. AlphaFold-2 (AF2), an AI system developed by DeepMind, Inc., utilizes attention-based deep learning to predict models of protein structures with high accuracy relative to structures determined by X-ray crystallography and cryo-electron microscopy (cryoEM). Comparing AF2 models to structures determined using solution NMR data, both high similarities and distinct differences have been observed. Since AF2 was trained on X-ray crystal and cryoEM structures, we assessed how accurately AF2 can model small, monomeric, solution protein NMR structures which (i) were not used in the AF2 training data set, and (ii) did not have homologous structures in the Protein Data Bank at the time of AF2 training. We identified nine open source protein NMR data sets for such "blind" targets, including chemical shift, raw NMR FID data, NOESY peak lists, and (for 1 case) 15 N- 1 H residual dipolar coupling data. For these nine small (70 - 108 residues) monomeric proteins, we generated AF2 prediction models and assessed how well these models fit to these experimental NMR data, using several well-established NMR structure validation tools. In most of these cases, the AF2 models fit the NMR data nearly as well, or sometimes better than, the corresponding NMR structure models previously deposited in the Protein Data Bank. These results provide benchmark NMR data for assessing new NMR data analysis and protein structure prediction methods. They also document the potential for using AF2 as a guiding tool in protein NMR data analysis, and more generally for hypothesis generation in structural biology research.

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.

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 β-barrel fold comprising five β-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.

Currently used biomarkers for acute kidney injury (AKI), namely Ngal, SCr, and BUN, are inadequate for timely detection of AKI in preterm infants.

Adenoviruses (Ads) are used in numerous preclinical and clinical studies for delivery of anti-cancer therapeutic genes. Unfortunately, Ad has a poor ability to distribute throughout a tumor mass after intratumoral injection, and infects cells primarily within the immediate area of the injection tract. Thus, Ad-encoded transgene expression is typically limited to only a small percentage of cells within the tumor. One method to increase the proportion of the tumor impacted by Ad is through expression of fusogenic proteins. Infection of a single cell with an Ad vector encoding a fusogenic protein should lead to syncytium formation with adjacent cells, effectively spreading the effect of Ad and Ad-encoded therapeutic transgenes to a greater percentage of the tumor mass. Moreover, syncytium formation can be cytotoxic, suggesting that such proteins may be effective sole therapeutics. We show that an early region 1 (E1)-deleted Ad expressing reptilian reovirus p14 fusion-associated small transmembrane (FAST) protein caused extensive cell fusion in the replication-permissive 293 cell line and at high multiplicity of infection in non-permissive human lung adenocarcinoma A549 cells in vitro. FAST protein expression in the A549 cancer cell line led to a loss of cellular metabolic activity and membrane integrity, which correlated with induction of apoptosis. However, in an A549 xenograft CD-1 nude mouse cancer model, Ad-mediated FAST gene delivery did not induce detectable cell fusion, reduce tumor burden nor enhance mouse survival compared to controls. Taken together, our results show that, although AdFAST can enhance cancer cell killing in vitro, it is not effective as a sole therapeutic in the A549 tumor model in vivo.

Hsp90 is a homodimeric ATP-dependent molecular chaperone that remodels its substrate 'client' proteins, facilitating their folding and activating them for biological function. Despite decades of research, the mechanism connecting ATP hydrolysis and chaperone function remains elusive. Particularly puzzling has been the apparent lack of cooperativity in hydrolysis of the ATP in each protomer. A crystal structure of the mitochondrial Hsp90, TRAP1, revealed that the catalytically active state is closed in a highly strained asymmetric conformation. This asymmetry, unobserved in other Hsp90 homologs, is due to buckling of one of the protomers and is most pronounced at the broadly conserved client-binding region. Here, we show that rather than being cooperative or independent, ATP hydrolysis on the two protomers is sequential and deterministic. Moreover, dimer asymmetry sets up differential hydrolysis rates for each protomer, such that the buckled conformation favors ATP hydrolysis. Remarkably, after the first hydrolysis, the dimer undergoes a flip in the asymmetry while remaining in a closed state for the second hydrolysis. From these results, we propose a model where direct coupling of ATP hydrolysis and conformational flipping rearranges client-binding sites, providing a paradigm of how energy from ATP hydrolysis can be used for client remodeling.

CV_2116 is a small hypothetical protein of 82 amino acids from the Gram-negative coccobacillus Chromobacterium violaceum. A PSI-BLAST search using the CV_2116 sequence as a query identified only one hit (E = 2e(-07)) corresponding to a hypothetical protein OR16_04617 from Cupriavidus basilensis OR16, which failed to provide insight into the function of CV_2116. The CV_2116 gene was cloned into the p15TvLic expression plasmid, transformed into E. coli, and (13)C- and (15)N-labeled NMR samples of CV_2116 were overexpressed in E. coli and purified for structure determination using NMR spectroscopy. The resulting high-quality solution NMR structure of CV_2116 revealed a novel α + β fold containing two anti-parallel β-sheets in the N-terminal two-thirds of the protein and one α-helix in the C-terminal third of the protein. CV_2116 does not belong to any known protein sequence family and a Dali search indicated that no similar structures exist in the protein data bank. Although no function of CV_2116 could be derived from either sequence or structural similarity searches, the neighboring genes of CV_2116 encode various proteins annotated as similar to bacteriophage tail assembly proteins. Interestingly, C. violaceum exhibits an extensive network of bacteriophage tail-like structures that likely result from lateral gene transfer by incorporation of viral DNA into its genome (prophages) due to bacteriophage infection. Indeed, C. violaceum has been shown to contain four prophage elements and CV_2116 resides in the fourth of these elements. Analysis of the putative operon in which CV_2116 resides indicates that CV_2116 might be a component of the bacteriophage tail-like assembly that occurs in C. violaceum.

To determine if spatial variations in gut permeability play a role in regulating type 1 diabetes (T1D) progression.

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.

Necrotizing enterocolitis (NEC) is a devastating intestinal disease that afflicts 10% of extremely preterm infants. The contribution of early intestinal colonization to NEC onset is not understood, and predictive biomarkers to guide prevention are lacking. We analyzed banked stool and urine samples collected prior to disease onset from infants <29 weeks gestational age, including 11 infants who developed NEC and 21 matched controls who survived free of NEC. Stool bacterial communities were profiled by 16S rRNA gene sequencing. Urinary metabolomic profiles were assessed by NMR.

Calicheamicin γ1I (1) is an enediyne antitumor compound produced by Micromonospora echinospora spp. calichensis, and its biosynthetic gene cluster has been previously reported. Despite extensive analysis and biochemical study, several genes in the biosynthetic gene cluster of 1 remain functionally unassigned. Using a structural genomics approach and biochemical characterization, two proteins encoded by genes from the 1 biosynthetic gene cluster assigned as "unknowns", CalU16 and CalU19, were characterized. Structure analysis revealed that they possess the STeroidogenic Acute Regulatory protein related lipid Transfer (START) domain known mainly to bind and transport lipids and previously identified as the structural signature of the enediyne self-resistance protein CalC. Subsequent study revealed calU16 and calU19 to confer resistance to 1, and reminiscent of the prototype CalC, both CalU16 and CalU19 were cleaved by 1 in vitro. Through site-directed mutagenesis and mass spectrometry, we identified the site of cleavage in each protein and characterized their function in conferring resistance against 1. This report emphasizes the importance of structural genomics as a powerful tool for the functional annotation of unknown proteins.

The P[19] genotype belongs to the P[II] genogroup of group A rotaviruses (RVs). However, unlike the other P[II] RVs, which mainly infect humans, P[19] RVs commonly infect animals (pigs), making P[19] unique for the study of RV diversity and host ranges. Through in vitro binding assays and saturation transfer difference (STD) nuclear magnetic resonance (NMR), we found that P[19] could bind mucin cores 2, 4, and 6, as well as type 1 histo-blood group antigens (HBGAs). The common sequences of these glycans serve as minimal binding units, while additional residues, such as the A, B, H, and Lewis epitopes of the type 1 HBGAs, can further define the binding outcomes and therefore likely the host ranges for P[19] RVs. This complex binding property of P[19] is shared with the other three P[II] RVs (P[4], P[6], and P[8]) in that all of them recognized the type 1 HBGA precursor, although P[4] and P[8], but not P[6], also bind to mucin cores. Moreover, while essential for P[4] and P[8] binding, the addition of the Lewis epitope blocked P[6] and P[19] binding to type 1 HBGAs. Chemical-shift NMR of P[19] VP8* identified a ligand binding interface that has shifted away from the known RV P-genotype binding sites but is conserved among all P[II] RVs and two P[I] RVs (P[10] and P[12]), suggesting an evolutionary connection among these human and animal RVs. Taken together, these data are important for hypotheses on potential mechanisms for RV diversity, host ranges, and cross-species transmission.

Metabolic profiles of hydrophilic and lipophilic cell extracts from three cancer cell lines, Miapaca-2, Panc-1 and AsPC-1, and a non-cancerous pancreatic ductal epithelial cell line, H6C7, were examined by proton nuclear magnetic resonance spectroscopy. Over twenty five hydrophilic metabolites were identified by principal component and statistical significance analyses as distinguishing the four cell types. Fifteen metabolites were identified with significantly altered concentrations in all cancer cells, e.g. absence of phosphatidylgrycerol and phosphatidylcholine, and increased phosphatidylethanolamine and cholesterols. Altered concentrations of metabolites involved in glycerophospholipid metabolism, lipopolysaccharide and fatty acid biosynthesis indicated differences in cellular membrane composition between non-cancerous and cancer cells. In addition to cancer specific metabolites, several metabolite changes were unique to each cancer cell line. Increased N-acetyl groups in AsPC-1, octanoic acids in Panc-1, and UDP species in Miapaca-2 indicated differences in cellular membrane composition between the cancer cell lines. Induced glutamine metabolism and protein synthesis in cancer cells were indicated by absence of glutamine other metabolites such as acetate, lactate, serine, branched amino acids, and succinate. Knowledge of the specifically altered metabolic pathways identified in these pancreatic cancer cell lines may be useful for identifying new therapeutic targets and studying the effects of potential new therapeutic drugs.

Fundamental changes in the composition and distribution of lipids within the brain are believed to contribute to the cognitive decline associated with Alzheimer's disease (AD). The mechanisms by which these changes in lipid composition affect cellular function and ultimately cognition are not well understood. Although "candidate gene" approaches can provide insight into the effects of dysregulated lipid metabolism they require a preexisting understanding of the molecular targets of individual lipid species. In this report we combine unbiased gene expression profiling with a genome-wide chemogenomic screen to identify the mitochondria as an important downstream target of PC(O-16:0/2:0), a neurotoxic lipid species elevated in AD. Further examination revealed that PC(O-16:0/2:0) similarly promotes a global increase in ceramide accumulation in human neurons which was associated with mitochondrial-derived reactive oxygen species (ROS) and toxicity. These findings suggest that PC(O-16:0/2:0)-dependent mitochondrial dysfunction may be an underlying contributing factor to the ROS production associated with AD.

The HGMA1 architectural transcription factor is highly overexpressed in many human cancers. Because HMGA1 is a hub for regulation of many oncogenes, its overexpression in cancer plays a central role in cancer progression and therefore HMGA1 is gaining increasing attention as a target for development of therapeutic approaches to suppress either its expression or action in cancer cells. We have developed the strategy of introducing decoy hyper binding sites for HMGA1 into the nucleus of cancer cells with the goal of competetively sequestering overexpressed HMGA1 and thus suppressing its oncogenic action. Towards achieving this goal, we have introduced an HMGA1 decoy hyper binding site composed of six copies of a high affinity HMGA1 binding site into the genome of the replication defective adenovirus serotype 5 genome and shown that the engineered virus effectively reduces the viability of human pancreatic and cancer cells. Here we report the first pre-clinical measures of toxicity and biodistribution of the engineered virus in C57BL/6J Black 6 mice. The immune response to exposure of the engineered virus was determined by assaying the serum levels of key cytokines, IL-6 and TNF-α. Toxicity due to exposure to the virus was determined by measuring the serum levels of the liver enzymes aspartate aminotransferase and alanine aminotransferase. Biodistribution was measured following direct injection into the pancreas or liver by quantifying viral loads in the pancreas, liver, spleen and brain.

Formamidopyrimidine-DNA glycosylase (Fpg) is a base excision repair (BER) protein that removes oxidative DNA lesions. Recent crystal structures of Fpg bound to DNA revealed residues involved in damage recognition and enzyme catalysis, but failed to shed light on the dynamic nature of the processes. To examine the structural and dynamic changes that occur in solution when Fpg binds DNA, NMR spectroscopy was used to study Escherichia coli Fpg free in solution and bound to a double-stranded DNA oligomer containing 1,3-propanediol (13-PD), a non-hydrolyzable abasic-site analogue. Only 209 out of a possible 251 (83%) free-precession 15N/1H HSQC cross peaks were observed and 180 of these were assignable, indicating that approximately 30% of the residues undergo intermediate motion on the NMR timescale, broadening the resonances beyond detection or making them intractable in backbone assignment experiments. The majority of these affected residues were in the polypeptide linker region and the interface between the N- and C-terminal domains. DNA titration experiments revealed line broadening and chemical shift perturbations for backbone amides nearby and distant from the DNA binding surface, but failed to quench the intermediate timescale motion observed for free Fpg, including those residues directly involved in DNA binding, notwithstanding a nanomolar dissociation constant for 13-PD binding. Indeed, after binding to 13-PD, at least approximately 40% of the Fpg residues undergo intermediate timescale motion even though all other residues exhibit tight DNA binding characteristic of slow exchange. CPMG-HSQC experiments revealed millisecond to microsecond motion for the backbone amides of D91 and H92 that were quenched upon binding 13-PD. In free Fpg, heteronuclear 1H-15N NOE experiments detected picosecond timescale backbone motion in the alphaF-beta9 loop, the region primarily responsible for chemically discriminating 8-oxoguanine (8-oxoG) over normal guanine, that was quenched after binding 13-PD. Collectively, these observations reveal that, in solution, Fpg is a very dynamic molecule even after binding damaged DNA. Such motion, especially at the DNA binding surface, may be key to its processive search for DNA damage and its catalytic functions once it recognizes damaged DNA.