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The massive genomic data from The Cancer Genome Atlas (TCGA), including proteomics data from Clinical Proteomic Tumor Analysis Consortium (CPTAC), provides a unique opportunity to study cancer systematically. While most observations are made from a single type of genomics data, we apply big data analytics and systems biology approaches by simultaneously analyzing DNA amplification, mRNA and protein abundance. Using multiple genomic profiles, we have discovered widespread dosage compensation for the extensive aneuploidy observed in TCGA breast cancer samples. We do identify 11 genes that show strong correlation across all features (DNA/mRNA/protein) analogous to that of the well-known oncogene HER2 (ERBB2). These genes are generally less well-characterized regarding their role in cancer and we advocate their further study. We also discover that shRNA knockdown of these genes has an impact on cancer cell growth, suggesting a vulnerability that could be used for cancer therapy. Our study shows the advantages of systematic big data methodologies and also provides future research directions.

Modern humans have populated Europe for more than 45,000 years 1,2 . Our knowledge of the genetic relatedness and structure of ancient hunter-gatherers is however limited, owing to the scarceness and poor molecular preservation of human remains from that period 3 . Here we analyse 356 ancient hunter-gatherer genomes, including new genomic data for 116 individuals from 14 countries in western and central Eurasia, spanning between 35,000 and 5,000 years ago. We identify a genetic ancestry profile in individuals associated with Upper Palaeolithic Gravettian assemblages from western Europe that is distinct from contemporaneous groups related to this archaeological culture in central and southern Europe 4 , but resembles that of preceding individuals associated with the Aurignacian culture. This ancestry profile survived during the Last Glacial Maximum (25,000 to 19,000 years ago) in human populations from southwestern Europe associated with the Solutrean culture, and with the following Magdalenian culture that re-expanded northeastward after the Last Glacial Maximum. Conversely, we reveal a genetic turnover in southern Europe suggesting a local replacement of human groups around the time of the Last Glacial Maximum, accompanied by a north-to-south dispersal of populations associated with the Epigravettian culture. From at least 14,000 years ago, an ancestry related to this culture spread from the south across the rest of Europe, largely replacing the Magdalenian-associated gene pool. After a period of limited admixture that spanned the beginning of the Mesolithic, we find genetic interactions between western and eastern European hunter-gatherers, who were also characterized by marked differences in phenotypically relevant variants., (© 2023. The Author(s).)

Hepatic failure due to hepatitis C is the leading indicator for orthotopic liver transplantation (OLT) in the United States. Unfortunately, recurrent hepatitis C virus infection is essentially universal following orthotopic liver transplantation. Although significant advances have been made in the past decade for the treatment of hepatitis C, a similar level of success has not yet been achieved for most hepatitis C virus-infected liver transplant recipients. In addition, deleterious side effects of the currently available antiviral agents continue to significantly hamper their use. Several recent reports, however, indicate that newer immunosuppressive regimens combined with novel modifications of existing treatment paradigms will likely lead to improved clinical outcomes for the hepatitis C virus-infected liver transplant recipient.

Clostridium difficile causes an intense inflammatory colitis through the actions of two large exotoxins, toxin A and toxin B. IL-8 is believed to play an important role in the pathophysiology of C. difficile-mediated colitis, although the mechanism whereby the toxins up-regulate the release of IL-8 from target cells is not well understood. In this study, we investigated the mechanisms through which toxin A induces IL-8 secretion in human monocytes. We found that cellular uptake of toxin A is required for the up-regulation of IL-8, an effect that is not duplicated by a recombinant toxin fragment comprising the cell-binding domain alone. Toxin A induced IL-8 expression at the level of gene transcription and this effect occurred through a mechanism requiring intracellular calcium and calmodulin activation. Additionally, the effects of toxin A were inhibited by the protein tyrosine kinase inhibitor genistein, but were unaffected by inhibitors of protein kinase C and phosphatidylinositol-3 kinase. We determined that toxin A activates nuclear translocation of the transcription factors NF-kappa B and AP-1, but not NF-IL-6. NF-kappa B inhibitors blocked the ability of toxin A to induce IL-8 secretion, and supershift analysis indicated that the major isoform of NF-kappa B activated by the toxin is a p50-p65 heterodimer. This study is the first to identify intracellular signaling pathways and transcription factors involved in the C. difficile toxin-mediated up-regulation of IL-8 synthesis and release by target cells. This information should increase our understanding of the pathogenesis of C. difficile colitis and the nature of IL-8 gene regulation as well.

An improved understanding of how intestinal bacteria cause disease has become increasingly important because of the emergence of new enteric pathogens, increasing threats of drug resistance, and a growing awareness of their importance in malnutrition and diarrhea. Reviewed here are the varied ways that intestinal bacteria cause disease, which provide fundamental lessons about microbial pathogenesis as well as cell signaling. Following colonization, enteric pathogens may adhere to or invade the epithelium or may produce secretory exotoxins or cytotoxins. In addition, by direct or indirect effects, they may trigger secondary mediator release of cytokines that attract inflammatory cells, which release further products, such as prostaglandins or platelet-activating factor, which can also trigger secretion. An improved understanding of pathogenesis not only opens new approaches to treatment and control but may also suggest improved simple means of diagnosis and even vaccine development.

The Rho family small GTPases are members of the Ras superfamily of small GTPases. Rho proteins were first determined to act as key regulators of many types of actin cytoskeletal-dependent cellular functions. Recent work by several investigators indicates that Rho GTPases are also critical modulators of several important intracellular and nuclear signal transduction pathways. Certain clostridial toxins and exoenzymes covalently modify, and thereby inactivate, specific types of Rho family GTPases. As such, these microbial enzymes have proven invaluable in helping to identify structural and functional attributes of Rho GTPases.

Rho family GTPases are known to be involved in cytoskeletal reorganization. We examined the possibility that these functions may be dictated by a balance of Rho family GTPase signaling. Using transient viral expression of RhoA, Rac1, Cdc42 and their mutants, as well as C3 exoenzyme, we altered cytoskeletal organization under normal growth conditions. Overexpression of wild-type or constitutively active forms of the Rho family GTPases led to their respective activation phenotypes. Overexpression of dominant negative forms of given Rho family GTPases led to a phenotype consistent with activation of the other Rho family GTPase. Treatment with C. difficile toxin A, that inactivates all Rho family GTPases, led to the transient appearance of a variety of activation phenotypes. Previously, we reported that inactivation of Rho led to induction of apoptosis, implying that Rho may play an important role in cell survival signaling. This signaling, however, is not affected by expression of any forms of Rac1 or Cdc42, and only inactivation of Rho led to induction of apoptosis. Rho family GTPases appear to coordinate cytoskeletal organization by a balance of signaling, while cell survival is regulated by a distinct Rho-mediated signaling pathway.

Rho small GTPases regulate a variety of cellular signaling pathways involved in cell growth and transformation. In this study, we examined potential roles for Rho in adhesion-dependent and -independent pathways regulating apoptosis. Rho GTPases are specifically inactivated by exoenzyme C3 (C3) of Clostridium botulinum. Using a novel Sindbis virus-based gene expression system, we created a double subgenomic recombinant (dsSIN:C3) capable of expressing active C3 in intact cells. Infection of L929 fibroblasts with dsSIN:C3 caused essentially complete ADP-ribosylation of intracellular Rho within 1 h. dsSIN:C3-infected cells also became rounded within 1-2 h and detached by 5 h post-infection. Infection of L929 in suspension with dsSIN:C3 disrupted the ability for normal cellular attachment and spreading. Infection of primary cell explants of chicken embryo fibroblasts (CEF) and rat aortic smooth muscle cells (RSM) with dsSIN:C3 caused cytoskeletal effects similar to those seen in L929. We also observed that C3 markedly decreased the basal phosphorylation state of focal adhesion kinase (FAK). Most intriguingly, we found that dsSIN-based expression of C3 or loss of function mutants of Rho could each induce apoptosis and, in RSM, this effect was observed to be adhesion-independent. Rho GTPases, therefore, appear to regulate signal pathways that are required for cell survival and growth that are separate from, but likely overlap with, Rho-dependent pathways involved in cellular adhesion.

A group of transplantation antigens, referred to as tum- antigens, were identified in mouse tumor cells that had been mutagenized to produce variant cells and were recognized by clonal cytolytic T lymphocytes (CTL). Alterations in these variant cells that were recognized by CTL resulted from point mutations in the genes of specific proteins. We have isolated human and bovine cDNA clones that encode the homologs of the mouse tum- antigen P198. This 23.6-kDa protein is highly basic with a predicted pI of 11.55. p23/P198 is highly conserved across mammalian species, with > 94% identity (97% including conservative substitutions) among the human, bovine, and mouse deduced amino acid sequences. The nucleotide sequences of both the coding and 5'- and 3'-untranslated regions from human, bovine, and mouse are also highly conserved with > 88% identity in the coding regions. Hybridization of poly(A)+ RNA from various mammalian sources with cDNA and oligonucleotides specific for the coding region identified two mRNAs of 1.2 and 0.8 kb, whereas probes specific for the 3'-untranslated region between two consensus polyadenylation signals hybridized with the 1.2-kb, but not the 0.8-kb, mRNA. The abundance of the 1.2-kb mRNA relative to that of the 0.8-kb species varied depending upon the cell type. A single predominant transcription initiation site was mapped by primer extension. These studies indicate that this highly basic 23.6-kDa protein is encoded by two major mRNA species that differ only in the length of their 3'-untranslated regions and that the mechanism that gives rise to these two mRNAs, utilization of alternative polyadenylation sites, is conserved across species.(ABSTRACT TRUNCATED AT 250 WORDS)

ADP-ribosylation factors (ARFs) are a family of approximately 20-kDa guanine nucleotide-binding proteins initially identified by their ability to enhance cholera toxin ADP-ribosyltransferase activity in the presence of GTP. ARFs have been purified from both membrane and cytosolic fractions. ARF purified from bovine brain cytosol requires phospholipid plus detergent for high affinity guanine nucleotide binding and for optimal enhancement of cholera toxin ADP-ribosyltransferase activity. The phospholipid requirements, combined with a putative role for ARF in vesicular transport, suggested that the soluble protein might interact reversibly with membranes. A polyclonal antibody against purified bovine ARF (sARF II) was used to detect ARF by immunoblot in membrane and soluble fractions from rat pheochromocytoma (PC-12) cell homogenates. ARF was predominantly cytosolic but increased in membranes during incubation of homogenates with nonhydrolyzable GTP analogues guanosine 5'-O-(3-thiotriphosphate), guanylyl-(beta gamma-imido)-diphosphate, and guanylyl-(beta gamma-methylene)-diphosphate, and to a lesser extent, adenosine 5'-O-(3-thiotriphosphate). GTP, GDP, GMP, and ATP were inactive. Cytosolic ARF similarly associated with added phosphatidylserine, phosphatidylinositol, or cardiolipin in GTP gamma S-dependent fashion. ARF binding to phosphatidylserine was reversible and coincident with stimulation of cholera toxin-catalyzed ADP-ribosylation. These observations may reflect a mechanism by which ARF could cycle between soluble and membrane compartments in vivo.

Activation of adenylyl cyclase by cholera toxin A subunit (CT-A) results from the ADP-ribosylation of the stimulatory guanine nucleotide binding protein (GS alpha). This process requires GTP and an endogenous guanine nucleotide binding protein known as ADP-ribosylation factor (ARF). One membrane (mARF) and two soluble forms (sARF I and sARF II) of ARF have been purified from bovine brain. Because the conditions reported to enhance the binding of guanine nucleotides by ARF differ from those observed to promote optimal activity, we sought to characterize the determinants influencing the functional interaction of guanine nucleotides with ARF. High-affinity GTP binding by sARF II (apparent KD of approximately 70 nM) required Mg2+, DMPC, and sodium cholate. sARF II, in DMPC/cholate, also enhanced CT-A ADP-ribosyltransferase activity (apparent EC50 for GTP of approximately 50 nM), although there was a delay before achievement of a maximal rate of sARF II stimulated toxin activity. The delay was abolished by incubation of sARF II with GTP at 30 degrees C before initiation of the assay. In contrast, a maximal rate of activation of toxin by sARF II, in 0.003% SDS, occurred without delay (apparent EC50 for GTP of approximately 5 microM). High-affinity GTP binding by sARF II was not detectable in SDS. Enhancement of CT-A ADP-ribosyltransferase activity by sARF II, therefore, can occur under conditions in which sARF II exhibits either a relatively low affinity or a relatively high affinity for GTP. The interaction of GTP with ARF under these conditions may reflect ways in which intracellular membrane and cytosolic environments modulate GTP-mediated activation of ARF.

The adherence of human monocytes and culture-derived macrophages to surfaces coated with complement subcomponent C1q has been previously shown to enhance Fc receptor (FcR)-mediated phagocytosis by these cells. We examined the effects of C1q on C3b/C4b receptor (CR1)-mediated phagocytosis by mononuclear phagocytes. A small percentage of human monocytes cultured in the presence of serum became competent to ingest sheep erythrocytes bearing IgM and C4b (EAC4b). This phagocytic activity was enhanced when these cultured-derived macrophages were adhered to C1q-coated surfaces. However, when cultured in a defined serum-free medium, these cells did not ingest EAC4b, even in the presence of C1q. To investigate this differential responsiveness, we studied the effects of C1q in conjunction with cell-activating agents on CR1 activation. Treatment of serum-free cultured monocytes with phorbol dibutyrate (PDBu), prior to addition of the targets, induced these cells to ingest EAC4b. In addition, when exposed to C1q, both the percentage of these PDBu mononuclear phagocytes ingesting EAC4b and the number of targets ingested increased threefold over the level achieved by macrophages treated with PDBu alone. The chemoattractant N-formyl-methionyl-leucyl-phenylalanine did not activate CR1-mediated phagocytosis and did not substitute for PDBu in causing synergy with C1q. Freshly isolated monocytes adhered to human serum albumin-coated glass slides in the absence or presence of PDBu did not phagocytose EAC4b. Also C1q did not stimulate monocyte CR1-mediated phagocytosis. However, addition of PDBu to cells adherent to the C1q surface triggered phagocytosis of EAC4b. The concentration of PDBu and the time of addition of PDBu relative to addition of the EAC4b targets were found to be important parameters for the achievement of maximal synergy in both the freshly isolated and cultured cell systems. This enhanced phagocytic activity was also seen with cells adhered to the purified collagen-like, pepsin-resistant, fragment of C1q. Since this region was previously shown to interact with C1q surface receptors, it appears that occupancy of this receptor is triggering events contributing to the enhanced cellular function. These experiments suggest that C1q and PDBu promote ingestion via CR1 by different but synergistic mechanisms. These data also demonstrate that the CR1-mediated enhancement of phagocytosis is not specific for FcR-mediated ingestion, but also applies to phagocytosis via CR1.

Cholera toxin catalyzes the ADP-ribosylation that results in activation of the stimulatory guanine nucleotide-binding protein of the adenylyl cyclase system, known as Gs. The toxin also ADP-ribosylates other proteins and simple guanidino compounds and auto-ADP-ribosylates its AI protein (CTA1). All of the ADP-ribosyltransferase activities of CTAI are enhanced by 19-21-kDa guanine nucleotide-binding proteins known as ADP-ribosylation factors, or ARFs. CTAI contains a single cysteine located near the carboxy terminus. CTAI was immobilized through this cysteine by reaction with iodoacetyl-N-biotinyl-hexylenediamine and binding of the resulting biotinylated protein to avidin-agarose. Immobilized CTAI catalyzed the ARF-stimulated ADP-ribosylation of agmatine. The reaction was enhanced by detergents and phospholipid, but the fold stimulation by purified sARF-II from bovine brain was considerably less than that observed with free CTA. ADP-ribosylation of Gsa by immobilized CTAI, which was somewhat enhanced by sARF-II, was much less than predicted on the basis of the NAD:agmatine ADP-ribosyltransferase activity. Immobilized CTAI catalyzed its own auto-ADP-ribosylation as well as the ADP-ribosylation of the immobilized avidin and CTA2, with relatively little stimulation by sARF-II. ADP-ribosylation of CTA2 by free CTAI is minimal. These observations are consistent with the conclusion that the cysteine near the carboxy terminus of the toxin is not critical for ADP-ribosyltransferase activity or for its regulation by sARF-II. Biotinylation and immobilization of the toxin through this cysteine may, however, limit accessibility to Gsa or SARF-II, or perhaps otherwise reduce interaction with these proteins whether as substrates or activator.