Human Pleural Fluid Elicits Pyruvate and Phenylalanine Metabolism in Acinetobacter baumannii to Enhance Cytotoxicity and Immune Evasion

The CCAAT box-harboring proteins represent a family of heterotrimeric transcription factors which is highly conserved in eukaryotes. In fungi, one of the particularly important homologs of this family is the Hap complex that separates the DNA-binding domain from the activation domain and imposes essential impacts on regulation of a wide range of cellular functions. So far, a comprehensive summary of this complex has been described in filamentous fungi but not in the yeast. In this review, we summarize a number of studies related to the structure and assembly mode of the Hap complex in a list of representative yeasts. Furthermore, we emphasize recent advances in understanding the regulatory functions of this complex, with a special focus on its role in regulating respiration, production of reactive oxygen species (ROS) and iron homeostasis.Fil: Nyah, Rodman. California State University; Estados UnidosFil: Martinez, Jasmine. California State University; Estados UnidosFil: Fung, Sammie. California State University; Estados UnidosFil: Nakanouchi, Jun. California State University; Estados UnidosFil: Myers, Amber L.. California State University; Estados UnidosFil: Harris, Caitlin M.. California State University; Estados UnidosFil: Dang, Emily. California State University; Estados UnidosFil: Fernandez, Jennifer. California State University; Estados UnidosFil: Liu, Christine. California State University; Estados UnidosFil: Mendoza, Anthony M.. California State University; Estados UnidosFil: Jimenez, Verónica. California State University; Estados UnidosFil: Nikolaidis, Nikolas. California State University; Estados UnidosFil: Brennan, Catherine A.. California State University; Estados UnidosFil: Bonomo, Robert A.. Louis Stokes Cleveland Department of Veterans Affairs Medical Cente; Estados Unidos. Center for Antimicrobial Resistance and Epidemiology; Estados Unidos. Case Western Reserve University School of Medicine; Estados UnidosFil: Sieira, Rodrigo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Ramirez, Maria Soledad. California State University; Estados Unido

Spatial analysis of Cdc42 activity reveals a role for plasma membrane–associated Cdc42 in centrosome regulation

The ability of the small GTPase Cdc42 to regulate diverse cellular processes depends on tight spatial control of its activity. Cdc42 function is best understood at the plasma membrane (PM), where it regulates cytoskeletal organization and cell polarization. Active Cdc42 has also been detected at the Golgi, but its role and regulation at this organelle are only partially understood. Here we analyze the spatial distribution of Cdc42 activity by moni­toring the dynamics of the Cdc42 FLARE biosensor using the phasor approach to FLIM-FRET. Phasor analysis revealed that Cdc42 is active at all Golgi cisternae and that this activity is controlled by Tuba and ARHGAP10, two Golgi-associated Cdc42 regulators. To our surprise, FGD1, another Cdc42 GEF at the Golgi, was not required for Cdc42 regulation at the Golgi, although its depletion decreased Cdc42 activity at the PM. Similarly, changes in Golgi morphology did not affect Cdc42 activity at the Golgi but were associated with a substantial reduction in PM-associated Cdc42 activity. Of interest, cells with reduced Cdc42 activity at the PM displayed altered centrosome morphology, suggesting that centrosome regulation may be mediated by active Cdc42 at the PM. Our study describes a novel quantitative approach to determine Cdc42 activity at specific subcellular locations and reveals new regulatory principles and functions of this small GTPase

Spatial analysis of Cdc42 activity reveals a role for plasma membrane–associated Cdc42 in centrosome regulation

The ability of the small GTPase Cdc42 to regulate diverse cellular processes depends on tight spatial control of its activity. Cdc42 function is best understood at the plasma membrane (PM), where it regulates cytoskeletal organization and cell polarization. Active Cdc42 has also been detected at the Golgi, but its role and regulation at this organelle are only partially understood. Here we analyze the spatial distribution of Cdc42 activity by monitoring the dynamics of the Cdc42 FLARE biosensor using the phasor approach to FLIM-FRET. Phasor analysis revealed that Cdc42 is active at all Golgi cisternae and that this activity is controlled by Tuba and ARHGAP10, two Golgi-associated Cdc42 regulators. To our surprise, FGD1, another Cdc42 GEF at the Golgi, was not required for Cdc42 regulation at the Golgi, although its depletion decreased Cdc42 activity at the PM. Similarly, changes in Golgi morphology did not affect Cdc42 activity at the Golgi but were associated with a substantial reduction in PM-associated Cdc42 activity. Of interest, cells with reduced Cdc42 activity at the PM displayed altered centrosome morphology, suggesting that centrosome regulation may be mediated by active Cdc42 at the PM. Our study describes a novel quantitative approach to determine Cdc42 activity at specific subcellular locations and reveals new regulatory principles and functions of this small GTPase

ViroFind: A novel target-enrichment deep-sequencing platform reveals a complex JC virus population in the brain of PML patients

Deep nucleotide sequencing enables the unbiased, broad-spectrum detection of viruses in clinical samples without requiring an a priori hypothesis for the source of infection. However, its use in clinical research applications is limited by low cost-effectiveness given that most of the sequencing information from clinical samples is related to the human genome, which renders the analysis of viral genomes challenging. To overcome this limitation we developed ViroFind, an in-solution target-enrichment platform for virus detection and discovery in clinical samples. ViroFind comprises 165,433 viral probes that cover the genomes of 535 selected DNA and RNA viruses that infect humans or could cause zoonosis. The ViroFind probes are used in a hybridization reaction to enrich viral sequences and therefore enhance the detection of viral genomes via deep sequencing. We used ViroFind to detect and analyze all viral populations in the brain of 5 patients with progressive multifocal leukoencephalopathy (PML) and of 18 control subjects with no known neurological disease. Compared to direct deep sequencing, by using ViroFind we enriched viral sequences present in the clinical samples up to 127-fold. We discovered highly complex polyoma virus JC populations in the PML brain samples with a remarkable degree of genetic divergence among the JC virus variants of each PML brain sample. Specifically for the viral capsid protein VP1 gene, we identified 24 single nucleotide substitutions, 12 of which were associated with amino acid changes. The most frequent (4 of 5 samples, 80%) amino acid change was D66H, which is associated with enhanced tissue tropism, and hence likely a viral fitness advantage, compared to other variants. Lastly, we also detected sparse JC virus sequences in 10 of 18 (55.5%) of control samples and sparse human herpes virus 6B (HHV6B) sequences in the brain of 11 of 18 (61.1%) control subjects. In sum, ViroFind enabled the in-depth analysis of all viral genomes in PML and control brain samples and allowed us to demonstrate a high degree of JC virus genetic divergence in vivo that has been previously underappreciated. ViroFind can be used to investigate the structure of the virome with unprecedented depth in health and disease state

Comparison of Proteomic Assessment Methods in Multiple Cohort Studies

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/155922/1/pmic13292_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/155922/2/pmic13292.pd

Opioids exacerbate inflammation in people with well-controlled HIV

IntroductionPeople with HIV (PWH) are known to have underlying inflammation and immune activation despite virologic control. Substance use including opioid dependence is common in this population and is associated with increased morbidity and reduced lifespan. The primary objective of the present study termed opioid immunity study (OPIS), was to investigate the impact of chronic opioids in PWH.MethodsThe study recruited people with and without HIV who had opioid use disorder (OUD). Study participants (n=221) were categorized into four groups: HIV+OP+, n=34; HIV-OP+, n=66; HIV+OP-, n=55 and HIV-OP-, n=62 as controls. PWH were virally suppressed on ART and those with OUD were followed in a syringe exchange program with confirmation of OP use by urine drug screening. A composite cytokine score was developed for 20 plasma cytokines that are linked to inflammation. Cellular markers of immune activation (IA), exhaustion, and senescence were determined in CD4 and CD8 T cells. Regression models were constructed to examine the relationships of HIV status and opioid use, controlling for other confounding factors.ResultsHIV+OP+ participants exhibited highest inflammatory cytokines and cellular IA, followed by HIV-OP+ for inflammation and HIV+OP- for IA. Inflammation was found to be driven more by opioid use than HIV positivity while IA was driven more by HIV than opioid use. In people with OUD, expression of CD38 on CD28-CD57+ senescent-like T cells was elevated and correlated positively with inflammation.DiscussionGiven the association of inflammation with a multitude of adverse health outcomes, our findings merit further investigations to understand the mechanistic pathways involved

T-regulatory cell modulation: the future of cancer immunotherapy?

T-regulatory cells suppress anti-tumour immunity in cancer patients and in murine tumour models. Furthermore, their activity is likely to have an effect on the effectiveness of immunotherapeutic treatments for cancer. Here we describe the current status of developing clinical strategies for modulating Treg activity in cancer patients

MUC1 alters oncogenic events and transcription in human breast cancer cells

INTRODUCTION: MUC1 is an oncoprotein whose overexpression correlates with aggressiveness of tumors and poor survival of cancer patients. Many of the oncogenic effects of MUC1 are believed to occur through interaction of its cytoplasmic tail with signaling molecules. As expected for a protein with oncogenic functions, MUC1 is linked to regulation of proliferation, apoptosis, invasion, and transcription. METHODS: To clarify the role of MUC1 in cancer, we transfected two breast cancer cell lines (MDA-MB-468 and BT-20) with small interfering (si)RNA directed against MUC1 and analyzed transcriptional responses and oncogenic events (proliferation, apoptosis and invasion). RESULTS: Transcription of several genes was altered after transfection of MUC1 siRNA, including decreased MAP2K1 (MEK1), JUN, PDGFA, CDC25A, VEGF and ITGAV (integrin α(v)), and increased TNF, RAF1, and MMP2. Additional changes were seen at the protein level, such as increased expression of c-Myc, heightened phosphorylation of AKT, and decreased activation of MEK1/2 and ERK1/2. These were correlated with cellular events, as MUC1 siRNA in the MDA-MB-468 line decreased proliferation and invasion, and increased stress-induced apoptosis. Intriguingly, BT-20 cells displayed similar levels of apoptosis regardless of siRNA, and actually increased proliferation after MUC1 siRNA. CONCLUSION: These results further the growing knowledge of the role of MUC1 in transcription, and suggest that the regulation of MUC1 in breast cancer may be more complex than previously appreciated. The differences between these two cell lines emphasize the importance of understanding the context of cell-specific signaling events when analyzing the oncogenic functions of MUC1, and caution against generalizing the results of individual cell lines without adequate confirmation in intact biological systems