Liver Bacterial Dysbiosis With Non-Tuberculosis Mycobacteria Occurs in SIV-Infected Macaques and Persists During Antiretroviral Therapy

Liver disease is a significant contributor to morbidity and mortality in HIV-infected individuals, even during successful viral suppression with combination antiretroviral therapy (cART). Similar to HIV infection, SIV infection of rhesus macaques is associated with gut microbiome dysbiosis and microbial translocation that can be detected systemically in the blood. As microbes leaving the intestines must first pass through the liver via the portal vein, we evaluated the livers of both SIV-infected (SIV+) and SIV-infected cART treated (SIV+cART) rhesus macaques for evidence of microbial changes compared to uninfected macaques. Dysbiosis was observed in both the SIV+ and SIV+cART macaques, encompassing changes in the relative abundance of several genera, including a reduction in the levels of Lactobacillus and Staphylococcus. Most strikingly, we found an increase in the relative abundance and absolute quantity of bacteria within the Mycobacterium genus in both SIV+ and SIV+cART macaques. Multi-gene sequencing identified a species of atypical mycobacteria similar to the opportunistic pathogen M. smegmatis. Phosphatidyl inositol lipoarabinomannan (PILAM) (a glycolipid cell wall component found in atypical mycobacteria) stimulation in primary human hepatocytes resulted in an upregulation of inflammatory transcriptional responses, including an increase in the chemokines associated with neutrophil recruitment (CXCL1, CXCL5, and CXCL6). These studies provide key insights into SIV associated changes in hepatic microbial composition and indicate a link between microbial components and immune cell recruitment in SIV+ and SIV+cART treated macaques.

Lipid Alterations in African American Men with Prostate Cancer

African-American (AA) men are more than twice as likely to die of prostate cancer (PCa) than European American (EA) men. Previous in silico analysis revealed enrichment of altered lipid metabolic pathways in pan-cancer AA tumors. Here, we performed global unbiased lipidomics profiling on 48 matched localized PCa and benign adjacent tissues (30 AA, 24 ancestry-verified, and 18 EA, 8 ancestry verified) and quantified 429 lipids belonging to 14 lipid classes. Significant alterations in long chain polyunsaturated lipids were observed between PCa and benign adjacent tissues, low and high Gleason tumors, as well as associated with early biochemical recurrence, both in the entire cohort, and within AA patients. Alterations in cholesteryl esters, and phosphatidyl inositol classes of lipids delineated AA and EA PCa, while the levels of lipids belonging to triglycerides, phosphatidyl glycerol, phosphatidyl choline, phosphatidic acid, and cholesteryl esters distinguished AA and EA PCa patients with biochemical recurrence. These first-in-field results implicate lipid alterations as biological factors for prostate cancer disparities.

High Dimensional Immune Profiling Reveals Different Response Patterns in Active and Latent Tuberculosis Following Stimulation With Mycobacterial Glycolipids

Upon infection with Mycobacterium tuberculosis (Mtb) the host immune response might clear the bacteria, control its growth leading to latent tuberculosis (LTB), or fail to control its growth resulting in active TB (ATB). There is however no clear understanding of the features underlying a more or less effective response. Mtb glycolipids are abundant in the bacterial cell envelope and modulate the immune response to Mtb, but the patterns of response to glycolipids are still underexplored. To identify the CD45+ leukocyte activation landscape induced by Mtb glycolipids in peripheral blood of ATB and LTB, we performed a detailed assessment of the immune response of PBMCs to the Mtb glycolipids lipoarabinomannan (LAM) and its biosynthetic precursor phosphatidyl-inositol mannoside (PIM), and purified-protein derivate (PPD). At 24 h of stimulation, cell profiling and secretome analysis was done using mass cytometry and high-multiplex immunoassay. PIM induced a diverse cytokine response, mainly affecting antigen-presenting cells to produce both pro-inflammatory and anti-inflammatory cytokines, but not IFN-γ, contrasting with PPD that was a strong inducer of IFN-γ. The effect of PIM on the antigen-presenting cells was partly TLR2-dependent. Expansion of monocyte subsets in response to PIM or LAM was reduced primarily in LTB as compared to healthy controls, suggesting a hyporesponsive/tolerance pattern derived from Mtb infection.

Mass Spectrometry and Computer Simulation Predict the Interactions of AGPS and HNRNPK in Glioma

Ether lipids are overexpressed in malignant tumor and play an important role in tumor process. Glioma is the most common malignant central nervous system tumor, and the content of ether lipids is higher than that of normal tissues. Alkylglycerone phosphate synthase (AGPS) is a key enzyme in the synthesis of ether esters and plays a vital role in maintaining the morphology and pathogenic properties of tumor cells. The cell proliferation and the content of tumor-related lipid such as monoalkylglycerol ether (MAGe), lysophosphatidic acid ether (LPAe), lysophosphatidylcholine ether (LPCe), lysophosphatidylethanolamine ether (LPEe), phosphatidyl inositol (PI), phosphatidylcholine (PC), and phosphatidylserine (PS) were suppressed after AGPS silencing in U251, H4, and TJ905 cells; however, heterogeneous nuclear ribonucleoprotein K (HNRNPK) could reverse the above phenomenon such as cellar proliferation and ether lipid secretion. We found that HNRNPK was the target protein of AGPS by coimmunoprecipitation and mass spectrometry assay and verified by western blot assay in U251 cells. It confirmed that AGPS and HNRNPK are coexpressed in the cellular nucleus by a confocal laser microscope. The main protein-protein interaction mechanism between AGPS and HNRNPK is hydrogen bond, conjugation bond, hydrophobic bond, and electrostatic force by computer simulation prediction.

Molecular determinants of the modulation of the VSD-PD coupling mechanism of the KV7.1 channel by the KCNE1 ancillary subunits

The IKS current is diffused through the plasma membranes of cardiomyocytes during the last phase of the cardiac action potential. This repolarization current is conducted by a tetrameric protein complex derived from the co-expression of four voltage-gated potassium channel KV7.1 α-subunits and KCNE1 ancillary subunits from KCNQ1 and KCNE1 genes, respectively. We studied here the conformational space of KV7.1 in presence and absence of KCNE1, by building transmembrane models of their known Resting, Intermediate, and Activated states. We conducted Molecular Dynamics simulations of these models in lipid bilayers including the phosphatidyl-inositol-4,5-bisphosphate (PIP2) lipids. The comparative analysis of MD trajectories obtained for the KV7.1 and IKS models reveals how KCNE1 shifts the coupling mechanism between the activation state of the Voltage Sensor Domain of the channel and the conformation (open or closed) of its Pore Domain.

Kazrin C entraps early endosomes at the pericentriolar region and facilitates endocytic recycling

We identified kazrin C as a human protein that inhibits clathrin-mediated endocytosis when overexpressed. We now generated kazrin knock out and GFP-kazrin C expressing MEF lines to investigate in detail its function in endocytic traffic. We find that kazrin depletion delays recycling of internalized material and causes accumulation and dispersal of early endosomes (EE), indicating a role in transport from the early to the perinuclear recycling endosomes (RE). Consistently, we found that the C-terminal domain of kazrin C, predicted to be an intrinsically disordered region (IDR), specifically interacts with several endosomal components, including Epsin Homology Domain (EHD) proteins, γ-adaptin, and phosphatidyl-inositol-3 phosphate. Further, kazrin C shares homology with dynein/dynactin adaptors, it directly interacts with the dynactin complex and the dynein light intermediate chain LIC1, and overexpressed GFP-kazrin C forms condensates that entrap EE in the vicinity of the centrosome, in a microtubule-dependent manner. Altogether, the data indicates that kazrin C facilitates cargo recycling by trapping EE or EE-derived transport intermediates at the perinuclear region, where transfer of cargo to the RE might occur.

Simulating PIP2-Induced Gating Transitions in Kir6.2 Channels

ATP-sensitive potassium (KATP) channels consist of an inwardly rectifying K+ channel (Kir6.2) pore, to which four ATP-sensitive sulfonylurea receptor (SUR) domains are attached, thereby coupling K+ permeation directly to the metabolic state of the cell. Dysfunction is linked to neonatal diabetes and other diseases. K+ flux through these channels is controlled by conformational changes in the helix bundle region, which acts as a physical barrier for K+ permeation. In addition, the G-loop, located in the cytoplasmic domain, and the selectivity filter might contribute to gating, as suggested by different disease-causing mutations. Gating of Kir channels is regulated by different ligands, like Gβγ, H+, Na+, adenosine nucleotides, and the signaling lipid phosphatidyl-inositol 4,5-bisphosphate (PIP2), which is an essential activator for all eukaryotic Kir family members. Although molecular determinants of PIP2 activation of KATP channels have been investigated in functional studies, structural information of the binding site is still lacking as PIP2 could not be resolved in Kir6.2 cryo-EM structures. In this study, we used Molecular Dynamics (MD) simulations to examine the dynamics of residues associated with gating in Kir6.2. By combining this structural information with functional data, we investigated the mechanism underlying Kir6.2 channel regulation by PIP2.

Novel Mechanism for an Old Drug: Phenazopyridine is a Kinase Inhibitor Affecting Autophagy and Cellular Differentiation

Phenazopyridine is a widely used drug against urinary tract pain. The compound has also been shown to enhance neural differentiation of pluripotent stem cells. However, its mechanism of action is not understood. Based on its chemical structure, we hypothesized that phenazopyridine could be a kinase inhibitor. Phenazopyridine was investigated in the following experimental systems: 1) activity of kinases in pluripotent stem cells; 2) binding to recombinant kinases, and 3) functional impact on pluripotent stem cells. Upon addition to pluripotent stem cells, phenazopyridine induced changes in kinase activities, particularly involving Mitogen-Activated Protein Kinases, Cyclin-Dependent Kinases, and AKT pathway kinases. To identify the primary targets of phenazopyridine, we screened its interactions with 401 human kinases. Dose-inhibition curves showed that three of these kinases interacted with phenazopyridine with sub-micromolar binding affinities: cyclin-G-associated kinase, and the two phosphatidylinositol kinases PI4KB and PIP4K2C, the latter being known for participating in pain induction. Docking revealed that phenazopyridine forms strong H-bonds with the hinge region of the ATP-binding pocket of these kinases. As previous studies suggested increased autophagy upon inhibition of the phosphatidyl-inositol/AKT pathway, we also investigated the impact of phenazopyridine on this pathway and found an upregulation. In conclusion, our study demonstrates for the first time that phenazopyridine is a kinase inhibitor, impacting notably phosphatidylinositol kinases involved in nociception.

Structural insights into the role of DNA-PK as a master regulator in NHEJ

DNA-dependent protein kinase catalytic subunit DNA-PKcs/PRKDC is the largest serine/threonine protein kinase of the phosphatidyl inositol 3-kinase-like protein kinase (PIKK) family and is the most highly expressed PIKK in human cells. With its DNA-binding partner Ku70/80, DNA-PKcs is required for regulated and efficient repair of ionizing radiation-induced DNA double-strand breaks via the non-homologous end joining (NHEJ) pathway. Loss of DNA-PKcs or other NHEJ factors leads to radiation sensitivity and unrepaired DNA double-strand breaks (DSBs), as well as defects in V(D)J recombination and immune defects. In this review, we highlight the contributions of the late Dr. Carl W. Anderson to the discovery and early characterization of DNA-PK. We furthermore build upon his foundational work to provide recent insights into the structure of NHEJ synaptic complexes, an evolutionarily conserved and functionally important YRPD motif, and the role of DNA-PKcs and its phosphorylation in NHEJ. The combined results identify DNA-PKcs as a master regulator that is activated by its detection of two double-strand DNA ends for a cascade of phosphorylation events that provide specificity and efficiency in assembling the synaptic complex for NHEJ.

Gordonia jinghuaiqii sp. nov. and Gordonia zhaorongruii sp. nov., isolated from Tibetan Plateau wildlife

Four mesophilic and Gram-stain-positive strains (zg-686T/zg-691 and HY186T/HY189) isolated from Tibetan Plateau wildlife (PR China) belong to the genus Gordonia according to 16S rRNA gene and genomic sequence-based phylogenetic/genomic results. They have a DNA G+C content range of 67.4–68.3 mol% and low DNA relatedness (19.2–27.6 %) with all available genomes in the genus Gordonia . Strains zg-686T/zg-691 and HY186T/HY189 had C18 : 1ω9c, C18 : 0 10-methyl, C16 : 1 ω7c/C16 : 1ω6c and C16 : 0 as major cellular fatty acids. The polar lipids detected in strains zg-686T and HY186T included diphosphatidylglycerol, phosphatidylethanolamine, phosphatidyl inositol mannoside and phosphatidylinositol. The respiratory quinones comprised MK8(H2) (10.8 %) and MK9(H2) (89.2 %) for strain zg-686T, and MK6 (7.7 %), MK8(H2) (8.4 %), MK8(H4) (3.1 %) and MK9(H2) (80.8 %) for strain HY186T. Optimal growth conditions were pH 7.0, 35–37 °C and 0.5–1.5 % NaCl (w/v) for strains pair zg-686T/zg-691, and pH 7.0, 28 °C and 1.5 % (w/v) NaCl for strains pair HY186T/HY189. Based on these genotypic and phenotypic results, these four strains could be classified as two different novel species in the genus Gordonia , for which the names Gordonia jinghuaiqii sp. nov. and Gordonia zhaorongruii sp. nov. are proposed. The type strains are zg-686T (=GDMCC 1.1715T =JCM 33890T) and HY186T (=CGMCC 4.7607T =JCM 33466T), respectively.