Microbiology/Infectious Disease

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    Impairment of HIV-1 replication by UBE3A- and HIV-1 Nef-mediated regulation of proteasomal degradation.
    (2019-03-05) PARK, INWOO
    To date, regulation of HIV-1 life cycle has been mainly explained by the stage-specific expression of HIV-1 viral genes, even if elimination processes of the synthesized proteins after completion of their duties in the infected cells were quintessential for understanding of the molecular processes of the virus life cycle and thereby HIV-1 pathogenesis. Several lines of our experiments demonstrated that a key pathogenic HIV-1 viral protein, Nef, interacted with ubiquitin (Ub)-protein ligase E3A (UBE3A/E6AP), an important E3 Ub ligase in the proteasomal degradation processes, suggesting that interaction between Nef and UBE3A is integral in regulation of viral and cellular protein decay and thereby competition for survival between the entered HIV-1 and the infected host cells. In fact, Nef and UBE3A degraded reciprocally, and UBE3A-mediated degradation of Nef was significantly more potent than Nef-triggered degradation of UBE3A. Further, UBE3A degraded not only Nef but also HIV-1 structural proteins, Gag, thereby significantly inhibited HIV-1 replication in Jurkat T cells. However, UBE3A failed to induce decay of Gag in ∆nef-HIV-1 replicating cells, indicating that interaction between Nef and UBE3Awas pivotal for UBE3A-mediated degradation of the viral proteins. In contrast, Gag and Env did not degrade UBE3A. Mechanistic study showed that Nef and UBE3A were specific and antagonistic each other in regulating proteasome activity and ubiquitination of cellular proteins in general. Further, excess, not stoichiometric, amount of Nef reduced the amount of intracellular Gag as well as degraded a cardinal transcription regulator, Tat, demonstrating a broad role of Nef in regulation of HIV-1 life cycle. Structure and function analysis of Nef indicated that specific domains of Nef overlapping with LTR were essential for the observed actions. Taken together, these data indicated that the Nef and UBE3A complex play a pivotal role in coordinating viral protein degradation and hence HIV-1 replication, providing insights as to the nature of pathobiologic and defense strategies of HIV-1 and HIV-infected host cells and major clues for physical knockout of key viral pathogenic element, Nef, using UBE3A by the ubiquitin proteasome system.
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    A Systematic Review of the Utility of Procalcitonin in Bacterial Meningitis
    (2019-03-05) Gaviola, Dr. Marian; Mai, Steven
    Purpose/Objective: To review the utility of procalcitonin (PCT) in the management of bacterial meningitis (BM). Methods: An English-language MEDLINE search from 1964 through June 20, 2018 was completed using the following search terms: calcitonin, calcitonin gene-related peptides, meningitis: bacterial/diagnosis, and meningitis, bacterial/blood. Primary literature that evaluated the diagnostic value of PCT in adult patients for separating BM from viral or aseptic meningitis and studies comparing PCT with other biomarkers were included. Studies that did not consider antibiotic pretreatment as an exclusion criterion were excluded. Results: A total of 15 studies were identified for inclusion in this review. Ten studies evaluated the utility of PCT in distinguishing BM from non-bacterial meningitis (NBM) in a total of 1022 patients. Eight of these were prospective studies and two were retrospective. All 10 studies showed that PCT is significantly elevated in cases of BM compared to NBM, with the average elevation ranging from 0.5 ng/mL to 4.714 ng/mL in the serum and 0.2 ng/mL to 1.88 ng/mL in the cerebrospinal fluid (CSF). Three studies compared the diagnostic value of serum PCT versus non-serum PCT. All three studies were prospective clinical studies and included a total 502 patients. Two studies showed that serum PCT had a higher diagnostic value compared to CSF PCT and only one showed that CSF PCT is superior. Finally, no studies reviewed the use of PCT in guiding antibiotic de-escalation and no studies reviewed whether to use PCT in parallels or in series. However, two studies considered the use of PCT in guiding antibiotic usage and one study examined the use of PCT in conjunction with lactate. These studies showed that there are higher levels of CSF PCT in patients with gram-negative infections compared to those with gram-positive infections and explored the possibility of using PCT in addition to glucose to guide antibiotic therapy. Lastly, one study showed that there is higher specificity when using PCT with lactate in series and higher sensitivity when used in parallel. Conclusions: PCT is a useful biomarker in identifying cases of BM and serum PCT may be better than CSF PCT in identifying BM. However, larger studies are necessary to confirm these results and identify a standardized threshold. Additionally, more studies are necessary to explore the best way to utilize PCT and in diagnosing BM and the utility of PCT in guiding antibiotic therapy for BM.
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    The HIV-1 negative regulatory factor (Nef) is important for efficient virus production from astrocytes
    (2019-03-05) He, Johnny; KANDEL, SURESH
    The HIV-1 negative regulatory factor (Nef) is important for efficient virus production from astrocytes Suresh Kandel, MS and Johnny J. He, PhD Email: Johnny.He@unthsc.edu; Suresh.kandel@my.unthsc.edu Department of Microbiology, Immunology, and Genetics, University of North Texas Health Science Center, Fort Worth, TX 76107 Purpose: The HIV-1 negative regulatory factor (Nef) is a membrane associated myristoylated protein with a molecular weight of 27-32 KDa. It downregulates immune molecules such as MHC-I/CD4 receptors on CD4+ T lymphocytes and is indispensable for AIDS progression and high level of viremia. Additionally, Nef plays important roles in the virus production from immune cells and the infectivity of viruses released from these cells. However, much remained undefined about the involvement of Nef for virus production and infectivity in the context of CNS, particularly in astrocytes. Astrocytes are the most abundant long- lived cell types in the brain. HIV-1 infection leads to restrictive virus replication and establishes latency in these cells. Therefore, the main objective of this study is to determine the role of HIV-1 Nef in the virus production from astrocytes and the infectivity of viruses released from astrocytes. Methods: We transfected pNL4-3 and pNL4-3Nef- plasmids in astrocytic cell line LN299. Non-astrocytic cell line HEK 293T was used as a control. The virus production was determined using reverse transcriptase assay, while the infectivity of viruses was determined using a LTR-driven luciferase stably expressing cell line, TZM-BL. Results: In HEK 293T, the virus production was high and showed no difference between NL4-3 and NL4-3Nef-. In comparison to HEK 293T, the virus production was lower in LN299. There was more virus production in pNL4-3 transfected cells than pNL4-3Nef- transfected cells. There were no differences in infectivity of the viruses produced between pNL4-3-transfected cells and pNL4-3Nef- transfected cells in both HEK 293T and LN299. Conclusion: The results demonstrated that Nef expression gave rise to differences in virus production in astrocytes and suggest that Nef plays an important regulatory role in HIV gene expression in astrocytes. Further studies are underway to investigate the underlying mechanisms. Keywords: Nef, HIV-1, Astrocytes, budding, Infectivity
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    Metformin Enhanced HIV Gene Expression and Production
    (2019-03-05) He, Johnny; Rezaei, Sahar
    Purpose: HIV-1 adopts several factors of host machinery to generate a permissive environment for viral replication and transmission. HIV-1 enhances the activity of mTORC1 which appears to be necessary for the optimal expression of the structural viral protein Gag. In addition, HIV-1 hijacks the Rag GTPase/mTORC1 complex to modulate host cell function for optimal virus trafficking, assembly and/or budding. Exosomal release of HIV proteins primes cells for new infection. Metformin (1,1-dimethyl biguanide hydrochloride) is a USA Food and Drug Administration (FDA)-approved biguanide derivative and the most widely prescribed antihyperglycemic drug which is used as first-line therapy for diabetes mellitus type 2. Metformin inhibits mTORC1 by activation of AMPK. It has recently been shown that metformin can exert antiviral effects against Hepatitis B, Dengue, and Zika viruses. In this project, we aim to determine the effect of metformin on HIV replication/release using HIV transfected/infected cells. Methods: MTT (Methylthiazolyldiphenyl-tetrazolium bromide) was used to determine cell viability. Reverse transcriptase assay (RT) activity assay was used to determine the levels of virus replication and production in cells. Western blotting was used to determine intracellular protein expression. Western blotting followed by semiquantitative protein band detection was performed using a Bio-Rad ChemiDoc imaging system (Bio-Rad). Band intensities were calculated by measuring the ratio between the protein of interest to beta-actin. HIV LTR promoter-driven luciferase reporter cell line TZM-bl was used to determine the LTR promoter activity. Results: No effects on cell proliferation were noted in both 293T and TZM-bl treated up to 4 mM Metformin. Metformin did not alter HIV promoter activity. Metformin increased HIV virus production. Consistent with this finding, Metformin increased intracellular HIV gene expression, specifically Gag expression and Tat expression. In addition, Metformin did not appear to have any effects on the activity of HIV reverse transcriptase. Conclusion: These findings demonstrated that Metformin enhances HIV gene expression and production and suggest that Metformin may regulate steps of the HIV life cycle other than reverse transcription and HIV LTR promoter transcription. These findings also provide evidence to support metformin as a potential, low cost supplementary therapeutic agent for the elimination of latent viral reservoirs.