Sirtuin 3 Mediated by Spinal cMyc-Enhancer of Zeste Homology 2 Pathway Plays an Important Role in Human Immunodeficiency Virus-Related Neuropathic Pain Model.

BACKGROUND: Clinical data demonstrate that chronic use of opioid analgesics increases neuropathic pain in people living with human immunodeficiency virus (HIV). Therefore, it is important to elucidate the molecular mechanisms of HIV-related chronic pain. In this study, we investigated the role of the transcription factor cMyc, epigenetic writer enhancer of zeste homology 2 (EZH2), and sirtuin 3 (Sirt3) pathway in HIV glycoprotein gp120 with morphine (gp120M)-induced neuropathic pain in rats. METHODS: Neuropathic pain was induced by intrathecal administration of recombinant gp120 with morphine. Mechanical withdrawal threshold was measured using von Frey filaments, and thermal latency using the hotplate test. Spinal expression of cMyc, EZH2, and Sirt3 were measured using Western blots. Antinociceptive effects of intrathecal administration of antisense oligodeoxynucleotide against cMyc, a selective inhibitor of EZH2, or recombinant Sirt3 were tested. RESULTS: In the spinal dorsal horn, gp120M upregulated expression of cMyc (ratio of gp120M versus control, 1.68 ± 0.08 vs 1.00 ± 0.14, P = .0132) and EZH2 (ratio of gp120M versus control, 1.76 ± 0.05 vs 1.00 ± 0.16, P = .006), and downregulated Sirt3 (ratio of control versus gp120M, 1.00 ± 0.13 vs 0.43 ± 0.10, P = .0069) compared to control. Treatment with intrathecal antisense oligodeoxynucleotide against cMyc, GSK126 (EZH2 selective inhibitor), or recombinant Sirt3 reduced mechanical allodynia and thermal hyperalgesia in this gp120M pain model. Knockdown of cMyc reduced spinal EZH2 expression in gp120M treated rats. Chromatin immunoprecipitation (ChIP) assay showed that enrichment of cMyc binding to the ezh2 gene promoter region was increased in the gp120M-treated rat spinal dorsal horn, and that intrathecal administration of antisense ODN against cMyc (AS-cMyc) reversed the increased enrichment of cMyc. Enrichment of trimethylation of histone 3 on lysine residue 27 (H3K27me3; an epigenetic mark associated with the downregulation of gene expression) binding to the sirt3 gene promoter region was upregulated in the gp120M-treated rat spinal dorsal horn; that intrathecal GSK126 reversed the increased enrichment of H3K27me3 in the sirt3 gene promoter. Luciferase reporter assay demonstrated that cMyc mediated ezh2 gene transcription at the ezh2 gene promoter region, and that H3K27me3 silenced sirt3 gene transcription at the gene promoter region. CONCLUSION: These results demonstrated that spinal Sirt3 decrease in gp120M-induced neuropathic pain was mediated by cMyc-EZH2/H3K27me3 activity in an epigenetic manner. This study provided new insight into the mechanisms of neuropathic pain in HIV patients with chronic opioids.

Role of Tumor Necrosis Factor Receptor 1-Reactive Oxygen Species-Caspase 11 Pathway in Neuropathic Pain Mediated by HIV gp120 With Morphine in Rats.

BACKGROUND: Recent clinical research suggests that repeated use of opioid pain medications can increase neuropathic pain in people living with human immunodeficiency virus (HIV; PLWH). Therefore, it is significant to elucidate the exact mechanisms of HIV-related chronic pain. HIV infection and chronic morphine induce proinflammatory factors, such as tumor necrosis factor (TNF)α acting through tumor necrosis factor receptor I (TNFRI). HIV coat proteins and/or chronic morphine increase mitochondrial superoxide in the spinal cord dorsal horn (SCDH). Recently, emerging cytoplasmic caspase-11 is defined as a noncanonical inflammasome and can be activated by reactive oxygen species (ROS). Here, we tested our hypothesis that HIV coat glycoprotein gp120 with chronic morphine activates a TNFRI-mtROS-caspase-11 pathway in rats, which increases neuroinflammation and neuropathic pain. METHODS: Neuropathic pain was induced by repeated administration of recombinant gp120 with morphine (gp120/M) in rats. Mechanical allodynia was assessed using von Frey filaments, and thermal latency using hotplate test. Protein expression of spinal TNFRI and cleaved caspase-11 was examined using western blots. The image of spinal mitochondrial superoxide was examined using MitoSox Red (mitochondrial superoxide indicator) image assay. Immunohistochemistry was used to examine the location of TNFRI and caspase-11 in the SCDH. Intrathecal administration of antisense oligodeoxynucleotide (AS-ODN) against TNFRI, caspase-11 siRNA, or a scavenger of mitochondrial superoxide was given for antinociceptive effects. Statistical tests were done using analysis of variance (1- or 2-way), or 2-tailed t test. RESULTS: Intrathecal gp120/M induced mechanical allodynia and thermal hyperalgesia lasting for 3 weeks ( P < .001). Gp120/M increased the expression of spinal TNFRI, mitochondrial superoxide, and cleaved caspase-11. Immunohistochemistry showed that TNFRI and caspase-11 were mainly expressed in the neurons of the SCDH. Intrathecal administration of antisense oligonucleotides against TNFRI, Mito-Tempol (a scavenger of mitochondrial superoxide), or caspase-11 siRNA reduced mechanical allodynia and thermal hyperalgesia in the gp120/M neuropathic pain model. Spinal knockdown of TNFRI reduced MitoSox profile cell number in the SCDH; intrathecal Mito-T decreased spinal caspase-11 expression in gp120/M rats. In the cultured B35 neurons treated with TNFα, pretreatment with Mito-Tempol reduced active caspase-11 in the neurons. CONCLUSIONS: These results suggest that spinal TNFRI-mtROS-caspase 11 signal pathway plays a critical role in the HIV-associated neuropathic pain state, providing a novel approach to treating chronic pain in PLWH with opioids.

Hypertriglyceridemia-Induced Pancreatitis and a Lipemic Blood Sample: A Case Report and Brief Clinical Review.

Hypertriglyceridemia is the third most common cause of acute pancreatitis after gallstones and long-term alcohol use. There are specific therapeutic options unique to hyperglyceridemia-induced pancreatitis, such as continuous insulin therapy and plasmapheresis, emphasizing the importance of identifying hypertriglyceridemia as the cause. Triglyceride levels > 1000 mg/dL may result in a visibly lipemic blood sample. Lipemic samples may interfere with laboratory equipment, resulting in erroneous levels or the inability to measure several serum blood tests. Consider hypertriglyceridemia as a cause for acute pancreatitis in the setting of a lipemic blood sample or when gallstones have been excluded.

A lethargic child with COVID-19 infection.

ABSTRACT: COVID-19 infection in children is less understood than COVID-19 infection in adults, and although it is believed to cause mild or asymptomatic infections, several cases of severe or atypical presentations have been reported. Children presenting with gastrointestinal symptoms, even those without respiratory symptoms, should raise the suspicion for possible COVID-19 infection. This case report describes a 20-month-old girl with a clear history of COVID-19 exposure whose acute abdomen and lethargy were diagnosed as COVID-19 infection complicated by intussusception.

Impact of the Coronavirus Disease Pandemic on Mental Health among Local Residents in Korea: a Cross Sectional Study.

BACKGROUND: This study aimed to evaluate traumatic stress and mental health problems associated with the prolonged coronavirus disease pandemic and to determine the differences across different age groups. METHODS: A total of 1,151 individuals who visited Gwangmyeong City Mental Health Welfare Center, South Korea, or accessed the website from September 1 to December 31, 2020, were included in the study. Mental health problems such as traumatic stress (Primary Care Posttraumatic Stress Disorder Screen for the Diagnostic and Statistical Manual of Mental Disorder-5); depression (Patient Health Questionnaire-9 and Children's Depression Inventory); anxiety (Generalized Anxiety Disorder-7 and Penn State Worry Questionnaire for Children); suicide risk (P4 Screener); and demographic information were evaluated. The participants were divided into three groups based on age group: children and adolescents, adults, and the elderly. RESULTS: The results showed that 24.7%, 20.9%, 16.8%, and 20.5% of the participants were at high-risk for traumatic stress, depression, anxiety, and suicide, respectively. The difference in the proportion of high-risk groups by age of all participants was significant for traumatic stress, depression, anxiety, and suicide risk. In particular, the percentage of high-risk groups in all areas was the highest in the adult group. Also, in most areas, the ratio of the high-risk groups for children and adolescent group was the lowest, but the suicide risk-related ratio was not (adolescent group: 20.9%, adult group: 25%, elderly group 9.3%). CONCLUSION: These results suggest that there is a need for continued interest in the mental health of the general population even after the initial period of coronavirus disease. Additionally, this study may be helpful when considering the resilience or risk factors of mental health in a prolonged disaster situation.

Identification of Ebola Virus Inhibitors Targeting GP2 Using Principles of Molecular Mimicry.

A key step in the Ebola virus (EBOV) replication cycle involves conformational changes in viral glycoprotein 2 (GP2) which facilitate host-viral membrane fusion and subsequent release of the viral genome. Ebola GP2 plays a critical role in virus entry and has similarities in mechanism and structure to the HIV gp41 protein for which inhibitors have been successfully developed. In this work, a putative binding pocket for the C-terminal heptad repeat in the N-terminal heptad repeat trimer was targeted for identification of small molecules that arrest EBOV-host membrane fusion. Two computational structure-based virtual screens of ∼1.7 M compounds were performed (DOCK program) against a GP2 five-helix bundle, resulting in 165 commercially available compounds purchased for experimental testing. Based on assessment of inhibitory activity, cytotoxicity, and target specificity, four promising candidates emerged with 50% inhibitory concentration values in the 3 to 26 µM range. Molecular dynamics simulations of the two most potent candidates in their DOCK-predicted binding poses indicate that the majority of favorable interactions involve seven highly conserved residues that can be used to guide further inhibitor development and refinement targeting EBOV.IMPORTANCE The most recent Ebola virus disease outbreak, from 2014 to 2016, resulted in approximately 28,000 individuals becoming infected, which led to over 12,000 causalities worldwide. The particularly high pathogenicity of the virus makes paramount the identification and development of promising lead compounds to serve as inhibitors of Ebola infection. To limit viral load, the virus-host membrane fusion event can be targeted through the inhibition of the class I fusion glycoprotein of Ebolavirus In the current work, several promising small-molecule inhibitors that target the glycoprotein GP2 were identified through systematic application of structure-based computational and experimental drug design procedures.

Total FDG lesion number on PET/CT predicts survival of esophageal carcinoma patients with recurrence following curative surgery.

BACKGROUND: Total lesion number is a prognostic determinant in recurrent esophageal cancer, but this requires multiple tests. Here, we investigated the prognostic value of total FDG lesion number obtained from a single PET/CT study. METHODS: Subjects were 153 esophageal squamous cell carcinoma patients with loco-regional or distant recurrence following curative surgery. FDG PET/CT performed within 30 days was inspected for abnormal FDG uptake lesions using a SUVmax of 3.0 as threshold for significance. Prognostic associations were assessed by Cox proportional hazards regression and Kaplan-Meier analysis. RESULTS: PET/CT showed significant local FDG lesions in 49.0%, regional lesions in 78.4%, and distant lesions in 44.4% of patients. Among 73 patients with loco-regional recurrence, 54 had 0-3 and 19 had ≥4 FDG lesions. Among 80 patients with distant recurrence, 31 had 0-3 and 49 had ≥4 FDG lesions. During a median follow-up of 11.8 months, 99 deaths occurred. Univariate variables associated with poor survival included ≥4 FDG lesions and no treatment for loco-regional recurrence and no treatment for distant recurrence. Kaplan Meier analysis showed worse survival for ≥4 than 0-3 FDG lesions in patients with loco-regional recurrence (15.6 vs. 32.1 months; P=0.009), but not in those with distant recurrence. Significant independent predictors of poor survival were ≥4 FDG lesions and no treatment for loco-regional recurrence and no treatment for distant recurrence. CONCLUSIONS: Total FDG lesion number assessed by PET/CT is a significant independent prognostic factor in esophageal cancer patients with loco-regional recurrence following curative surgery.

Phosphorylated CCAAT/Enhancer Binding Protein ß Contributes to Rat HIV-Related Neuropathic Pain: In Vitro and In Vivo Studies.

Chronic pain is increasingly recognized as an important comorbidity of HIV-infected patients, however, the exact molecular mechanisms of HIV-related pain are still elusive. CCAAT/enhancer binding proteins (C/EBPs) are expressed in various tissues, including the CNS. C/EBPß, one of the C/EBPs, is involved in the progression of HIV/AIDS, but the exact role of C/EBPß and its upstream factors are not clear in HIV pain state. Here, we used a neuropathic pain model of perineural HIV envelope glycoprotein gp120 application onto the rat sciatic nerve to test the role of phosphorylated C/EBPß (pC/EBPß) and its upstream pathway in the spinal cord dorsal horn (SCDH). HIV gp120 induced overexpression of pC/EBPß in the ipsilateral SCDH compared with contralateral SCDH. Inhibition of C/EBPß using siRNA against C/EBPß reduced mechanical allodynia. HIV gp120 also increased TNFα, TNFRI, mitochondrial superoxide (mtO2·-), and pCREB in the ipsilateral SCDH. ChIP-qPCR assay showed that pCREB enrichment on the C/EBPß gene promoter regions in rats with gp120 was higher than that in sham rats. Intrathecal TNF soluble receptor I (functionally blocking TNFα bioactivity) or knockdown of TNFRI using antisense oligodeoxynucleotide against TNFRI reduced mechanical allodynia, and decreased mtO2·-, pCREB and pC/EBPß. Intrathecal Mito-tempol (a mitochondria-targeted O2·-scavenger) reduced mechanical allodynia and decreased pCREB and pC/EBPß. Knockdown of CREB with antisense oligodeoxynucleotide against CREB reduced mechanical allodynia and lowered pC/EBPß. These results suggested that the pathway of TNFα/TNFRI-mtO2·--pCREB triggers pC/EBPß in the HIV gp120-induced neuropathic pain state. Furthermore, we confirmed the pathway using both cultured neurons treated with recombinant TNFα in vitro and repeated intrathecal injection of recombinant TNFα in naive rats. This finding provides new insights in the understanding of the HIV neuropathic pain mechanisms and treatment.SIGNIFICANCE STATEMENT Painful HIV-associated sensory neuropathy is a neurological complication of HIV infection. Phosphorylated C/EBPß (pC/EBPß) influences AIDS progression, but it is still not clear about the exact role of pC/EBPß and the detailed upstream factors of pC/EBPß in HIV-related pain. In a neuropathic pain model of perineural HIV gp120 application onto the sciatic nerve, we found that pC/EBPß was triggered by TNFα/TNFRI-mtO2·--pCREB signaling pathway. The pathway was confirmed by using cultured neurons treated with recombinant TNFα in vitro, and by repeated intrathecal injection of recombinant TNFα in naive rats. The present results revealed the functional significance of TNFα/TNFRI-mtO2·--pCREB-pC/EBPß signaling in HIV neuropathic pain, and should help in the development of more specific treatments for neuropathic pain.

Ryanodine Receptor to Mitochondrial Reactive Oxygen Species Pathway Plays an Important Role in Chronic Human Immunodeficiency Virus gp120MN-Induced Neuropathic Pain in Rats.

BACKGROUND: Chronic pain is one of the most common complaints in patients with human immunodeficiency virus (HIV)-associated sensory neuropathy. Ryanodine receptor (RyR) and mitochondrial oxidative stress are involved in neuropathic pain induced by nerve injury. Here, we investigated the role of RyR and mitochondrial superoxide in neuropathic pain induced by repeated intrathecal HIV glycoprotein 120 (gp120) injection. METHODS: Recombinant HIV glycoprotein gp120MN was intrathecally administered to induce neuropathic pain. Mechanical threshold was tested using von Frey filaments. Peripheral nerve fiber was assessed by the quantification of the intraepidermal nerve fiber density in the skin of the hindpaw. The expression of spinal RyR was examined using Western blots. Colocalization of RyR with neuronal nuclei (NeuN; neuron marker), glial fibrillary acidic protein (GFAP; astrocyte marker), or ionizing calcium-binding adaptor molecule 1 (Iba1; microglia marker) in the spinal cord was examined using immunohistochemistry. MitoSox-positive profiles (a mitochondrial-targeted fluorescent superoxide indicator) were examined. The antiallodynic effects of intrathecal administration of RyR antagonist, dantrolene (a clinical drug for malignant hyperthermia management), or selective mitochondrial superoxide scavenger, Mito-Tempol, were evaluated in the model. RESULTS: We found that repeated but not single intrathecal injection of recombinant protein gp120 induced persistent mechanical allodynia. Intraepidermal nerve fibers in repeated gp120 group was lower than that in sham at 2 weeks, and the difference in means (95% confidence interval) was 8.495 (4.79-12.20), P = .0014. Repeated gp120 increased expression of RyR, and the difference in means (95% confidence interval) was 1.50 (0.504-2.495), P = .007. Repeated gp120 also increased mitochondrial superoxide cell number in the spinal cord, and the difference in means (95% confidence interval) was 6.99 (5.99-8.00), P < .0001. Inhibition of spinal RyR or selective mitochondrial superoxide scavenger dose dependently reduced mechanical allodynia induced by repeated gp120 injection. RyR and mitochondrial superoxide were colocalized in the neuron, but not glia. Intrathecal injection of RyR inhibitor lowered mitochondrial superoxide in the spinal cord dorsal horn in the gp120 neuropathic pain model. CONCLUSIONS: These data suggest that repeated intrathecal HIV gp120 injection induced an acute to chronic pain translation in rats, and that neuronal RyR and mitochondrial superoxide in the spinal cord dorsal horn played an important role in the HIV neuropathic pain model. The current results provide evidence for a novel approach to understanding the molecular mechanisms of HIV chronic pain and treating chronic pain in patients with HIV.

Role of nociceptin/orphanin FQ and nociceptin opioid peptide receptor in depression and antidepressant effects of nociceptin opioid peptide receptor antagonists.

Nociceptin/orphanin FQ (N/OFQ) and its receptor, nociceptin opioid peptide (NOP) receptor, are localized in brain areas implicated in depression including the amygdala, bed nucleus of the stria terminalis, habenula, and monoaminergic nuclei in the brain stem. N/OFQ inhibits neuronal excitability of monoaminergic neurons and monoamine release from their terminals by activation of G protein-coupled inwardly rectifying K+ channels and inhibition of voltage sensitive calcium channels, respectively. Therefore, NOP receptor antagonists have been proposed as a potential antidepressant. Indeed, mounting evidence shows that NOP receptor antagonists have antidepressant-like effects in various preclinical animal models of depression, and recent clinical studies again confirmed the idea that blockade of NOP receptor signaling could provide a novel strategy for the treatment of depression. In this review, we describe the pharmacological effects of N/OFQ in relation to depression and explore the possible mechanism of NOP receptor antagonists as potential antidepressants.

Structure-based identification of inhibitors targeting obstruction of the HIVgp41 N-heptad repeat trimer.

The viral protein HIVgp41 is an attractive and validated drug target that proceeds through a sequence of conformational changes crucial for membrane fusion, which facilitates viral entry. Prior work has identified inhibitors that interfere with the formation of a required six-helix bundle, composed of trimeric C-heptad (CHR) and N-heptad (NHR) repeat elements, through blocking association of an outer CHR helix or obstructing formation of the inner NHR trimer itself. In this work, we employed similarity-based scoring to identify and experimentally characterize 113 compounds, related to 2 small-molecule inhibitors recently reported by Allen et al. (Bioorg. Med. Chem Lett.2015, 25 2853-59), proposed to act via the NHR trimer obstruction mechanism. The compounds were first tested in an HIV cell-cell fusion assay with the most promising evaluated in a second, more biologically relevant viral entry assay. Of the candidates, compound #11 emerged as the most promising hit (IC50=37.81µM), as a result of exhibiting activity in both assays with low cytotoxicity, as was similarly seen with the known control peptide inhibitor C34. The compound also showed no inhibition of VSV-G pseudotyped HIV entry compared to a control inhibitor suggesting it was specific for HIVgp41. Molecular dynamics simulations showed the predicted DOCK pose of #11 interacts with HIVgp41 in an energetic fashion (per-residue footprints) similar to the four native NHR residues (IQLT) which candidate inhibitors were intended to mimic.

Inhibition of Mitochondrial Fission Protein Reduced Mechanical Allodynia and Suppressed Spinal Mitochondrial Superoxide Induced by Perineural Human Immunodeficiency Virus gp120 in Rats.

BACKGROUND: Mitochondria play an important role in many cellular and physiologic functions. Mitochondria are dynamic organelles, and their fusion and fission regulate cellular signaling, development, and mitochondrial homeostasis. The most common complaint of human immunodeficiency virus (HIV)-sensory neuropathy is pain on the soles in patients with HIV, but the exact molecular mechanisms of HIV neuropathic pain are not clear. In the present study, we investigated the role of mitochondrial dynamin-related protein 1 (Drp1, a GTPase that mediates mitochondrial fission) in the perineural HIV coat glycoprotein gp120-induced neuropathic pain state. METHODS: Neuropathic pain was induced by the application of recombinant HIV-1 envelope protein gp120 into the sciatic nerve. Mechanical threshold was tested using von Frey filaments. The mechanical threshold response was assessed over time using the area under curves. Intrathecal administration of antisense oligodeoxynucleotide (ODN) against Drp1, mitochondrial division inhibitor-1 (mdivi-1), or phenyl-N-tert-butylnitrone (a reactive oxygen species scavenger) was given. The expression of spinal Drp1 was examined using western blots. The expression of mitochondrial superoxide in the spinal dorsal horn was examined using MitoSox imaging. RESULTS: Intrathecal administration of either antisense ODN against Drp1 or mdivi-1 decreased mechanical allodynia (a sensation of pain evoked by nonpainful stimuli) in the gp120 model. Intrathecal ODN or mdivi-1 did not change basic mechanical threshold in sham surgery rats. Intrathecal Drp1 antisense ODN decreased the spinal expression of increased Drp1 protein induced by peripheral gp120 application. Intrathecal phenyl-N-tert-butylnitrone reduced mechanical allodynia. Furthermore, both intrathecal Drp1 antisense ODN and mdivi-1 reversed the upregulation of mitochondrial superoxide in the spinal dorsal horn in the gp120 neuropathic pain state. CONCLUSIONS: These data suggest that mitochondrial division plays a substantial role in the HIV gp120-related neuropathic pain state through mitochondrial reactive oxygen species and provides evidence for a novel approach to treating chronic pain in patients with HIV.

The membrane proximal external regions of gp41 from HIV-1 strains HXB2 and JRFL have different sensitivities to alanine mutation.

The transmembrane subunit (gp41) of the HIV envelope protein complex (Env) mediates the viral fusion step of HIV entry. The membrane proximal external region (MPER), one of the functional domains of gp41, has been the focus of a great deal of research because it is a target for neutralizing antibodies. In this study, we examined 23 amino acid residues in the MPER (660-683) in both a CXCR4 coreceptor-utilizing strain (HXB2) and a CCR5-utilizing strain (JRFL) by alanine scanning mutagenesis. Despite the high degree of gp41 sequence conservation, the effects of alanine mutation in the MPER were different between the two strains. Most mutations in HXB2 had fusogenicity and protein expression levels not less than 50% of that of the wild type in the case of cell-cell fusion. However, ∼30% of the mutants in HXB2 showed a severe defect in fusogenicity in viral entry. Mutations in the MPER of strain JRFL had more dramatic effects than that in HXB2 in cell-cell fusion and viral entry. The fact that there are large differences in the effects of mutation between two strains suggests the potential for the interaction of the MPER with nonconserved sequences such as the fusion peptide and/or other NHR domains as well as potential long-range structural effects on the conformational changes that occur with the Env complex during membrane fusion.

Small molecule inhibitors of HIVgp41 N-heptad repeat trimer formation.

Identification of mechanistically novel anti-HIV fusion inhibitors was accomplished using a computer-aided structure-based design approach with the goal of blocking the formation of the N-heptad repeat (NHR) trimer of the viral protein gp41. A virtual screening strategy that included per-residue interaction patterns (footprints) was employed to identify small molecules compatible with putative binding pockets at the internal interface of the NHR helices at the core native viral six-helix bundle. From a screen of ∼2.8 million compounds using the DOCK program, 120 with favorable energetic and footprint overlap characteristics were purchased and experimentally tested leading to two compounds with favorable cell-cell fusion (IC50) and cytotoxicity profiles. Importantly, both hits were identified on the basis of scores containing footprint overlap terms and would not have been identified using the standard DOCK energy function alone. To our knowledge, these compounds represent the first reported small molecules that inhibit viral entry via the proposed NHR-trimer obstruction mechanism.

Gene Transfer of Glutamic Acid Decarboxylase 67 by Herpes Simplex Virus Vectors Suppresses Neuropathic Pain Induced by Human Immunodeficiency Virus gp120 Combined with ddC in Rats.

BACKGROUND: Human immunodeficiency virus (HIV)-related painful sensory neuropathies primarily consist of the HIV infection-related distal sensory polyneuropathy and antiretroviral toxic neuropathies. Pharmacotherapy provides only partial relief of pain in patients with HIV/acquired immune deficiency syndrome because little is known about the exact neuropathological mechanisms for HIV-associated neuropathic pain (NP). Hypofunction of γ-aminobutyric acid (GABA) GABAergic inhibitory mechanisms has been reported after peripheral nerve injury. In this study, we tested the hypothesis that HIV gp120 combined with antiretroviral therapy reduces spinal GABAergic inhibitory tone and that restoration of GABAergic inhibitory tone will reduce HIV-related NP in a rat model. METHODS: The application of recombinant HIV-1 envelope protein gp120 into the sciatic nerve plus systemic ddC (one antiretroviral drug) induced mechanical allodynia. The hind paws of rats were inoculated with replication-defective herpes simplex virus (HSV) vectors genetically encoding gad1 gene to express glutamic acid decarboxylase 67 (GAD67), an enzyme that catalyzes the decarboxylation of glutamate to GABA. Mechanical threshold was tested using von Frey filaments before and after treatments with the vectors. The expression of GAD67 in both the lumbar spinal cord and the L4-5 dorsal root ganglia was examined using western blots. The expression of mitochondrial superoxide in the spinal dorsal horn was examined using MitoSox imaging. The immunoreactivity of spinal GABA, pCREB, and pC/EBPß was tested using immunohistochemistry. RESULTS: In the gp120 with ddC-induced neuropathic pain model, GAD67 expression mediated by the HSV vector caused an elevation of mechanical threshold that was apparent on day 3 after vector inoculation. The antiallodynic effect of the single HSV vector inoculation expressing GAD67 lasted >28 days. The area under the time-effect curves in the HSV vector expressing GAD67 was increased compared with that in the control vectors (P = 0.0005). Intrathecal GABA-A/B agonists elevated mechanical threshold in the pain model. The HSV vectors expressing GAD67 reversed the lowered GABA immunoreactivity in the spinal dorsal horn in the neuropathic rats. HSV vectors expressing GAD67 in the neuropathic rats reversed the increased signals of mitochondrial superoxide in the spinal dorsal horn. The vectors expressing GAD67 reversed the upregulated immunoreactivity expression of pCREB and pC/EBPß in the spinal dorsal horn in rats exhibiting NP. CONCLUSIONS: Based on our results, we suggest that GAD67 mediated by HSV vectors acting through the suppression of mitochondrial reactive oxygen species and transcriptional factors in the spinal cord decreases pain in the HIV-related neuropathic pain model, providing preclinical evidence for gene therapy applications in patients with HIV-related pain states.

Mechanical allodynia induced by nucleoside reverse transcriptase inhibitor is suppressed by p55TNFSR mediated by herpes simplex virus vector through the SDF1α/CXCR4 system in rats.

BACKGROUND: In the human immunodeficiency virus (HIV)-associated sensory neuropathy, neuropathic pain associated with the use of nucleoside reverse transcriptase inhibitors (NRTIs) in patients with HIV/acquired immunodeficiency syndrome is clinically common. While evidence demonstrates that neuropathic pain is influenced by neuroinflammatory events that include the proinflammatory molecules, tumor necrosis factor-α (TNF-α), stromal cell-derived factor 1-α (SDF1-α), and C-X-C chemokine receptor type 4 (CXCR4), the detailed mechanisms by which NRTIs contribute to the development of neuropathic pain are not known. In this study, we investigated the role of these proinflammatory molecules in the dorsal root ganglion (DRG) and the spinal dorsal horn in NRTIs-mediated neuropathic pain state. METHODS: Neuropathic pain was induced by intraperitoneal administration of 2',3'-dideoxycytidine (ddC, one of the NRTIs). Mechanical threshold was tested using von Frey filament fibers. Nonreplicating herpes simplex virus (HSV) vectors expressing p55 TNF soluble receptor (p55TNFSR) were inoculated into hindpaw of rats. The expression of TNF-α, SDF1-α, and CXCR4 in both the lumbar spinal cord and the L4/5 DRG was examined using Western blots. Intrathecal CXCR4 antagonist was administered. RESULTS: The present study demonstrated that (1) systemic ddC induced upregulation of TNF-α, SDF1-α, and CXCR4 in both the lumbar spinal cord and the L4/5 DRG; (2) p55TNFSR mediated by a nonreplicating HSV vector reversed mechanical allodynia induced by systemic ddC; (3) intrathecal administration of the CXCR4 antagonist AMD3100 increased mechanical threshold; and (4) HSV vector expressing p55TNFSR reversed upregulation of TNF-α, SDF1-α, and CXCR4 induced by ddC in the lumbar spinal dorsal horn and the DRG. CONCLUSIONS: Our studies demonstrate that TNF-α through the SDF1/CXCR4 system is involved in the NRTIs-related neuropathic pain state and that blocking the signaling of these proinflammatory molecules is able to reduce NRTIs-related neuropathic pain. These results provide a novel mechanism-based approach (gene therapy) to treating HIV-associated neuropathic pain.

Effects of signal sequences and folding accessory proteins on extracellular expression of carboxypeptidase Y in recombinant Saccharomyces cerevisiae.

Carboxypeptidase Y (CPY) is a yeast vacuolar protease with useful applications including C-terminal sequencing of peptides and terminal modification of target proteins. To overexpress CPY with the pro-sequence (proCPY) encoded by the Saccharomyces cerevisiae PRC1 gene in recombinant S. cerevisiae, the proCPY gene was combined with the gene coding for a signal sequence of S. cerevisiae mating factor α (MFα), invertase (SUC2), or Kluyveromyces marxianus inulinase (INU1). Among the three constructs, the MFα signal sequence gave the best specific activity of extracellular CPY. To enhance the CPY expression level, folding accessory proteins of Kar2p, Pdi1p and Ero1p located in the S. cerevisiae endoplasmic reticulum were expressed individually and combinatorially. A single expression of Kar2p led to a 28 % enhancement in extracellular CPY activity, relative to the control strain of S. cerevisiae CEN.PK2-1D/p426Gal1-MFαCPY. Coexpression of Kar2p, Pdi1p and Ero1p gave a synergistic effect on CPY expression, of which activity was 1.7 times higher than that of the control strain. This work showed that engineering of signal sequences and protein-folding proteins would be helpful to overexpress yeast proteins of interest.

Sargassum horneri extract fermented by Lactiplantibacillus pentosus SH803 mediates adipocyte metabolism in 3T3-L1 preadipocytes by regulating oxidative damage and inflammation.

Sargassum horneri (S. horneri), a brown seaweed excessively proliferating along Asian coastlines, are damaging marine ecosystems. Thus, this study aimed to enhance nutritional value of S. horneri through lactic acid bacteria fermentation to increase S. horneri utilization as a functional food supplement, and consequently resolve coastal S. horneri accumulation. S. horneri supplemented fermentation was most effective with Lactiplantibacillus pentosus SH803, thus this product (F-SHWE) was used for further in vitro studies. F-SHWE normalized expressions of oxidative stress related genes NF-κB, p53, BAX, cytochrome C, caspase 9, and caspase 3, while non-fermented S. horneri (SHWE) did not, in a H2O2-induced HT-29 cell model. Moreover, in an LPS-induced HT-29 cell model, F-SHWE repaired expressions of inflammation marker genes ZO1, IL1ß, IFNγ more effectively than SHWE. For further functional assessment, F-SHWE was also treated in 3T3-L1 adipocytes. As a result, F-SHWE decreased lipid accumulation, along with gene expression of adipogenesis markers PPARγ, C/EBPα, C/EBPß, aP2, and Lpl; lipogenesis markers Lep, Akt, SREBP1, Acc, Fas; inflammation markers IFN-γ and NF-κB. Notably, gene expression of C/EBPß, IFN-γ and NF-κB were suppressed only by F-SHWE, suggesting the enhancing effect of fermentation on obesity-related properties. Compositional analysis attributed the protective effects of F-SHWE to acetate, an organic acid significantly higher in F-SHWE than SHWE. Therefore, F-SHWE is a novel potential anti-obesity agent, providing a strategy to reduce excess S. horneri populations along marine ecosystems.

Significant differences in cell-cell fusion and viral entry between strains revealed by scanning mutagenesis of the C-heptad repeat of HIV gp41.

The transmembrane subunit, gp41, of the HIV envelope mediates the viral fusion step of entry into the host cell. The protein consists of an extracellular domain, a transmembrane domain, and a cytoplasmic tail. The extracellular domain contains a fusion peptide, an N-terminal heptad repeat, a loop region, a C-terminal heptad repeat (CHR), and a membrane-proximal external region. For this study, we examined each amino acid in the CHR (residues 623-659) by alanine scanning mutagenesis in two HIV strains: one CCR5-utilizing strain (JRFL) and one CXCR4-utilizing strain (HXB2). We studied the functional importance of each amino acid residue by measuring mutational effects in both cell-cell fusion and viral entry and assessing envelope expression and gp120-gp41 proteolytic processing. The transmembrane subunit of the HIV envelope, gp41, is very sensitive to subtle changes, like alanine substitution, which severely affect envelope function at multiple sites. Two important general findings are apparent when the entire data set from this study is taken into account. (1) Strain HXB2 is much more stable to mutagenesis than strain JRFL, and (2) viral entry is much more stable to mutagenesis than cell-cell fusion. These findings strengthen our notion that gp41 is a vulnerable target for therapeutic and prophylactic intervention. Further structural studies aimed at gaining a full understanding of the intermediate states that drive HIV membrane fusion are imperative.

Perception and Experience of Sexual and Gender Minority Korean Youth in School Counseling.

To comprehensively explore the school counseling experience of sexual and gender minority (SGM) youths in South Korea, we interviewed 14 SGM youths about their school counseling experience and the climate of their schools toward SGM. Results showed that a hostile school climate and the accessibility, confidentiality, trustworthiness, and LGBTQ competency of school counselors drove the reluctance of these youths to receive school counseling services. Implications for the role of school counseling services for SGM youths were discussed based on the findings of this work and the suggestions offered by the interviewed SGM youths. Supplementary Information: The online version contains supplementary material available at 10.1007/s10447-022-09490-0.