Analysis of SARS-CoV-2 variant mutations reveals neutralization escape mechanisms and the ability to use ACE2 receptors from additional species.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continue to emerge during the global pandemic and may facilitate escape from current antibody therapies and vaccine protection. Here we showed that the South African variant B.1.351 was the most resistant to current monoclonal antibodies and convalescent plasma from coronavirus disease 2019 (COVID-19)-infected individuals, followed by the Brazilian variant P.1 and the United Kingdom variant B.1.1.7. This resistance hierarchy corresponded with Y144del and 242-244del mutations in the N-terminal domain and K417N/T, E484K, and N501Y mutations in the receptor-binding domain (RBD) of SARS-CoV-2. Crystal structure analysis of the B.1.351 triple mutant (417N-484K-501Y) RBD complexed with the monoclonal antibody P2C-1F11 revealed the molecular basis for antibody neutralization and escape. B.1.351 and P.1 also acquired the ability to use mouse and mink ACE2 receptors for entry. Our results demonstrate major antigenic shifts and potential broadening of the host range for B.1.351 and P.1 variants, which poses serious challenges to current antibody therapies and vaccine protection.

Brain endothelial GSDMD activation mediates inflammatory BBB breakdown.

The blood-brain barrier (BBB) protects the central nervous system from infections or harmful substances1; its impairment can lead to or exacerbate various diseases of the central nervous system2-4. However, the mechanisms of BBB disruption during infection and inflammatory conditions5,6 remain poorly defined. Here we find that activation of the pore-forming protein GSDMD by the cytosolic lipopolysaccharide (LPS) sensor caspase-11 (refs. 7-9), but not by TLR4-induced cytokines, mediates BBB breakdown in response to circulating LPS or during LPS-induced sepsis. Mice deficient in the LBP-CD14 LPS transfer and internalization pathway10-12 resist BBB disruption. Single-cell RNA-sequencing analysis reveals that brain endothelial cells (bECs), which express high levels of GSDMD, have a prominent response to circulating LPS. LPS acting on bECs primes Casp11 and Cd14 expression and induces GSDMD-mediated plasma membrane permeabilization and pyroptosis in vitro and in mice. Electron microscopy shows that this features ultrastructural changes in the disrupted BBB, including pyroptotic endothelia, abnormal appearance of tight junctions and vasculature detachment from the basement membrane. Comprehensive mouse genetic analyses, combined with a bEC-targeting adeno-associated virus system, establish that GSDMD activation in bECs underlies BBB disruption by LPS. Delivery of active GSDMD into bECs bypasses LPS stimulation and opens the BBB. In CASP4-humanized mice, Gram-negative Klebsiella pneumoniae infection disrupts the BBB; this is blocked by expression of a GSDMD-neutralizing nanobody in bECs. Our findings outline a mechanism for inflammatory BBB breakdown, and suggest potential therapies for diseases of the central nervous system associated with BBB impairment.

Mitochondrial disturbance related to increased caspase-1 of CD4+T cells in HIV-1 infection.

BACKGROUND: In HIV-1 infection, more than 95% of CD4+T cells die of caspase-1 mediated pyroptosis. What governs the increased susceptibility of CD4+T cells to pyroptosis is poorly understood. METHODS: Blood samples were obtained from 31 untreated HIV-infected patients (UNT), 29 antiretroviral therapy treated HIV-infected patients (ART), and 21 healthy control donors (HD). Plasma levels of IL-18 and IL-1ß, caspase-1 expression, mitochondrial mass (MM) and mitochondrial fusion/fisson genes of CD4+T subsets were measured. RESULTS: A significantly higher IL-18 level in plasma and MM level of CD4+T cells were found in HIV-infected patients (UNT and ART) compared to HD, and the MMhigh phenotype was manifested, related to increased caspase-1 expression. Moreover, the increased MM was more pronounced in the early differentiated and inactivated CD4+T cells. However, higher MM was not intrinsically linked to T cell differentiation disorder or excessive activation of the CD4+T cells. Mechanistically, the increased MM was significantly correlated with an elevated level of expression of the mitochondrial fusion gene mitofusin1. CONCLUSION: An increase in MM was associated with heightened sensitivity of CD4+T cells to pyroptosis, even in early differentiated and inactivated CD4+T cells, in patients with HIV-1 infection, regardless of whether patients were on antiretroviral therapy or not. These new revelations have uncovered a previously unappreciated challenge to immune reconstitution with antiretroviral therapy.

Incidence of low-level viremia and its impact on virologic failure among people living with HIV who started an integrase strand transfer inhibitors: a longitudinal cohort study.

BACKGROUND: Low-level viremia (LLV) has been identified as a potential precursor to virologic failure (VF), yet its clinical implications, particularly within the context of Integrase Strand Transfer Inhibitors (INSTIs)-based regimens, remain insufficiently explored. The study aimed to investigate the relationship between LLV and VF within ART-naïve patients on INSTIs-based regimens in China. METHODS: A longitudinal cohort study was conducted with ART-naïve patients aged ≥ 18 years at Beijing Ditan Hospital, under the Chinese National Free Antiretroviral Treatment Program (NFATP). The LLV was defined as a viral load (VL) ranging from 50 to 199 copies/mL after six months of ART initiation, and VF as a VL ≥ 200 copies/mL. Sensitive analyses were also performed, defining LLV as 50-999 copies/mL and VF as exceeding 1000 copies/mL. Multivariate logistic regression, Kaplan-Meier (KM) curve, and Generalized Estimating Equation (GEE) models were used to evaluate the risk factors associated with LLV and VF events. RESULTS: The study involved 830 ART-naïve patients, comprising 600 in the INSTIs group and 230 in the protease inhibitors (PIs) group. LLV events were observed in 10.4% of patients on PIs-based regimens and and 3.2% on INSTIs-based regimens (P < 0.001). INSTIs-based regimens demonstrated a protective effect against LLV events (aHR = 0.27, 95% CI 0.137-0.532). VF events occurred in 10.9% of patients on PIs-based regimens and 2.0% on INSTIs-based regimens, respectively (P < 0.001). The occurrence of LLV events significantly increased the risk of VF by 123.5% (95% CI 7.5%-364.4%), while the integrase inhibitors were associated with a 76.9% (95% CI 59.1%-86.9%) reduction in VF risk. CONCLUSION: Our findings indicate that INSTIs-based regimens are critical protective factors against LLV and subsequent VF. These results underscore the importance of HIV viral load monitoring to ensuring effective treatment outcomes, highlighting the necessity for prompt and precise monitoring to refine HIV treatment methodologies.

Drug resistance profiles and influencing factors among HIV-infected children and adolescents receiving long-term ART: a multicentre observational study in China.

OBJECTIVES: To analyse the characteristics and determinants of drug resistance mutations (DRMs) in HIV-infected children and adolescents on long-term ART in China. METHODS: An observational cohort study was conducted in five centres. All participants younger than 15 years at ART initiation were screened, and those identified as having virological failure (VF) with viral load (VL) ≥ 400 copies/mL were included for genotypic resistance testing. Logistic regression analysis was performed and the accumulation of major mutations was analysed in a subgroup of resistant individuals with complete VL results since HIV diagnosis. RESULTS: Among 562 eligible participants, protease and RT regions were successfully amplified for 93 who failed treatment with a median of 10.0 years ART. Sixty-eight (73.1%) harboured ≥1 major mutations. NRTI, NNRTI and dual-class resistance accounted for 48.4%, 63.4% and 38.7%, respectively. Only 3.2% were resistant to PIs. Age at ART initiation [adjusted OR (aOR) = 0.813, 95% CI 0.690-0.957], subtype B (aOR = 4.378, 95% CI 1.414-13.560) and an initial NNRTI-based regimen (aOR = 3.331, 95% CI 1.180-9.402) were independently associated with DRMs. Among 40 resistant participants with additional VL data, 55.0% had continued VF on a suboptimal regimen and the estimated duration of VF was positively correlated with the total number of major mutations (r = 0.504, P = 0.001). CONCLUSIONS: The development of DRMs was common in children and adolescents receiving long-term treatment, and continued VF was prevalent in those with resistance. Timely genotypic testing and new child-friendly formulations are therefore urgently required.

Prevalence of doravirine cross-resistance in HIV-infected adults who failed first-line ART in China, 2014-18.

OBJECTIVES: To evaluate the prevalence and characteristics of doravirine resistance and cross-resistance in patients who failed first-line ART in China. METHODS: From 2014 to 2108, 4132 patients from five provinces were tested for drug resistance by genotypic resistance testing. Drug resistance mutations were assessed using the Stanford HIVdb algorithm Version 9.0. Sequences classified as having low-level, intermediate and high-level resistance were defined as having drug resistance. RESULTS: Overall, the prevalence of doravirine and other NNRTIs cross-resistance was 69.5%, with intermediate and high-level resistance accounting for 56.4%. Doravirine resistance highly correlated with efavirenz (r = 0.720) and nevirapine (r = 0.721) resistance and moderately correlated with etravirine (r = 0.637) and rilpivirine (r = 0.692) resistance. The most frequent doravirine-associated resistance mutations were V106M (8.7%), K101E (6.8%) and P225H (5.1%). High-level resistance was mainly due to Y188L (3.2%) and M230L (2.7%). There were significant differences between genotypes and provinces. Compared with CRF01_AE, CRF07_BC (OR = 0.595, 95% CI = 0.546-0.648) and CRF08_BC (OR = 0.467, 95% CI = 0.407-0.536) were associated with lower risks of doravirine resistance. Conversely, genotype A (OR = 3.003, 95% CI = 1.806-4.991) and genotype B (OR = 1.250, 95% CI = 1.021-1.531) were associated with higher risks of doravirine resistance. The risk of doravirine resistance was significantly lower in Xinjiang compared with other provinces. CONCLUSIONS: In China, the prevalence of doravirine cross-resistance among patients who have failed first-line ART is high. Therefore, doravirine should not be used blindly without genotypic resistance testing and is not recommended for people who have failed first-line NNRTI-based ART.

Quantitative Detection and Viral Load Analysis of SARS-CoV-2 in Infected Patients.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has become a public health emergency. The widely used reverse transcription-polymerase chain reaction (RT-PCR) method has limitations for clinical diagnosis and treatment. METHODS: A total of 323 samples from 76 COVID-19-confirmed patients were analyzed by droplet digital PCR (ddPCR) and RT-PCR based 2 target genes (ORF1ab and N). Nasal swabs, throat swabs, sputum, blood, and urine were collected. Clinical and imaging data were obtained for clinical staging. RESULTS: In 95 samples that tested positive by both methods, the cycle threshold (Ct) of RT-PCR was highly correlated with the copy number of ddPCR (ORF1ab gene, R2 = 0.83; N gene, R2 = 0.87). Four (4/161) negative and 41 (41/67) single-gene positive samples tested by RT-PCR were positive according to ddPCR with viral loads ranging from 11.1 to 123.2 copies/test. The viral load of respiratory samples was then compared and the average viral load in sputum (17 429 ±â€…6920 copies/test) was found to be significantly higher than in throat swabs (2552 ±â€…1965 copies/test, P < .001) and nasal swabs (651 ±â€…501 copies/test, P < .001). Furthermore, the viral loads in the early and progressive stages were significantly higher than that in the recovery stage (46 800 ±â€…17 272 vs 1252 ±â€…1027, P < .001) analyzed by sputum samples. CONCLUSIONS: Quantitative monitoring of viral load in lower respiratory tract samples helps to evaluate disease progression, especially in cases of low viral load.

Evaluation of the Diagnostic Accuracy of the CareStart™ Glucose-6-Phosphate Dehydrogenase Deficiency Rapid Diagnostic Test among Chinese Newborns.

Previous studies have shown that the CareStart™ G6PD Deficiency rapid diagnostic test has high diagnostic accuracy on G6PD deficiency in Africa and Thailand, but not in China. As there are regional differences of G6PD genotype distribution, we are attending to verify the effectiveness of the kit in Chinese population. The study cohort included 247 newborns admitted to our hospital for jaundice. The quantitative detection of G6PD enzyme activity and G6PD gene mutations analysis was used to classify the status of G6PD deficiency. The performance of CareStart™ assays was verified by calculating the sensitivity, specificity and area under the receiver operating characteristic curve (AUC) based on the corrected G6PD deficiency status. In male newborns, the sensitivity of the CareStart™ assay was 98.9%, the specificity was 94.2% and the AUC was 0.97. In female newborns, the sensitivity was 58.5% when the cutoff value of residual enzyme activity was 100%; however, the sensitivity was 100% when the cutoff value was 60%. Therefore, the CareStart™ test can effectively screen G6PD deficiency in male newborns and female infants with less than 60% residual enzyme activity, female infants with residual enzyme activities of 60-100% are more likely to be missed diagnosed among Chinese newborns.

Thirteen-year viral suppression and immunologic recovery of LPV/r-based regimens in pediatric HIV treatment: a multicenter cohort study in resource-constrained settings of China.

Background: Antiretroviral Therapy (ART) in children remains challenging due to resource-constrained settings. We conducted a 13-year, prospective, multicenter cohort study on the effectiveness and safety of LPV/r-based regimens in ART-naive and ART-experienced children. Methods: From January 2008 to May 2021, children living with HIV-1 were recruited with LPV/r-based regimens from 8 clinical research sites in 6 provinces in China. Effectiveness outcomes were virologic failure (defined as at least two consecutive measurements of VL > 200 copies/mL after 6 months of ART) and immune response (defined as CD4% recovered to more than 25% after 12 months of treatment). The safety outcomes were treatment-related grade 2-4 adverse events and abnormal laboratory test results. Results: A total of 345 ART-naïve children and 113 ART-experienced children were included in this cohort study. The median follow-up time was 7.3 (IQR 5.5-10.5) years. The incidence density of virologic failure was 4.1 (95% CI 3.3-4.9) per 100 person-years in ART-naïve children and 5.0 (95% CI 3.5-6.5) per 100 person-years in ART-experienced children. Kaplan Meyer (KM) curve analysis showed children with ART experience were at a higher risk of virologic failure (p < 0.05). The risk factors of virologic failure in ART-naïve children were clinic setting in rural hospitals (aHR = 2.251, 1.108-4.575), annual missed dose times >5 days of LPV intake (aHR = 1.889, 1.004-3.554); The risk factor of virologic failure in ART-experienced children was missed dose times >5 days (aHR = 2.689, 1.299-5.604) and mother as caregivers for ART administration (aHR = 0.475, 0.238-0.948). However, during long-term treatment, viral suppression rates between ART-naïve and ART-experienced children remained similar. No significant differences were observed in the immune response, treatment-related grade 2-4 events, and abnormal laboratory test results between ART-naïve children and ART-experienced children. Conclusion: Our research underscores that with consistent, long-term treatment of LPV/r-based regimens, ART-experienced children can achieve therapeutic outcomes comparable to ART-naïve children. It provides crucial insights on LPV/r-based regimens in pediatric HIV treatment, especially in resource-limited settings where high-cost Integrase Strand Transfer Inhibitors (INSTs) are inaccessible. This evidence-based understanding provides an essential addition to the global therapeutic strategies for pediatric HIV treatment.

Distribution pattern, molecular transmission networks, and phylodynamic of hepatitis C virus in China.

In China, few molecular epidemiological data on hepatitis C virus (HCV) are available and all previous studies were limited by small sample sizes or specific population characteristics. Here, we report characterization of the epidemic history and transmission dynamics of HCV strains in China. We included HCV sequences of individuals belonging to three HCV surveillance programs: 1) patients diagnosed with HIV infection at the Beijing HIV laboratory network, most of whom were people who inject drugs and former paid blood donors, 2) men who have sex with men, and 3) the general population. We also used publicly available HCV sequences sampled in China in our study. In total, we obtained 1,603 Ns5b and 865 C/E2 sequences from 1,811 individuals. The most common HCV strains were subtypes 1b (29.1%), 3b (25.5%) and 3a (15.1%). In transmission network analysis, factors independently associated with clustering included the region (OR: 0.37, 95% CI: 0.19-0.71), infection subtype (OR: 0.23, 95% CI: 0.1-0.52), and sampling period (OR: 0.43, 95% CI: 0.27-0.68). The history of the major HCV subtypes was complex, which coincided with some important sociomedical events in China. Of note, five of eight HCV subtype (1a, 1b, 2a, 3a, and 3b), which constituted 81.8% HCV strains genotyped in our study, showed a tendency towards decline in the effective population size during the past decade until present, which is a good omen for the goal of eliminating HCV by 2030 in China.

Efficacy and safety of ainuovirine versus efavirenz combination therapies with lamivudine/tenofovir disoproxil fumarate for medication of treatment-naïve HIV-1-positive adults: week 48 results of a randomized controlled phase 3 clinical trial followed by an open-label setting until week 96.

Background: Ainuovirine (ANV) is a new non-nucleoside reverse transcriptase inhibitor (NNRTI), which was initially synthesized in Korea and later further developed in both Korea and China. Methods: A randomized, double-blind, double-dummy, positive parallel group, non-inferiority, phase 3 trial was conducted in 7 sites across China. Eligible HIV-1-positive antiretroviral therapy (ART)-naïve adults aged 18-65 years were randomly assigned in a 1:1 ratio to receive tenofovir disoproxil fumarate and lamivudine (TDF+3TC) in combination with either ANV (ANV group) or efavirenz (EFV group) for up to 48 weeks. Subsequently, participants in both groups received one of the two drug combinations according to their choice until week 96 in an observational study under an open-label setting. The primary endpoint was the proportion of participants achieving HIV RNA <50 copies/mL at week 48, with non-inferiority pre-specified at a margin of 10%. The secondary efficacy endpoints were logarithmic changes in HIV RNA, percentage of participants with HIV RNA levels ≤400 copies/mL and changes in the CD4 T-cell count after 48 and 96 weeks of treatment, as well as the percentage of participants with HIV RNA levels <50 copies/mL at 96 weeks of treatment. Safety endpoints were the incidence of adverse events and laboratory abnormalities evaluated according to the Division of AIDS criteria. This study was registered with the Chinese Clinical Trial Registry (Registration number: ChiCTR1800019041). Findings: Between November 27, 2018 and March 11, 2021, a total of 826 participants were screened, and 630 were finally enrolled and randomly assigned (1:1) to either ANV (n = 315) or EFV (n = 315) groups. The mean age was 30.6 ± 9.4 years and most participants were male (94.6%). At week 48, 274 (87.0%) of 315 participants in the ANV group and 288 (91.7%) of 314 in the EFV group achieved HIV-1 RNA <50 copies/mL and non-inferiority was established (difference: -4.7%, 95% CI: -9.6 to 0.1%). In the period, 293 participants continued to take the ANV regimen and 287 switched from the EFV to the ANV regimen. During the open-label period, 92.5% (271/293) of participants in the continued ANV group and 95.1% (273/287) in the ANV to EFV transfer group remained virologically suppressed (HIV-1 RNA <50 copies/mL) at week 96 (p = 0.189). The incidence of NNRTI treatment-related adverse events (TEAEs) at week 48 was 67.6% in 315 participants in the ANV group, which was significantly lower than in 91.4% of 314 participants in the EFV group (p < 0.001). The most common TEAEs (weeks 0-48) were dizziness (10.5%) and dyslipidemia (22.2%) in the ANV group vs. 51.0% and 34.4% in the EFV group, respectively, followed by transaminase elevation (9.2% vs. 29.0%), γ-glutamyl transferase elevation (8.3% vs. 19.1%), and rash (7.9% vs. 18.8%) (all p < 0.001). After switching from EFV to ANV, TEAEs in the former EFV participants were significantly reduced in the following observational period of 48-96 weeks. Interpretation: The week 48 results indicated that the efficacy of ANV was non-inferior to EFV when combined with two NRTIs. The per-protocol risk difference at week 48 for the primary endpoint also supported non-inferiority. TEAEs in ANV treated participants were less frequent with regard to liver toxicity, dyslipidemia, neuropsychiatric symptoms and rash compared to the EFV group during the first 48 weeks of therapy. The effects were maintained during the 48-96 weeks of therapy. Funding: Jiangsu Aidea Pharmaceutical Co., Ltd.

Alterations in circulating markers in HIV/AIDS patients with poor immune reconstitution: Novel insights from microbial translocation and innate immunity.

After long-term anti-retroviral therapy (ART) treatment, most human immunodeficiency virus (HIV)/Acquired Immure Deficiency Syndrome (AIDS) patients can achieve virological suppression and gradual recovery of CD4+ T-lymphocyte (CD4+ T cell) counts. However, some patients still fail to attain normal CD4+ T cell counts; this group of patients are called immune non-responders (INRs), and these patients show severe immune dysfunction. The potential mechanism of poor immune reconstitution (PIR) remains unclear and the identification of uniform biomarkers to predict the occurrence of PIR is particularly vital. But limited information is available on the relationship between circulating markers of INRs and immune recovery. Hence, this review summarises alterations in the intestine microbiota and associated markers in the setting of PIR to better understand host-microbiota-metabolite interactions in HIV immune reconstitution and to identify biomarkers that can predict recovery of CD4+ T cell counts in INRs.

Elucidating the Gene Signatures and Immune Cell Types in HIV-Infected Immunological Non-Responders by Bioinformatics Analyses.

Objective: Numerous studies have reported on the pathogenesis of poor immune reconstitution (PIR) after antiretroviral treatment in human immunodeficiency virus (HIV) patients. However, fewer studies focused on both immune-related genes (IRGs) and immune cells, and the correlation between IRGs and immune cells was evaluated via bioinformatics analyses. Methods: Gene expression profiling of GSE143742 from the Gene Expression Omnibus (GEO) database was analyzed to get differentially expressed immune-related genes (DEIRGs). The enrichment analysis and protein-protein interaction (PPI) networks of DEIRGs were established. The relative fractions of 22 immune cell types were detected using the "CIBERSORT". The correlation analysis between DEIRGs and immune cells was constructed to discover the potential IRGs associated with immune cells. A logistic regression diagnostic model was built, and a receiver operating characteristic (ROC) curve was performed to evaluate the model's diagnostic efficacy. The CMap database was used to find molecules with therapeutic potential. RT-qPCR was used to verify the expression of the hub DEIRGs. Results: We identified eight types of significantly changed immune cells and five hub IRGs in INRs. The DEIRGs were mainly enriched in lymphocyte activation, receptor-ligand activity, and T cell receptor signaling pathway. The correlation analysis showed that the expression of TNF, CXCR4 and TFRC correlate with CD8 cells, resting mast cells, activated NK cells, and naïve CD4 cells in INRs. Meanwhile, TFRC and IL7R relate to activated NK cells and resting memory CD4 cells respectively in IRs. A diagnostic model was constructed using multiple logistic regression and nine small molecules were identified as possible drugs. Conclusion: In this study, we suggested that the process of PIR might be related to TNF, CXCR4, TFRC, CD48, and IL7R. And these IRGs play roles in regulating immune-competent cells. And our constructed diagnostic model has excellent effectiveness. Moreover, some small-molecule drugs are screened to alleviate PIR.

Evaluation of a Novel In-house HIV-1 Genotype Drug Resistance Assay using Clinical Samples in China.

BACKGROUND: HIV drug resistance poses a major challenge for anti-retroviral treatment (ART) and the prevention and control of HIV epidemic. OBJECTIVE: The study aims to establish a novel in-house assay with high efficiency, named AP inhouse method, that would be suitable for HIV-1 drug resistance detection in China. METHODS: An in-house HIV-1 genotyping method was used to sequence the partial pol gene from 60 clinical plasma samples; the results of our test were compared with a commercial ViroSeq HIV-1 genotyping system. RESULTS: Among sixty samples, 58(96.7%) were successfully amplified by AP in-house method, five of them harbored viral load below 1,000 copies/ml. The genotype distribution was 43.1% CRF07_ BC (25/58), 39.7% CRF01_AE (23/58), 6.9% CRF55_01B (4/58), 5.2% subtype B (3/58) and 5.2% CRF08_BC (3/58). Compared with that of the ViroSeq system, the consistent rate of these nucleotides and amino acids obtained by AP in-house method was up to 99.5 ± 0.4% and 99.5 ± 0.4%, respectively. A total of 290 HIV-1 drug resistance mutations were identified by two methods, including 126 nucleoside reverse transcriptase inhibitors (NRTIs), 145 non-nucleoside reverse transcriptase inhibitors (NNRTIs) and 19 protease inhibitors (PIs) resistance mutations. Out of them, 94.1% (273/290) were completely concordant between the AP in-house method and the ViroSeq system. CONCLUSION: Overall, the evaluation of AP in-house method provided comparable results to those of the ViroSeq system on diversified HIV-1 subtypes in China.

Comprehensive quantitative method for neurotransmitters to study the activity of a sedative-hypnotic candidate using microdialysis and LC×LC-MS/MS.

Neurotransmitters (NTs) and their metabolites play crucial roles in the regulation of the sleep-wake cycle. Thus, a comprehensive quantitative analysis of NTs would be useful in elucidating the potential mechanisms involved in sedative-hypnotic activities. In this study, we developed a high-throughput quantitative method based on a two-dimensional chromatography-mass spectrometry technique to simultaneously analyze 63 NTs and their metabolites in rat plasma, brain homogenate, and microdialysis samples from five different sleep-associated regions of the brain. Moreover, this method was used to study the neurochemical mechanism of an adenosine analog sedative-hypnotic candidate YZG-331. Most of the correlations between NTs were lost after the administration of the sedative, particularly in the caudate putamen (CPu) and dorsal raphe nucleus (DRN), indicating that the sleep-wake balance was affected. Administration of the adenosine analog YZG-331 could act similar as accumulation of adenosine, inducing adenosine and its metabolite adenine were decreased significantly in the CPu, accompanying with GABA, aspartate, and glutamate changed slightly by the communications between different neurons to further promote sleep. In addition, YZG-331 affected the metabolism of tryptophan and serotonin (5-HT) in the DRN and orbital frontal cortex (OFC). Melatonin and 5-hydroxyindole-3-acetic acid (a metabolite of 5-HT) were significantly increased in the OFC, and the levels of glutamate/glutamine, asparagine, and adrenaline were altered. Sleep homeostasis is a balance between the duration of sleep and wakefulness and is coordinated by all NTs. The high-throughput quantitative method introduced in this study may aid in revealing the temporal cohesion among NTs, evaluating sleep homeostasis, and determining the effects of sedative-hypnotic drugs.

The mechanism study of YZG-331 on sedative and hypnotic effects.

YZG-331 is a synthetic novel derivates of N6-(4-hydroxybenzyl) adenine riboside (NHBA), which has potent sedative and hypnotic effects based on our previous study. We are now aiming to investigate the mechanism of YZG-331. In this research, the behavioral studies showed that YZG-331 (4, 8, 16 mg/kg, i.g.) could reduce the spontaneous locomotor activity in mice, which could be blocked by AM (non-selective adenosine receptor antagonist), DPCPX (adenosine A1 receptor (A1R) antagonist), and SCH58261 (adenosine A2a receptor (A2aR) antagonist). Moreover, YZG-331 no longer exerted sedative effect in A1R or A2aR knockdown mice. YZG-331 (2.5, 5, 10 mg/kg, i.g.) prolonged sleeping time in pentobarbital sodium treated mice, which can be prevented by DPCPX or SCH58261. The above results demonstrated that YZG-331 exerted sedative and hypnotic effects through A1R and A2aR. In addition, it was found that YZG-331 (25, 50, 100 µM) decreased intracellular calcium level and YZG-331 (10 mg/kg, i.g.) decreased CaMKII phosphorylation (pCaMKII) level in mouse hypothalamus and cortex. In summary, this study indicated that activation of A1R/ A2aR and down regulation of Ca2+-CaMKII signaling pathway were involved in the sedative and hypnotic effects of YZG-331.

Accelerated Aging of T-Cell Subsets Among ART-Naïve HIV-Infected Chinese Men Who have Sex with Men: A Case-Control Study.

BACKGROUND: Evidence of lymphopoiesis, exhaustion, and premature aging in Chinese patients with human immunodeficiency virus (HIV) is very limited. OBJECTIVE: To assess biological aging and immune senescence in Chinese healthy controls (HC) and ART-naïve HIV-infected men who have sex with men (MSM). METHODS: This case-control study was conducted in Beijing Ditan Hospital from March 2018 to June 2019. The percentages of naïve (TN), central memory (TCM), effector memory (TEM), and terminally differentiated memory (TemRA) subsets of CD4 and CD8 T cells were studied, along with markers of senescence (CD28-CD57+) and activation (HLA-DR+). Telomere length of naïve (CD45RA+) and memory (CD45RO+) CD8 T cells were quantified by real-time PCR. RESULTS: A total of 26 HIV-infected and 20 age-matched HC MSM were included. Compared to the HC group, the CD4/CD8 ratio of the HIV-infected group was significantly reduced (0.30 vs. 1.70, P<0.001); significant differences emerged among all CD8 but not CD4 T cell subsets (all P<0.05). In the HIV-infected group, the percentages of senescent cells (CD28-CD57+) in TN, TCM, TEM, and TemRA subsets of CD8 T cells were higher (all P<0.05); while a significant difference was only found in naïve CD4 T cells (P<0.05). HLA-DR expression was increased significantly in all CD4 and CD8 T cell subsets. Both naïve (CD45RA+) and memory (CD45RO+) CD8 T cells in this population had significantly shorter telomere lengths (P<0.01) compared to the HC group. CONCLUSION: HIV-infected MSM exhibit signs of accelerated immune senescence and biological aging, which particularly affects the CD8 T-cell subsets.

Drug Resistance to HIV-1 Integrase Inhibitors Among Treatment-Naive Patients in Beijing, China.

Introduction: Integrase strand transfer inhibitors (INSTIs) are important drugs that are currently used as the first line treatment for HIV-1 patients. The aim of this study was to characterize HIV-1 INSTI mutations among ART-naive patients in Beijing from 2019-2021. Methods: 865 ART-naive patients were enrolled in this study between January 2019 and June 2021 in Beijing. The amplification of the entire pol gene containing the reverse transcriptase, protease and integrase regions was performed using a validated In-house SBS method. HIV-1 subtypes and circulating recombinant forms (CRFs) were determined using the COMET online tool (http://comet.retrovirology.lu). Stanford HIV-1 drug resistance database (HIVdb version 8.9) was used to analyze the mutations. Results: 865 HIV-1 pol sequences were successfully amplified and sequenced. Among them, no major INSTI-related mutations were identified, but 12 polymorphic accessory mutations were found. Two patients have E138A and G163R mutations respectively and both could cause low-level resistance to RAL and EVG. Furthermore, one patient having S230R mutation resulted in low-level resistance to RAL, EVG, DTG and BIC. Conclusion: The prevalence of INSTIs mutations remains low, which demonstrated that INSTIs have good applicability currently in our city. Nevertheless, it is very important to monitor the INSTI-related mutations in Beijing.