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Circulation Research Jun 2024Calcium (Ca) uptake by mitochondria occurs via the mitochondrial Ca uniporter. Mitochondrial Ca uniporter exists as a complex, regulated by 3 MICU (mitochondrial Ca...
BACKGROUND
Calcium (Ca) uptake by mitochondria occurs via the mitochondrial Ca uniporter. Mitochondrial Ca uniporter exists as a complex, regulated by 3 MICU (mitochondrial Ca uptake) proteins localized in the intermembrane space: MICU1, MICU2, and MICU3. Although MICU3 is present in the heart, its role is largely unknown.
METHODS
We used CRISPR-Cas9 to generate a mouse with global deletion of MICU3 and an adeno-associated virus (AAV9) to overexpress MICU3 in wild-type mice. We examined the role of MICU3 in regulating mitochondrial calcium ([Ca]) in ex vivo hearts using an optical method following adrenergic stimulation in perfused hearts loaded with a Ca-sensitive fluorophore. Additionally, we studied how deletion and overexpression of MICU3, respectively, impact cardiac function in vivo by echocardiography and the molecular composition of the mitochondrial Ca uniporter complex via Western blot, immunoprecipitation, and Blue native-PAGE analysis. Finally, we measured MICU3 expression in failing human hearts.
RESULTS
MICU3 knock out hearts and cardiomyocytes exhibited a significantly smaller increase in [Ca] than wild-type hearts following acute isoproterenol infusion. In contrast, heart with overexpression of MICU3 exhibited an enhanced increase in [Ca] compared with control hearts. Echocardiography analysis showed no significant difference in cardiac function in knock out MICU3 mice relative to wild-type mice at baseline. However, mice with overexpression of MICU3 exhibited significantly reduced ejection fraction and fractional shortening compared with control mice. We observed a significant increase in the ratio of heart weight to tibia length in hearts with overexpression of MICU3 compared with controls, consistent with hypertrophy. We also found a significant decrease in MICU3 protein and expression in failing human hearts.
CONCLUSIONS
Our results indicate that increased and decreased expression of MICU3 enhances and reduces, respectively, the uptake of [Ca] in the heart. We conclude that MICU3 plays an important role in regulating [Ca] physiologically, and overexpression of MICU3 is sufficient to induce cardiac hypertrophy, making MICU3 a possible therapeutic target.
Topics: Animals; Humans; Calcium-Binding Proteins; Mitochondria, Heart; Mice; Myocytes, Cardiac; Mice, Knockout; Male; Mitochondrial Membrane Transport Proteins; Calcium; Cardiomegaly; Mice, Inbred C57BL; Calcium Channels; Calcium Signaling; Heart Failure; Cation Transport Proteins; Female
PubMed: 38747181
DOI: 10.1161/CIRCRESAHA.123.324026 -
Frontiers in Immunology 2023Influenza virus is responsible for a large global burden of disease, especially in children. Multiple Organ Dysfunction Syndrome (MODS) is a life-threatening and fatal...
BACKGROUND
Influenza virus is responsible for a large global burden of disease, especially in children. Multiple Organ Dysfunction Syndrome (MODS) is a life-threatening and fatal complication of severe influenza infection.
METHODS
We measured RNA expression of 469 biologically plausible candidate genes in children admitted to North American pediatric intensive care units with severe influenza virus infection with and without MODS. Whole blood samples from 191 influenza-infected children (median age 6.4 years, IQR: 2.2, 11) were collected a median of 27 hours following admission; for 45 children a second blood sample was collected approximately seven days later. Extracted RNA was hybridized to NanoString mRNA probes, counts normalized, and analyzed using linear models controlling for age and bacterial co-infections (FDR q<0.05).
RESULTS
Comparing pediatric samples collected near admission, children with Prolonged MODS for ≥7 days (n=38; 9 deaths) had significant upregulation of nine mRNA transcripts associated with neutrophil degranulation ( compared to those who recovered more rapidly from MODS (n=27). These neutrophil transcripts present in early samples predicted Prolonged MODS or death when compared to patients who recovered, however in paired longitudinal samples, they were not differentially expressed over time. Instead, five genes involved in protein metabolism and/or adaptive immunity signaling pathways (, ) were associated with MODS recovery within a week.
CONCLUSION
Thus, early increased expression of neutrophil degranulation genes indicated worse clinical outcomes in children with influenza infection, consistent with reports in adult cohorts with influenza, sepsis, and acute respiratory distress syndrome.
Topics: Humans; Multiple Organ Failure; Influenza, Human; Transcriptome; Phenotype; Hospitalization; Bacterial Infections
PubMed: 37533854
DOI: 10.3389/fimmu.2023.1220028 -
Nature Aug 2023HIV-1 remains a global health crisis, highlighting the need to identify new targets for therapies. Here, given the disproportionate HIV-1 burden and marked human genome...
HIV-1 remains a global health crisis, highlighting the need to identify new targets for therapies. Here, given the disproportionate HIV-1 burden and marked human genome diversity in Africa, we assessed the genetic determinants of control of set-point viral load in 3,879 people of African ancestries living with HIV-1 participating in the international collaboration for the genomics of HIV. We identify a previously undescribed association signal on chromosome 1 where the peak variant associates with an approximately 0.3 log-transformed copies per ml lower set-point viral load per minor allele copy and is specific to populations of African descent. The top associated variant is intergenic and lies between a long intergenic non-coding RNA (LINC00624) and the coding gene CHD1L, which encodes a helicase that is involved in DNA repair. Infection assays in iPS cell-derived macrophages and other immortalized cell lines showed increased HIV-1 replication in CHD1L-knockdown and CHD1L-knockout cells. We provide evidence from population genetic studies that Africa-specific genetic variation near CHD1L associates with HIV replication in vivo. Although experimental studies suggest that CHD1L is able to limit HIV infection in some cell types in vitro, further investigation is required to understand the mechanisms underlying our observations, including any potential indirect effects of CHD1L on HIV spread in vivo that our cell-based assays cannot recapitulate.
Topics: Humans; Cell Line; DNA Helicases; DNA-Binding Proteins; Genetic Variation; HIV Infections; HIV-1; Viral Load; Africa; Chromosomes, Human, Pair 1; Alleles; RNA, Long Noncoding; Virus Replication
PubMed: 37532928
DOI: 10.1038/s41586-023-06370-4 -
Oncoimmunology 2023Lung cancer remains among the most difficult-to-treat malignancies and is the leading cause of cancer-related deaths worldwide. The introduction of targeted therapies...
Lung cancer remains among the most difficult-to-treat malignancies and is the leading cause of cancer-related deaths worldwide. The introduction of targeted therapies and checkpoint inhibitors has improved treatment outcomes; however, most patients with advanced-stage non-small cell lung cancer (NSCLC) eventually fail these therapies. Therefore, there is a major unmet clinical need for checkpoint refractory/resistant NSCLC. Here, we tested the combination of aPD-1 and adenovirus armed with TNFα and IL-2 (Ad5-CMV-mTNFα/mIL-2) in an immunocompetent murine NSCLC model. Moreover, although local delivery has been standard for virotherapy, treatment was administered intravenously to facilitate clinical translation and putative routine use. We showed that treatment of tumor-bearing animals with aPD-1 in combination with intravenously injected armed adenovirus significantly decreased cancer growth, even in the presence of neutralizing antibodies. We observed an increased frequency of cytotoxic tumor-infiltrating lymphocytes, including tumor-specific cells. Combination treatment led to a decreased percentage of immunosuppressive tumor-associated macrophages and an improvement in dendritic cell maturation. Moreover, we observed expansion of the tumor-specific memory T cell compartment in secondary lymphoid organs in the group that received aPD-1 with the virus. However, although the non-replicative Ad5-CMV-mTNFα/mIL-2 virus allows high transgene expression in the murine model, it does not fully reflect the clinical outcome in humans. Thus, we complemented our findings using NSCLC ex vivo models fully permissive for the TNFα and IL-2- armed oncolytic adenovirus TILT-123. Overall, our data demonstrate the ability of systemically administered adenovirus armed with TNFα and IL-2 to potentiate the anti-tumor efficacy of aPD-1 and warrant further investigation in clinical trials.
Topics: Animals; Humans; Mice; Adenoviridae; Carcinoma, Non-Small-Cell Lung; Interleukin-2; Lung Neoplasms; Tumor Necrosis Factor-alpha; Immune Checkpoint Inhibitors
PubMed: 37546696
DOI: 10.1080/2162402X.2023.2241710 -
Virus Research Aug 2024The human JC polyomavirus (JCV) is a widespread, neurotropic, opportunistic pathogen responsible for progressive multifocal leukoencephalopathy (PML) as well as other...
The human JC polyomavirus (JCV) is a widespread, neurotropic, opportunistic pathogen responsible for progressive multifocal leukoencephalopathy (PML) as well as other diseases in immunosuppressed individuals, including granule cell neuronopathy, JCV-associated nephropathy, encephalitis, and meningitis in rare cases. JCV classification is still unclear, where the ICTV (International Committee on Taxonomy of Viruses) has grouped all the strains into human polyomavirus 2, with no classification on clade and subclade levels. Therefore, JCV strains were previously classified using different genomic regions, e.g., full-length, VP1, and the V-T intergenic region etc., and the strains were grouped into several types related to various geographic locations and human ethnicities. However, neither of these classifications and nomenclature contemplates all the groups described so far. Herein, we evaluated all the available full-length coding genomes, VP1, and large T antigen nucleotide sequences of JCV reported during 1993-2023 and classified them into four major phylogenetic clades, i.e., GI-GIV, where GI is further grouped into two types GI.1 and GI.2 with five sub-clades each (GI.1/GI.2 a-e), GII into three (GII a-c), GIII as a separate clade, and GIV into seven sub-clades (GIV a-g). Similarly, the phylogeographic network analysis indicated four major clusters corresponding to GI-GIV clades, each with multiple subclusters and mutational sub-branches corresponding to the subclades. GI and GIV clusters are connected via GI.1-e reported from Europe and America, GII, GIII and GIV clusters are connected by GII-b and GII-c strains reported from Africa, while GIV cluster strains are connected to the Russia-Italy JCV haplotype. Furthermore, we identified JCV-variant-GS/B-Germany-1997 (GenBank ID: AF004350.1) as an inter-genotype recombinant having major and minor parents in the GI.1-e and GII-a clades, respectively. Additionally, the amino acid variability analysis revealed high entropy across all proteins. The large T antigen exhibited the highest variability, while the small t antigen showed the lowest variability. Our phylogenetic and phylogeographic analyses provide a new approach to genotyping and sub-genotyping and present a comprehensive classification system of JCV strains based on their genetic characteristics and geographic distribution, while the genetic recombination and amino acid variability can help identify pathogenicity and develop effective preventive and control measures against JCV infections.
Topics: JC Virus; Phylogeography; Humans; Phylogeny; Genome, Viral; Leukoencephalopathy, Progressive Multifocal; Polyomavirus Infections; Genetic Variation; Cluster Analysis
PubMed: 38848817
DOI: 10.1016/j.virusres.2024.199414 -
Frontiers in Cellular and Infection... 2023Banna virus (BAV), a potential pathogen that may cause human encephalitis, is the prototype species of genus within the family , and has been isolated from a variety of...
INTRODUCTION
Banna virus (BAV), a potential pathogen that may cause human encephalitis, is the prototype species of genus within the family , and has been isolated from a variety of blood-sucking insects and mammals in Asia.
METHODS
, , and were collected overnight in Yunnan, China, during 2016-2023 using light traps. Virus was isolated from these collected blood-sucking insects and grown using (C6/36) cells. Preliminary identification of the virus was performed by agarose gel electrophoresis (AGE). The full genome sequences of the BAVs were determined by full-length amplification of cDNAs (FLAC) and sequenced using next-generation sequencing.
RESULTS
In this study, 13 strains BAV were isolated from , and . Their viral genome consisted of 12 segments of double-stranded RNA (dsRNA), and with three distinct distribution patterns. Sequence analysis showed that Seg-5 of four strains (SJ_M46, SJ_M49, JC_M19-13 and JC_C24-13) has 435 bases nucleotide sequence insertions in their ORF compared to other BAVs, resulting in the length of Seg-5 up to 2128 nt. There are 34 bases sequence deletion in Seg-9 of 3 strains (WS_T06, MS_M166 and MS_M140). Comparison of the coding sequences of VP1, VP2, VP5, VP9 and VP12 of the 13 BAV strains, the results show that VP1, VP2 and VP12 are characterised by high levels of sequence conservation, while VP9 is highly variable, under great pressure to adapt and may be correlated with serotype. While also variable, VP5 appears to be under less adaptive pressure than VP9. Additionally, phylogenetic analysis indicates that the 13 BAV strains locate in the same evolutionary cluster as BAVs isolated from various blood-sucking insects, and are clustered according to geographical distribution.
CONCLUSION
The data obtained herein would be beneficial for the surveillance of evolutionary characteristics of BAV in China and neighboring countries as well as extend the knowledge about its genomic diversity and geographic distribution.
Topics: Animals; Aedes; Ceratopogonidae; China; Coltivirus; Genome, Viral; Mammals; Phylogeny; Ticks
PubMed: 38035340
DOI: 10.3389/fcimb.2023.1283580 -
IScience Dec 2023Infection with West Nile virus (WNV) drives a wide range of responses, from asymptomatic to flu-like symptoms/fever or severe cases of encephalitis and death. To...
Infection with West Nile virus (WNV) drives a wide range of responses, from asymptomatic to flu-like symptoms/fever or severe cases of encephalitis and death. To identify cellular and molecular signatures distinguishing WNV severity, we employed systems profiling of peripheral blood from asymptomatic and severely ill individuals infected with WNV. We interrogated immune responses longitudinally from acute infection through convalescence employing single-cell protein and transcriptional profiling complemented with matched serum proteomics and metabolomics as well as multi-omics analysis. At the acute time point, we detected both elevation of pro-inflammatory markers in innate immune cell types and reduction of regulatory T cell activity in participants with severe infection, whereas asymptomatic donors had higher expression of genes associated with anti-inflammatory CD16 monocytes. Therefore, we demonstrated the potential of systems immunology using multiple cell-type and cell-state-specific analyses to identify correlates of infection severity and host cellular activity contributing to an effective anti-viral response.
PubMed: 38047068
DOI: 10.1016/j.isci.2023.108387 -
Clinical Infectious Diseases : An... Aug 2023There are conflicting data regarding baseline determinants of virological nonsuppression outcomes in persons with human immunodeficiency virus (HIV) starting...
Plasma Human Immunodeficiency Virus 1 RNA and CD4+ T-Cell Counts Are Determinants of Virological Nonsuppression Outcomes With Initial Integrase Inhibitor-Based Regimens: A Prospective RESPOND Cohort Study.
BACKGROUND
There are conflicting data regarding baseline determinants of virological nonsuppression outcomes in persons with human immunodeficiency virus (HIV) starting antiretroviral treatment (ART). We evaluated the impact of different baseline variables in the RESPOND cohort.
METHODS
We included treatment-naive participants aged ≥18 who initiated 3-drug ART, in 2014-2020. We assessed the odds of virological suppression (VS) at weeks 48 and 96 using logistic regression. Viral blips, low-level viremia (LLV), residual viremia (RV), and virological failure (VF) rates were assessed using Cox regression.
RESULTS
Of 4310 eligible participants, 72% started integrase strand transfer inhibitor (INSTI)-based regimens. At 48 and 96 weeks, 91.0% and 93.3% achieved VS, respectively. At 48 weeks, Kaplan-Meier estimates of rates were 9.6% for viral blips, 2.1% for LLV, 22.2% for RV, and 2.1% for VF. Baseline HIV-1 RNA levels >100 000 copies/mL and CD4+ T-cell counts ≤200/µL were negatively associated with VS at weeks 48 (adjusted odds ratio, 0.51 [95% confidence interval, .39-.68] and .40 [.27-.58], respectively) and 96 and with significantly higher rates of blips, LLV, and RV. CD4+ T-cell counts ≤200/µL were associated with higher risk of VF (adjusted hazard ratio, 3.12 [95% confidence interval, 2.02-4.83]). Results were consistent in those starting INSTIs versus other regimens and those starting dolutegravir versus other INSTIs.
CONCLUSIONS
Initial high HIV-1 RNA and low CD4+ T-cell counts are associated with lower rates of VS at 48 and 96 weeks and higher rates of viral blips, LLV, and RV. Low baseline CD4+ T-cell counts are associated with higher VF rates. These associations remain with INSTI-based and specifically with dolutegravir-based regimens. These findings suggest that the impact of these baseline determinants is independent of the ART regimen initiated.
Topics: Humans; CD4-Positive T-Lymphocytes; Cohort Studies; HIV Infections; HIV Integrase Inhibitors; HIV-1; Prospective Studies; Viral Load; Viremia; RNA, Viral
PubMed: 37052343
DOI: 10.1093/cid/ciad219 -
Communications Biology Oct 2023The respiratory syncytial virus polymerase complex, consisting of the polymerase (L) and phosphoprotein (P), catalyzes nucleotide polymerization, cap addition, and cap...
The respiratory syncytial virus polymerase complex, consisting of the polymerase (L) and phosphoprotein (P), catalyzes nucleotide polymerization, cap addition, and cap methylation via the RNA dependent RNA polymerase, capping, and Methyltransferase domains on L. Several nucleoside and non-nucleoside inhibitors have been reported to inhibit this polymerase complex, but the structural details of the exact inhibitor-polymerase interactions have been lacking. Here, we report a non-nucleoside inhibitor JNJ-8003 with sub-nanomolar inhibition potency in both antiviral and polymerase assays. Our 2.9 Å resolution cryo-EM structure revealed that JNJ-8003 binds to an induced-fit pocket on the capping domain, with multiple interactions consistent with its tight binding and resistance mutation profile. The minigenome and gel-based de novo RNA synthesis and primer extension assays demonstrated that JNJ-8003 inhibited nucleotide polymerization at the early stages of RNA transcription and replication. Our results support that JNJ-8003 binding modulates a functional interplay between the capping and RdRp domains, and this molecular insight could accelerate the design of broad-spectrum antiviral drugs.
Topics: Respiratory Syncytial Virus, Human; RNA-Dependent RNA Polymerase; Protein Binding; RNA; Nucleotides
PubMed: 37865687
DOI: 10.1038/s42003-023-05451-4 -
Clinical Infectious Diseases : An... Oct 2023Patients with antibody deficiency respond poorly to coronavirus disease 2019 (COVID-19) vaccination and are at risk of severe or prolonged infection. They are given...
BACKGROUND
Patients with antibody deficiency respond poorly to coronavirus disease 2019 (COVID-19) vaccination and are at risk of severe or prolonged infection. They are given long-term immunoglobulin replacement therapy (IRT) prepared from healthy donor plasma to confer passive immunity against infection. Following widespread COVID-19 vaccination alongside natural exposure, we hypothesized that immunoglobulin preparations will now contain neutralizing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike antibodies, which confer protection against COVID-19 disease and may help to treat chronic infection.
METHODS
We evaluated anti-SARS-CoV-2 spike antibody in a cohort of patients before and after immunoglobulin infusion. Neutralizing capacity of patient samples and immunoglobulin products was assessed using in vitro pseudovirus and live-virus neutralization assays, the latter investigating multiple batches against current circulating Omicron variants. We describe the clinical course of 9 patients started on IRT during treatment of COVID-19.
RESULTS
In 35 individuals with antibody deficiency established on IRT, median anti-spike antibody titer increased from 2123 to 10 600 U/mL postinfusion, with corresponding increase in pseudovirus neutralization titers to levels comparable to healthy donors. Testing immunoglobulin products directly in the live-virus assay confirmed neutralization, including of BQ1.1 and XBB variants, but with variation between immunoglobulin products and batches.Initiation of IRT alongside remdesivir in patients with antibody deficiency and prolonged COVID-19 infection (median 189 days, maximum >900 days with an ancestral viral strain) resulted in clearance of SARS-CoV-2 at a median of 20 days.
CONCLUSIONS
Immunoglobulin preparations now contain neutralizing anti-SARS-CoV-2 antibodies that are transmitted to patients and help to treat COVID-19 in individuals with failure of humoral immunity.
Topics: Humans; Antibodies, Neutralizing; Spike Glycoprotein, Coronavirus; COVID-19 Vaccines; SARS-CoV-2; COVID-19; Antibodies, Viral
PubMed: 37338118
DOI: 10.1093/cid/ciad368