ABSTRACT
In higher eukaryotes, tRNA methyltransferase 10A (TRMT10A) is responsible for N1-methylguanosine modification at position nine of various cytoplasmic tRNAs. Pathogenic mutations in TRMT10A cause intellectual disability, microcephaly, diabetes, and short stature in humans, and generate cytotoxic tRNA fragments in cultured cells; however, it is not clear how TRMT10A supports codon translation or brain functions. Here, we generated Trmt10a null mice and showed that tRNAGln(CUG) and initiator methionine tRNA levels were universally decreased in various tissues; the same was true in a human cell line lacking TRMT10A. Ribosome profiling of mouse brain revealed that dysfunction of TRMT10A causes ribosome slowdown at the Gln(CAG) codon and increases translation of Atf4 due to higher frequency of leaky scanning of its upstream open reading frames. Broadly speaking, translation of a subset of mRNAs, especially those for neuronal structures, is perturbed in the mutant brain. Despite not showing discernable defects in the pancreas, liver, or kidney, Trmt10a null mice showed lower body weight and smaller hippocampal postsynaptic densities, which is associated with defective synaptic plasticity and memory. Taken together, our study provides mechanistic insight into the roles of TRMT10A in the brain, and exemplifies the importance of universal tRNA modification during translation of specific codons.
ABSTRACT
MTU1 controls intramitochondrial protein synthesis by catalyzing the 2-thiouridine modification of mitochondrial transfer RNAs (mt-tRNAs). Missense mutations in the MTU1 gene are associated with life-threatening reversible infantile hepatic failure. However, the molecular pathogenesis is not well understood. Here, we investigated 17 mutations associated with this disease, and our results showed that most disease-related mutations are partial loss-of-function mutations, with three mutations being particularly severe. Mutant MTU1 is rapidly degraded by mitochondrial caseinolytic peptidase (CLPP) through a direct interaction with its chaperone protein CLPX. Notably, knockdown of CLPP significantly increased mutant MTU1 protein expression and mt-tRNA 2-thiolation, suggesting that accelerated proteolysis of mutant MTU1 plays a role in disease pathogenesis. In addition, molecular dynamics simulations demonstrated that disease-associated mutations may lead to abnormal intermolecular interactions, thereby impairing MTU1 enzyme activity. Finally, clinical data analysis underscores a significant correlation between patient prognosis and residual 2-thiolation levels, which is partially consistent with the AlphaMissense predictions. These findings provide a comprehensive understanding of MTU1-related diseases, offering prospects for modification-based diagnostics and novel therapeutic strategies centered on targeting CLPP.
Subject(s)
Mitochondria , Mitochondrial Proteins , Peptide Hydrolases , tRNA Methyltransferases , Humans , Endopeptidase Clp/genetics , Endopeptidase Clp/metabolism , Mitochondria/genetics , Mitochondria/metabolism , Mutation , Peptide Hydrolases/genetics , Proteolysis , RNA, Mitochondrial/metabolism , RNA, Transfer/metabolism , tRNA Methyltransferases/genetics , Mitochondrial Proteins/metabolismABSTRACT
Naked mole-rats (NMRs) have exceptional longevity and are resistant to age-related physiological decline and diseases. Given the role of cellular senescence in aging, we postulated that NMRs possess unidentified species-specific mechanisms to prevent senescent cell accumulation. Here, we show that upon induction of cellular senescence, NMR fibroblasts underwent delayed and progressive cell death that required activation of the INK4a-retinoblastoma protein (RB) pathway (termed "INK4a-RB cell death"), a phenomenon not observed in mouse fibroblasts. Naked mole-rat fibroblasts uniquely accumulated serotonin and were inherently vulnerable to hydrogen peroxide (H2 O2 ). After activation of the INK4a-RB pathway, NMR fibroblasts increased monoamine oxidase levels, leading to serotonin oxidization and H2 O2 production, which resulted in increased intracellular oxidative damage and cell death activation. In the NMR lung, induction of cellular senescence caused delayed, progressive cell death mediated by monoamine oxidase activation, thereby preventing senescent cell accumulation, consistent with in vitro results. The present findings indicate that INK4a-RB cell death likely functions as a natural senolytic mechanism in NMRs, providing an evolutionary rationale for senescent cell removal as a strategy to resist aging.
Subject(s)
Cellular Senescence , Serotonin , Animals , Mice , Serotonin/metabolism , Cellular Senescence/physiology , Aging/metabolism , Cell Death , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Mole Rats/metabolismABSTRACT
Understanding the physiology of human-induced pluripotent stem cells (iPSCs) is necessary for directed differentiation, mimicking embryonic development, and regenerative medicine applications. Pluripotent stem cells (PSCs) exhibit unique abilities such as self-renewal and pluripotency, but they lack some functions that are associated with normal somatic cells. One such function is the circadian oscillation of clock genes; however, whether or not PSCs demonstrate this capability remains unclear. In this study, the reason why circadian rhythm does not oscillate in human iPSCs was examined. This phenomenon may be due to the transcriptional repression of clock genes resulting from the hypermethylation of histone H3 at lysine 27 (H3K27), or it may be due to the low levels of brain and muscle ARNT-like 1 (BMAL1) protein. Therefore, BMAL1-overexpressing cells were generated and pre-treated with GSK126, an inhibitor of enhancer of zest homologue 2 (EZH2), which is a methyltransferase of H3K27 and a component of polycomb repressive complex 2. Consequently, a significant circadian rhythm following endogenous BMAL1, period 2 (PER2), and other clock gene expression was induced by these two factors, suggesting a candidate mechanism for the lack of rhythmicity of clock gene expression in iPSCs.
Subject(s)
ARNTL Transcription Factors , Circadian Clocks , Induced Pluripotent Stem Cells , Polycomb Repressive Complex 2 , Humans , ARNTL Transcription Factors/genetics , ARNTL Transcription Factors/metabolism , Brain/metabolism , Circadian Clocks/genetics , Circadian Rhythm/physiology , CLOCK Proteins/genetics , CLOCK Proteins/metabolism , Induced Pluripotent Stem Cells/metabolism , Muscles/metabolism , Polycomb Repressive Complex 2/metabolismABSTRACT
Previous studies have revealed that age-related hearing loss (AHL) in Cdk5 regulatory subunit-associated protein 1 (Cdk5rap1)-knockout mice is associated with pathology in the cochlea. Here, we aimed to identify mitochondrial alterations in the cochlea of Cdk5rap1-knockout mice with AHL. Mitochondria in the spiral ganglion neurons (SGNs) and hair cells (HCs) were normal despite senescence; however, the mitochondria of types I, II, and IV spiral ligament fibrocytes were ballooned, damaged, and ballooned, respectively, in the stria vascularis. Our results suggest that the accumulation of dysfunctional mitochondria in the lateral wall, rather than the loss of HCs and SGNs, leads to the onset of AHL. Our results provide valuable information regarding the underlying mechanisms of AHL and the relationship between aberrant tRNA modification-induced hearing loss and mitochondrial dysfunction.
Subject(s)
Cochlea , Hearing Loss , Animals , Mice , Cochlea/metabolism , Cochlea/pathology , Hearing Loss/genetics , Hearing Loss/metabolism , Hearing Loss/pathology , Mice, Knockout , Mitochondria/genetics , Mitochondria/metabolismABSTRACT
In mammalian mitochondria, translation of the AUA codon is supported by 5-formylcytidine (f5C) modification in the mitochondrial methionine tRNA anticodon. The 5-formylation is initiated by NSUN3 methylase. Human NSUN3 mutations are associated with mitochondrial diseases. Here we show that Nsun3 is essential for embryonic development in mice with whole-body Nsun3 knockout embryos dying between E10.5 and E12.5. To determine the functions of NSUN3 in adult tissue, we generated heart-specific Nsun3 knockout (Nsun3HKO) mice. Nsun3HKO heart mitochondria were enlarged and contained fragmented cristae. Nsun3HKO resulted in enhanced heart contraction and age-associated mild heart enlargement. In the Nsun3HKO hearts, mitochondrial mRNAs that encode respiratory complex subunits were not down regulated, but the enzymatic activities of the respiratory complexes decreased, especially in older mice. Our study emphasizes that mitochondrial tRNA anticodon modification is essential for mammalian embryonic development and shows that tissue-specific loss of a single mitochondrial tRNA modification can induce tissue aberration that worsens in later adulthood.
Subject(s)
Anticodon , RNA, Transfer, Met , Humans , Animals , Mice , Adult , RNA, Transfer, Met/genetics , Codon , Mitochondria/genetics , Mammals/genetics , Methyltransferases/geneticsABSTRACT
Cancer stem-like cells (CSCs) have a unique translation mode, but little is understood about the process of elongation, especially the contribution of tRNA modifications to the maintenance of CSCs properties. Here, it is reported that, contrary to the initial aim, a tRNA-modifying methylthiotransferase CDKAL1 promotes CSC-factor SALL2 synthesis by assembling the eIF4F translation initiation complex. CDKAL1 expression is upregulated in patients with worse prognoses and is essential for maintaining CSCs in rhabdomyosarcoma (RMS) and common cancers. Translatome analysis reveals that a group of mRNAs whose translation is CDKAL1-dependent contains cytosine-rich sequences in the 5' untranslated region (5'UTR). Mechanistically, CDKAL1 promotes the translation of such mRNAs by organizing the eIF4F translation initiation complex. This complex formation does not require the enzyme activity of CDKAL1 but requires only the NH2 -terminus domain of CDKAL1. Furthermore, sites in CDKAL1 essential for forming the eIF4F complex are identified and discovered candidate inhibitors of CDKAL1-dependent translation.
Subject(s)
Eukaryotic Initiation Factor-4F , Neoplasms , Humans , Eukaryotic Initiation Factor-4F/genetics , Eukaryotic Initiation Factor-4F/metabolism , Protein Biosynthesis/genetics , RNA, Messenger/genetics , tRNA Methyltransferases/genetics , tRNA Methyltransferases/metabolismABSTRACT
Myeloid malignancies with DDX41 mutations are often associated with bone marrow failure and cytopenia before overt disease manifestation. However, the mechanisms underlying these specific conditions remain elusive. Here, we demonstrate that loss of DDX41 function impairs efficient RNA splicing, resulting in DNA replication stress with excess R-loop formation. Mechanistically, DDX41 binds to the 5' splice site (5'SS) of coding RNA and coordinates RNA splicing and transcriptional elongation; loss of DDX41 prevents splicing-coupled transient pausing of RNA polymerase II at 5'SS, causing aberrant R-loop formation and transcription-replication collisions. Although the degree of DNA replication stress acquired in S phase is small, cells undergo mitosis with under-replicated DNA being remained, resulting in micronuclei formation and significant DNA damage, thus leading to impaired cell proliferation and genomic instability. These processes may be responsible for disease phenotypes associated with DDX41 mutations.
Subject(s)
RNA Splice Sites , RNA Splicing , Cell Line , RNA Splicing/genetics , Mutation , DNA ReplicationABSTRACT
SARS-CoV-2 infection alters cellular RNA content. Cellular RNAs are chemically modified and eventually degraded, depositing modified nucleosides into extracellular fluids such as serum and urine. Here we searched for COVID-19-specific changes in modified nucleoside levels contained in serum and urine of 308 COVID-19 patients using liquid chromatography-mass spectrometry (LC-MS). We found that two modified nucleosides, N6-threonylcarbamoyladenosine (t6A) and 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A), were elevated in serum and urine of COVID-19 patients. Moreover, these levels were associated with symptom severity and decreased upon recovery from COVID-19. In addition, the elevation of similarly modified nucleosides was observed regardless of COVID-19 variants. These findings illuminate specific modified RNA nucleosides in the extracellular fluids as biomarkers for COVID-19 infection and severity.
Subject(s)
COVID-19 , Nucleosides , Adenosine/analogs & derivatives , Biomarkers , COVID-19/diagnosis , Humans , Nucleosides/chemistry , RNA , SARS-CoV-2 , Threonine/analogs & derivativesABSTRACT
Human endogenous retroviruses (HERVs) occupy approximately 8% of the human genome. HERVs, transcribed in early embryos, are epigenetically silenced in somatic cells, except under pathological conditions. HERV-K is thought to protect embryos from exogenous viral infection. However, uncontrolled HERV-K expression in somatic cells has been implicated in several diseases. Here, we show that SOX2, which plays a key role in maintaining the pluripotency of stem cells, is critical for HERV-K LTR5Hs. HERV-K undergoes retrotransposition within producer cells in the absence of Env expression. Furthermore, we identified new HERV-K integration sites in long-term culture of induced pluripotent stem cells that express SOX2. These results suggest that the strict dependence of HERV-K on SOX2 has allowed HERV-K to protect early embryos during evolution while limiting the potentially harmful effects of HERV-K retrotransposition on host genome integrity in these early embryos. IMPORTANCE Human endogenous retroviruses (HERVs) account for approximately 8% of the human genome; however, the physiological role of HERV-K remains unknown. This study found that HERV-K LTR5Hs and LTR5B were transactivated by SOX2, which is essential for maintaining and reestablishing pluripotency. HERV-K can undergo retrotransposition within producer cells without env expression, and new integration sites may affect cell proliferation. In induced pluripotent stem cells (iPSCs), genomic impairment due to HERV-K retrotransposition has been identified, but it is a rare event. Considering the retention of SOX2-responsive elements in the HERV-K long terminal repeat (LTR) for over 20 million years, we conclude that HERV-K may play important physiological roles in SOX2-expressing cells.
Subject(s)
Endogenous Retroviruses , Induced Pluripotent Stem Cells , SOXB1 Transcription Factors , Endogenous Retroviruses/genetics , Humans , Induced Pluripotent Stem Cells/virology , SOXB1 Transcription Factors/genetics , Terminal Repeat Sequences/genetics , Virus IntegrationABSTRACT
Male sexual activity reduces the level of depression through oxytocin (OT) release within the brain. In this study, we showed that male mating behavior reduces depression-like behavior through OT-induced cAMP response element binding protein (CREB) signaling in the hippocampus. Moreover, we showed that mating behavior in wild-type (WT) male mice increased CREB phosphorylation in hippocampus, whereas that OT receptor knockout (OTR KO) male mice had no effect on CREB phosphorylation. CREB phosphorylation in hippocampus was also increased after OT induction in hippocampal slices prepared from WT mice. In addition, male mating behavior induced the expression of brain-derived neurotrophic factor (BDNF), which was not observed in OTR KO mice. Antidepressant-like effect of mating behavior had no effect in OTR KO mice. These findings suggest that male sexual activity has antidepressant effects through OT-induced CREB signaling in the hippocampus.
Subject(s)
Cyclic AMP Response Element-Binding Protein , Oxytocin , Animals , Antidepressive Agents/metabolism , Antidepressive Agents/pharmacology , Brain-Derived Neurotrophic Factor/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , Cyclic AMP Response Element-Binding Protein/pharmacology , Depression/drug therapy , Hippocampus/metabolism , Male , Mice , Oxytocin/metabolism , Oxytocin/pharmacology , Signal TransductionABSTRACT
N6 -isopentenyladenosine (i6A), a modified adenosine monomer, is known to induce cell death upon its addition to the culture medium. However, the molecular fate of extracellularly added i6A has yet to be identified. Here we show that i6A addition to cell culture medium results in i6A incorporation into cellular RNA in several cell lines, including the 5-fluorouracil (5-FU)-resistant human oral squamous cell carcinoma cell line FR2-SAS and its parental 5-FU-sensitive cell line SAS. i6A was predominantly incorporated into 18S and 28S rRNAs, and i6A incorporation into total RNA was mostly suppressed by treating these cell lines with an RNA polymerase I (Pol I) inhibitor. i6A was incorporated into RNA even upon inactivation of TRIT1, the only cellular i6A-modifying enzyme. These results indicate that upon cellular uptake of i6A, it is anabolized to be used for Pol I transcription. Interestingly, at lower i6A concentrations, the cytotoxic effect of i6A was substantially more pronounced in FR2-SAS cells than in SAS cells. Moreover, in FR2-SAS cells, i6A treatment decreased the rate of cellular protein synthesis and increased intracellular protein aggregation, and these effects were more pronounced than in SAS cells. Our work provides insights into the molecular fate of extracellularly applied i6A in the context of intracellular nucleic acid anabolism and suggests investigation of i6A as a candidate for a chemotherapy agent against 5-FU-resistant cancer cells.
Subject(s)
Antineoplastic Agents , Carcinoma, Squamous Cell , Mouth Neoplasms , Cell Line, Tumor , Fluorouracil/metabolism , Fluorouracil/pharmacology , Humans , Isopentenyladenosine , RNA , RNA, Ribosomal/metabolismABSTRACT
Naked mole-rats (NMRs) have a very low spontaneous carcinogenesis rate, which has prompted studies on the responsible mechanisms to provide clues for human cancer prevention. However, it remains unknown whether and how NMR tissues respond to experimental carcinogenesis induction. Here, we show that NMRs exhibit extraordinary resistance against potent chemical carcinogenesis induction through a dampened inflammatory response. Although carcinogenic insults damaged skin cells of both NMRs and mice, NMR skin showed markedly lower immune cell infiltration. NMRs harbour loss-of-function mutations in RIPK3 and MLKL genes, which are essential for necroptosis, a type of necrotic cell death that activates strong inflammation. In mice, disruption of Ripk3 reduced immune cell infiltration and delayed carcinogenesis. Therefore, necroptosis deficiency may serve as a cancer resistance mechanism via attenuating the inflammatory response in NMRs. Our study sheds light on the importance of a dampened inflammatory response as a non-cell-autonomous cancer resistance mechanism in NMRs.
Subject(s)
Mole Rats , Necroptosis , Animals , Carcinogenesis , Inflammation , Mice , SkinABSTRACT
BACKGROUND: Rapid increase in COVID-19 suspected cases has rendered disease diagnosis challenging, mainly depending upon RT-qPCR. Reliable, rapid, and cost-effective diagnostic assays that complement RT-qPCR should be introduced after thoroughly evaluating their performance upon various disease phases, viral load, and sample storage conditions. OBJECTIVE: We investigated the correlation of cycle threshold (Ct) value, which implies the viral load and infection phase, and the storage condition of the clinical specimen with the diagnosis of SARS-CoV-2 through our newly developed in-house rapid enzyme-linked immunosorbent assay (ELISA) system. METHOD: Naso-oropharyngeal samples of 339 COVID-19 suspected cases were collected and evaluated through RT-qPCR that were stored up to 30 days in different conditions (i.e. -80°C, -20°C and initially at 4°C followed by -80°C). The clinical specimens were evaluated with our in-house ELISA system after finalizing the assay method through checkerboard assay and minimizing the signal/noise ratio. RESULT: The ELISA system showed the highest sensitivity (92.9%) for samples with Ct ≤30 and preserving at -80°C temperature. The sensitivity reduced proportionally with increasing Ct value and preserving temperature. However, the specificity ranged between 98.3% and 100%. CONCLUSION: The results indicate the necessity of early infection phase diagnosis and lower temperature preservation of samples to perform rapid antigen ELISA tests.
Subject(s)
COVID-19 , SARS-CoV-2 , Specimen Handling , Viral Load , COVID-19/diagnosis , Enzyme-Linked Immunosorbent Assay , Humans , SARS-CoV-2/isolation & purification , Sensitivity and SpecificityABSTRACT
Retroviral infection requires reverse transcription, and the reverse transcriptase (RT) uses cellular tRNA as its primer. In humans, the TRMT6-TRMT61A methyltransferase complex incorporates N1-methyladenosine modification at tRNA position 58 (m1A58); however, the role of m1A58 as an RT-stop site during retroviral infection has remained questionable. Here, we constructed TRMT6 mutant cells to determine the roles of m1A in HIV-1 infection. We confirmed that tRNA3Lys m1A58 was required for in vitro plus-strand strong-stop by RT. Accordingly, infectivity of VSV-G pseudotyped HIV-1 decreased when the virus contained m1A58-deficient tRNA3Lys instead of m1A58-modified tRNA3Lys. In TRMT6 mutant cells, the global protein synthesis rate was equivalent to that of wild-type cells. However, unexpectedly, plasmid-derived HIV-1 expression showed that TRMT6 mutant cells decreased accumulation of HIV-1 capsid, integrase, Tat, Gag, and GagPol proteins without reduction of HIV-1 RNAs in cells, and fewer viruses were produced. Moreover, the importance of 5,2'-O-dimethyluridine at U54 of tRNA3Lys as a second RT-stop site was supported by conservation of retroviral genome-tRNALys sequence-complementarity, and TRMT6 was required for efficient 5-methylation of U54. These findings illuminate the fundamental importance of tRNA m1A58 modification in both the early and late steps of HIV-1 replication, as well as in the cellular tRNA modification network.
Subject(s)
HIV-1/physiology , RNA Processing, Post-Transcriptional , RNA, Transfer, Lys/metabolism , Virus Replication , Animals , HEK293 Cells , HeLa Cells , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Methylation , Mice , Mutation , RNA, Transfer, Lys/chemistryABSTRACT
BACKGROUND: Vaccination with the Oxford-AstraZeneca COVID-19 vaccine (AZD1222) initially started in the UK and quickly implemented around the Globe, including Bangladesh. Up to date, more than nine million doses administrated to the Bangladeshi public. METHOD: Herein, we studied the antibody response to the first dose of AZD1222 in 86 Bangladeshi individuals using in-house ELISA kits. Study subjects were categorized into two groups, convalescent and uninfected, based on prior infection history and SARS-CoV-2 nucleocapsid-IgG profiles. RESULTS: All the convalescent individuals presented elevated spike-1-IgG compared to 90% of uninfected ones after the first dose. Day >28 post-vaccination, the convalescent group showed six times higher antibody titer than the uninfected ones. The most elevated antibody titers for the former and later group were found at Day 14 and Days >28 post-vaccination, respectively. The spike-1-IgA titer showed a similar pattern as spike-1-IgG, although in a low-titer. In contrast, the IgM titer did not show any significant change in either group. CONCLUSION: High antibody titer in the convalescent group, signify the importance of the first dose among the uninfected group. This study advocates the integration of antibody tests in vaccination programs in the healthcare system for maximizing benefit.
Subject(s)
Antibodies, Viral/blood , Antibody Formation , COVID-19 Vaccines/immunology , COVID-19 , Bangladesh , ChAdOx1 nCoV-19 , Humans , Immunoglobulin A/blood , Immunoglobulin G/blood , Immunoglobulin M/bloodABSTRACT
RNA contains a wide variety of posttranscriptional modifications covalently attached to its base or sugar group. These modified nucleosides are liberated from RNA molecules as the consequence of RNA catabolism and released into extracellular space, but the molecular mechanism of extracellular transport and its pathophysiological implications have been unclear. In the present study, we discovered that RNA-derived modified nucleosides are exported to extracellular space through equilibrative nucleoside transporters 1 and 2 (ENT1 and ENT2), with ENT1 showing higher preference for modified nucleosides than ENT2. Pharmacological inhibition or genetic deletion of ENT1 and ENT2 significantly attenuated export of modified nucleosides thereby resulting in their accumulation in cytosol. Using mutagenesis strategy, we identified an amino acid residue in ENT1 that is involved in the discrimination of unmodified and modified nucleosides. In ENTs-deficient cells, the elevated levels of intracellular modified nucleosides were closely associated with an induction of autophagy response as evidenced by increased LC3-II level. Importantly, we performed a screening of modified nucleosides capable of inducing autophagy and found that 1-methylguanosine (m1G) was sufficient to induce LC3-II levels. Pathophysiologically, defective export of modified nucleosides drastically induced Zika virus replication in an autophagy-dependent manner. In addition, we also found that pharmacological inhibition of ENTs by dilazep significantly induced Zika virus replication. Collectively, our findings highlight RNA-derived modified nucleosides as important signaling modulators that activate autophagy response and indicate that defective export of these modified nucleoside can have profound consequences for pathophysiology.
Subject(s)
Autophagy , Equilibrative Nucleoside Transporter 1/metabolism , Equilibrative-Nucleoside Transporter 2/metabolism , Nucleosides/metabolism , RNA/metabolism , Zika Virus Infection/virology , Zika Virus/physiology , Active Transport, Cell Nucleus , Equilibrative Nucleoside Transporter 1/genetics , Equilibrative-Nucleoside Transporter 2/genetics , Humans , Nucleosides/chemistry , Nucleosides/genetics , RNA/genetics , Tumor Cells, Cultured , Virus Replication , Zika Virus Infection/genetics , Zika Virus Infection/pathologyABSTRACT
BACKGROUND: Serological tests detecting severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are widely used in seroprevalence studies and evaluating the efficacy of the vaccination program. Some of the widely used serological testing techniques are enzyme-linked immune-sorbent assay (ELISA), chemiluminescence immunoassay (CLIA), and lateral flow immunoassay (LFIA). However, these tests are plagued with low sensitivity or specificity, time-consuming, labor-intensive, and expensive. We developed a serological test implementing flow-through dot-blot assay (FT-DBA) for SARS-CoV-2 specific IgG detection, which provides enhanced sensitivity and specificity while being quick to perform and easy to use. METHODS: SARS-CoV-2 antigens were immobilized on nitrocellulose membrane to capture human IgG, which was then detected with anti-human IgG conjugated gold nanoparticle (hIgG-AuNP). A total of 181 samples were analyzed in-house. Within which 35 were further evaluated in US FDA-approved CLIA Elecsys SARS-CoV-2 assay. The positive panel consisted of RT-qPCR positive samples from patients with both <14 days and >14 days from the onset of clinical symptoms. The negative panel contained samples collected from the pre-pandemic era dengue patients and healthy donors during the pandemic. Moreover, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of FT-DBA were evaluated against RT-qPCR positive sera. However, the overall efficacies were assessed with sera that seroconverted against either nucleocapsid (NCP) or receptor-binding domain (RBD). RESULTS: In-house ELISA selected a total of 81 true seropositive and 100 seronegative samples. The sensitivity of samples with <14 days using FT-DBA was 94.7%, increasing to 100% for samples >14 days. The overall detection sensitivity and specificity were 98.8% and 98%, respectively, whereas the overall PPV and NPV were 99.6% and 99%. Moreover, comparative analysis between in-house ELISA assays and FT-DBA revealed clinical agreement of Cohen's Kappa value of 0.944. The FT-DBA showed sensitivity and specificity of 100% when compared with commercial CLIA kits. CONCLUSION: The assay can confirm past SARS-CoV-2 infection with high accuracy within 2 minutes compared to commercial CLIA or in-house ELISA. It can help track SARS-CoV-2 disease progression, population screening, and vaccination response. The ease of use of the assay without requiring any instruments while being semi-quantitative provides the avenue of its implementation in remote areas around the globe, where conventional serodiagnosis is not feasible.
Subject(s)
Gold/chemistry , Immunoblotting/methods , Immunoglobulin G/analysis , Metal Nanoparticles/chemistry , Nucleocapsid/analysis , SARS-CoV-2/isolation & purification , Adult , Antibodies, Viral/blood , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Male , Predictive Value of Tests , SARS-CoV-2/immunology , Sensitivity and Specificity , Seroepidemiologic StudiesABSTRACT
BACKGROUND: Dynamics and persistence of neutralizing and non-neutralizing antibodies can give us the knowledge required for serodiagnosis, disease management, and successful vaccine design and development. The disappearance of antibodies, absence of humoral immunity activation, and sporadic reinfection cases emphasize the importance of longitudinal antibody dynamics against variable structural antigens. METHODS: In this study, twenty-five healthy subjects working in a SARS-COV-2 serodiagnostic assay development project were enrolled, and their sign and symptoms were followed up to six months. Three subjects showed COVID-19-like symptoms, and three subjects' antibody dynamics were followed over 120 days by analyzing 516 samples. We have developed 12 different types of in-house ELISAs to observe the kinetics of IgG, IgM, and IgA against four SARS-CoV-2 proteins, namely nucleocapsid, RBD, S1, and whole spike (S1+S2). For the development of these assays, 30-104 pre-pandemic samples were taken as negative controls and 83 RT-qPCR positive samples as positive ones. RESULTS: All three subjects presented COVID-19-like symptoms twice, with mild symptoms in the first episode were severe in the second, and RT-qPCR confirmed the latter. The initial episode did not culminate with any significant antibody development, while a multifold increase in IgG antibodies characterized the second episode. Interestingly, IgG antibody development concurrent with IgM and IgA and persisted, whereas the latter two weans off rather quickly if appeared. CONCLUSION: Antibody kinetics observed in this study can provide a pathway to the successful development of sero-diagnostics and epidemiologists to predict the fate of vaccination currently in place.
ABSTRACT
Mitochondrial dysfunction is associated with aging and age-related hearing loss (AHL). However, the precise mechanisms underlying the pathophysiology of hearing loss remain unclear. Cdk5 regulatory subunit-associated protein 1 (CDK5RAP1) enables efficient intramitochondrial translation by catalyzing the deposition of 2-methylthio modifications on mitochondrial tRNAs. Here we investigated the effect of defective mitochondrial protein translation on hearing and AHL in a Cdk5rap1 deficiency C57BL/6 mouse model. Compared to control C57BL/6 mice, Cdk5rap1-knockout female mice displayed hearing loss phenotypically similar to AHL from an early age. The premature hearing loss in Cdk5rap1-knockout mice was associated with the degeneration of the spiral ligament and reduction of endocochlear potentials following the loss of auditory sensory cells. Furthermore, cultured primary mouse embryonic fibroblasts displayed early onset of cellular senescence associated with high oxidative stress and cell death. These results indicate that the CDK5RAP1 deficiency-induced defective mitochondrial translation might cause early hearing loss through the induction of cellular senescence and cochlear dysfunction in the inner ear. Our results suggest that the accumulation of dysfunctional mitochondria might promote AHL progression. Furthermore, our findings suggest that mitochondrial dysfunction and dysregulated mitochondrial tRNA modifications mechanistically cause AHL. Understanding the mechanisms underlying AHL will guide future clinical investigations and interventions in the attempt to mitigate the consequences of AHL.