The mosaic oat genome gives insights into a uniquely healthy cereal crop.

Cultivated oat (Avena sativa L.) is an allohexaploid (AACCDD, 2n = 6x = 42) thought to have been domesticated more than 3,000 years ago while growing as a weed in wheat, emmer and barley fields in Anatolia1,2. Oat has a low carbon footprint, substantial health benefits and the potential to replace animal-based food products. However, the lack of a fully annotated reference genome has hampered efforts to deconvolute its complex evolutionary history and functional gene dynamics. Here we present a high-quality reference genome of A. sativa and close relatives of its diploid (Avena longiglumis, AA, 2n = 14) and tetraploid (Avena insularis, CCDD, 2n = 4x = 28) progenitors. We reveal the mosaic structure of the oat genome, trace large-scale genomic reorganizations in the polyploidization history of oat and illustrate a breeding barrier associated with the genome architecture of oat. We showcase detailed analyses of gene families implicated in human health and nutrition, which adds to the evidence supporting oat safety in gluten-free diets, and we perform mapping-by-sequencing of an agronomic trait related to water-use efficiency. This resource for the Avena genus will help to leverage knowledge from other cereal genomes, improve understanding of basic oat biology and accelerate genomics-assisted breeding and reanalysis of quantitative trait studies.

The nuclear pore Y-complex functions as a platform for transcriptional regulation of FLOWERING LOCUS C in Arabidopsis.

The nuclear pore complex (NPC) has multiple functions beyond the nucleo-cytoplasmic transport of large molecules. Subnuclear compartmentalization of chromatin is critical for gene expression in animals and yeast. However, the mechanism by which the NPC regulates gene expression is poorly understood in plants. Here we report that the Y-complex (Nup107-160 complex, a subcomplex of the NPC) self-maintains its nucleoporin homeostasis and modulates FLOWERING LOCUS C (FLC) transcription via changing histone modifications at this locus. We show that Y-complex nucleoporins are intimately associated with FLC chromatin through their interactions with histone H2A at the nuclear membrane. Fluorescence in situ hybridization assays revealed that Nup96, a Y-complex nucleoporin, enhances FLC positioning at the nuclear periphery. Nup96 interacted with HISTONE DEACETYLASE 6 (HDA6), a key repressor of FLC expression via histone modification, at the nuclear membrane to attenuate HDA6-catalyzed deposition at the FLC locus and change histone modifications. Moreover, we demonstrate that Y-complex nucleoporins interact with RNA polymerase II to increase its occupancy at the FLC locus, facilitating transcription. Collectively, our findings identify an attractive mechanism for the Y-complex in regulating FLC expression via tethering the locus at the nuclear periphery and altering its histone modification.

Controlling laser-dressed resonance line shape using attosecond extreme-ultraviolet pulse with a spectral minimum.

High-harmonic generation from a gas target exhibits sharp spectral features and rapid phase variation near the Cooper minimum. By applying spectral filtering, shaped isolated attosecond pulses can be generated where the pulse is split into two in the time domain. Using such shaped extreme-ultraviolet (XUV) pulses, we theoretically study attosecond transient absorption (ATA) spectra of helium [Formula: see text] autoionizing state which is resonantly coupled to the [Formula: see text] dark state by a time-delayed infrared laser. Our simulations show that the asymmetric [Formula: see text] Fano line shape can be readily tuned into symmetric Lorentzian within the time delay of a few tens of attoseconds. Such efficient control is due to the destructive interference in the generation of the [Formula: see text] state when it is excited by a strongly shaped XUV pulse. This is to be compared to prior experiments where tuning the line shape of a Fano resonance would take tens of femtoseconds. We also show that the predicted ATA spectral line shape can be observed experimentally after propagation in a gas medium. Our results suggest that strongly shaped attosecond XUV pulses offer the opportunity for controlling and probing fine features of narrow resonances on the few-ten attoseconds timescale.

Blockade of C5a receptor unleashes tumor-associated macrophage antitumor response and enhances CXCL9-dependent CD8+ T cell activity.

Macrophages play a crucial role in shaping the immune state within the tumor microenvironment (TME) and are often influenced by tumors to hinder antitumor immunity. However, the underlying mechanisms are still elusive. Here, we observed abnormal expression of complement 5a receptor (C5aR) in human ovarian cancer (OC), and identified high levels of C5aR expression on tumor-associated macrophages (TAMs), which led to the polarization of TAMs toward an immunosuppressive phenotype. C5aR knockout or inhibitor treatment restored TAM antitumor response and attenuated tumor progression. Mechanistically, C5aR deficiency reprogrammed macrophages from a protumor state to an antitumor state, associating with the upregulation of immune response and stimulation pathways, which in turn resulted in the enhanced antitumor response of cytotoxic T cells in a manner dependent on chemokine (C-X-C motif) ligand 9 (CXCL9). The pharmacological inhibition of C5aR also improved the efficacy of immune checkpoint blockade therapy. In patients, C5aR expression associated with CXCL9 production and infiltration of CD8+ T cells, and a high C5aR level predicted poor clinical outcomes and worse benefits from anti-PD-1 therapy. Thus, our study sheds light on the mechanisms underlying the modulation of TAM antitumor immune response by the C5a-C5aR axis and highlights the potential of targeting C5aR for clinical applications.

Deleterious and Adaptive Mutations in Plant Germplasm Conserved Ex Situ.

Conserving more than 7 million plant germplasm accessions in 1,750 genebanks worldwide raises the hope of securing the food supply for humanity for future generations. However, there is a genetic cost for such long-term germplasm conservation, which has been largely unaccounted for before. We investigated the extent and variation of deleterious and adaptive mutations in 490 individual plants representing barley, wheat, oat, soybean, maize, rapa, and sunflower collections in a seed genebank using RNA-Seq technology. These collections were found to have a range of deleterious mutations detected from 125 (maize) to 83,695 (oat) with a mean of 13,537 and of the averaged sample-wise mutation burden per deleterious locus from 0.069 to 0.357 with a mean of 0.200. Soybean and sunflower collections showed that accessions acquired earlier had increased mutation burdens. The germplasm with more years of storage in several collections carried more deleterious and fewer adaptive mutations. The samples with more cycles of germplasm regeneration revealed fewer deleterious and more adaptive mutations. These findings are significant for understanding mutational dynamics and genetic cost in conserved germplasm and have implications for long-term germplasm management and conservation.

Bone-derived PDGF-BB enhances hippocampal non-specific transcytosis through microglia-endothelial crosstalk in HFD-induced metabolic syndrome.

BACKGROUND: It is well known that high-fat diet (HFD)-induced metabolic syndrome plays a crucial role in cognitive decline and brain-blood barrier (BBB) breakdown. However, whether the bone-brain axis participates in this pathological process remains unknown. Here, we report that platelet-derived growth factor-BB (PDGF-BB) secretion by preosteoclasts in the bone accelerates neuroinflammation. The expression of alkaline phosphatase (ALPL), a nonspecific transcytosis marker, was upregulated during HFD challenge. MAIN BODY: Preosteoclast-specific Pdgfb transgenic mice with high PDGF-BB concentrations in the circulation recapitulated the HFD-induced neuroinflammation and transcytosis shift. Preosteoclast-specific Pdgfb knockout mice were partially rescued from hippocampal neuroinflammation and transcytosis shifts in HFD-challenged mice. HFD-induced PDGF-BB elevation aggravated microglia-associated neuroinflammation and interleukin-1ß (IL-1ß) secretion, which increased ALPL expression and transcytosis shift through enhancing protein 1 (SP1) translocation in endothelial cells. CONCLUSION: Our findings confirm the role of bone-secreted PDGF-BB in neuroinflammation and the transcytosis shift in the hippocampal region during HFD challenge and identify a novel mechanism of microglia-endothelial crosstalk in HFD-induced metabolic syndrome.

The amino acid sensor MetRS is required for methionine-induced milk protein synthesis in a domestic pigeon model.

This study was conducted to investigate whether methionyl-tRNA synthetase (MetRS) is a mediator of Met-induced crop milk protein synthesis via the janus kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) signalling pathway in breeding pigeons. In Experiment 1, a total of 216 pairs of breeding pigeons were divided into 3 groups (control, Met-deficient, and Met-rescue groups). In Experiments 2 and 3, forty pairs of breeding pigeons from each experiment were allocated into 4 groups. The 2nd experiment included a control group and 3 MetRS inhibitor (REP8839) groups. The 3rd experiment included a Met-deficient group, Met-sufficient group, REP8839 + Met-deficient group, and REP8839 + Met-sufficient group. Experiment 1 showed that Met supplementation increased crop development, crop milk protein synthesis, the protein expression of MetRS and JAK2/STAT5 signalling pathway, and improved squab growth. Experiment 2 showed that crop development, crop milk protein synthesis, and the protein expression of MetRS and the JAK2/STAT5 signalling pathway were decreased, and squab growth was inhibited by the injection of 1.0 mg/kg BW REP8839, which was the selected dose for the 3rd experiment. These results showed that Met supplementation increased crop development, crop milk protein synthesis, and the expression of MetRS and JAK2/STAT5 signalling pathway and rescued squab growth after the injection of REP8839. Moreover, the Co-IP results showed that there was an interaction between MetRS and JAK2. Taken together, these findings indicate that MetRS mediates Met-induced crop milk protein synthesis via the JAK2/STAT5 signalling pathway, resulting in improved squab growth in breeding pigeons.

A deafness-associated mitochondrial DNA mutation caused pleiotropic effects on DNA replication and tRNA metabolism.

In this report, we investigated the molecular mechanism underlying a deafness-associated m.5783C > T mutation that affects the canonical C50-G63 base-pairing of TΨC stem of tRNACys and immediately adjacent to 5' end of light-strand origin of mitochondrial DNA (mtDNA) replication (OriL). Two dimensional agarose gel electrophoresis revealed marked decreases in the replication intermediates including ascending arm of Y-fork arcs spanning OriL in the mutant cybrids bearing m.5783C > T mutation. mtDNA replication alterations were further evidenced by decreased levels of PolγA, Twinkle and SSBP1, newly synthesized mtDNA and mtDNA contents in the mutant cybrids. The m.5783C > T mutation altered tRNACys structure and function, including decreased melting temperature, conformational changes, instability and deficient aminoacylation of mutated tRNACys. The m.5783C > T mutation impaired the 5' end processing efficiency of tRNACys precursors and reduced the levels of tRNACys and downstream tRNATyr. The aberrant tRNA metabolism impaired mitochondrial translation, which was especially pronounced effects in the polypeptides harboring higher numbers of cysteine and tyrosine codons. These alterations led to deficient oxidative phosphorylation including instability and reduced activities of the respiratory chain enzyme complexes I, III, IV and intact supercomplexes overall. Our findings highlight the impact of mitochondrial dysfunction on deafness arising from defects in mitochondrial DNA replication and tRNA metabolism.

Chronic Toxoplasma gondii infection enhances susceptibility to colitis.

Oral infection with Toxoplasma gondii results in dysbiosis and enteritis, both of which revert to normal during chronic infection. However, whether infection leaves a lasting impact on mucosal responses remains uncertain. Here we examined the effect of the chemical irritant dextran sodium sulfate (DSS) on intestinal damage and wound healing in chronically infected mice. Our findings indicate that prior infection with T. gondii exacerbates damage to the colon caused by DSS and impairs wound healing by suppressing stem cell regeneration of the epithelium. Enhanced tissue damage was attributable to inflammatory monocytes that emerge preactivated from bone marrow, migrate to the intestine, and release inflammatory mediators, including nitric oxide. Tissue damage was reversed by neutralization of inflammatory monocytes or nitric oxide, revealing a causal mechanism for tissue damage. Our findings suggest that chronic infection with T. gondii enhances monocyte activation to increase inflammation associated with a secondary environmental insult.

Patterns of the Predicted Mutation Burden in 19,778 Domesticated Barley Accessions Conserved Ex Situ.

Long-term conservation of more than 7 million plant germplasm accessions in 1750 genebanks worldwide is a challenging mission. The extent of deleterious mutations present in conserved germplasm and the genetic risk associated with accumulative mutations are largely unknown. This study took advantage of published barley genomic data to predict sample-wise mutation burdens for 19,778 domesticated barley (Hordeum vulgare L.) accessions conserved ex situ. It was found that the conserved germplasm harbored 407 deleterious mutations and 337 (or 82%) identified deleterious alleles were present in 20 (or 0.1%) or fewer barley accessions. Analysis of the predicted mutation burdens revealed significant differences in mutation burden for several groups of barley germplasm (landrace > cultivar (or higher burden estimate in landrace than in cultivar); winter barley > spring barley; six-rowed barley > two-rowed barley; and 1000-accession core collection > non-core germplasm). Significant differences in burden estimate were also found among seven major geographical regions. The sample-wise predicted mutation burdens were positively correlated with the estimates of sample average pairwise genetic difference. These findings are significant for barley germplasm management and utilization and for a better understanding of the genetic risk in conserved plant germplasm.

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Multisystem inflammatory syndrome in children (MIS-C) is a complex syndrome characterized by multi-organ involvement that has emerged in the context of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak. The clinical presentation of MIS-C is similar to Kawasaki disease but predominantly presents with fever and gastrointestinal symptoms, and severe cases can involve toxic shock and cardiac dysfunction. Epidemiological findings indicate that the majority of MIS-C patients test positive for SARS-CoV-2 antibodies. The pathogenesis and pathophysiology of MIS-C remain unclear, though immune dysregulation following SARS-CoV-2 infection is considered a major contributing factor. Current treatment approaches for MIS-C primarily involve intravenous immunoglobulin therapy and symptomatic supportive care. This review article provides a comprehensive overview of the definition, epidemiology, pathogenesis, clinical presentation, diagnosis, treatment, and prognosis of MIS-C.

Dual-Wave Acoustofluidic Centrifuge for Ultrafast Concentration of Nanoparticles and Extracellular Vesicles.

Extracellular vesicles (EVs) are secreted nanostructures that play various roles in critical cancer processes. They operate as an intercellular communication system, transferring complex sets of biomolecules from cell to cell. The concentration of EVs is difficult to decipher, and there is an unmet technological need for improved (faster, simpler, and gentler) approaches to isolate EVs from complex matrices. Herein, an acoustofluidic concentration of extracellular vesicles (ACEV) is presented, based on a thin-film printed circuit board with interdigital electrodes mounted on a piezoelectric substrate. An angle of 120° is identified between the electrodes and the reference flat of the piezoelectric substrate for simultaneous generation of Rayleigh and shear horizontal waves. The dual waves create a complex acoustic field in a droplet, resulting in effective concentration of nanoparticles and EVs. The ACEV is able to concentrate 20 nm nanospheres within 105 s and four EV dilutions derived from the human prostate cancer (Du145) cell line in approximately 30 s. Cryo-electron microscopy confirmed the preservation of EV integrity. The ACEV device holds great potential to revolutionize investigations of EVs. Its faster, simpler, and gentler approach to EV isolation and concentration can save time and effort in phenotypic and functional studies of EVs.

Nomogram to Predict Recurrence and Guide a Pragmatic Surveillance Strategy After Resection of Hepatoid Adenocarcinoma of the Stomach: A Retrospective Multicenter Study.

BACKGROUND: An accurate recurrence risk assessment system and surveillance strategy for hepatoid adenocarcinoma of the stomach (HAS) remain poorly defined. This study aimed to develop a nomogram to predict postoperative recurrence of HAS and guide individually tailored surveillance strategies. METHODS: The study enrolled all patients with primary HAS who had undergone curative-intent resection at 14 institutions from 2004 to 2019. Clinicopathologic variables with statistical significance in the multivariate Cox regression were incorporated into a nomogram to build a recurrence predictive model. RESULTS: The nomogram of recurrence-free survival (RFS) based on independent prognostic factors, including age, preoperative carcinoembryonic antigen, number of examined lymph nodes, perineural invasion, and lymph node ratio, achieved a C-index of 0.723 (95% confidence interval [CI], 0.674-0.772) in the whole cohort, which was significantly higher than those of the eighth American Joint Committed on Cancer (AJCC) staging system (C-index, 0.629; 95% CI, 0.573-0.685; P < 0.001). The nomogram accurately stratified patients into low-, middle-, and high-risk groups of postoperative recurrence. The postoperative recurrence risk rates for patients in the middle- and high-risk groups were respectively 3 and 10 times higher than for the low-risk group. The patients in the middle- and high-risk groups showed more recurrence and metastasis, particularly multiple site metastasis, within 36 months after the operation than those in the low-risk group (low, 2.2%; middle, 8.6%; high, 24.0%; P = 0.003). CONCLUSIONS: The nomogram achieved good prediction of postoperative recurrence for the patients with HAS after radical resection. For the middle- and high-risk patients, more active surveillance and targeted examination methods should be adopted within 36 months after the operation, particularly for liver and multiple metastases.

Control of the annular spatial profile of high harmonics using a Bessel-Gaussian beam carrying the nonzero orbital angular momentum.

We propose to generate vortex high harmonics in the extreme ultraviolet (XUV) with a controllable spatial profile by using a Bessel-Gaussian (BG) beam carrying a nonzero orbital angular momentum (OAM). Such BG beam has quite a different intensity profile at the focus compared to the generally used BG beam without carrying the OAM. We show that the BG beam is capable of generating single-ring structured high harmonics, which is quite different from an Laguerre-Gaussian (LG) beam with a similar intensity distribution at the laser focus. We reveal that favorable phase-matching conditions can be achieved off-axis and away from the laser focus because a single-atom intrinsic phase due to the short electron trajectory can be well compensated by a geometric phase of the BG beam. We thus give a general rule that vortex high harmonics with a single annular profile can be efficiently generated when a gas medium is located at 1.5zred to 2.0zred before or after the laser focus of the BG beam, here zred is a reduced length. We also show the validity of this rule when the BG beam carries a higher OAM. This work is expected to be useful for synthesizing attosecond vortex pulses.

Nup96 and HOS1 Are Mutually Stabilized and Gate CONSTANS Protein Level, Conferring Long-Day Photoperiodic Flowering Regulation in Arabidopsis.

The nuclear pore complex profoundly affects the timing of flowering; however, the underlying mechanisms are poorly understood. Here, we report that Nucleoporin96 (Nup96) acts as a negative regulator of long-day photoperiodic flowering in Arabidopsis (Arabidopsis thaliana). Through multiple approaches, we identified the E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE1 (HOS1) and demonstrated its interaction in vivo with Nup96. Nup96 and HOS1 mainly localize and interact on the nuclear membrane. Loss of function of Nup96 leads to destruction of HOS1 proteins without a change in their mRNA abundance, which results in overaccumulation of the key activator of long-day photoperiodic flowering, CONSTANS (CO) proteins, as previously reported in hos1 mutants. Unexpectedly, mutation of HOS1 strikingly diminishes Nup96 protein level, suggesting that Nup96 and HOS1 are mutually stabilized and thus form a novel repressive module that regulates CO protein turnover. Therefore, the nup96 and hos1 single and nup96 hos1 double mutants have highly similar early-flowering phenotypes and overlapping transcriptome changes. Together, this study reveals a repression mechanism in which the Nup96-HOS1 repressive module gates the level of CO proteins and thereby prevents precocious flowering in long-day conditions.

Sarcopenia as a risk factor of progression-free survival in patients with metastases: a systematic review and meta-analysis.

BACKGROUND: Metastasis of cancer causes more than 90% of cancer deaths and is severely damaging to human health. In recent years, several studies have linked sarcopenia to shorter survival in patients with metastatic cancer. Several predictive models exist to predict mortality in patients with metastatic cancer, but have reported limited accuracy. METHODS: We systematically searched Medline, EMBASE, and the Cochrane Library for articles published on or before October 14, 2022. Pooled Hazard Ratio (HR) estimates with 95% confidence intervals (CIs) were calculated using a random effects model. The primary outcome was an increased risk of death or tumor progression in patients with metastatic cancer, which is expressed as progression-free survival (PFS). In addition, we performed subgroup analyses and leave-one-out sensitivity analyses to explore the main sources of heterogeneity and the stability of the results. RESULTS: Sixteen retrospective cohort studies with 1,675 patients were included in the 888 papers screened. The results showed that sarcopenia was associated with lower progression-free survival (HR = 1.56, 95% CI = 1.19-2.03, I2 = 76.3%, P < 0.001). This result was further confirmed by trim-and-fill procedures and leave-one-out sensitivity analysis. CONCLUSIONS: This study suggests that sarcopenia may be a risk factor for reduced progression-free survival in patients with metastatic cancer. Further studies are still needed to explain the reason for this high heterogeneity in outcome. TRIAL REGISTRATION: CRD42022325910.

Impact Dynamics of Non-Newtonian Droplets on Superhydrophobic Surfaces.

Droplet impact behavior on a solid surface is critical for many industrial applications such as spray coating, food production, printing, and agriculture. For all of these applications, a common challenge is to modify and control the impact regime and contact time of the droplets. This challenge becomes more critical for non-Newtonian liquids with complex rheology. In this research, we explored the impact dynamics of non-Newtonian liquids (by adding different concentrations of Xanthan into water) on superhydrophobic surfaces. Our experimental results show that by increasing the Xanthan concentration in water, the shapes of the bouncing droplet are dramatically altered, e.g., its shape at the separation moment is changed from a conventional vertical jetting into a "mushroom"-like one. As a result, the contact time of the non-Newtonian droplet could be reduced by up to ∼50%. We compare the impact scenarios of Xanthan liquids with those of glycerol solutions having a similar apparent viscosity, and results show that the differences in the elongation viscosity induce different impact dynamics of the droplets. Finally, we show that by increasing the Weber number for all of the liquids, the contact time is reduced, and the maximum spreading radius is increased.

The distinctive role of membrane fibrinogen-like protein 2 in the liver stage of rodent malaria infections.

Viral infection often induce the expression of murine fibrinogen-like protein 2 (mFGL2) triggering immune coagulation, which causes severe liver pathogenesis via increased fibrin deposition and thrombosis in the microvasculature. We aimed to investigate the role of mFGL2 in the liver stage of malaria infections. We reveal that infection with malaria sporozoites also induces increased expression of mFGL2 and that this expression is primarily located within the liver Kupffer and endothelial cells. In addition, we report that inhibition of FGL2 has no significant effect on immune coagulation but increases the expression of inflammatory cytokines in the livers of infected mice. Interestingly, FGL2 deficiency had no significant impact on the development of liver stage malaria parasites or the pathogenesis of the infected liver. In contrast to viral infections, we conclude that mFGL2 does not contribute to either parasite development or liver pathology during these infections, revealing the unique features of this protein in liver-stage malaria infections.

Optimization of single-tube nested PCR for the detection of Echinococcus spp.

Echinococcosis is a serious zoonotic life-threatening parasitic disease caused by metacestodes of Echinococcus spp., and appropriate sensitive diagnosis and genotyping techniques are required to detect infections and study the genetic characterization of Echinococcus spp. isolates. In this study, a single-tube nested PCR (STNPCR) method was developed and evaluated for the detection of Echinococcus spp. DNA based on the COI gene. STNPCR was 100 times more sensitive than conventional PCR and showed the same sensitivity to common nested PCR (NPCR); but with a lower risk of cross-contamination. The limit of detection of the developed STNPCR method was estimated to be 10 copies/µL of the recombinant standard plasmids of Echinococcus spp. COI gene. In clinical application, 8 cyst tissue samples and 12 calcification tissue samples were analysed by conventional PCR with outer and inner primers and resulted in 100.00% (8/8) and 8.33% (1/12), 100.00% (8/8) and 16.67% (2/12) positive reactions, respectively, while STNPCR and NPCR were all able to identify the presence of genomic DNA in 100.00% (8/8) and 83.33% (10/12) of the same samples. Due to its high sensitivity combined with the potential for the elimination of cross-contamination, the STNPCR method was suitable for epidemiological investigations and characteristic genetic studies of Echinococcus spp. tissue samples. The STNPCR method can effectively amplify low concentrations of genomic DNA from calcification samples and cyst residues infected with Echinococcus spp. Subsequently, the sequences of positive PCR products were obtained, which were useful for haplotype analysis, genetic diversity, and evolution studies of Echinococcus spp., and understanding of Echinococcus spp. dissemination and transmission among the hosts.

A deafness-associated tRNA mutation caused pleiotropic effects on the m1G37 modification, processing, stability and aminoacylation of tRNAIle and mitochondrial translation.

Defects in the posttranscriptional modifications of mitochondrial tRNAs have been linked to human diseases, but their pathophysiology remains elusive. In this report, we investigated the molecular mechanism underlying a deafness-associated tRNAIle 4295A>G mutation affecting a highly conserved adenosine at position 37, 3' adjacent to the tRNA's anticodon. Primer extension and methylation activity assays revealed that the m.4295A>G mutation introduced a tRNA methyltransferase 5 (TRMT5)-catalyzed m1G37 modification of tRNAIle. Molecular dynamics simulations suggested that the m.4295A>G mutation affected tRNAIle structure and function, supported by increased melting temperature, conformational changes and instability of mutated tRNA. An in vitro processing experiment revealed that the m.4295A>G mutation reduced the 5' end processing efficiency of tRNAIle precursors, catalyzed by RNase P. We demonstrated that cybrid cell lines carrying the m.4295A>G mutation exhibited significant alterations in aminoacylation and steady-state levels of tRNAIle. The aberrant tRNA metabolism resulted in the impairment of mitochondrial translation, respiratory deficiency, decreasing membrane potentials and ATP production, increasing production of reactive oxygen species and promoting autophagy. These demonstrated the pleiotropic effects of m.4295A>G mutation on tRNAIle and mitochondrial functions. Our findings highlighted the essential role of deficient posttranscriptional modifications in the structure and function of tRNA and their pathogenic consequence of deafness.