Targeting Pin1 renders pancreatic cancer eradicable by synergizing with immunochemotherapy.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by notorious resistance to current therapies attributed to inherent tumor heterogeneity and highly desmoplastic and immunosuppressive tumor microenvironment (TME). Unique proline isomerase Pin1 regulates multiple cancer pathways, but its role in the TME and cancer immunotherapy is unknown. Here, we find that Pin1 is overexpressed both in cancer cells and cancer-associated fibroblasts (CAFs) and correlates with poor survival in PDAC patients. Targeting Pin1 using clinically available drugs induces complete elimination or sustained remissions of aggressive PDAC by synergizing with anti-PD-1 and gemcitabine in diverse model systems. Mechanistically, Pin1 drives the desmoplastic and immunosuppressive TME by acting on CAFs and induces lysosomal degradation of the PD-1 ligand PD-L1 and the gemcitabine transporter ENT1 in cancer cells, besides activating multiple cancer pathways. Thus, Pin1 inhibition simultaneously blocks multiple cancer pathways, disrupts the desmoplastic and immunosuppressive TME, and upregulates PD-L1 and ENT1, rendering PDAC eradicable by immunochemotherapy.

A transcription-independent mechanism determines rapid periodic fluctuations of BRCA1 expression.

BRCA1 expression is highly regulated to prevent genomic instability and tumorigenesis. Dysregulation of BRCA1 expression correlates closely with sporadic basal-like breast cancer and ovarian cancer. The most significant characteristic of BRCA1 regulation is periodic expression fluctuation throughout the cell cycle, which is important for the orderly progression of different DNA repair pathways throughout the various cell cycle phases and for further genomic stability. However, the underlying mechanism driving this phenomenon is poorly understood. Here, we demonstrate that RBM10-mediated RNA alternative splicing coupled to nonsense-mediated mRNA decay (AS-NMD), rather than transcription, determines the periodic fluctuations in G1/S-phase BRCA1 expression. Furthermore, AS-NMD broadly regulates the expression of period genes, such as DNA replication-related genes, in an uneconomical but more rapid manner. In summary, we identified an unexpected posttranscriptional mechanism distinct from canonical processes that mediates the rapid regulation of BRCA1 as well as other period gene expression during the G1/S-phase transition and provided insights into potential targets for cancer therapy.

Unambiguous discrimination of all 20 proteinogenic amino acids and their modifications by nanopore.

Natural proteins are composed of 20 proteinogenic amino acids and their post-translational modifications (PTMs). However, due to the lack of a suitable nanopore sensor that can simultaneously discriminate between all 20 amino acids and their PTMs, direct sequencing of protein with nanopores has not yet been realized. Here, we present an engineered hetero-octameric Mycobacterium smegmatis porin A (MspA) nanopore containing a sole Ni2+ modification. It enables full discrimination of all 20 proteinogenic amino acids and 4 representative modified amino acids, Nω,N'ω-dimethyl-arginine (Me-R), O-acetyl-threonine (Ac-T), N4-(ß-N-acetyl-D-glucosaminyl)-asparagine (GlcNAc-N) and O-phosphoserine (P-S). Assisted by machine learning, an accuracy of 98.6% was achieved. Amino acid supplement tablets and peptidase-digested amino acids from peptides were also analyzed using this strategy. This capacity for simultaneous discrimination of all 20 proteinogenic amino acids and their PTMs suggests the potential to achieve protein sequencing using this nanopore-based strategy.

Proline isomer-specific antibodies reveal the early pathogenic tau conformation in Alzheimer's disease.

cis-trans isomerization of proteins phosphorylated by proline-directed kinases is proposed to control numerous signaling molecules and is implicated in the pathogenesis of Alzheimer's and other diseases. However, there is no direct evidence for the existence of cis-trans protein isomers in vivo or for their conformation-specific function or regulation. Here we develop peptide chemistries that allow the generation of cis- and trans-specific antibodies and use them to raise antibodies specific for isomers of phosphorylated tau. cis, but not trans, p-tau appears early in the brains of humans with mild cognitive impairment, accumulates exclusively in degenerated neurons, and localizes to dystrophic neurites during Alzheimer's progression. Unlike trans p-tau, the cis isomer cannot promote microtubule assembly, is more resistant to dephosphorylation and degradation, and is more prone to aggregation. Pin1 converts cis to trans p-tau to prevent Alzheimer's tau pathology. Isomer-specific antibodies and vaccines may therefore have value for the early diagnosis and treatment of Alzheimer's disease.

C1QBP Promotes Homologous Recombination by Stabilizing MRE11 and Controlling the Assembly and Activation of MRE11/RAD50/NBS1 Complex.

MRE11 nuclease forms a trimeric complex (MRN) with RAD50 and NBS1 and plays a central role in preventing genomic instability. When DNA double-strand breaks (DSBs) occur, MRN is quickly recruited to the damage site and initiates DNA end resection; accordingly, MRE11 must be tightly regulated to avoid inefficient repair or nonspecific resection. Here, we show that MRE11 and RAD50 form a complex (MRC) with C1QBP, which stabilizes MRE11/RAD50, while inhibiting MRE11 nuclease activity by preventing its binding to DNA or chromatin. Upon DNA damage, ATM phosphorylates MRE11-S676/S678 to quickly dissociate the MRC complex. Either excess or insufficient C1QBP impedes the recruitment of MRE11 to DSBs and impairs the DNA damage response. C1QBP is highly expressed in breast cancer and positively correlates with MRE11 expression, and the inhibition of C1QBP enhances tumor regression with chemotherapy. By influencing MRE11 at multiple levels, C1QBP is, thus, an important player in the DNA damage response.

The ARK2N-CK2 complex initiates transcription-coupled repair through enhancing the interaction of CSB with lesion-stalled RNAPII.

Transcription is extremely important for cellular processes but can be hindered by RNA polymerase II (RNAPII) pausing and stalling. Cockayne syndrome protein B (CSB) promotes the progression of paused RNAPII or initiates transcription-coupled nucleotide excision repair (TC-NER) to remove stalled RNAPII. However, the specific mechanism by which CSB initiates TC-NER upon damage remains unclear. In this study, we identified the indispensable role of the ARK2N-CK2 complex in the CSB-mediated initiation of TC-NER. The ARK2N-CK2 complex is recruited to damage sites through CSB and then phosphorylates CSB. Phosphorylation of CSB enhances its binding to stalled RNAPII, prolonging the association of CSB with chromatin and promoting CSA-mediated ubiquitination of stalled RNAPII. Consistent with this finding, Ark2n-/- mice exhibit a phenotype resembling Cockayne syndrome. These findings shed light on the pivotal role of the ARK2N-CK2 complex in governing the fate of RNAPII through CSB, bridging a critical gap necessary for initiating TC-NER.

Hydrogen Embrittlement as a Conspicuous Material Challenge─Comprehensive Review and Future Directions.

Hydrogen is considered a clean and efficient energy carrier crucial for shaping the net-zero future. Large-scale production, transportation, storage, and use of green hydrogen are expected to be undertaken in the coming decades. As the smallest element in the universe, however, hydrogen can adsorb on, diffuse into, and interact with many metallic materials, degrading their mechanical properties. This multifaceted phenomenon is generically categorized as hydrogen embrittlement (HE). HE is one of the most complex material problems that arises as an outcome of the intricate interplay across specific spatial and temporal scales between the mechanical driving force and the material resistance fingerprinted by the microstructures and subsequently weakened by the presence of hydrogen. Based on recent developments in the field as well as our collective understanding, this Review is devoted to treating HE as a whole and providing a constructive and systematic discussion on hydrogen entry, diffusion, trapping, hydrogen-microstructure interaction mechanisms, and consequences of HE in steels, nickel alloys, and aluminum alloys used for energy transport and storage. HE in emerging material systems, such as high entropy alloys and additively manufactured materials, is also discussed. Priority has been particularly given to these less understood aspects. Combining perspectives of materials chemistry, materials science, mechanics, and artificial intelligence, this Review aspires to present a comprehensive and impartial viewpoint on the existing knowledge and conclude with our forecasts of various paths forward meant to fuel the exploration of future research regarding hydrogen-induced material challenges.

Eukaryotic AlaX provides multiple checkpoints for quality and quantity of aminoacyl-tRNAs in translation.

Translational fidelity relies critically on correct aminoacyl-tRNA supply. The trans-editing factor AlaX predominantly hydrolyzes Ser-tRNAAla, functioning as a third sieve of alanyl-tRNA synthetase (AlaRS). Despite extensive studies in bacteria and archaea, the mechanism of trans-editing in mammals remains largely unknown. Here, we show that human AlaX (hAlaX), which is exclusively distributed in the cytoplasm, is an active trans-editing factor with strict Ser-specificity. In vitro, both hAlaX and yeast AlaX (ScAlaX) were capable of hydrolyzing nearly all Ser-mischarged cytoplasmic and mitochondrial tRNAs; and robustly edited cognate Ser-charged cytoplasmic and mitochondrial tRNASers. In vivo or cell-based studies revealed that loss of ScAlaX or hAlaX readily induced Ala- and Thr-to-Ser misincorporation. Overexpression of hAlaX impeded the decoding efficiency of consecutive Ser codons, implying its regulatory role in Ser codon decoding. Remarkably, yeast cells with ScAlaX deletion responded differently to translation inhibitor treatment, with a gain in geneticin resistance, but sensitivity to cycloheximide, both of which were rescued by editing-capable ScAlaX, alanyl- or threonyl-tRNA synthetase. Altogether, our results demonstrated the previously undescribed editing peculiarities of eukaryotic AlaXs, which provide multiple checkpoints to maintain the speed and fidelity of genetic decoding.

Multifaceted roles of t6A biogenesis in efficiency and fidelity of mitochondrial gene expression.

N 6-Threonylcarbamoyladenosine at A37 (t6A37) of ANN-decoding transfer RNAs (tRNAs) is a universal modification whose functions have been well documented in bacteria and lower eukaryotes; however, its role in organellar translation is not completely understood. In this study, we deleted the mitochondrial t6A37-modifying enzyme OSGEPL1 in HEK293T cells. OSGEPL1 is dispensable for cell viability. t6A37 hypomodification selectively stimulated N1-methyladenosine at A9 (m1A9) and N2-methylguanosine at G10 (m2G10) modifications and caused a substantial reduction in the aminoacylation of mitochondrial tRNAThr and tRNALys, resulting in impaired translation efficiency. Multiple types of amino acid misincorporation due to the misreading of near-cognate codons by t6A37-unmodified tRNAs were detected, indicating a triggered translational infidelity. Accordingly, the alterations in mitochondrial structure, function, and the activated mitochondrial unfolded protein response were observed. Mitochondrial function was efficiently restored by wild-type, but not by tRNA-binding-defective OSGEPL1. Lastly, in Osgepl1 deletion mice, disruption to mitochondrial translation was evident but resulted in no observable deficiency under physiological conditions in heart, which displays the highest Osgepl1 expression. Taken together, our data delineate the multifaceted roles of mitochondrial t6A37 modification in translation efficiency and quality control in mitochondria.

Activity reconstitution of Kre33 and Tan1 reveals a molecular ruler mechanism in eukaryotic tRNA acetylation.

RNA acetylation is a universal post-transcriptional modification that occurs in various RNAs. Transfer RNA (tRNA) acetylation is found at position 34 (ac4C34) in bacterial tRNAMet and position 12 (ac4C12) in eukaryotic tRNASer and tRNALeu. The biochemical mechanism, structural basis and functional significance of ac4C34 are well understood; however, despite being discovered in the 1960s and identification of Kre33/NAT10 and Tan1/THUMPD1 as modifying apparatuses, ac4C12 modification activity has never been reconstituted for nearly six decades. Here, we successfully reconstituted the ac4C12 modification activity of yeast Kre33 and Tan1. Biogenesis of ac4C12 is primarily dependent on a minimal set of elements, including a canonical acceptor stem, the presence of the 11CCG13 motif and correct D-arm orientation, indicating a molecular ruler mechanism. A single A13G mutation conferred ac4C12 modification to multiple non-substrate tRNAs. Moreover, we were able to introduce ac4C modifications into small RNAs. ac4C12 modification contributed little to tRNA melting temperature and aminoacylation in vitro and in vivo. Collectively, our results realize in vitro activity reconstitution, delineate tRNA substrate selection mechanism for ac4C12 biogenesis and develop a valuable system for preparing acetylated tRNAs as well as non-tRNA RNA species, which will advance the functional interpretation of the acetylation in RNA structures and functions.

Mammalian mitochondrial translation infidelity leads to oxidative stress-induced cell cycle arrest and cardiomyopathy.

Proofreading (editing) of mischarged tRNAs by cytoplasmic aminoacyl-tRNA synthetases (aaRSs), whose impairment causes neurodegeneration and cardiac diseases, is of high significance for protein homeostasis. However, whether mitochondrial translation needs fidelity and the significance of editing by mitochondrial aaRSs have been unclear. Here, we show that mammalian cells critically depended on the editing of mitochondrial threonyl-tRNA synthetase (mtThrRS, encoded by Tars2), disruption of which accumulated Ser-tRNAThr and generated a large abundance of Thr-to-Ser misincorporated peptides in vivo. Such infidelity impaired mitochondrial translation and oxidative phosphorylation, causing oxidative stress and cell cycle arrest in the G0/G1 phase. Notably, reactive oxygen species (ROS) scavenging by N-acetylcysteine attenuated this abnormal cell proliferation. A mouse model of heart-specific defective mtThrRS editing was established. Increased ROS levels, blocked cardiomyocyte proliferation, contractile dysfunction, dilated cardiomyopathy, and cardiac fibrosis were observed. Our results elucidate that mitochondria critically require a high level of translational accuracy at Thr codons and highlight the cellular dysfunctions and imbalance in tissue homeostasis caused by mitochondrial mistranslation.

Clonorchiasis and opisthorchiasis: epidemiology, transmission, clinical features, morbidity, diagnosis, treatment, and control.

Clonorchis sinensis, Opisthorchis viverrini, and Opisthorchis felineus are important liver flukes that cause a considerable public health burden in eastern Asia, southeastern Asia, and eastern Europe, respectively. The life cycles are complex, involving humans, animal reservoirs, and two kinds of intermediate hosts. An interplay of biological, cultural, ecological, economic, and social factors drives transmission. Chronic infections are associated with liver and biliary complications, most importantly cholangiocarcinoma. With regard to diagnosis, stool microscopy is widely used in epidemiologic surveys and for individual diagnosis. Immunologic techniques are employed for screening purposes, and molecular techniques facilitate species differentiation in reference laboratories. The mainstay of control is preventive chemotherapy with praziquantel, usually combined with behavioral change through information, education and communication, and environmental control. Tribendimidine, a drug registered in the People's Republic of China for soil-transmitted helminth infections, shows potential against both C. sinensis and O. viverrini and, hence, warrants further clinical development. Novel control approaches include fish vaccine and biological control. Considerable advances have been made using multi-omics which may trigger the development of new interventions. Pressing research needs include mapping the current distribution, disentangling the transmission, accurately estimating the disease burden, and developing new diagnostic and treatment tools, which would aid to optimize control and elimination measures.

IEPAPI: a method for immune epitope prediction by incorporating antigen presentation and immunogenicity.

CD8+ T cells can recognize peptides presented by class I human leukocyte antigen (HLA-I) of nucleated cells. Exploring this immune mechanism is essential for identifying T-cell vaccine targets in cancer immunotherapy. Over the past decade, the wealth of data generated by experiments has spawned many computational approaches for predicting HLA-I binding, antigen presentation and T-cell immune responses. Nevertheless, existing HLA-I binding and antigen presentation prediction approaches suffer from low precision due to the absence of T-cell receptor (TCR) recognition. Direct modeling of T-cell immune responses is less effective as TCR recognition's mechanism still remains underexplored. Therefore, directly applying these existing methods to screen cancer neoantigens is still challenging. Here, we propose a novel immune epitope prediction method termed IEPAPI by effectively incorporating antigen presentation and immunogenicity. First, IEPAPI employs a transformer-based feature extraction block to acquire representations of peptides and HLA-I proteins. Second, IEPAPI integrates the prediction of antigen presentation prediction into the input of immunogenicity prediction branch to simulate the connection between the biological processes in the T-cell immune response. Quantitative comparison results on an independent antigen presentation test dataset exhibit that IEPAPI outperformed the current state-of-the-art approaches NetMHCpan4.1 and mhcflurry2.0 on 100 (25/25) and 76% (19/25) of the HLA subtypes, respectively. Furthermore, IEPAPI demonstrates the best precision on two independent neoantigen datasets when compared with existing approaches, suggesting that IEPAPI provides a vital tool for T-cell vaccine design.

Circular mRNA-based TCR-T offers a safe and effective therapeutic strategy for treatment of cytomegalovirus infection.

Circular mRNA (cmRNA) is particular useful due to its high resistance to degradation by exonucleases, resulting in greater stability and protein expression compared to linear mRNA. T cell receptor (TCR)-engineered T cells (TCR-T) represent a promising means of treating viral infections and cancer. This study aimed to evaluate the feasibility and efficacy of cmRNA in antigen-specific-TCR discovery and TCR-T therapy. Using human cytomegalovirus (CMV) pp65 antigen as a model, we found that the expansion of pp65-responsive T cells was induced more effectively by monocyte-derived dendritic cells transfected with pp65-encoding cmRNA compared with linear mRNA. Subsequently, we developed cmRNA-transduced pp65-TCR-T (cm-pp65-TCR-T) that specifically targets the CMV-pp65 epitope. Our results showed that pp65-TCR could be expressed on primary T cells for more than 7 days. Moreover, both in vitro killing and in vivo CDX models demonstrated that cm-pp65-TCR-T cells specifically and persistently kill pp65-and HLA-expressing tumor cells, significantly prolonging the survival of mice. Collectively, our results demonstrated that cmRNA can be used as a more effective technical approach for antigen-specific TCR isolation and identification, and cm-pp65-TCR-T may provide a safe, non-viral, non-integrated therapeutic approach for controlling CMV infection, particularly in patients who have undergone allogeneic hematopoietic stem cell transplantation.

PEBP4 deficiency aggravates LPS-induced acute lung injury and alveolar fluid clearance impairment via modulating PI3K/AKT signaling pathway.

Acute lung injury (ALI) is a common clinical syndrome, which often results in pulmonary edema and respiratory distress. It has been recently reported that phosphatidylethanolamine binding protein 4 (PEBP4), a basic cytoplasmic protein, has anti-inflammatory and hepatoprotective effects, but its relationship with ALI remains undefined so far. In this study, we generated PEBP4 knockout (KO) mice to investigate the potential function of PEBP4, as well as to evaluate the capacity of alveolar fluid clearance (AFC) and the activity of phosphatidylinositide 3-kinases (PI3K)/serine-theronine protein kinase B (PKB, also known as AKT) signaling pathway in lipopolysaccharide (LPS)-induced ALI mice models. We found that PEBP4 deficiency exacerbated lung pathological damage and edema, and increased the wet/dry weight ratio and total protein concentration of bronchoalveolar lavage fluid (BALF) in LPS-treated mice. Meanwhile, PEBP4 KO promoted an LPS-induced rise in the pulmonary myeloperoxidase (MPO) activity, serum interleuin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α levels, and pulmonary cyclooxygenase-2 (COX-2) expression. Mechanically, PEBP4 deletion further reduced the protein expression of Na+ transport markers, including epithelial sodium channel (ENaC)-α, ENaC-γ, Na,K-ATPase α1, and Na,K-ATPase ß1, and strengthened the inhibition of PI3K/AKT signaling in LPS-challenged mice. Furthermore, we demonstrated that selective activation of PI3K/AKT with 740YP or SC79 partially reversed all of the above effects caused by PEBP4 KO in LPS-treated mice. Altogether, our results indicated the PEBP4 deletion has a deterioration effect on LPS-induced ALI by impairing the capacity of AFC, which may be achieved through modulating the PI3K/AKT pathway.

Origin for electrochemically driven phase transformation in the oxygen electrode for a solid oxide cell.

The next generation of fuel cells, electrolyzers, and batteries requires higher power, faster kinetics, and larger energy density, which necessitate the use of compositionally complex oxides to achieve multifunctionalities and activity. These compositionally complex oxides may change their phases and structures during an electrochemical process-a so-called "electrochemically driven phase transformation." The origin for such a phase change has remained obscure. The aim of this paper is to present an experimental study and a theoretical analysis of phase evolution in praseodymium nickelates. Nickelate-based electrodes show up to 60 times greater phase transformation during operation when compared with thermally annealed ones. Theoretical analysis suggests that the presence of a reduced oxygen partial pressure at the interface between the oxygen electrode and the electrolyte is the origin for the phase change in an oxygen electrode. Guided by the theory, the addition of the electronic conduction in the interface layer leads to the significant suppression of phase change while improving cell performance and performance stability.

Identification of Fenton-like active Cu sites by heteroatom modulation of electronic density.

Developing heterogeneous catalysts with atomically dispersed active sites is vital to boost peroxymonosulfate (PMS) activation for Fenton-like activity, but how to controllably adjust the electronic configuration of metal centers to further improve the activation kinetics still remains a great challenge. Herein, we report a systematic investigation into heteroatom-doped engineering for tuning the electronic structure of Cu-N4 sites by integrating electron-deficient boron (B) or electron-rich phosphorus (P) heteroatoms into carbon substrate for PMS activation. The electron-depleted Cu-N4/C-B is found to exhibit the most active oxidation capacity among the prepared Cu-N4 single-atom catalysts, which is at the top rankings of the Cu-based catalysts and is superior to most of the state-of-the-art heterogeneous Fenton-like catalysts. Conversely, the electron-enriched Cu-N4/C-P induces a decrease in PMS activation. Both experimental results and theoretical simulations unravel that the long-range interaction with B atoms decreases the electronic density of Cu active sites and down-shifts the d-band center, and thereby optimizes the adsorption energy for PMS activation. This study provides an approach to finely control the electronic structure of Cu-N4 sites at the atomic level and is expected to guide the design of smart Fenton-like catalysts.

Prediction of Optical Properties in Particulate Media Using Double Optimization of Dependent Scattering and Particle Distribution.

The prediction of optical properties dominated by light scattering in particulate media composed of high-concentration and polydisperse particles is greatly important in various optical applications. However, the accuracy and efficiency of light propagation simulations are still limited by the huge computational burden and complex interactions between dense and polydisperse particles. Here, we proposed a new optimization strategy that can effectively and accurately predict optical properties based on Monte Carlo simulation with particle size and dependent scattering corrections. Both the scattering parameters of particles and the experimental reflectance spectrum are fully examined for verification. Furthermore, using the weighted solar reflectance of particulate media as a representative optical property, both numerical simulations and experiments confirm the superiority and universality of the proposed optimization approach in a variety of materials systems. Moreover, our work can guide the design of particulate media with specific optical features insightfully and will be applicable in many fields involving multiparticle scattering.

Loss of threonyl-tRNA synthetase-like protein Tarsl2 has little impact on protein synthesis but affects mouse development.

Aminoacyl-tRNA synthetases (aaRSs) are essential components for mRNA translation. Two sets of aaRSs are required for cytoplasmic and mitochondrial translation in vertebrates. Interestingly, TARSL2 is a recently evolved duplicated gene of TARS1 (encoding cytoplasmic threonyl-tRNA synthetase) and represents the only duplicated aaRS gene in vertebrates. Although TARSL2 retains the canonical aminoacylation and editing activities in vitro, whether it is a true tRNA synthetase for mRNA translation in vivo is unclear. In this study, we showed that Tars1 is an essential gene since homozygous Tars1 KO mice were lethal. In contrast, when Tarsl2 was deleted in mice and zebrafish, neither the abundance nor the charging levels of tRNAThrs were changed, indicating that cells relied on Tars1 but not on Tarsl2 for mRNA translation. Furthermore, Tarsl2 deletion did not influence the integrity of the multiple tRNA synthetase complex, suggesting that Tarsl2 is a peripheral member of the multiple tRNA synthetase complex. Finally, we observed that Tarsl2-deleted mice exhibited severe developmental retardation, elevated metabolic capacity, and abnormal bone and muscle development after 3 weeks. Collectively, these data suggest that, despite its intrinsic activity, loss of Tarsl2 has little influence on protein synthesis but does affect mouse development.

Elucidating the molecular mechanisms associated with TARS2-related mitochondrial disease.

TARS2 encodes human mitochondrial threonyl tRNA-synthetase that is responsible for generating mitochondrial Thr-tRNAThr and clearing mischarged Ser-tRNAThr during mitochondrial translation. Pathogenic variants in TARS2 have hitherto been reported in a pair of siblings and an unrelated patient with an early onset mitochondrial encephalomyopathy and a combined respiratory chain enzyme deficiency in muscle. We here report five additional unrelated patients with TARS2-related mitochondrial diseases, expanding the clinical phenotype to also include epilepsy, dystonia, hyperhidrosis and severe hearing impairment. In addition, we document seven novel TARS2 variants-one nonsense variant and six missense variants-that we demonstrate are pathogenic and causal of the disease presentation based on population frequency, homology modeling and functional studies that show the effects of the pathogenic variants on TARS2 stability and/or function.