Itaconate is a lysosomal inducer that promotes antibacterial innate immunity.

Lysosomes are the main organelles in macrophages for killing invading bacteria. However, the precise mechanism underlying lysosomal biogenesis upon bacterial infection remains enigmatic. We demonstrate here that LPS stimulation increases IRG1-dependent itaconate production, which promotes lysosomal biogenesis by activating the transcription factor, TFEB. Mechanistically, itaconate directly alkylates human TFEB at cysteine 212 (Cys270 in mice) to induce its nuclear localization by antagonizing mTOR-mediated phosphorylation and cytosolic retention. Functionally, abrogation of itaconate synthesis by IRG1/Irg1 knockout or expression of an alkylation-deficient TFEB mutant impairs the antibacterial ability of macrophages in vitro. Furthermore, knockin mice harboring an alkylation-deficient TFEB mutant display elevated susceptibility to Salmonella typhimurium infection, whereas in vivo treatment of OI, a cell-permeable itaconate derivative, limits inflammation. Our study identifies itaconate as an endogenous metabolite that functions as a lysosomal inducer in macrophages in response to bacterial infection, implying the potential therapeutic utility of itaconate in treating human bacterial infection.

Morphological diversity of single neurons in molecularly defined cell types.

Dendritic and axonal morphology reflects the input and output of neurons and is a defining feature of neuronal types1,2, yet our knowledge of its diversity remains limited. Here, to systematically examine complete single-neuron morphologies on a brain-wide scale, we established a pipeline encompassing sparse labelling, whole-brain imaging, reconstruction, registration and analysis. We fully reconstructed 1,741 neurons from cortex, claustrum, thalamus, striatum and other brain regions in mice. We identified 11 major projection neuron types with distinct morphological features and corresponding transcriptomic identities. Extensive projectional diversity was found within each of these major types, on the basis of which some types were clustered into more refined subtypes. This diversity follows a set of generalizable principles that govern long-range axonal projections at different levels, including molecular correspondence, divergent or convergent projection, axon termination pattern, regional specificity, topography, and individual cell variability. Although clear concordance with transcriptomic profiles is evident at the level of major projection type, fine-grained morphological diversity often does not readily correlate with transcriptomic subtypes derived from unsupervised clustering, highlighting the need for single-cell cross-modality studies. Overall, our study demonstrates the crucial need for quantitative description of complete single-cell anatomy in cell-type classification, as single-cell morphological diversity reveals a plethora of ways in which different cell types and their individual members may contribute to the configuration and function of their respective circuits.

SPINT2 mutations in the Kunitz domain 2 found in SCSD patients inactivate HAI-2 as prostasin inhibitor via abnormal protein folding and N-glycosylation.

Mutations in the Kunitz-type serine protease inhibitor HAI-2, encoded by SPINT2, are responsible for the pathogenesis of syndromic congenital sodium diarrhea (SCSD), an intractable secretory diarrhea of infancy. Some of the mutations cause defects in the functionally required Kunitz domain 1 and/or subcellular targeting signals. Almost all SCSD patients, however, harbor SPINT2 missense mutations that affect the functionally less important Kunitz domain 2. How theses single amino acid substitutions inactivate HAI-2 was, here, investigated by the doxycycline-inducible expression of three of these mutants in HAI-2-knockout Caco-2 human colorectal adenocarcinoma cells. Examining protein expressed from these HAI-2 mutants reveals that roughly 50% of the protein is synthesized as disulfide-linked oligomers that lose protease inhibitory activity due to the distortion of the Kunitz domains by disarrayed disulfide bonding. Although the remaining protein is synthesized as monomers, its glycosylation status suggests that the HAI-2 monomer remains in the immature, lightly glycosylated form, and is not converted to the heavily glycosylated mature form. Heavily glycosylated HAI-2 possesses full anti-protease activity and appropriate subcellular targeting signals, including the one embedded in the complex-type N-glycan. As predicted, these HAI-2 mutants cannot suppress the excessive prostasin proteolysis caused by HAI-2 deletion. The oligomerization and glycosylation defects have also been observed in a colorectal adenocarcinoma line that harbors one of these SPINT2 missense mutations. Our study reveals that the abnormal protein folding and N-glycosylation can cause widespread HAI-2 inactivation in SCSD patents.

Tanshinone analog inhibits castration-resistant prostate cancer cell growth by inhibiting glycolysis in an AR-dependent manner.

Androgen receptor (AR) is one of the key targets for the treatment of castration-resistant prostate cancer (CRPC). Current endocrine therapy can greatly improve patients with CRPC. However, with the change of pathogenic mechanism, acquired resistance often leads to the failure of treatment. Studies have shown that tanshinone IIA (TS-IIA) and its derivatives have significant antitumor activity, and have certain AR-targeting effects, but the mechanism is unknown. In this study, the TS-IIA analog TB3 was found to significantly inhibit the growth of CRPC in vitro and in vivo. Molecular docking, cellular thermal shift assay, and cycloheximide experiments confirmed that AR was the target of TB3 and promoted the degradation of AR. Furthermore, TB3 can significantly inhibit glycolysis metabolism by targeting the AR/PKM2 axis. The addition of pyruvic acid could significantly alleviate the inhibitory effect of TB3 on CRPC cells. Besides, the knockdown of AR or PKM2 also could reverse the effect of TB3 on CRPC cells. Taken together, our study suggests that TS-IIA derivative TB3 inhibits glycolysis to prevent the CRPC process by targeting the AR/PKM2 axis.

Deafness-associated tRNAPhe mutation impaired mitochondrial and cellular integrity.

Defects in mitochondrial RNA metabolism have been linked to sensorineural deafness that often occurs as a consequence of damaged or deficient inner ear hair cells. In this report, we investigated the molecular mechanism underlying a deafness-associated tRNAPhe 593T > C mutation that changed a highly conserved uracil to cytosine at position 17 of the DHU-loop. The m.593T > C mutation altered tRNAPhe structure and function, including increased melting temperature, resistance to S1 nuclease-mediated digestion, and conformational changes. The aberrant tRNA metabolism impaired mitochondrial translation, which was especially pronounced by decreases in levels of ND1, ND5, CYTB, CO1, and CO3 harboring higher numbers of phenylalanine. These alterations resulted in aberrant assembly, instability, and reduced activities of respiratory chain enzyme complexes I, III, IV, and intact supercomplexes overall. Furthermore, we found that the m.593T > C mutation caused markedly diminished membrane potential, and increased the production of reactive oxygen species in the mutant cell lines carrying the m.593T > C mutation. These mitochondrial dysfunctions led to the mitochondrial dynamic imbalance via increasing fission with abnormal mitochondrial morphology. Excessive fission impaired the process of autophagy including the initiation phase, formation, and maturation of the autophagosome. In particular, the m.593T > C mutation upregulated the PARKIN-dependent mitophagy pathway. These alterations promoted an intrinsic apoptotic process for the removal of damaged cells. Our findings provide critical insights into the pathophysiology of maternally inherited deafness arising from tRNA mutation-induced defects in mitochondrial and cellular integrity.

Nuclear modifier YARS2 allele correction restored retinal ganglion cells-specific deficiencies in Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy (LHON) is a maternally transmitted eye disease due to the degeneration of retinal ganglion cells (RGCs). Mitochondrial 11778G > A mutation is the most common LHON-associated mitochondrial DNA (mtDNA) mutation. Our recent studies demonstrated some LHON families manifested by synergic interaction between m.11778G > A mutation and YARS2 allele (c.572G > T, p.Gly191Val) encoding mitochondrial tyrosyl-tRNA synthetase. However, the RGC-specific effects of LHON-associated mtDNA mutations remain elusive and there is no highly effective therapy for LHON. Here, we generated patients-derived induced pluripotent stem cells (iPSCs) from fibroblasts derived from a Chinese LHON family (both m.11778G > A and c.572G > T mutations, only m.11778G > A mutation, and control subject). The c.572G > T mutation in iPSC lines from a syndromic individual was corrected by CRISPR/Cas9. Those iPSCs were differentiated into neural progenitor cells and subsequently induced RGC-like cells using a stepwise differentiation procedure. Those RGC-like cells derived from symptomatic individual harboring both m.11778G > A and c.572G > T mutations exhibited greater defects in neuronal differentiation, morphology including reduced area of soma, numbers of neurites and shortened length of axons, electrophysiological properties than those in cells bearing only m.11778G > A mutation. Furthermore, these RGC-like cells revealed more drastic reductions in oxygen consumption rates, levels of mitochondrial ATP and increasing productions of reactive oxygen species than those in other cell models. These mitochondrial dysfunctions promoted the apoptotic process for RGC degenerations. Correction of YARS2 c.572G > T mutation rescued deficiencies of patient-derived RGC-like cells. These findings provide new insights into pathophysiology of LHON arising from RGC-specific mitochondrial dysfunctions and step toward therapeutic intervention for this disease.

Genetic regulation of human brain proteome reveals proteins implicated in psychiatric disorders.

Psychiatric disorders are highly heritable yet polygenic, potentially involving hundreds of risk genes. Genome-wide association studies have identified hundreds of genomic susceptibility loci with susceptibility to psychiatric disorders; however, the contribution of these loci to the underlying psychopathology and etiology remains elusive. Here we generated deep human brain proteomics data by quantifying 11,608 proteins across 268 subjects using 11-plex tandem mass tag coupled with two-dimensional liquid chromatography-tandem mass spectrometry. Our analysis revealed 788 cis-acting protein quantitative trait loci associated with the expression of 883 proteins at a genome-wide false discovery rate <5%. In contrast to expression at the transcript level and complex diseases that are found to be mainly influenced by noncoding variants, we found protein expression level tends to be regulated by non-synonymous variants. We also provided evidence of 76 shared regulatory signals between gene expression and protein abundance. Mediation analysis revealed that for most (88%) of the colocalized genes, the expression levels of their corresponding proteins are regulated by cis-pQTLs via gene transcription. Using summary data-based Mendelian randomization analysis, we identified 4 proteins and 19 genes that are causally associated with schizophrenia. We further integrated multiple omics data with network analysis to prioritize candidate genes for schizophrenia risk loci. Collectively, our findings underscore the potential of proteome-wide linkage analysis in gaining mechanistic insights into the pathogenesis of psychiatric disorders.

Interaction of Filamin C With Actin Is Essential for Cardiac Development and Function.

BACKGROUND: FLNC (filamin C), a member of the filamin family predominantly expressed in striated muscles, plays a crucial role in bridging the cytoskeleton and ECM (extracellular matrix) in cardiomyocytes, thereby maintaining heart integrity and function. Although genetic variants within the N-terminal ABD (actin-binding domain) of FLNC have been identified in patients with cardiomyopathy, the precise contribution of the actin-binding capability to FLNC's function in mammalian hearts remains poorly understood. METHODS: We conducted in silico analysis of the 3-dimensional structure of mouse FLNC to identify key amino acid residues within the ABD that are essential for FLNC's actin-binding capacity. Subsequently, we performed coimmunoprecipitation and immunofluorescent assays to validate the in silico findings and assess the impact of these mutations on the interactions with other binding partners and the subcellular localization of FLNC. Additionally, we generated and analyzed knock-in mouse models in which the FLNC-actin interaction was completely disrupted by these mutations. RESULTS: Our findings revealed that F93A/L98E mutations completely disrupted FLNC-actin interaction while preserving FLNC's ability to interact with other binding partners ITGB1 (ß1 integrin) and γ-SAG (γ-sarcoglycan), as well as maintaining FLNC subcellular localization. Loss of FLNC-actin interaction in embryonic cardiomyocytes resulted in embryonic lethality and cardiac developmental defects, including ventricular wall malformation and reduced cardiomyocyte proliferation. Moreover, disruption of FLNC-actin interaction in adult cardiomyocytes led to severe dilated cardiomyopathy, enhanced lethality and dysregulation of key cytoskeleton components. CONCLUSIONS: Our data strongly support the crucial role of FLNC as a bridge between actin filaments and ECM through its interactions with actin, ITGB1, γ-SAG, and other associated proteins in cardiomyocytes. Disruption of FLN-actin interaction may result in detachment of actin filaments from the extracellular matrix, ultimately impairing normal cardiac development and function. These findings also provide insights into mechanisms underlying cardiomyopathy associated with genetic variants in FLNC ABD and other regions.

Role and mechanism of PVN-sympathetic-adipose circuit in depression and insulin resistance induced by chronic stress.

Chronic stress induces depression and insulin resistance, between which there is a bidirectional relationship. However, the mechanisms underlying this comorbidity remain unclear. White adipose tissue (WAT), innervated by sympathetic nerves, serves as a central node in the interorgan crosstalk through adipokines. Abnormal secretion of adipokines is involved in mood disorders and metabolic morbidities. We describe here a brain-sympathetic nerve-adipose circuit originating in the hypothalamic paraventricular nucleus (PVN) with a role in depression and insulin resistance induced by chronic stress. PVN neurons are labelled after inoculation of pseudorabies virus (PRV) into WAT and are activated under restraint stress. Chemogenetic manipulations suggest a role for the PVN in depression and insulin resistance. Chronic stress increases the sympathetic innervation of WAT and downregulates several antidepressant and insulin-sensitizing adipokines, including leptin, adiponectin, Angptl4 and Sfrp5. Chronic activation of the PVN has similar effects. ß-adrenergic receptors translate sympathetic tone into an adipose response, inducing downregulation of those adipokines and depressive-like behaviours and insulin resistance. We finally show that AP-1 has a role in the regulation of adipokine expression under chronic stress.

Kv1.1 preserves the neural stem cell pool and facilitates neuron maturation during adult hippocampal neurogenesis.

Adult hippocampal neurogenesis is critical for learning and memory, and aberrant adult neurogenesis has been implicated in cognitive decline associated with aging and neurological diseases [J. T. Gonçalves, S. T. Schafer, F. H. Gage, Cell 167, 897­914 (2016)]. In previous studies, we observed that the delayed-rectifier voltage-gated potassium channel Kv1.1 controls the membrane potential of neural stem and progenitor cells and acts as a brake on neurogenesis during neonatal hippocampal development [S. M. Chou et al., eLife 10, e58779 (2021)]. To assess the role of Kv1.1 in adult hippocampal neurogenesis, we developed an inducible conditional knockout mouse to specifically remove Kv1.1 from adult neural stem cells via tamoxifen administration. We determined that Kv1.1 deletion in adult neural stem cells causes overproliferation and depletion of radial glia-like neural stem cells, prevents proper adult-born granule cell maturation and integration into the dentate gyrus, and moderately impairs hippocampus-dependent contextual fear learning and memory. Taken together, these findings support a critical role for this voltage-gated ion channel in adult neurogenesis.

Graphene Failure under MPa: Nanowear of Step Edges Initiated by Interfacial Mechanochemical Reactions.

The low wear resistance of macroscale graphene coatings does not match the ultrahigh mechanical strength and chemical inertness of the graphene layer itself; however, the wear mechanism responsible for this issue at low mechanical stress is still unclear. Here, we demonstrate that the susceptibility of the graphene monolayer to wear at its atomic step edges is governed by the mechanochemistry of frictional interfaces. The mechanochemical reactions activated by chemically active SiO2 microspheres result in atomic attrition rather than mechanical damage such as surface fracture and folding by chemically inert diamond tools. Correspondingly, the threshold contact stress for graphene edge wear decreases more than 30 times to the MPa level, and mechanochemical wear can be described well with the mechanically assisted Arrhenius-type kinetic model, i.e., exponential dependence of the removal rate on the contact stress. These findings provide a strategy for improving the antiwear of graphene-based materials by reducing the mechanochemical interactions at tribological interfaces.

One-Dimensional Crystal-Structure Te-Se Alloy for Flexible Shortwave Infrared Photodetector and Imaging.

Flexible shortwave infrared detectors play a crucial role in wearable devices, bioimaging, automatic control, etc. Commercial shortwave infrared detectors face challenges in achieving flexibility due to the high fabrication temperature and rigid material properties. Herein, we develop a high-performance flexible Te0.7Se0.3 photodetector, resulting from the unique 1D crystal structure and small elastic modulus of Te-Se alloying. The flexible photodetector exhibits a broad-spectrum response ranging from 365 to 1650 nm, a fast response time of 6 µs, a broad linear dynamic range of 76 dB, and a specific detectivity of 4.8 × 1010 Jones at room temperature. The responsivity of the flexible detector remains at 93% of its initial value after bending with a small curvature of 3 mm. Based on the optimized flexible detector, we demonstrate its application in shortwave infrared imaging. These results showcase the great potential of Te0.7Se0.3 photodetectors for flexible electronics.

Identification of crosstalk genes relating to ECM-receptor interaction genes in MASH and DN using bioinformatics and machine learning.

This study aimed to identify genes shared by metabolic dysfunction-associated fatty liver disease (MASH) and diabetic nephropathy (DN) and the effect of extracellular matrix (ECM) receptor interaction genes on them. Datasets with MASH and DN were downloaded from the Gene Expression Omnibus (GEO) database. Pearson's coefficients assessed the correlation between ECM-receptor interaction genes and cross talk genes. The coexpression network of co-expression pairs (CP) genes was integrated with its protein-protein interaction (PPI) network, and machine learning was employed to identify essential disease-representing genes. Finally, immuno-penetration analysis was performed on the MASH and DN gene datasets using the CIBERSORT algorithm to evaluate the plausibility of these genes in diseases. We found 19 key CP genes. Fos proto-oncogene (FOS), belonging to the IL-17 signalling pathway, showed greater centrality PPI network; Hyaluronan Mediated Motility Receptor (HMMR), belonging to ECM-receptor interaction genes, showed most critical in the co-expression network map of 19 CP genes; Forkhead Box C1 (FOXC1), like FOS, showed a high ability to predict disease in XGBoost analysis. Further immune infiltration showed a clear positive correlation between FOS/FOXC1 and mast cells that secrete IL-17 during inflammation. Combining the results of previous studies, we suggest a FOS/FOXC1/HMMR regulatory axis in MASH and DN may be associated with mast cells in the acting IL-17 signalling pathway. Extracellular HMMR may regulate the IL-17 pathway represented by FOS through the Mitogen-Activated Protein Kinase 1 (ERK) or PI3K-Akt-mTOR pathway. HMMR may serve as a signalling carrier between MASH and DN and could be targeted for therapeutic development.

EEBR induces Caspase-1-dependent pyroptosis through the NF-κB/NLRP3 signalling cascade in non-small cell lung cancer.

Lung cancer is a leading cause of cancer-related deaths worldwide. Recent studies have identified pyroptosis, a type of programmed cell death, as a critical process in the development and progression of lung cancer. In this study, we investigated the effect of EEBR, a new compound synthesized by our team, on pyroptosis in non-small cell lung cancer cells (NSCLC) and the underlying molecular mechanisms. Our results demonstrated that EEBR significantly reduced the proliferation and metastasis of NSCLC cells in vitro. Moreover, EEBR-induced pyroptosis in NSCLC cells, as evidenced by cell membrane rupture, the release of cytokines such as interleukin-18 and interleukin-1 beta and the promotion of Gasdermin D cleavage in a Caspase-1-dependent manner. Furthermore, EEBR promoted the nuclear translocation of NF-κB and upregulated the protein level of NLRP3. Subsequent studies revealed that EEBR-induced pyroptosis was suppressed by the inhibition of NF-κB. Finally, EEBR effectively suppressed the growth of lung cancer xenograft tumours by promoting NSCLC pyroptosis in animal models. Taken together, our findings suggest that EEBR induces Caspase-1-dependent pyroptosis through the NF-κB/NLRP3 signalling cascade in NSCLC, highlighting its potential as a candidate drug for NSCLC treatment.

Hydrogen-Bonded Thiol Undergoes Unconventional Excited-State Intramolecular Proton-Transfer Reactions.

The chapter on the thiol-related hydrogen bond (H-bond) and its excited-state intramolecular proton-transfer (ESIPT) reaction was recently opened where compound 4'-diethylamino-3-mercaptoflavone (3NTF) undergoes ESIPT in both cyclohexane solution and solid, giving a 710 nm tautomer emission with an anomalously large Stokes shift of 12,230 cm-1. Considering the thiol H-bond to be unconventional compared to the conventional Pauling-type -OH or -NH H-bond, it is thus essential and timely to probe its fundamental difference between their ESIPT. However, thiol-associated ESIPT tends to be nonemissive due to the dominant nπ* character of the tautomeric lowest excited state. Herein, based on the 3-mercaptoflavone scaffold and π-elongation concept, a new series of 4'-substituted-7-diethylamino-3-mercaptoflavones, NTFs, was designed and synthesized with varied H-bond strength and 690-720 nm tautomeric emission upon ultraviolet (UV) excitation in cyclohexane. The order of their H-bonding strength was experimentally determined to be N-NTF < O-NTF < H-NTF < F-NTF, while the rate of -SH ESIPT measured by fluorescence upconversion was F-NTF (398 fs)-1 < H-NTF (232 fs)-1 < O-NTF (123 fs)-1 < N-NTF (101 fs)-1 in toluene. Unexpectedly, the strongest H-bonded F-NTF gives the slowest ESIPT, which does not conform to the traditional ESIPT model. The results are rationalized by the trend of carbonyl oxygen basicity rather than -SH acidity. Namely, the thiol acidity relevant to the H-bond strength plays a minor role in the driving force of ESIPT. Instead, the proton-accepting strength governs ESIPT. That is to say, the noncanonical thiol H-bonding system undergoes an unconventional type of ESIPT.

In Situ Tracking of Water Oxidation Generated Nanoscale Dynamics in Layered Double Hydroxides Nanosheets.

Layered double hydroxides (LDHs) are potential catalysts for water oxidation, and it is recognized that they undergo dynamic evolution during the operation. However, little is known about the interfacial behaviors at the nanoscale under working conditions nor the underlying effects on electrocatalytic performance. Herein, using electrochemical atomic force microscopy, we in situ visualize the heterogeneous evolution of LDH nanosheets during oxygen evolution reaction (OER). By further combining density functional theory calculations, we elucidate the origin of the heterogeneous dynamics and their impact on the OER efficiency. Our findings demonstrate that NiCo LDHs transform to the catalytically active NiCoOx(OH)2-x phase during OER, and the redox transition between is accompanied by compressive and tensile strain, leading to in-plane contraction and reversible expansion of the nanosheets. Nonisotropic strain and out-of-plane strain relaxation due to defects and interparticle interactions result in cracking and wrinkling in the nanostructure, which is responsible for the partial activation and long-term deterioration of LDH electrocatalysts toward the OER. With this knowledge, we suggest and validate that engineering defects can precisely tune these dynamic behaviors, improving the OER activity and stability among LDH-based electrocatalysts.

Angiotensin II acts through Rac1 to upregulate pendrin: role of NADPH oxidase.

Angiotensin II increases apical plasma membrane pendrin abundance and function. This study explored the role of the small GTPase Rac1 in the regulation of pendrin by angiotensin II. To do this, we generated intercalated cell (IC) Rac1 knockout mice and observed that IC Rac1 gene ablation reduced the relative abundance of pendrin in the apical region of intercalated cells in angiotensin II-treated mice but not vehicle-treated mice. Similarly, the Rac1 inhibitor EHT 1864 reduced apical pendrin abundance in angiotensin II-treated mice, through a mechanism that does not require aldosterone. This IC angiotensin II-Rac1 signaling cascade modulates pendrin subcellular distribution without significantly changing actin organization. However, NADPH oxidase inhibition with APX 115 reduced apical pendrin abundance in vivo in angiotensin II-treated mice. Moreover, superoxide dismutase mimetics reduced Cl- absorption in angiotensin II-treated cortical collecting ducts perfused in vitro. Since Rac1 is an NADPH subunit, Rac1 may modulate pendrin through NADPH oxidase-mediated reactive oxygen species production. Because pendrin gene ablation blunts the pressor response to angiotensin II, we asked if pendrin blunts the angiotensin II-induced increase in kidney superoxide. Although kidney superoxide was similar in vehicle-treated wild-type and pendrin knockout mice, it was lower in angiotensin II-treated pendrin-null kidneys than in wild-type kidneys. We conclude that angiotensin II acts through Rac1, independently of aldosterone, to increase apical pendrin abundance. Rac1 may stimulate pendrin, at least partly, through NADPH oxidase. This increase in pendrin abundance contributes to the increment in blood pressure and kidney superoxide content seen in angiotensin II-treated mice.NEW & NOTEWORTHY This study defines a new signaling mechanism by which angiotensin II modulates oxidative stress and blood pressure.

Genetic correction of TRMU allele restored the mitochondrial dysfunction-induced deficiencies in iPSCs-derived hair cells of hearing-impaired patients.

Sensorineural hearing loss often results from damaged or deficient inner ear hair cells. Mitochondrial 12S rRNA 1555A>G mutation has been associated with hearing loss in many families. The m.1555A>G mutation is a primary factor underlying the development of hearing loss and TRMU allele (c.28G>T, p.Ala10Sser) encoding tRNA thiouridylase interact with m.1555A>G mutation to cause hearing loss. However, the tissue specificity of mitochondrial dysfunction remains elusive and there is no highly effective therapy for mitochondrial deafness. We report here the generation of induced pluripotent stem cells (iPSCs) from lymphoblastoid cell lines derived from members of an Arab-Israeli family (asymptomatic individual carrying only m.1555A>G mutation, symptomatic individual bearing both m.1555A>G and c.28G>T mutations, and control subject). The c.28G>T mutation in iPSC lines from a hearing-impaired subject was corrected by CRISPR/Cas9. These iPSCs were differentiated into otic epithelial progenitor (OEP) cells and subsequent inner ear hair cell (HC)-like cells. The iPSCs bearing m.1555A>G mutation exhibited mildly deficient differentiation into OEP and resultant HC-like cells displayed mild defects in morphology and electrophysiological properties. Strikingly, those HC-like cells harboring m.1555A>G and TRMU c.28G>T mutations displayed greater defects in the development, morphology and functions than those in cells bearing only m.1555A>G mutation. Transcriptome analysis of patients-derived HC-like cells revealed altered expressions of genes vital for mechanotransduction of hair cells. Genetic correction of TRMU c.28G>T mutation yielded morphologic and functional recovery of patient derived HC-like cells. These findings provide new insights into pathophysiology of maternally inherited hearing loss and a step toward therapeutic interventions for this disease.

Altered hydroxymethylome in the substantia nigra of Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder, and aging and genetic and environmental exposure can contribute to its pathogenesis. DNA methylation has been suggested to play a pivotal role in neurodevelopment and neurodegenerative diseases. 5-hydroxymethylcytosine (5hmC) is generated through 5-methylcytosine (5mC) oxidization by ten-eleven translocation proteins and is particularly enriched in the brain. Although 5hmC has been linked to multiple neurological disorders, little is known about 5hmC alterations in the substantia nigra of patients with PD. To determine the specific alterations in DNA methylation and hydroxymethylation in PD brain samples, we examined the genome-wide profiles of 5mC and 5hmC in the substantia nigra of patients with PD and Alzheimer's disease (ad). We identified 4119 differentially hydroxymethylated regions (DhMRs) and no differentially methylated regions (DMRs) in the postmortem brains of patients with PD compared with those of controls. These DhMRs were PD-specific when compared with the results of AD. Gene ontology analysis revealed that several signaling pathways, such as neurogenesis and neuronal differentiation, were significantly enriched in PD DhMRs. KEGG enrichment analysis revealed substantial alterations in multiple signaling pathways, including phospholipase D (PLD), cAMP and Rap1. In addition, using a PD Drosophila model, we found that one of the 5hmC-modulated genes, PLD1, modulated α-synuclein toxicity. Our analysis suggested that 5hmC may act as an independent epigenetic marker and contribute to the pathogenesis of PD.

International Multicenter Study of Ultra-Low GRWR Grafts in Adult Living Donor Liver Transplantation.

Decreasing the graft size in living donor liver transplantation (LDLT) increases the risk of early allograft dysfunction. Graft-to-recipient-weight-ratio (GRWR) of 0.8 is considered the threshold. There is evidence that smaller volume grafts may also provide equally good outcomes, the cut-off of which remains unknown. In this retrospective multi-center study, 92 adult LDLT with a final GRWR<=0.6 performed at 12 international liver transplant (LT) centers over a 3-year period were included. Perioperative data including preoperative status, portal flow hemodynamics (PFH) and portal flow modulation (PFM), development of SFSS, morbidity and mortality was collated and analyzed. Thirty-two (36.7%) patients developed SFSS and this was associated with increased 30-day, 90-day and one-year mortality. Pre-operative MELD and inpatient status were independent predictors for SFSS (p<0.05). Pre-LT renal dysfunction was an independent predictor of survival (Hazard ratio- 3.1;95% ci 1.1,8.9, p=0.035). PFH or PFM were not predictive of SFSS or survival. We report the largest ever multi-center study of LDLT outcomes using ultralow-GRWR grafts and for the first-time validate the ILTS-iLDLT-LTSI consensus definition and grading of SFSS. Pre-operative recipient condition rather than GRWR and PFH were independent predictors of SFSS. Algorithms to predict SFSS and LT outcomes should incorporate recipient factors along with GRWR.