NADH improves AIF dimerization and inhibits apoptosis in iPSCs-derived neurons from patients with auditory neuropathy spectrum disorder.

Auditory neuropathy spectrum disorder (ANSD) is a hearing impairment involving disruptions to inner hair cells (IHCs), ribbon synapses, spiral ganglion neurons (SGNs), and/or the auditory nerve itself. The outcomes of cochlear implants (CI) for ANSD are variable and dependent on the location of lesion sites. Discovering a potential therapeutic agent for ANSD remains an urgent requirement. Here, 293T stable transfection cell lines and patient induced pluripotent stem cells (iPSCs)-derived auditory neurons carrying the apoptosis inducing factor (AIF) p.R422Q variant were used to pursue a therapeutic regent for ANSD. Nicotinamide adenine dinucleotide (NADH) is a main electron donor in the electron transport chain (ETC). In 293T stable transfection cells with the p.R422Q variant, NADH treatment improved AIF dimerization, rescued mitochondrial dysfunctions, and decreased cell apoptosis. The effects of NADH were further confirmed in patient iPSCs-derived neurons. The relative level of AIF dimers was increased to 150.7 % (P = 0.026) from 59.2 % in patient-neurons upon NADH treatment. Such increased AIF dimerization promoted the mitochondrial import of coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4), which further restored mitochondrial functions. Similarly, the content of mitochondrial calcium (mCa2+) was downregulated from 136.7 % to 102.3 % (P = 0.0024) in patient-neurons upon NADH treatment. Such decreased mCa2+ levels inhibited calpain activity, ultimately reducing the percentage of apoptotic cells from 30.5 % to 21.1 % (P = 0.021). We also compared the therapeutic effects of gene correction and NADH treatment on hereditary ANSD. NADH treatment had comparable restorative effects on functions of ANSD patient-specific cells to that of gene correction. Our findings offer evidence of the molecular mechanisms of ANSD and introduce NADH as a potential therapeutic agent for ANSD therapy.

Outcomes after ORIF are similar in young and elderly patients with tibial plateau fractures: A minimum 2-year follow-up study.

BACKGROUND: This study aimed to compare the outcomes of open reduction internal fixation in young and elderly patients with tibial plateau fractures. METHODS: A total of 224 patients with tibial plateau fractures treated with open reduction internal fixation at a level I trauma center from 2014 to 2019 were reviewed. The patients with a minimum follow-up of 2 years were divided into two groups, with those aged 60 years and older divided into the elderly group and those under 60 years divided into the young group. The mean follow-up time was 55 months (range: 24-80), and the primary outcomes were quality of reduction and function. The secondary outcomes included complications, fracture healing time, and conversion to total knee arthroplasty. RESULTS: The elderly group had a higher proportion of women than the young group (61.1% vs. 23.9%, p < 0.001). Diabetes was more prevalent in the elderly cohort than in the young cohort (18.9% vs. 9.0%, p = 0.030). The rate of bone grafts was higher in the elderly group (57.8% vs. 41.8%, p = 0.019), but no significant differences were found between the groups regarding fracture characteristics, the operative time or intraoperative blood loss. The reduction quality, knee function, postoperative complications, healing time, and total knee arthroplasty conversion rate were not significantly different (p > 0.05) between the groups. CONCLUSIONS: Open reduction internal fixation remains a satisfactory technique to treat tibial plateau fractures in the elderly. Although the rate of bone grafts is higher in elderly patients, they had comparable outcomes compared with their younger counterparts.

Regulation of potential denitrification rates in sediments by microbial-driven elemental coupled metabolisms.

Microbial driven coupled processes between denitrification and methane/sulfur metabolism play a very substantial role in accelerating nitrogen removal in river sediments. Until now, little is known about how element coupling processes alter nitrogen metabolism by the microbial functional communities. The primary objective of this research was to clarify the contributory role of microbial-mediated coupled processes in controlling denitrification. Specifically, the study sought to identify the key bioindicators (or metabolic pathway) for preferably regulating and predicting potential denitrification rate (PDR). Here, a total of 40 sediment samples were collected from the inflow rivers of Chaohu Lake under nitrogen stress. The results revealed the ecological importance of methanogens and sulfate reducing bacteria in the microbial interaction network. Correlations between quantitative or predicted genes showed that the methanogenic gene (mcrA) was synergistic with denitrifying genes, further unraveling that the key role of methanogenesis in denitrification process for facilitating nitrogen removal. The PDR of sediments ranged from 0.03 to 133.21 µg N·g-1·h-1. The study uncovered specific environmental factors (NH4+ and OM) and microbial indicators (nosZ, mcrA, Paracoccus, Thauera, Methanobrevibacter and Desulfomicrobium) as potential contributors to the variations in PDR. Structural Equation Model (SEM) analysis revealed a significant direct effect of NH4+ on PDR, evidenced by a standardized coefficient (λ) of 0.77 (P < 0.001). Additionally, the findings also emphasized the salient role of methanogens (Methanobrevibacter) and methanogenic gene (mcrA) in indicating PDR. The research's aforementioned findings shed light on the substantial consequences of methanogenesis on nitrogen metabolism in coupled processes, enabling improved control of nitrogen pollution in river sediments. This study provided fresh perspectives on the effects of multiple functional taxa on denitrification, and reinforces the significance of coupling processes for nitrogen removal.

Impaired AIF-CHCHD4 interaction and mitochondrial calcium overload contribute to auditory neuropathy spectrum disorder in patient-iPSC-derived neurons with AIFM1 variant.

Auditory neuropathy spectrum disorder (ANSD) is a hearing impairment caused by dysfunction of inner hair cells, ribbon synapses, spiral ganglion neurons and/or the auditory nerve itself. Approximately 1/7000 newborns have abnormal auditory nerve function, accounting for 10%-14% of cases of permanent hearing loss in children. Although we previously identified the AIFM1 c.1265 G > A variant to be associated with ANSD, the mechanism by which ANSD is associated with AIFM1 is poorly understood. We generated induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells (PBMCs) via nucleofection with episomal plasmids. The patient-specific iPSCs were edited via CRISPR/Cas9 technology to generate gene-corrected isogenic iPSCs. These iPSCs were further differentiated into neurons via neural stem cells (NSCs). The pathogenic mechanism was explored in these neurons. In patient cells (PBMCs, iPSCs, and neurons), the AIFM1 c.1265 G > A variant caused a novel splicing variant (c.1267-1305del), resulting in AIF p.R422Q and p.423-435del proteins, which impaired AIF dimerization. Such impaired AIF dimerization then weakened the interaction between AIF and coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4). On the one hand, the mitochondrial import of ETC complex subunits was inhibited, subsequently leading to an increased ADP/ATP ratio and elevated ROS levels. On the other hand, MICU1-MICU2 heterodimerization was impaired, leading to mCa2+ overload. Calpain was activated by mCa2+ and subsequently cleaved AIF for its translocation into the nucleus, ultimately resulting in caspase-independent apoptosis. Interestingly, correction of the AIFM1 variant significantly restored the structure and function of AIF, further improving the physiological state of patient-specific iPSC-derived neurons. This study demonstrates that the AIFM1 variant is one of the molecular bases of ANSD. Mitochondrial dysfunction, especially mCa2+ overload, plays a prominent role in ANSD associated with AIFM1. Our findings help elucidate the mechanism of ANSD and may lead to the provision of novel therapies.

1-Deoxynojirimycin promotes cardiac function and rescues mitochondrial cristae in mitochondrial hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most prominent cause of sudden cardiac death in young people. Due to heterogeneity in clinical manifestations, conventional HCM drugs have limitations for mitochondrial hypertrophic cardiomyopathy. Discovering more effective compounds would be of substantial benefit for further elucidating the pathogenic mechanisms of HCM and treating patients with this condition. We previously reported the MT-RNR2 variant associated with HCM that results in mitochondrial dysfunction. Here, we screened a mitochondria-associated compound library by quantifying the mitochondrial membrane potential of HCM cybrids and the survival rate of HCM-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) in galactose media. 1-Deoxynojirimycin (DNJ) was identified to rescue mitochondrial function by targeting optic atrophy protein 1 (OPA1) to promote its oligomerization, leading to reconstruction of the mitochondrial cristae. DNJ treatment further recovered the physiological properties of HCM iPSC-CMs by improving Ca2+ homeostasis and electrophysiological properties. An angiotensin II-induced cardiac hypertrophy mouse model further verified the efficacy of DNJ in promoting cardiac mitochondrial function and alleviating cardiac hypertrophy in vivo. These results demonstrated that DNJ could be a potential mitochondrial rescue agent for mitochondrial hypertrophic cardiomyopathy. Our findings will help elucidate the mechanism of HCM and provide a potential therapeutic strategy.

KLF14 regulates the growth of hepatocellular carcinoma cells via its modulation of iron homeostasis through the repression of iron-responsive element-binding protein 2.

BACKGROUND: Hepatocellular carcinoma (HCC) is a multifactor-driven malignant tumor with rapid progression, which causes the difficulty to substantially improve the prognosis of HCC. Limited understanding of the mechanisms in HCC impedes the development of efficacious therapies. Despite Krüpple-Like factors (KLFs) were reported to be participated in HCC pathogenesis, the function of KLF14 in HCC remains largely unexplored. METHODS: We generated KLF14 overexpressed and silenced liver cancer cells, and nude mouse xenograft models for the in vitro and in vivo study. Luciferase reporter assay, ChIP-qPCR, Co-IP, immunofluorescence were performed for mechanism research. The expression of KLF14 in HCC samples was analyzed by quantitative RT-PCR, Western blotting, and immunohistochemistry (IHC) analysis. RESULTS: KLF14 was significantly downregulated in human HCC tissues, which was highly correlated with poor prognosis. Inhibition of KLF14 promoted liver cancer cells proliferation and overexpression of KLF14 suppressed cells growth. KLF14 exerts its anti-tumor function by inhibiting Iron-responsive element-binding protein 2 (IRP2), which then causes transferrin receptor-1(TfR1) downregulation and ferritin upregulation on the basis of IRP-IREs system. This then leading to cellular iron deficiency and HCC cells growth suppression in vitro and in vivo. Interestingly, KLF14 suppressed the transcription of IRP2 via recruiting SIRT1 to reduce the histone acetylation of the IRP2 promoter, resulting in iron depletion and cell growth suppression. More important, we found fluphenazine is an activator of KLF14, inhibiting HCC cells growth through inducing iron deficiency. CONCLUSION: KLF14 acts as a tumor suppressor which inhibits the proliferation of HCC cells by modulating cellular iron metabolism via the repression of IRP2. We identified Fluphenazine, as an activator of KLF14, could be a potential compound for HCC therapy. Our findings therefore provide an innovative insight into the pathogenesis of HCC and a promising therapeutic target.

SPINDLY interacts with EIN2 to facilitate ethylene signalling-mediated fruit ripening in tomato.

The post-translational modification of proteins enables cells to respond promptly to dynamic stimuli by controlling protein functions. In higher plants, SPINDLY (SPY) and SECRET AGENT (SEC) are two prominent O-glycosylation enzymes that have both unique and overlapping roles; however, the effects of their O-glycosylation on fruit ripening and the underlying mechanisms remain largely unknown. Here we report that SlSPY affects tomato fruit ripening. Using slspy mutants and two SlSPY-OE lines, we provide biological evidence for the positive role of SlSPY in fruit ripening. We demonstrate that SlSPY regulates fruit ripening by changing the ethylene response in tomato. To further investigate the underlying mechanism, we identify a central regulator of ethylene signalling ETHYLENE INSENSITIVE 2 (EIN2) as a SlSPY interacting protein. SlSPY promotes the stability and nuclear accumulation of SlEIN2. Mass spectrometry analysis further identified that SlEIN2 has two potential sites Ser771 and Thr821 of O-glycans modifications. Further study shows that SlEIN2 is essential for SlSPY in regulating fruit ripening in tomatoes. Collectively, our findings reveal a novel regulatory function of SlSPY in fruit and provide novel insights into the role of the SlSPY-SlEIN2 module in tomato fruit ripening.

Facile fabrication of ferroelectric poly(vinylidene fluoride) thin films with pure γ phase.

A simple heating-cooling procedure is employed to make pure polar γ-PVDF thin films. By controlling the relaxation state of the crystals, pure γ crystals are induced by two kinds of mechanism including self-seeding and self-nucleation upon cooling within 30 min. The methodology paves a new way for PVDF homopolymers in flexible ferroelectric device applications.

Acute toxicity of broflanilide on neurosecretory system and locomotory behavior of zebrafish (Danio rerio).

Broflanilide, a novel meta-diamide insecticide, possesses moderate acute toxicity to zebrafish, with a 96-h median lethal concentration (96-LC50) of 0.76 mg/L. However, its effect on fish behavior and the underlying mechanisms are still unclear. The present study evaluated the effects of broflanilide on the zebrafish brain over a 96-h exposure by comparing the histopathological changes and relative expression of targeted genes with the behavioral metrics. The results of the toxicity test showed that broflanilide could cause deformities, such as deformation of the operculum and spinal curvature, at 0.6, 0.82 and 1.15 mg/L. Results also showed tissue damage and apoptosis in the cerebellum under 0.27 and 0.6 mg/L exposure. Additionally, broflanilide affected the neurotransmitters, metabolites and transcripts of genes associated with dopamine, gamma-aminobutyric acid expression. and the signaling pathways in zebrafish brains at 0.60 mg/L after 1 h and 96 h of exposure, while the levels of glutamate, glutamate decarboxylase, GABA transaminase, nicotinamide adenine dinucleotide (NADH) and adenosine triphosphate (ATP) were also inhibited at 0.27 mg/L after 96 h of exposure. The accumulated swimming distance was significantly longer and the average speed was significantly faster than the control at 0.27 and 0.6 mg/L after 1-h of exposure, while these metrics were lowered at 0.6 mg/L after 96 h of exposure. The study results demonstrates that broflanilide affects the zebrafish brain, neurotransmitters and associated fish behaviors. This study also provides deeper insight into the mechanistic understanding of the effects of broflanilide on the zebrafish brain.

Alpha-2 Heremans Schmid Glycoprotein (AHSG) promotes the proliferation of bladder cancer cells by regulating the TGF-ß signalling pathway.

Bladder cancer (BC) is one of the most common urinary tract malignancies and is the tenth most common cancer globally. Alpha-2 Heremans Schmid Glycoprotein (AHSG) is a multifunctional protein that plays different roles in the progression of multiple tumors. However, the role and mechanism of AHSG in the development and progression of BC are unknown. AHSG expression was assessed in BC cells and tissues using western blot and immunohistochemistry. Using plasmid and siRNA, overexpressed and knocked down AHSG in BC cells were constructed. A series of functional experiments, including CCK8, plate clone formation, and flow cytometry, were performed to evaluate cell proliferation and cycle. AHSG was expressed higher in BC cells and tissues than in normal bladder epithelial cells and non-tumor tissues. Functionally, the overexpression of AHSG significantly increased the proliferation of BC cells and promoted the cell cycle from G1 to the S phase, whereas the knockdown of AHSG gave the opposite result.Additionally, western blot results revealed that AHSG expression level was negatively correlated with the phosphorylation level of Smad2/3 protein, a key downstream molecule of the traditional TGF-ß signaling pathway, suggesting that AHSG could antagonize the traditional TGF-ß signaling pathway. Finally, the expression level of AHSG in the urine of BC patients was significantly higher than that of healthy subjects by ELISA, with specificity. Our study concluded that AHSG might be a novel marker of BC that promotes the proliferation of BC cells by regulating the TGF-ß signaling pathway.

The miR164a-NAM3 module confers cold tolerance by inducing ethylene production in tomato.

Because of a high sensitivity to cold, both the yield and quality of tomato (Solanum lycopersicum L.) are severely restricted by cold stress. The NAC transcription factor (TF) family has been characterized as an important player in plant growth, development, and the stress response, but the role of NAC TFs in cold stress and their interaction with other post-transcriptional regulators such as microRNAs in cold tolerance remains elusive. Here, we demonstrated that SlNAM3, the predicted target of Sl-miR164a/b-5p, improved cold tolerance as indicated by a higher maximum quantum efficiency of photosystem II (Fv/Fm), lower relative electrolyte leakage, and less wilting in SlNAM3-overexpression plants compared to wild-type. Further genetic and molecular confirmation revealed that Sl-miR164a/b-5p functioned upstream of SlNAM3 by inhibiting the expression of the latter, thus playing a negative role in cold tolerance. Interestingly, this role is partially mediated by an ethylene-dependent pathway because either Sl-miR164a/b-5p silencing or SlNAM3 overexpression improved cold tolerance in the transgenic lines by promoting ethylene production. Moreover, silencing of the ethylene synthesis genes, SlACS1A, SlACS1B, SlACO1, and SlACO4, resulted in a significant decrease in cold tolerance. Further experiments demonstrated that NAM3 activates SlACS1A, SlACS1B, SlACO1, and SlACO4 transcription by directly binding to their promoters. Taken together, the present study identified the miR164a-NAM3 module conferring cold tolerance in tomato plants via the direct regulation of SlACS1A, SlACS1B, SlACO1, and SlACO4 expression to induce ethylene synthesis.

Systems metabolic engineering of Corynebacterium glutamicum for high-level production of 1,3-propanediol from glucose and xylose.

Corynebacterium glutamicum is a versatile chassis which has been widely used to produce various amino acids and organic acids. In this study, we report the development of an efficient C. glutamicum strain to produce 1,3-propanediol (1,3-PDO) from glucose and xylose by systems metabolic engineering approaches, including (1) construction and optimization of two different glycerol synthesis modules; (2) combining glycerol and 1,3-PDO synthesis modules; (3) reducing 3-hydroxypropionate accumulation by clarifying a mechanism involving 1,3-PDO re-consumption; (4) reducing the accumulation of toxic 3-hydroxypropionaldehyde by pathway engineering; (5) engineering NADPH generation pathway and anaplerotic pathway. The final engineered strain can efficiently produce 1,3-PDO from glucose with a titer of 110.4 g/L, a yield of 0.42 g/g glucose, and a productivity of 2.30 g/L/h in fed-batch fermentation. By further introducing an optimized xylose metabolism module, the engineered strain can simultaneously utilize glucose and xylose to produce 1,3-PDO with a titer of 98.2 g/L and a yield of 0.38 g/g sugars. This result demonstrates that C. glutamicum is a potential chassis for the industrial production of 1,3-PDO from abundant lignocellulosic feedstocks.

The high thermal stability of white emitting phosphor Mg2 Y2 Al2 Si2 O12 :Dy3+ ,Eu3+ ,La3+ /Lu3+ for white light emitting diodes.

A series of Mg2 Y2 Al2 Si2 O12 :Dy3+ ,Eu3+ was prepared using a solid-state method, and the phosphor emitted white light by tuning the ratio of Dy3+ /Eu3+ . The effects of La3+ /Lu3+ on the structure and luminescence properties of Mg2 Y2 Al2 Si2 O12 :Dy3+ ,Eu3+ were explored. Under the influence of bond length and twist, the luminescence intensity of the materials increased first and then decreased under excitation with ultraviolet light. The lattice distortion of the trivalent cation La3+ -substituted Mg2 Y2 Al2 Si2 O12 :Dy3+ and Eu3+ phosphors was reduced, the symmetry of polyhedron occupied by the luminescence centre improved, and the thermal stability of the luminescence centre improved to a certain extent. White light emitting diodes (LEDs) were fabricated by combining a 370 nm LED chip and the Mg2 Y2 Al2 Si2 O12 :Dy3+ ,Eu3+ ,La3+ (Mg2 Y2 Al2 Si2 O12 :Dy3+ ,Eu3+ ,Lu3+ ) phosphor. The results showed that Mg2 Y2 Al2 Si2 O12 :Dy3+ ,Eu3+ ,La3+ /Lu3+ may have potential application in the area of white LEDs.

Fabrication and investigation on Ag nanowires/TiO2 nanosheets/graphene hybrid nanocomposite and its water treatment performance.

In this paper, a novel Ag nanowires/TiO2 nanosheets/graphene nanocomposite was fabricated via a facile method of hydrothermal and calcination, and then the water treatment performance of it was evaluated for methylene blue (MB) and Escherichia coli removal. The as-prepared Ag nanowires/TiO2 nanosheets/graphene nanocomposite was characterized by Fourier-transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), field-emission scanning electron microscopy (FE-SEM), energy-dispersive X-ray spectroscopy (EDX), UV-visible diffuse reflection spectroscopy (DRS), molecular dynamics simulation, and gas chromatography-mass spectrometry (GC-MS). All data revealed that the Ag/TiO2/graphene nanocomposite showed a rich cell structure. The photocatalytic activity of Ag/TiO2/graphene nanocomposite is higher than those of pristine TiO2 nanosheets and TiO2/graphene nanocomposite. Under optimized conditions, the degradation efficiency was 100% and 71% for MB (30 mg/L) and with 10 mg Ag/TiO2/graphene nanocomposite under UV and visible light irradiation for 2 h, respectively. Ag/TiO2/graphene also showed excellent bacteria-killing activity. Meanwhile, the Ag/TiO2/graphene nanocomposite exhibited microstructure stability and cyclic stability. The water treatment performance was enhanced mainly attributed to the excellent adsorption performance of graphene and the high efficiency in separation of electron-hole pairs induced by the remarkable synergistic effects of TiO2, Ag, and graphene. On the basis of the experimental results, the photocatalytic mechanism and MB degradation mechanism were proposed. It is hoped that our work could avert the misleading message to the readership, hence offering a valuable source of reference on fabricating composite photocatalyst with stable microstructure and excellent performance for their application in the environment clean-up. Graphical abstract.

Self-polarized Poly(vinylidene fluoride) Ultrathin Film and Its Piezo/Ferroelectric Properties.

Organic nonvolatile memory with ultralow power consumption is a critical research demand for next-generation memory applications. However, obtaining a large-area, highly oriented ferroelectric ultrathin film with low leakage current and stable ferroelectric switching remains a challenge for achieving low operation voltage in ferroelectric memory transistors. Here, an ideal ferroelectric neat PVDF ultrathin film with a high degree of orientation is fabricated by a melt-draw technique without post-thermal treatment and assisted stabilization process. The PVDF ultrathin film is self-polarized with predominantly vertical orientation of dipole moments, exhibiting a d33 of 25 pm V-1 and the ultralow coercive voltage of approximately 3 V characterized by piezoresponse force microscopy. A remnant polarization of 6.3 µC cm-2 is identified based on a PVDF capacitor with an active layer formed by six layers of melt-drawn thin films. By employing a single-layer melt-drawn PVDF ultrathin film as an insulation layer, a bottom-gate-top-contact ferroelectric field-effect transistor is fabricated with a very low operation voltage of 5 V. It exhibits a memory window with an on/off current ratio of 103 at zero gate bias and threshold voltage shift of around 2 V.