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K-joints have been commonly used in steel structures benefiting from excellent connection performance. In this study, a combined experimental and nonlinear analysis was performed on CFDST-K joints to investigate the displacement, strain evolution, bearing capacity degradation, impact force, and energy absorption capacity in detail with different structure-related parameters such as hollow rate and steel tube thickness. The finite element model was developed and validated by experimental results. Results indicated that the CFDST-K joint can effectively enhance impact resistance with a more reasonable energy distribution. To improve the anti-impact property, the optimal hollow rate is 0.63 and the diameter-to-thickness ratio of the outer tube should be less than 30. Meanwhile, the inner tube thickness had little effect on impact resistance under low-impact energy. Finally, simplified calculation methods for the deformation and impact force were derived based on a parametric study on the dynamic response by discussing the influence of the impact energy, hollow rate, diameter-to-thickness ratio, and axial compression ratio. The findings can serve as a foundation for the development of more reliable CFDST-K joints for steel structures.

Abstract High‐fat diet (HFD)‐induced obesity is a crucial risk factor for metabolic syndrome, mainly due to adipose tissue dysfunctions associated with it. However, the underlying mechanism remains unclear. This study has used genetic screening to identify an obesity‐associated human lncRNA LINK‐A as a critical molecule bridging the metabolic microenvironment and energy expenditure in vivo by establishing the HFD‐induced obesity knock‐in (KI) mouse model. Mechanistically, HFD LINK‐A KI mice induce the infiltration of inflammatory factors, including IL‐1β and CXCL16, through the LINK‐A/HB‐EGF/HIF1α feedback loop axis in a self‐amplified manner, thereby promoting the adipose tissue microenvironment remodeling and adaptive thermogenesis disorder, ultimately leading to obesity and insulin resistance. Notably, LINK‐A expression is positively correlated with inflammatory factor expression in individuals who are overweight. Of note, targeting LINK‐A via nucleic acid drug antisense oligonucleotides (ASO) attenuate HFD‐induced obesity and metabolic syndrome, pointing out LINK‐A as a valuable and effective therapeutic target for treating HFD‐induced obesity. Briefly, the results reveale the roles of lncRNAs (such as LINK‐A) in remodeling tissue inflammatory microenvironments to promote HFD‐induced obesity.

Abstract Fibrous red phosphorus (RP) has triggered growing attention as an emerging quasi-one-dimensional (quasi-1D) van der Waals crystal recently. Unfortunately, it is difficult to achieve substrate growth of high-quality fibrous RP flakes due to their inherent quasi-1D structure, which impedes their fundamental property exploration and device integration. Herein, we demonstrate a bottom-up approach for the growth of fibrous RP flakes with (001)-preferred orientation via a chemical vapor transport (CVT) reaction in the P/Sn/I2 system. The formation of fibrous RP flakes can be attributed to the synergistic effect of Sn-mediated P4 partial pressure and the SnI2 capping layer-directed growth. Moreover, we investigate the optical anisotropy of the as-grown flakes, demonstrating their potential application as micro phase retarders in polarization conversion. Our developed bottom-up approach lays the foundation for studying the anisotropy and device integration of fibrous red phosphorus, opening up possibilities for the two-dimensional growth of quasi-1D van der Waals materials.

Abstract 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.

Abstract Iron metabolism dysregulation is tightly associated with cancer development. But the underlying mechanisms remain poorly understood. Increasing evidence has shown that long noncoding RNAs (lncRNAs) participate in various metabolic processes via integrating signaling pathway. In this study, we revealed one iron-triggered lncRNA, one target of YAP, LncRIM (LncRNA Related to Iron Metabolism, also named ZBED5-AS1 and Loc729013), which effectively links the Hippo pathway to iron metabolism and is largely independent on IRP2. Mechanically, LncRIM directly binds NF2 to inhibit NF2-LATS1 interaction, which causes YAP activation and increases intracellular iron level via DMT1 and TFR1. Additionally, LncRIM-NF2 axis mediates cellular iron metabolism dependent on the Hippo pathway. Clinically, high expression of LncRIM correlates with poor patient survival, suggesting its potential use as a biomarker and therapeutic target. Taken together, our study demonstrated a novel mechanism in which LncRIM-NF2 axis facilitates iron-mediated feedback loop to hyperactivate YAP and promote breast cancer development.

Abstract 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.

Cancer cells exploit immune checkpoint pathways, such as PD-1/PD-L1, to evade elimination by the immune system. Here, the authors demonstrate that TMUB1 regulates post-translational modifications of PD-L1 and that targeting the TMUB1/PD-L1 interaction promotes anti-tumour T cells responses

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.

Background & Aims: Alcohol-associated liver disease (AALD) is one of the most common causes of liver injury and failure. Limited knowledge of the mechanisms underlying AALD impedes the development of efficacious therapies. Bile acid (BA) signaling was shown to participate in the progression of AALD. However, the mechanisms remain poorly understood. Methods: C57BL/6J wild-type (WT), Takeda G-protein–coupled bile acid receptor 5 (TGR5) knockout (KO) and brown adipose tissue (BAT)-specific TGR5 knockdown mice were subjected to ethanol feeding–induced AALD. Liver samples from alcoholic hepatitis patients were used to examine the BA circulation signaling. Human Embryonic Kidney Cells 293 were used for the TGR5 reporter assay. 23(S)-methyl-lithocholic acid was used as a molecular tool to confirm the regulatory functions of BAT in the AALD mouse model. Results: Ethanol feeding increased the expression of the thermogenesis genes downstream of TGR5 in BAT of WT, but not TGR5 KO, mice. TGR5 deficiency significantly blocked BAT activity and energy expenditure in mice after ethanol feeding. Alcohol increased serum BA levels in mice and human beings through altering BA transportation, and the altered BAs activated TGR5 signaling to regulate metabolism. Compared with ethanol-fed WT mice, ethanol-fed TGR5 KO mice showed less free fatty acid (FFA) β-oxidation in BAT, leading to higher levels of FFA in the circulation, increased liver uptake of FFAs, and exacerbated AALD. BAT-specific TGR5 knockdown mice showed similar results with TGR5 KO mice in AALD. Agonist treatment significantly activated TGR5 signaling in BAT, increased thermogenesis, reduced serum FFA level, and ameliorated hepatic steatosis and injury in AALD mice, while these effects were lost in TGR5 KO mice. Conclusions: BA signaling plays a protective role in AALD by enhancing BAT thermogenesis. Targeting TGR5 in BAT may be a promising approach for the treatment of AALD.

Summary: Neurofibromatosis type 2 is an autosomal dominant multiple neoplasia syndrome and is usually caused by mutations in the neurofibromin 2 (NF2) gene, which encodes a tumor suppressor and initiates the Hippo pathway. However, the mechanism by which NF2 functions in the Hippo pathway isn’t fully understood. Here we identified a NF2 c.770-784del mutation from a neurofibromatosis type 2 family. MD simulations showed that this mutation significantly changed the structure of the F3 module of the NF2-FERM domain. Functional assays indicated that the NF2 c.770-784del variant formed LLPS in the cytoplasm with LATS to restrain LATS plasma membrane localization and inactivated the Hippo pathway. Besides, this deletion partly caused a skipping of exon 8 and reduced the protein level of NF2, collectively promoting proliferation and tumorigenesis of meningeal cells. We identified an unrecognized mechanism of LLPS and splicing skipping for the NF2-induced Hippo pathway, which provided new insight into the pathogenesis of neurofibromatosis type 2.

BackgroundAarskog–Scott syndrome, a rare X-linked genetic disorder, is identified by combined clinical manifestations of short stature, facial, skeletal, and genital anomalies. Annually, two or three new cases are diagnosed with Aarskog–Scott syndrome, which is associated with FGD1 variants. However, there is no specific treatment for Aarskog–Scott syndrome due to its unclear mechanism.MethodsClinical data were collected when the patient first visited the hospital. Trio whole-exome sequencing and Sanger sequencing were performed for the genetic cause of disease. To evaluate the pathogenicity of the variants in vitro, stable cell lines were constructed using lentivirus infection in 143B cell. Furthermore, Western blot was used to verify the expression of signaling pathway-related proteins, and the transcription levels of osteogenic-related genes were verified by luciferase reporter gene assay.ResultsA 7-year-old boy was manifested with facial abnormalities, intellectual disability, and short stature (−3.98 SDS) while the growth hormone level of stimulation test was normal. Trio whole-exome sequencing and Sanger sequencing identified a variant (c.1270A>G, p.Asn424Asp) in FGD1 gene. The Asn424 residue was highly conserved and the hydrogen bond in the FGD1 variant protein has changed, which led to decrease in the interaction with CDC42 protein. In vitro study showed that the Asn424Asp variant significantly decreased the transcription levels of OCN, COL1A1, and ALP activity, and it activated the phosphorylation of JNK1.ConclusionMolecular biological mechanisms between abnormal expression of FGD1and Aarskog–Scott syndrome remain poorly understood. In our study, c.1270A>G variant of FGD1 resulted in Aarskog–Scott syndrome, and the analysis of pathogenicity supports the deleterious effect of the variant. Furthermore, we demonstrated the weakened affinity of the mutant FGD1 and CDC42. Decreased expression of osteogenic-related gene and abnormal activation of JNK1 were also shown in this work.

Background: 46,XY disorders/differences of sex development (46,XY DSD) are congenital conditions that result from abnormal gonadal development (gonadal dysgenesis) or abnormalities in androgen synthesis or action. During early embryonic development, several genes are involved in regulating the initiation and maintenance of testicular or ovarian-specific pathways. Recent reports have shown that MAP3K1 genes mediate the development of the 46,XY DSD, which present as complete or partial gonadal dysgenesis. Previous functional studies have demonstrated that some MAP3K1 variants result in the gain of protein function. However, data on possible mechanisms of MAP3K1 genes in modulating protein functions remain scant.Methods: This study identified a Han Chinese family with the 46,XY DSD. To assess the history and clinical manifestations for the 46,XY DSD patients, the physical, operational, ultra-sonographical, pathological, and other examinations were performed for family members. Variant analysis was conducted using both trio whole-exome sequencing (trio WES) and Sanger sequencing. On the other hand, we generated transiently transfected testicular teratoma cells (NT2/D1) and ovary-derived granular cells (KGN), with mutant or wild-type MAP3K1 gene. We then performed functional assays such as determination of steady-state levels of gender related factors, protein interaction and luciferase assay system.Results: Two affected siblings were diagnosed with 46,XY DSD. Our analysis showed a missense c.556A > G/p.R186G variant in the MAP3K1 gene. Functional assays demonstrated that the MAP3K1R186G variant was associated with significantly decreased affinity to ubiquitin (Ub; 43–49%) and increased affinity to RhoA, which was 3.19 ± 0.18 fold, compared to MAP3K1. The MAP3K1R186G led to hyperphosphorylation of p38 and GSK3β, and promoted hyperactivation of the Wnt4/β-catenin signaling. In addition, there was increased recruitment of β-catenin into the nucleus, which enhanced the expression of pro-ovarian transcription factor FOXL2 gene, thus contributing to the 46,XY DSD.Conclusion: Our study identified a missense MAP3K1 variant associated with 46,XY DSD. We demonstrated that MAP3K1R186G variant enhances binding to the RhoA and improves its own stability, resulting in the activation of the Wnt4/β-catenin/FOXL2 pathway. Taken together, these findings provide novel insights into the molecular mechanisms of 46,XY DSD and promotes better clinical evaluation.

Abstract Background Drosophila Prominin-like is a homolog of mammalian CD133, which is recognized as a biomarker for stem cells. The interacting proteins of CD133 and their biological functions remain elusive. Results In this study, we using yeast two-hybrid assays, GST pull-down assay and co-immunoprecipitation (Co-IP) methods found that Drosophila Prominin-like interacts with ND20, a subunit of mitochondrial respiratory complex I. Bioinformatics analysis suggests that Prominin-like is a six-transmembrane glycoprotein which localizes on cellular membranes. Immunostaining and mitochondrial fractionation indicate that Drosophila Prominin-like could localize in the mitochondria. The knockdown of prominin-like in S2 cells resulted in transient mitochondrial dysfunctions as evidenced by reduced ATP production, elevated ROS generation and an accompanied reduction in mitochondrial proteins. Mitochondrial dysfunctions were detected in aged prominin-like mutant flies. Conclusion Our data indicates that Prominin-like acts to maintain mitochondrial function through its interaction with ND20 which, itself, is active in the mitochondrial electron transport chain. Our study provides insights into a novel molecular mechanism of Drosophila prominin-like and suggests a similar function of CD133 in mammals.

Summary: Hypertrophic cardiomyopathy (HCM) is the most common cause of sudden cardiac death in young individuals. A potential role of mtDNA mutations in HCM is known. However, the underlying molecular mechanisms linking mtDNA mutations to HCM remain poorly understood due to lack of cell and animal models. Here, we generated induced pluripotent stem cell-derived cardiomyocytes (HCM-iPSC-CMs) from human patients in a maternally inherited HCM family who carry the m.2336T>C mutation in the mitochondrial 16S rRNA gene (MT-RNR2). The results showed that the m.2336T>C mutation resulted in mitochondrial dysfunctions and ultrastructure defects by decreasing the stability of 16S rRNA, which led to reduced levels of mitochondrial proteins. The ATP/ADP ratio and mitochondrial membrane potential were also reduced, thereby elevating the intracellular Ca2+ concentration, which was associated with numerous HCM-specific electrophysiological abnormalities. Our findings therefore provide an innovative insight into the pathogenesis of maternally inherited HCM. : In this article, Yan Q, Liu Z, Huang W, and colleagues show that patient-specific iPSCs as well as their derived cardiomyocytes carrying the m.2336T>C mutation in MT-RNR2 were generated to understand the pathogenic mechanism of maternally inherited HCM. MT-RNR2 mutation resulted in mitochondrial dysfunctions and ultrastructure defects, which induced abnormal Ca2+ homeostasis, then HCM-specific cellular and electrophysiological characteristics in iPSC-CMs. Keywords: mitochondrion, hypertrophic cardiomyopathy, induced pluripotent stem cells, MT-RNR2, maternal inheritance

Large-scale two-dimensional air-gap photonic crystals (PhCs) were embedded in an n-type GaN layer to enhance the light emission of GaN-based light-emitting diodes (LEDs). The embedded-PhCs were obtained through overgrowth on n-GaN templates, which had air holes array previously fabricated by using nanosphere lithography technology. The effects of the distance D from active region to PhCs on the light extraction efficiency (LEE) of LEDs are investigated by experiments and finite difference-time domain simulation. The PhC LEDs with D = 2370 nm showed an improved light output power by 80.8% on average at forward current of 350 mA without degradation of electrical performance compared to conventional LEDs. It is mainly attributed to the improved LEE by enhancing the interaction of the guided modes and the embedded air-gap PhCs.

Background: Congenital adrenal hyperplasia (CAH) encompasses a group of autosomal recessive diseases characterized by enzyme deficiencies, within steroid hormone anabolism, which lead to disorders in cortisol synthesis. The 17α-hydroxylase/17,20-lyase deficiency (17-OHD) is an uncommon form of CAH caused by variants in the CYP17A1 gene.Aims: We report a novel compound heterozygous CYP17A1 variant and its association with the pathogenesis of 17-OHD.Methods: The patient was assessed for medical history, clinical manifestations, physical examination, laboratory examination, karyotype analysis, and adrenal computed tomography. Mutation screening was conducted using whole-exome sequencing (WES) and Sanger sequencing. The wild-type and mutant CYP17A1 complementary DNAs (cDNAs) were amplified and cloned into a pcDNA3.1(+) vector. These plasmids were transfected transiently into HEK-293T cells. Quantitative PCR and Western blotting analysis were performed to measure the expression level of P450c17. An enzymatic activity assay was conducted to measure the content of 17-hydroxyprogesterone (17-OHP) and dehydroepiandrosterone (DHEA) in medium using liquid chromatography–tandem mass spectrometry (LC-MS/MS).Results: The proband was characterized by 17-OHD with rhabdomyolysis, hypokalemia, and adrenal insufficiency. Novel compound heterozygous variants of the CYP17A1 gene (c.1304T > C/p.Phe435Ser and c.1228delG/p.Asp410Ilefs*9) were identified. The enzymatic activity assay revealed that this variant resulted in a complete deficiency of 17α-hydroxylase and 17,20-lyase activity. This was consistent with the hormonal characteristics of the proband’s blood.Conclusions: These results suggest that the compound heterozygous variant of c.1304T > C and c.1228delG of the CYP17A1 gene can lead to 17-OHD. Our findings thus provide a novel insight into the clinical evaluations and molecular basis of 17-OHD.

Lead halide perovskites have gained more and more attention because of their ease of synthesis and excellent photoelectric properties including a large absorption coefficient, long carrier lifetime, long carrier diffusion length, and high carrier mobility. However, their toxicity, instability, and phase degradation in ambient environments impede their large-scale applications. To address these concerns, it is desirable to find stable alternative halide perovskites without toxicity and with comparable optoelectronic properties to lead-based perovskites. Over the years, a considerable number of lead-free halide perovskites have been added to this family of materials, including A2B’B’’X6, A2BX6, and A3B2X9 type perovskites. Among these, double perovskites with the general formula A2B’B’’X6 are deemed to be a potential alternative to lead halide perovskites as they possess good stability under ambient conditions and excellent optoelectronic properties. In this review, recent progress in exploring Pb-free halide double perovskites is highlighted. The synthesis, composition-tuning, physical properties, and applications of representative 3D, 2D, and nanocrystal A2B’B’’X6 double perovskites are introduced. In addition, perspectives about current challenges and solutions in this field are also provided.

Ferroelectric solid solutions usually exhibit enhanced functional properties at the morphotropic phase boundary separating two ferroelectric phases with different orientations of polarization. The underlying mechanism is generally associated with polarization rotational instability and the flattened free energy profile. In this work we show that the polarization extensional instability can also be induced at the morphotropic phase boundary beyond the reported polar-nonpolar phase boundary. The piezoelectricity enhanced by this mechanism exhibits excellent thermal stability, which helps to develop high performance piezoelectric materials with good temperature stability.

Relaxor [011]c PMN-0.35PT single crystal phase transition characteristics are investigated through various methods including variable temperature dielectric properties, X-ray diffraction, bipolar ferroelectric hysteresis loops (P-E) and electric-field-induced strain (S-E) hysteresis loops measurements. The results reveal that two phase transitions exist within the range from room temperature to 250 °C: orthorhombic (O)-tetragonal (T)-cubic (C). The O-to-T and T-to-C phase transition temperatures have been identified as 84 °C and 152 °C, respectively. Diffuseness degree of the T-to-C phase transition for the unpoled single crystal has been calculated to be 1.56, implying an intermediate state between normal and relaxor ferroelectrics. Temperature-dependent remanent polarization (Pr), coercive field (Ec), saturation polarization (Ps), hysteresis loop squareness (Rsq), and longitudinal piezoelectric constant (d* 33) are also explored to learn the details of the phase transitions. Variable temperature unipolar Suni-E hysteresis loops avail additional evidence for the microstructure change in the as-measured single crystal.

MTO1, together with MSS1 and MTO2, is a gene involved in the pathway of encoding a mitochondria-specific RNA-modifying enzyme related to the post-transcriptional modification of mitochondrial tRNAs. We have previously shown that a mutation of the MTO2 or MSS1 gene can suppress the neomycin-sensitive phenotype of yeast carrying a mitochondrial 15S rRNA C1477G mutation. Here we report that a null mutation of MTO1 also can inhibit the aminoglycoside-sensitivity of yeast carrying mitochondrial 15S rRNA C1477G mutation. The C1477G mutation corresponds to the human 12S rRNA A1555G mutation. Yeast with an mtDNA C1477G mutation exhibits hypersensitivity to neomycin and displays mitochondrial function impairment beyond neomycin treatment. When the mto1 null mutation and mitochondrial C1477G mutation coexist, the yeast strain shows growth recovery. The deletion of the nuclear gene MTO1 regulates neomycin sensitivity in yeast carrying the mitochondrial 15S rRNA C1477G mutation. MTO1 deletion causes the expression levels of the key glycolytic genes HXK2, PFK1 and PYK1 to become significantly up-regulated. The energy deficit due to impaired mitochondrial function was partially compensated by the energy generated by glycolysis. Being in the same pathway, the regulation of MTO1, MSS1 and MTO2 to the neomycin-sensitivity of yeast showed difference in the growth activity of strains, mitochondrial function and the expression level of glycolytic genes.

The phenotypic manifestation of mitochondrial DNA (mtDNA) mutations can be modulated by nuclear genes and environmental factors. However, neither the interaction among these factors nor their underlying mechanisms are well understood. The yeast Saccharomyces cerevisiae mtDNA 15S rRNA C1477G mutation (PR) corresponds to the human 12S rRNA A1555G mutation. Here we report that a nuclear modifier gene mss1 mutation suppresses the neomycin-sensitivity phenotype of a yeast C1477G mutant in fermentable YPD medium. Functional assays show that the mitochondrial function of the yeast C1477G mutant was impaired severely in YPD medium with neomycin. Moreover, the mss1 mutation led to a significant increase in the steady-state level of HAP5 (heme activated protein), which greatly up-regulated the expression of glycolytic transcription factors RAP1, GCR1, and GCR2 and thus stimulated glycolysis. Furthermore, the high expression of the key glycolytic enzyme genes HXK2, PFK1 and PYK1 indicated that enhanced glycolysis not only compensated for the ATP reduction from oxidative phosphorylation (OXPHOS) in mitochondria, but also ensured the growth of the mss1(PR) mutant in YPD medium with neomycin. This study advances our understanding of the phenotypic manifestation of mtDNA mutations.

BACKGROUND: Peritoneal fluid (PF) from patients with endometriosis can inhibit early embryo development via probable functional changes of embryo mitochondria in the early stage of embryo development. The purpose of this study was to determine the effect of PF from patients with endometriosis on mitochondrial function and development of early mouse embryos. METHODOLOGY/PRINCIPAL FINDINGS: PF was collected from patients with infertility and endometriosis, infertility due to tubal factors, and normal control subjects, and the level of NO was measured. Early murine embryos were then cultured with PF from normal control subjects, those with endometriosis, and with human tubal fluid (HTF), respectively. Cleavage and blastulation rates, mitochondrial DNA (mtDNA) copy numbers, adenosine triphosphate (ATP) level, and mitochondrial membrane potential (ΔΨm) of the different groups were compared. The NO level in the PF of patients with endometriosis was significantly greater than in those without endometriosis and control patients. The embryos cultures with PF from patients with endometriosis had a lower cleavage rate and blastulation rate, and higher ATP and ΔΨm level at the 2- and 4-cell stages. No significant difference was found in mtDNA copies among the 3 groups. CONCLUSIONS/SIGNIFICANCE: PF from patients with endometriosis can inhibit early embryo development via probable functional changes of embryo mitochondria in the early stage of embryo development. Understanding the effects of PF on embryo development may assist in developing new methods of treatment for infertility.

The phenotypic manifestations of mitochondrial DNA (mtDNA) mutations are modulated by mitochondrial DNA haplotypes, nuclear modifier genes and environmental factors. The yeast mitochondrial 15S rRNA C1477G (P(R) or P(R) 454) mutation corresponds to the human 12S rRNA C1494T and A1555G mutations, which are well known as primary factors for aminoglycoside-induced nonsyndromic deafness. Here we report that the deletion of the nuclear modifier gene MTO2 suppressed the aminoglycoside-sensitivity of mitochondrial 15S rRNA C1477G mutation in Saccharomyces cerevisiae. First, the strain with a single mtDNA C1477G mutation exhibited hypersensitivity to neomycin. Functional assays indicated that the steady-state transcription level of mitochondrial DNA, the mitochondrial respiratory rate, and the membrane potential decreased significantly after neomycin treatment. The impaired mitochondria could not produce sufficient energy to maintain cell viability. Second, when the mto2 null and the mitochondrial C1477G mutations co-existed (mto2(P(R))), the oxygen consumption rate in the double mutant decreased markedly compared to that of the control strains (MTO2(P(S)), mto2(P(S)) and MTO2(P(R))). The expression levels of the key glycolytic genes HXK2, PFK1 and PYK1 in the mto2(P(R)) strain were stimulated by neomycin and up-regulated by 89%, 112% and 55%, respectively. The enhanced glycolysis compensated for the respiratory energy deficits, and could be inhibited by the glycolytic enzyme inhibitor. Our findings in yeast will provide a new insight into the pathogenesis of human deafness.

Abstract SARS-CoV-2-contributes to sickness and death in COVID-19 patients partly by inducing a hyper-proinflammatory immune response in the host airway. This hyper-proinflammatory state involves activation of signaling by NFκB, and unexpectedly, ENaC, the epithelial sodium channel. Post-infection inflammation may also contribute to "Long COVID"/PASC. Enhanced signaling by NFκB and ENaC also marks the airway of patients suffering from cystic fibrosis, a life-limiting proinflammatory genetic disease due to inactivating mutations in the CFTR gene. We therefore hypothesized that inflammation in the COVID-19 airway might similarly be due to inhibition of CFTR signaling by SARS-CoV-2 spike protein, and therefore activation of both NFκB and ENaC signaling. We used western blot and electrophysiological techniques, and an organoid model of normal airway epithelia, differentiated on an air–liquid-interface (ALI). We found that CFTR protein expression and CFTR cAMP-activated chloride channel activity were lost when the model epithelium was exposed to SARS-CoV-2 spike proteins. As hypothesized, the absence of CFTR led to activation of both TNFα/NFκB signaling and α and γ ENaC. We had previously shown that the cardiac glycoside drugs digoxin, digitoxin and ouabain blocked interaction of spike protein and ACE2. Consistently, addition of 30 nM concentrations of the cardiac glycoside drugs, prevented loss of both CFTR protein and CFTR channel activity. ACE2 and CFTR were found to co-immunoprecipitate in both basal cells and differentiated epithelia. Thus spike-dependent CFTR loss might involve ACE2 as a bridge between Spike and CFTR. In addition, spike exposure to the epithelia resulted in failure of endosomal recycling to return CFTR to the plasma membrane. Thus, failure of CFTR recovery from endosomal recycling might be a mechanism for spike-dependent loss of CFTR. Finally, we found that authentic SARS-CoV-2 virus infection induced loss of CFTR protein, which was rescued by the cardiac glycoside drugs digitoxin and ouabain. Based on experiments with this organoid model of small airway epithelia, and comparisons with 16HBE14o- and other cell types expressing normal CFTR, we predict that inflammation in the COVID-19 airway may be mediated by inhibition of CFTR signaling by the SARS-CoV-2 spike protein, thus inducing a cystic fibrosis-like clinical phenotype. To our knowledge this is the first time COVID-19 airway inflammation has been experimentally traced in normal subjects to a contribution from SARS-CoV-2 spike-dependent inhibition of CFTR signaling.