Reactive oxygen species regulation by NCF1 governs ferroptosis susceptibility of Kupffer cells to MASH.

Impaired self-renewal of Kupffer cells (KCs) leads to inflammation in metabolic dysfunction-associated steatohepatitis (MASH). Here, we identify neutrophil cytosolic factor 1 (NCF1) as a critical regulator of iron homeostasis in KCs. NCF1 is upregulated in liver macrophages and dendritic cells in humans with metabolic dysfunction-associated steatotic liver disease and in MASH mice. Macrophage NCF1, but not dendritic cell NCF1, triggers KC iron overload, ferroptosis, and monocyte-derived macrophage infiltration, thus aggravating MASH progression. Mechanistically, elevated oxidized phospholipids induced by macrophage NCF1 promote Toll-like receptor (TLR4)-dependent hepatocyte hepcidin production, leading to increased KC iron deposition and subsequent KC ferroptosis. Importantly, the human low-functional polymorphic variant NCF190H alleviates KC ferroptosis and MASH in mice. In conclusion, macrophage NCF1 impairs iron homeostasis in KCs by oxidizing phospholipids, triggering hepatocyte hepcidin release and KC ferroptosis in MASH, highlighting NCF1 as a therapeutic target for improving KC fate and limiting MASH progression.

Gene Expression, Morphology, and Electrophysiology During the Dynamic Development of Human Induced Pluripotent Stem Cell-Derived Atrial- and Ventricular-Like Cardiomyocytes.

Background and Objectives: Gene expression, morphology, and electrophysiological combination are essential for assessing the dynamic development of human induced pluripotent stem cell-derived atrial- and ventricular-like cardiomyocytes (iPS-AM and iPS-VM, respectively). Methods: For iPS-AM/VM differentiation, we performed the small molecule-based temporal modulation of the retinoic acid and bone morphogenetic protein signaling pathways. We investigated the gene expression and morphology using immunofluorescence, quantitative real-time polymerase chain reaction, flow cytometry, and transmission electron microscopy as well as registered electrophysiological functions using a whole-cell patch clamp on days 20, 30, and 60 post-differentiations. Results: Pan-cardiomyocyte marker, including troponin T2 (TNNT2) and alpha-actinin-2 (ACTN2), expressions increased both in iPS-AMs and iPS-VMs. Similarly, the mRNA expression of both iPS-AM-specific markers, ie, natriuretic peptide A (NPPA), myosin light chain 7 (MYL7), and K+ channel Kir3.4 (KCNJ5), and iPS-VM-specific markers, ie, gap junction α-1 (GJA1), myosin light chain 2 (MYL2), and alpha-1-subunit of a voltage-dependent L-type calcium channel (CACNA1C), increased from 0 to 20 days, and then decreased from 30 to 60 days. Concerning morphology, cardiac troponin-T (cTnT) arrangement was progressively organized and developed from a disorderly myofibrillar distribution to an organized sarcomere pattern both in iPS-AMs and iPS-VMs. Mitochondrial numbers gradually increased and those of lipid droplets decreased during dynamic development. Regarding physiological function, the resting and action potential amplitudes remained statistically indifferent in both cell types, and the action potential duration was prolonged during the development. Conclusion: IPS-AMs/VMs displayed dynamic development concerning their gene expression, morphology, and electrophysiological function. The discoveries of this study could provide novel insights into heart development and encourage further research.

Exploring therapeutic targets for molecular therapy of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis is a chronic and progressive interstitial lung disease with a poor prognosis. Idiopathic pulmonary fibrosis is characterized by repeated alveolar epithelial damage leading to abnormal repair. The intercellular microenvironment is disturbed, leading to continuous activation of fibroblasts and myofibroblasts, deposition of extracellular matrix, and ultimately fibrosis. Moreover, pulmonary fibrosis was also found as a COVID-19 complication. Currently, two drugs, pirfenidone and nintedanib, are approved for clinical therapy worldwide. However, they can merely slow the disease's progression rather than rescue it. These two drugs have other limitations, such as lack of efficacy, adverse effects, and poor pharmacokinetics. Consequently, a growing number of molecular therapies have been actively developed. Treatment options for IPF are becoming increasingly available. This article reviews the research platform, including cell and animal models involved in molecular therapy studies of idiopathic pulmonary fibrosis as well as the promising therapeutic targets and their development progress during clinical trials. The former includes patient case/control studies, cell models, and animal models. The latter includes transforming growth factor-beta, vascular endothelial growth factor, platelet-derived growth factor, fibroblast growth factor, lysophosphatidic acid, interleukin-13, Rho-associated coiled-coil forming protein kinase family, and Janus kinases/signal transducers and activators of transcription pathway. We mainly focused on the therapeutic targets that have not only entered clinical trials but were publicly published with their clinical outcomes. Moreover, this work provides an outlook on some promising targets for further validation of their possibilities to cure the disease.

Mitochondrial dysfunction is associated with hypertrophic cardiomyopathy in Pompe disease-specific induced pluripotent stem cell-derived cardiomyocytes.

Pompe disease (PD) is a rare autosomal recessive disorder that presents with progressive hypertrophic cardiomyopathy. However, the detailed mechanism remains clarified. Herein, PD patient-specific induced pluripotent stem cells were differentiated into cardiomyocytes (PD-iCMs) that exhibited cardiomyopathic features of PD, including decreased acid alpha-glucosidase activity, lysosomal glycogen accumulation and hypertrophy. The defective mitochondria were involved in the cardiac pathology as shown by the significantly decreased number of mitochondria and impaired respiratory function and ATP production in PD-iCMs, which was partially due to elevated levels of intracellular reactive oxygen species produced from depolarized mitochondria. Further analysis showed that impaired fusion and autophagy of mitochondria and declined expression of mitochondrial complexes underlies the mechanism of dysfunctional mitochondria. This was alleviated by supplementation with recombinant human acid alpha-glucosidase that improved the mitochondrial function and concomitantly mitigated the cardiac pathology. Therefore, this study suggests that defective mitochondria underlie the pathogenesis of cardiomyopathy in patients with PD.

The implication of infection with respiratory syncytial virus in pediatric recurrent wheezing and asthma: knowledge expanded post-COVID-19 era.

BACKGROUND: Respiratory syncytial virus (RSV) infection has been identified to serve as the primary cause of acute lower respiratory infectious diseases in children under the age of one and a significant risk factor for the emergence and development of pediatric recurrent wheezing and asthma, though the exact mechanism is still unknown. METHODS AND RESULTS: In this study, we discuss the key routes that lead to recurrent wheezing and bronchial asthma following RSV infection. It is interesting to note that following the coronavirus disease 2019 (COVID-19) epidemic, the prevalence of RSV changes significantly. This presents us with a rare opportunity to better understand the associated mechanism for RSV infection, its effects on the respiratory system, and the immunological response to RSV following the COVID-19 epidemic. To better understand the associated mechanisms in the occurrence and progression of pediatric asthma, we thoroughly described how the RSV infection directly destroys the physical barrier of airway epithelial tissue, promotes inflammatory responses, enhances airway hyper-responsiveness, and ultimately causes the airway remodeling. More critically, extensive discussion was also conducted regarding the potential impact of RSV infection on host pulmonary immune response. CONCLUSION: In conclusion, this study offers a comprehensive perspective to better understand how the RSV infection interacts in the control of the host's pulmonary immune system, causing recurrent wheezing and the development of asthma, and it sheds fresh light on potential avenues for pharmaceutical therapy in the future.

Successful Transfection of MicroRNA Mimics or Inhibitors in a Regular Cell Line and in Primary Cells Derived from Patients with Rheumatoid Arthritis.

The transfection of microRNA (miRNA) mimics and inhibitors can lead to the gain and loss of intracellular miRNA function, helping us better understand the role of miRNA during gene expression regulation under specific physical conditions. Our previous research has confirmed the efficiency and convenience of using liposomes to transfect miRNA mimics or inhibitors. This work uses miR-424 as an example, to provide a detailed introduction for the transfection process of miRNA mimics and inhibitors in the regular SW982 cell line and primary rheumatoid arthritis synovial fibroblasts (RASF) cells from patients by using lipofection, which can also serve as a reference to miRNA transfection in other cell lines. Key features • MiRNA mimics and inhibitors transfection in regular SW982 cell line and primary RASF cells. • Treatment and culture of RASF primary cells before transfection. • Using liposomes for transfection purposes.

Current updates on arrhythmia within Timothy syndrome: genetics, mechanisms and therapeutics.

Timothy syndrome (TS), characterised by multiple system malfunction especially the prolonged corrected QT interval and synchronised appearance of hand/foot syndactyly, is an extremely rare disease affecting early life with devastating arrhythmia. In this work, firstly, the various mutations in causative gene CACNA1C encoding cardiac L-type voltage-gated calcium channel (LTCC), regard with the genetic pathogeny and nomenclature of TS are reviewed. Secondly, the expression profile and function of CACNA1C gene encoding Cav1.2 proteins, and its gain-of-function mutation in TS leading to multiple organ disease phenotypes especially arrhythmia are discussed. More importantly, we focus on the altered molecular mechanism underlying arrhythmia in TS, and discuss about how LTCC malfunction in TS can cause disorganised calcium handling with excessive intracellular calcium and its triggered dysregulated excitation-transcription coupling. In addition, current therapeutics for TS cardiac phenotypes including LTCC blockers, beta-adrenergic blocking agents, sodium channel blocker, multichannel inhibitors and pacemakers are summarised. Eventually, the research strategy using patient-specific induced pluripotent stem cells is recommended as one of the promising future directions for developing therapeutic approaches. This review updates our understanding on the research progress and future avenues to study the genetics and molecular mechanism underlying the pathogenesis of devastating arrhythmia within TS, and provides novel insights for developing therapeutic measures.

Patient-specific induced pluripotent stem cell properties implicate Ca2+-homeostasis in clinical arrhythmia associated with combined heterozygous RYR2 and SCN10A variants.

We illustrate use of induced pluripotent stem cells (iPSCs) as platforms for investigating cardiomyocyte phenotypes in a human family pedigree exemplified by novel heterozygous RYR2-A1855D and SCN10A-Q1362H variants occurring alone and in combination. The proband, a four-month-old boy, presented with polymorphic ventricular tachycardia. Genetic tests revealed double novel heterozygous RYR2-A1855D and SCN10A-Q1362H variants inherited from his father (F) and mother (M), respectively. His father showed ventricular premature beats; his mother was asymptomatic. Molecular biological characterizations demonstrated greater TNNT2 messenger RNA (mRNA) expression in the iPSCs-induced cardiomyocytes (iPS-CMs) than in the iPSCs. Cardiac troponin Ts became progressively organized but cytoplasmic RYR2 and SCN10A aggregations occurred in the iPS-CMs. Proband-specific iPS-CMs showed decreased RYR2 and SCN10A mRNA expression. The RYR2-A1855D variant resulted in premature spontaneous sarcoplasmic reticular Ca2+ transients, Ca2+ oscillations and increased action potential durations. SCN10A-Q1362H did not confer any specific phenotype. However, the combined heterozygous RYR2-A1855D and SCN10A-Q1362H variants in the proband iPS-CMs resulted in accentuated Ca2+ homeostasis disorders, action potential prolongation and susceptibility to early afterdepolarizations at high stimulus frequencies. These findings attribute the clinical phenotype in the proband to effects of the heterozygous RYR2 variant exacerbated by heterozygous SCN10A modification. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Cardiac deficiency of P21-activated kinase 1 promotes atrial arrhythmogenesis in mice following adrenergic challenge.

P21-activated kinase 1 (Pak1) signalling plays a vital and overall protective role in the heart. However, the phenotypes of Pak1 deficiency in the cardiac atria have not been well explored. In this study, Pak1 cardiac-conditional knock-out (cKO) mice were studied under baseline and adrenergic challenge conditions. Pak1 cKO mice show atrial arrhythmias including atrial fibrillation (AF) in vivo, detected during anaesthetized electrocardiography without evidence of interstitial fibrosis upon Masson's trichrome staining. Optical mapping of left atrial preparations from Pak1 cKO mice revealed a higher incidence of Ca2+ and action potential alternans under isoprenaline challenge and differences in baseline action potential and calcium transient characteristics. Type-2 ryanodine receptor (RyR2) channels from Pak1 cKO hearts had a higher open probability than those from wild-type. Reverse transcription-quantitative polymerase chain reaction and Western blotting indicated that pCamkIIδ and RyR2 are highly phosphorylated at baseline in the atria of Pak1 cKO mice, while the expression of Slc8a2 and Slc8a3 as a Na+-Ca2+ exchanger, controlling the influx of Ca2+ from outside of the cell and efflux of Na+ from the cytoplasm, are augmented. Chromatin immunoprecipitation study showed that pCreb1 interacts with Slc8a2 and Slc8a3. Our study thus demonstrates that deficiency of Pak1 promotes atrial arrhythmogenesis under adrenergic stress, probably through post-translational and transcriptional modifications of key molecules that are critical to Ca2+ homeostasis. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Evaluation of immunoprotection against coronavirus disease 2019: Novel variants, vaccine inoculation, and complications.

The strikingly rapidly mutating nature of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome has been a constant challenge during the coronavirus disease 2019 (COVID-19) pandemic. In this study, various techniques, including reverse transcription-quantitative polymerase chain reaction, antigen-detection rapid diagnostic tests, and high-throughput sequencing were analyzed under different scenarios and spectra for the etiological diagnosis of COVID-19 at the population scale. This study aimed to summarize the latest research progress and provide up-to-date understanding of the methodology used for the evaluation of the immunoprotection conditions against future variants of SARS-CoV-2. Our novel work reviewed the current methods for the evaluation of the immunoprotection status of a specific population (endogenous antibodies) before and after vaccine inoculation (administered with biopharmaceutical antibody products). The present knowledge of the immunoprotection status regarding the COVID-19 complications was also discussed. Knowledge on the immunoprotection status of specific populations can help guide the design of pharmaceutical antibody products, inform practice guidelines, and develop national regulations with respect to the timing of and need for extra rounds of vaccine boosters.

Generation of one human induced pluripotent stem cell line (XACHi004-A) with heterozygous mutation of RYR2 gene from an atrial fibrillation patient.

As the most frequently diagnosed arrhythmia, atrial fibrillation (AF) has been recently reported to be closely related to ryanodine receptor (RyR2) dysfunction and calcium leak. Here, using non-integrating sendai viral method, we generated one iPSC line from peripheral blood mononuclear cells (PBMC) isolated a 10-year-old boy with simple atrial fibrillation which carries the heterozygous mutation in RYR2 gene (c.14638G > A, p.V4880I). The generated iPSC line was identified by the typical cell morphology, highly expressed pluripotent markers, normal karyotype, and in-vitro trilineage differentiation potential. It will provide a useful model for studying the pathophysiological consequences of RYR2 mutation on the AF pathogenesis.

Generation of two heterozygous GAA mutation-carrying human induced pluripotent stem cell lines (XACHi005-A, XACHi006-A) from parents of an infant with Pompe disease.

Pompe disease results from GAA mutations that leads to lysosomal glycogen accumulation and cardiac and skeletal muscle pathology. We have previously generated an infantile-onset Pompe disease patient-derived human-induced pluripotent stem cells (iPSCs) line carrying compound GAA mutations (R608X and E888X). Using his parents' peripheral blood mononuclear cells (PBMCs), we here generated two iPSCs lines which carry mutations of R608X E888X respectively. Both lines show typical cell morphology, high expressed pluripotent and self-renewal markers, normal karyotype, and trilineage differentiation potential. These two lines are valuable re-sources for studying the pathological mechanisms of GAA mutation-caused Pompe disease.

Real-world effectiveness and protection of SARS-CoV-2 vaccine among patients hospitalized for COVID-19 in Xi'an, China, December 8, 2021, to January 20, 2022: A retrospective study.

Introduction: In December 2021, a large-scale epidemic broke out in Xi'an, China, due to SARS-CoV-2 infection. This study reports the effect of vaccination on COVID-19 and evaluates the impact of different vaccine doses on routine laboratory markers. Methods: The laboratory data upon admission, of 231 cases with COVID-19 hospitalized from December 8, 2021 to January 20, 2022 in Xi'an, including blood routine, lymphocyte subtypes, coagulative function tests, virus specific antibodies and blood biochemical tests were collected and analyzed. Results: Of the 231 patients, 21 were not vaccinated, 158 were vaccinated with two doses and 52 with three doses. Unvaccinated patients had a higher proportion of moderate and severe symptoms than vaccinated patients, while two-dose vaccinated patients had a higher proportion than three-dose vaccinated patients. SARS-CoV-2 specific IgG levels were significantly elevated in vaccinated patients compared with unvaccinated patients. Particularly, unvaccinated patients had lower counts and percentages of lymphocytes, eosinophils and CD8+ T-lymphocytes, and elevated coagulation-related markers. In addition, vaccination had no effect on liver and kidney function. Conclusions: Vaccination against SARS-CoV-2, inducing high IgG level and increased CD8+ T cells and eosinophils, and regulating coagulation function, can significantly attenuate symptoms of COVID-19, suggesting that the vaccine remains protective against SARS-CoV-2.

Design, synthesis, and evaluation of fluoroquinolone derivatives as microRNA-21 small-molecule inhibitors.

MicroRNA-21 (miRNA-21) is highly expressed in various tumors. Small-molecule inhibition of miRNA-21 is considered to be an attractive novel cancer therapeutic strategy. In this study, fluoroquinolone derivatives A1-A43 were synthesized and used as miRNA-21 inhibitors. Compound A36 showed the most potent inhibitory activity and specificity for miRNA-21 in a dual-luciferase reporter assay in HeLa cells. Compound A36 significantly reduced the expression of mature miRNA-21 and increased the protein expression of miRNA-21 target genes, including programmed cell death protein 4 (PDCD4) and phosphatase and tensin homology deleted on chromosome ten (PTEN), at 10 µM in HeLa cells. The Cell Counting Kit-8 assay (CCK-8) was used to evaluate the antiproliferative activity of A36; the results showed that the IC50 value range of A36 against six tumor cell lines was between 1.76 and 13.0 µM. Meanwhile, A36 did not display cytotoxicity in BEAS-2B cells (lung epithelial cells from a healthy human donor). Furthermore, A36 significantly induced apoptosis, arrested cells at the G0/G1 phase, and inhibited cell-colony formation in HeLa cells. In addition, mRNA deep sequencing showed that treatment with A36 could generate 171 dysregulated mRNAs in HeLa cells, while the expression of miRNA-21 target gene dual-specificity phosphatase 5 (DUSP5) was significantly upregulated at both the mRNA and protein levels. Collectively, these findings demonstrated that A36 is a novel miRNA-21 inhibitor.

Integrated Excitatory/Inhibitory Imbalance and Transcriptomic Analysis Reveals the Association between Dysregulated Synaptic Genes and Anesthetic-Induced Cognitive Dysfunction.

Emerging evidence from human epidemiologic and animal studies has demonstrated that developmental anesthesia neurotoxicity could cause long-term cognitive deficits and behavioral problems. However, the underlying mechanisms remain largely unknown. We conducted an electrophysiological analysis of synapse activity and a transcriptomic assay of 24,881 mRNA expression on hippocampal tissues from postnatal day 60 (P60) mice receiving propofol exposure at postnatal day 7 (P7). We found that developmentally propofol-exposed P60 mouse hippocampal neurons displayed an E/I imbalance, compared with control mice as evidenced by the decreased excitation and increased inhibition. We found that propofol exposure at P7 led to the abnormal expression of 317 mRNAs in the hippocampus of P60 mice, including 23 synapse-related genes. Various bioinformatic analyses revealed that these abnormally expressed synaptic genes were associated with the function and development of synapse activity and plasticity, E/I balance, behavior, and cognitive impairment. Our findings suggest that the altered E/I balance may constitute a mechanism for propofol-induced long-term impaired learning and memory in mice. The transcriptomic and bioinformatic analysis of these dysregulated genes related to synaptic function paves the way for development of therapeutic strategies against anesthetic neurodegeneration through the restoration of E/I balance and the modification of synaptic gene expression.

Neuro-Immune Regulation in Inflammation and Airway Remodeling of Allergic Asthma.

Allergic asthma is a common chronic inflammation of the airways and causes airway remodeling eventually. For a long time, investigators have been focusing on the immunological mechanism of asthma. However, in recent years, the role of neuro-regulation in the occurrence of asthma has gradually attracted investigators' attention. In this review, we firstly describe neuro-immune regulation in inflammation of allergic asthma from two aspects: innate immunity and adaptive immunity. Secondly, we introduce neuro-immune regulation in airway remodeling of asthma. Finally, we prospect the role of pulmonary neuroendocrine cells in the development of asthma. In general, the amount of researches is limited. Further researches on the neural regulation during the occurrence of asthma will help us clarify the mechanism of asthma more comprehensively and find more effective ways to prevent and control asthma.

Identification of benzamides derivatives of norfloxacin as promising microRNA-21 inhibitors via repressing its transcription.

MicroRNA-21 is a carcinogenic microRNA, whose overexpression arises in a variety of tumor tissues. Hence, microRNA-21 a prospective target for cancer treatment, and regulation of microRNA-21 by small molecule inhibitors is deemed as a promising approach for tumor therapy. In this work, to discover potent microRNA-21 inhibitor, series of 4-(N-norfloxacin-acyl)aminobenzamides were designed and synthesized, and their inhibitory effects were appraised by utilizing dual luciferase reporter assays. The results indicated that compound A7 was the most efficient microRNA-21 small molecule inhibitor. What's more, A7 suppressed the migration of Hela cells and the colony formation of Hela and HCT-116 cells as well as promoted apoptosis of Hela cells. In the mechanism study, results of RT-qPCR certified that A7 could reduce the level of mature microRNA-21 via disrupting its expression at the transcriptional level of its primary form "pri-miR-21", which was distinct from most previous inhibitors directly binding with pre-miR-21. Noticeably, Western blotting and RT-qPCR uncovered A7 could upregulate the expression PTEN, EGR1 and SLIT2, which are the downstream functional targets of microRNA-21. These findings demonstrated that A7 was a promising microRNA-21 small molecule inhibitor and 4-(N-norfloxacin-acyl) aminobenzamide can serve as a new scaffold for discovery of potent microRNA-21 inhibitor.

The importance of non-coding RNAs in environmental stress-related developmental brain disorders: A systematic review of evidence associated with exposure to alcohol, anesthetic drugs, nicotine, and viral infections.

Brain development is a dynamic and lengthy process that includes cell proliferation, migration, neurogenesis, gliogenesis, synaptogenesis, and pruning. Disruption of any of these developmental events can result in long-term outcomes ranging from brain structural changes, to cognitive and behavioral abnormality, with the mechanisms largely unknown. Emerging evidence suggests non-coding RNAs (ncRNAs) as pivotal molecules that participate in normal brain development and neurodevelopmental disorders. NcRNAs such as long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) are transcribed from the genome but not translated into proteins. Many ncRNAs have been implicated as tuners of cell fate. In this review, we started with an introduction of the current knowledge of lncRNAs and miRNAs, and their potential roles in brain development in health and disorders. We then reviewed and discussed the evidence of ncRNA involvement in abnormal brain development resulted from alcohol, anesthetic drugs, nicotine, and viral infections. The complex connections among these ncRNAs were also discussed, along with potential overlapping ncRNA mechanisms, possible pharmacological targets for therapeutic/neuroprotective interventions, and potential biomarkers for brain developmental disorders.

Molecular detection of SARS-CoV-2 being challenged by virus variation and asymptomatic infection.

Coronavirus disease 2019 (COVID-19) has been a pandemic for more than a year. With the expanding second wave of the pandemic in winter, the continuous evolution of SARS-CoV-2 has brought new issues, including the significance of virus mutations in infection and the detection of asymptomatic infection. In this review, we first introduced several major SARS-CoV-2 mutations since the COVID-19 outbreak and then mentioned the widely used molecular detection techniques to diagnose COVID-19, primarily focusing on their strengths and limitations. We further discussed the effects of viral genetic variation and asymptomatic infection on the molecular detection of SARS-CoV-2 infection. The review finally summarized useful insights into the molecular diagnosis of COVID-19 under the special situation being challenged by virus mutation and asymptomatic infection.