Immunogenicity and safety in rabbits of a Clostridioides difficile vaccine combining novel toxoids and a novel adjuvant.

Clostridioides difficile infection (CDI) is a serious healthcare-associated disease, causing symptoms such as diarrhea and pseudomembranous colitis. The major virulence factors responsible for the disease symptoms are two secreted cytotoxic proteins, TcdA and TcdB. A parenteral vaccine based on formaldehyde-inactivated TcdA and TcdB supplemented with alum adjuvant, has previously been investigated in humans but resulted in an insufficient immune response. In search for an improved response, we investigated a novel toxin inactivation method and a novel, potent adjuvant. Inactivation of toxins by metal-catalyzed oxidation (MCO) was previously shown to preserve neutralizing epitopes and to annihilate reversion to toxicity. The immunogenicity and safety of TcdA and TcdB inactivated by MCO and combined with a novel carbohydrate fatty acid monosulphate ester-based (CMS) adjuvant were investigated in rabbits. Two or three intramuscular immunizations generated high serum IgG and neutralizing antibody titers against both toxins. The CMS adjuvant increased antibody responses to both toxins while an alum adjuvant control was effective only against TcdA. Systemic safety was evaluated by monitoring body weight, body temperature, and analysis of red and white blood cell counts shortly after immunization. Local safety was assessed by histopathologic examination of the injection site at the end of the study. Body weight gain was constant in all groups. Body temperature increased up to 1 ˚C one day after the first immunization but less after the second or third immunization. White blood cell counts, and percentage of neutrophils increased one day after immunization with CMS-adjuvanted vaccines, but not with alum. Histopathology of the injection sites 42 days after the last injection did not reveal any abnormal tissue reactions. From this study, we conclude that TcdA and TcdB inactivated by MCO and combined with CMS adjuvant demonstrated promising immunogenicity and safety in rabbits and could be a candidate for a vaccine against CDI.

Providing Behavioral Health Care in PACE - A Review of Federal and State Manual Regulations.

OBJECTIVES: Present analysis of the federal and state regulations that guide The Program of All-Inclusive Care for the Elderly (PACE) operations and core clinical features for direction on behavioral health (BH). DESIGN: Review and synthesize the federal (Centers for Medicare and Medicaid Services [CMS]) and all publicly available state manuals according to the BH-Serious Illness Care (SIC) model domains. SETTING AND PARTICIPANTS: The 155 PACE organizations operating in 32 states and the District of Columbia. METHODS: A multipronged search was conducted to identify official state and federal manuals guiding the implementation and functions of PACE organizations. The CMS PACE website was used to identify the federal PACE manual. State-level manuals for 32 states with PACE programs were identified through several sources, including official PACE websites, contacts through official websites, the National PACE Association (NPA), and public and academic search engines. The manuals were searched according to the BH-SIC model domains that pertain to integrating BH care with complex care individuals. RESULTS: According to the CMS Manual, the interdisciplinary team is responsible for holistic care of PACE enrollees, but a BH specialist is not a required member. The CMS Manual includes information on BH clinical functions, BH workforce, and structures for outcome measurement, quality, and accountability. Eight of 32 PACE-participating states offer publicly available state PACE manuals; of which 3 offer information on BH clinical functions. CONCLUSIONS AND IMPLICATIONS: Regarding BH, federal and state manual regulations establish limited guidance for comprehensive care service delivery at PACE organizations. The absence of clear directives weakens BH care delivery due to a limiting the ability to develop quality measures and accountability structures. This hinders incentivization and accountability to truly all-inclusive care. Clearer guidelines and regulatory parameters regarding BH care at federal and state levels may enable more PACE organizations to meet rising BH demands of aging communities.

Heat-responsive microRNAs participate in regulating the pollen fertility stability of CMS-D2 restorer line under high-temperature stress.

Anther development and pollen fertility of cytoplasmic male sterility (CMS) conditioned by Gossypium harknessii cytoplasm (CMS-D2) restorer lines are susceptible to continuous high-temperature (HT) stress in summer, which seriously hinders the large-scale application of "three-line" hybrids in production. Here, integrated small RNA, transcriptome, degradome, and hormone profiling was performed to explore the roles of microRNAs (miRNAs) in regulating fertility stability in mature pollens of isonuclear alloplasmic near-isogenic restorer lines NH and SH under HT stress at two environments. A total of 211 known and 248 novel miRNAs were identified, of which 159 were differentially expressed miRNAs (DEMs). Additionally, 45 DEMs in 39 miRNA clusters (PmCs) were also identified, and most highly expressed miRNAs were significantly induced in SH under extreme HT, especially four MIR482 and six MIR6300 family miRNAs. PmC28 was located in the fine-mapped interval of the Rf1 gene and contained two DEMs, gra-miR482_L-2R + 2 and gma-miR2118a-3p_R + 1_1ss18TG. Transcriptome sequencing identified 6281 differentially expressed genes, of which heat shock protein (HSP)-related genes, such as HSP70, HSP22, HSP18.5-C, HSP18.2 and HSP17.3-B, presented significantly reduced expression levels in SH under HT stress. Through integrating multi-omics data, we constructed a comprehensive molecular network of miRNA-mRNA-gene-KEGG containing 35 pairs of miRNA/target genes involved in regulating the pollen development in response to HT, among which the mtr-miR167a_R + 1, tcc-miR167c and ghr-miR390a, tcc-miR396c_L-1 and ghr-MIR169b-p3_1ss6AG regulated the pollen fertility by influencing ARF8 responsible for the auxin signal transduction, ascorbate and aldarate metabolism, and the sugar and lipid metabolism and transport pathways, respectively. Further combination with hormone analysis revealed that HT-induced jasmonic acid signaling could activate the expression of downstream auxin synthesis-related genes and cause excessive auxin accumulation, followed by a cascade of auxin signal transduction, ultimately resulting in pollen abortion. The results provide a new understanding of how heat-responsive miRNAs regulate the stability of fertility restoration for CMS-D2 cotton under heat stress.

Sex shapes cell-type-specific transcriptional signatures of stress exposure in the mouse hypothalamus.

Stress-related psychiatric disorders and the stress system show prominent differences between males and females, as well as strongly divergent transcriptional changes. Despite several proposed mechanisms, we still lack the understanding of the molecular processes at play. Here, we explore the contribution of cell types to transcriptional sex dimorphism using single-cell RNA sequencing. We identify cell-type-specific signatures of acute restraint stress in the paraventricular nucleus of the hypothalamus, a central hub of the stress response, in male and female mice. Further, we show that a history of chronic mild stress alters these signatures in a sex-specific way, and we identify oligodendrocytes as a major target for these sex-specific effects. This dataset, which we provide as an online interactive app, offers the transcriptomes of thousands of individual cells as a molecular resource for an in-depth dissection of the interplay between cell types and sex on the mechanisms of the stress response.

Assay for evaluation of proarrhythmic effects of herbal products: Case study with 12 Evodia preparations.

Guidelines for preclinical drug development reduce the occurrence of arrhythmia-related side effects. Besides ample evidence for the presence of arrhythmogenic substances in plants, there is no consensus on a research strategy for the evaluation of proarrhythmic effects of herbal products. Here, we propose a cardiac safety assay for the detection of proarrhythmic effects of plant extracts based on the experimental approaches described in the Comprehensive In vitro Proarrhythmia Assay (CiPA). Microelectrode array studies (MEAs) and voltage sensing optical technique on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were combined with ionic current measurements in mammalian cell lines, In-silico simulations of cardiac action potentials (APs) and statistic regression analysis. Proarrhythmic effects of 12 Evodia preparations, containing different amounts of the hERG inhibitors dehydroevodiamine (DHE) and hortiamine were analysed. Extracts produced different prolongation of the AP, occurrence of early after depolarisations and triangulation of the AP in hiPSC-CMs depending on the contents of the hERG inhibitors. DHE and hortiamine dose-dependently prolonged the field potential duration in hiPSC-CMs studied with MEAs. In-silico simulations of ventricular AP support a scenario where proarrhythmic effects of Evodia extracts are predominantly caused by the content of the selective hERG inhibitors. Statistic regression analysis revealed a high torsadogenic risk for both compounds that was comparable to drugs assigned to the high-risk category in a CiPA study.

Identification, cloning and characterization of AcMSH1 from Onion (Allium cepa L.).

BACKGROUND: MSH1 (MutS homolog1) is a nuclear-encoded protein that plays a crucial role in maintaining low mutation rates and stability of the organellar genome. While plastid MSH1 maintains nuclear epigenome plasticity and affects plant development patterns, mitochondrial MSH1 suppresses illegitimate recombination within the mitochondrial genome, affects mitochondrial genome substoichiometric shifting activity and induces cytoplasmic male sterility (CMS) in crops. However, a detailed functional investigation of onion MSH1 has yet to be achieved. MATERIALS AND RESULTS: The homology analysis of onion genome database identified a single copy of the AcMSH1 gene in the onion cv. Bhima Super. In silico analysis of AcMSH1 protein sequence revealed the presence of 6 conserved functional domains including a unique MSH1-specific GIY-YIG endonuclease domain at the C-terminal end. At N-terminal end, it has signal peptide sequences targeting chloroplast and mitochondria. The concentration of AcMSH1 was found to be highest in isolated mitochondria, followed by chloroplasts, and negligible in the cytoplasmic fraction; which proved its localization to the mitochondria and chloroplasts. Quantitative expression analysis revealed that AcMSH1 protein levels were highest in leaves, followed by flower buds, root tips, flowers, and umbels, with the lowest amount found in callus tissue. CONCLUSION: Onion genome has single copy of MSH1, with characteristic GIY-YIG endonuclease domain. AcMSH1 targeted towards both chloroplasts and mitochondria. The identification and characterisation of AcMSH1 may provide valuable insights into the development of CMS lines in onion.

Investigating the effect of Shenmai injection on cardiac electrophysiology and calcium signaling using human-induced pluripotent stem cell-derived cardiomyocytes.

Traditional Chinese medicine injection (TCMI) refers to the use of modern technology to make Chinese patent medicines in injectable forms, which shorten the onset time of the traditional Chinese medicine (TCM). Although there have been clinical cases in which Shenmai injection (SMI) was used to treat cardiovascular diseases (CVDs), there are no pharmacological experiments that investigate the efficacy of the drug in vitro or the underlying mechanisms. Aim of the study: We aimed to systemically evaluate the efficacy and investigate the mechanisms of SMI in modulating electrophysiology and calcium (Ca2+) signaling using a microelectrode array (MEA) and a genetically encoded Ca2+ indicator, GCaMP6s, respectively, in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Materials and methods: A MEA system was employed to record field potentials (FPs) in hiPSC-CMs. The QT interval is corrected by the RR interval, the reciprocal of the beating rate. GCaMP6s was used to measure Ca2+ signaling in hiPSC-CMs. Meanwhile, the transcriptome changes in hiPSC-CMs treated with 2% SMI were examined using RNAseq. In addition, the ingredients of SMI were investigated using liquid chromatography-mass spectrometry (LC-MS). Results: It was found that 0.5%, 1%, and 2% (v/v) SMIs could increase corrected QT (QTc) but did not change other FP parameters. GCaMP6s was successfully applied to measure the chronic function of SMI. The full width at half maximum (FWHM), rise time, and decay time significantly decreased after treatment with SMI for 1 h and 24 h, whereas an increased Ca2+ transient frequency was observed. Conclusions: We first used the Ca2+ indicator to measure the chronic effects of TCM. We found that SMI treatment can modulate electrophysiology and calcium signaling and regulate oxidative phosphorylation, cardiac muscle contraction, and the cell cycle pathway in hiPSC-CMs.

A review on qualifications and cost effectiveness of induced pluripotent stem cells (IPSCs)-induced cardiomyocytes in drug screening tests.

Human induced pluripotent stem cells (hIPSCs) have initiated a higher degree of successes in disease modelling, preclinical evaluation of drug therapy and pharmaco-toxicological testing. Since the discovery of iPSCs in 2006, many advanced techniques have been introduced to differentiate iPSCs to cardiomyocytes, which have been progressively improved. The disease models from iPSC-induced cardiomyocytes (iPSC-CM) have been successfully helping to study a variety of cardiac diseases such as long QT syndrome, drug-induced long QT, different cardiomyopathies related to mutations in mitochondria or desmosomal proteins and other rare genetic diseases. IPSC-CMs have also been used to screen the role of chemicals in cardiovascular drug discovery and individualisation of drug dosages. In this review, the quality of current procedures for characterisation and maturation of iPSC-CM lines will be discussed. Also, we will focus on time efficiency and cost of standard differentiation methods after reprogramming.

Impact of a mobile integrated healthcare and community paramedicine program on improving medication adherence in patients with heart failure and chronic obstructive pulmonary disease after hospital discharge: A pilot study.

Background: The mobile integrated health-community paramedicine (MIH-CP) program affiliated with the University of Maryland Medical Center focuses on improving patient transitions from hospital to home by addressing both medical and social determinants of health. Until recently, only self-contained health systems could integrate inpatient and outpatient medication data. Without some means to track patients in transition, there is a significant risk of medication-related problems and errors. Objective: To evaluate the impact of the MIH-CP program on medication adherence among patients with congestive heart failure (CHF) and/or chronic obstructive pulmonary disease (COPD). Methods: This is a pilot observational study designed to compare adherence to drug regimens prescribed at hospital discharge (measured by the proportion of days covered [PDC]) between patients enrolled in the MIH-CP program and a propensity-matched control group. Propensity scores were calculated using 11 demographic, diagnostic, third-party payer, and patient care-associated variables. Discharge medication details were obtained from electronic medical records. PDC for each of the medications were calculated from pharmacy claims data. Results: Eighty-three patients were included in the study; forty-three patients were placed in the intervention group and 40 were propensity-matched controls. After adjusting for age, sex, and third-party payer, findings indicated that medication adherence was higher among patients enrolled in the MIH-CP program compared with control during the first 30 days post-discharge, specifically among patients diagnosed with CHF (8% difference in PDC, 95% confidence interval [CI], -0.12-0.28%) and COPD (14% difference, 95% CI, -0.15-0.43%), although neither result achieved statistical significance. The differences in medication adherence between patients who were enrolled and those who were not enrolled in the MIH-CP program diminished after 30 days post-discharge. Conclusion: This pilot study demonstrated a trend toward improved medication adherence among patients enrolled in the MIH-CP program. Future research involving a larger patient cohort will be required to confirm these preliminary findings.

Metabolic Behaviors of Aconitum Alkaloids in Different Concentrations of Aconiti Lateralis Radix Praeparata and Effects of Aconitine in Healthy Human and Long QT Syndrome Cardiomyocytes.

Aconiti Lateralis Radix Praeparata (Fu Zi) is the processed lateral root of Aconitum carmichaelii Debx, which is widely used in emergency clinics. Poisoning incidents and adverse reactions occur with the improper intake of Fu Zi. Metabolic characteristics of aconitum alkaloids of Fu Zi may vary, and the effects of Fu Zi in healthy and Long QT syndrome (LQTS) patients is unknown. In this experiment, 24 Sprague Dawley rats were randomly divided into three groups: 2.0, 1.0, and 0.5 g/kg dose groups, and blood samples were collected after the oral administration of Fu Zi extract. We used an ultra-high performance liquid chromatography-tandem mass spectrometry system to detect the concentrations of six aconitum alkaloids. Cell toxicity, calcium imaging, and patch-clamp recordings of human induced pluripotent stem cells-cardiomyocytes (hiPSC-CMs) of aconitine in healthy and LQTS were observed. We found that the AUC(0-48h), Cmax, and t1/2 of the six compounds increased with the multiplicative dosages; those in the high group were significantly higher than those in the low group. Aconitine concentration-dependently decreased the amplitude, which has no significant effect on the cell index of normal hiPSC-CMs. Aconitine at 5.0 µM decreased the cell index between 5-30 min for LQTS hiPSC-CMs. Meanwhile, aconitine significantly increased the frequency of calcium transients in LQTS at 5 µM. Aconitine significantly shortened the action potential duration of human cardiomyocytes in both normal and LQTS groups. These results show metabolic behaviors of aconitum alkaloids in different concentrations of Fu Zi and effects of aconitine in healthy and LQTS patients.

Mushroom-Derived Novel Selenium Nanocomposites' Effects on Potato Plant Growth and Tuber Germination.

Multicomponent materials, where nanosized selenium (Se) is dispersed in polymer matrices, present as polymer nanocomposites (NCs), namely, selenium polymer nanocomposites (SeNCs). Selenium as an inorganic nanofiller in NCs has been extensively studied for its biological activity. More ecologically safe and beneficial approaches to obtain Se-based products are the current challenge. Biopolymers have attained great attention with perspective multifunctional and high-performance NCs exhibiting low environmental impact with unique properties, being abundantly available, renewable, and eco-friendly. Composites based on polysaccharides, including beta-glucans from edible and medicinal mushrooms, are bioactive, biocompatible, biodegradable, and have exhibited innovative potential. We synthesized SeNCs on the basis of the extracellular polysaccharides of several medicinal mushrooms. The influence of bio-composites from mushrooms on potato plant growth and tuber germination were studied in two potato cultivars: Lukyanovsky and Lugovskoi. Bio-composites based on Grifola umbellata demonstrated the strongest positive effect on the number of leaves and plant height in both cultivars, without negative effect on biomass of the vegetative part. Treatment of the potato tubers with SeNC from Gr. umbellata also significantly increased germ length. Potato plants exposed to Se-bio-composite from Ganoderma lucidum SIE1303 experienced an increase in the potato vegetative biomass by up to 55% versus the control. We found earlier that this bio-composite was the most efficient against biofilm formation by the potato ring rot causative agent Clavibacter sepedonicus (Cms). Bio-composites based on Pleurotus ostreatus promoted increase in the potato root biomass in the Lugovskoi cultivar by up to 79% versus the control. The phytostimulating ability of mushroom-based Se-containing bio-composites, together with their anti-phytopathogenic activity, testifies in favor of the bifunctional mode of action of these Se-biopreparations. The application of stimulatory green SeNCs for growth enhancement could be used to increase crop yield. Thus, by combining myco-nanotechnology with the intrinsic biological activity of selenium, an unexpectedly efficient tool for possible applications of SeNCs could be identified.

Clinical data mining reveals Gancao-Banxia as a potential herbal pair against moderate COVID-19 by dual binding to IL-6/STAT3.

BACKGROUND: Coronavirus disease 2019 (COVID-19) keeps spreading globally. Chinese medicine (CM) exerts a critical role for the prevention or therapy of COVID-19 in an integrative and holistic way. However, mining and development of early, efficient, multisite binding CMs that inhibit the cytokine storm are imminent. METHODS: The formulae were extracted retrospectively from clinical records in Hunan Province. Clinical data mining analysis and association rule analysis were employed for mining the high-frequency herbal pairs and groups from formulae. Network pharmacology methods were applied to initially explore the most critical pair's hub targets, active ingredients, and potential mechanisms. The binding power of active ingredients to the hub targets was verified by molecular docking. RESULTS: Eight hundred sixty-two prescriptions were obtained from 320 moderate COVID-19 through the Hunan Provincial Health Commission. Glycyrrhizae Radix et Rhizoma (Gancao) and Pinelliae Rhizoma (Banxia) were used with the highest frequency and support. There were 49 potential genes associated with Gancao-Banxia pair against moderate COVID-19 patients. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) indicated that Gancao-Banxia might act via inflammatory response, viral defense, and immune responses signaling pathways. IL-6 and STAT3 were the two most hub targets in the protein-protein interaction (PPI) network. The binding of five active ingredients originated from Gancao-Banxia to IL-6-STAT3 was verified by molecular docking, namely quercetin, coniferin, licochalcone a, Licoagrocarpin and (3S,6S)-3-(benzyl)-6-(4-hydroxybenzyl)piperazine-2,5-quinone, maximizing therapeutic efficacy. CONCLUSIONS: This work provided some potential candidate Chinese medicine formulas for moderate COVID-19. Among them, Gancao-Banxia was considered the most potential herbal pair. Bioinformatic data demonstrated that Gancao-Banxia pair may achieve dual inhibition of IL-6-STAT3 via directly interacting with IL-6 and STAT3, suppressing the IL-6 amplifier. SARS-CoV-2 models will be needed to validate this possibility in the future.

An integrative gene expression signature analysis identifies CMS4 KRAS-mutated colorectal cancers sensitive to combined MEK and SRC targeted therapy.

BACKGROUND: Over half of colorectal cancers (CRCs) are hard-wired to RAS/RAF/MEK/ERK pathway oncogenic signaling. However, the promise of targeted therapeutic inhibitors, has been tempered by disappointing clinical activity, likely due to complex resistance mechanisms that are not well understood. This study aims to investigate MEK inhibitor-associated resistance signaling and identify subpopulation(s) of CRC patients who may be sensitive to biomarker-driven drug combination(s). METHODS: We classified 2250 primary and metastatic human CRC tumors by consensus molecular subtypes (CMS). For each tumor, we generated multiple gene expression signature scores measuring MEK pathway activation, MEKi "bypass" resistance, SRC activation, dasatinib sensitivity, EMT, PC1, Hu-Lgr5-ISC, Hu-EphB2-ISC, Hu-Late TA, Hu-Proliferation, and WNT activity. We carried out correlation, survival and other bioinformatic analyses. Validation analyses were performed in two independent publicly available CRC tumor datasets (n = 585 and n = 677) and a CRC cell line dataset (n = 154). RESULTS: Here we report a central role of SRC in mediating "bypass"-resistance to MEK inhibition (MEKi), primarily in cancer stem cells (CSCs). Our integrated and comprehensive gene expression signature analyses in 2250 CRC tumors reveal that MEKi-resistance is strikingly-correlated with SRC activation (Spearman P < 10-320), which is similarly associated with EMT (epithelial to mesenchymal transition), regional metastasis and disease recurrence with poor prognosis. Deeper analysis shows that both MEKi-resistance and SRC activation are preferentially associated with a mesenchymal CSC phenotype. This association is validated in additional independent CRC tumor and cell lines datasets. The CMS classification analysis demonstrates the strikingly-distinct associations of CMS1-4 subtypes with the MEKi-resistance and SRC activation. Importantly, MEKi + SRCi sensitivities are predicted to occur predominantly in the KRAS mutant, mesenchymal CSC-like CMS4 CRCs. CONCLUSIONS: Large human tumor gene expression datasets representing CRC heterogeneity can provide deep biological insights heretofore not possible with cell line models, suggesting novel repurposed drug combinations. We identified SRC as a common targetable node--an Achilles' heel--in MEKi-targeted therapy-associated resistance in mesenchymal stem-like CRCs, which may help development of a biomarker-driven drug combination (MEKi + SRCi) to treat problematic subpopulations of CRC.

Deep learning detects cardiotoxicity in a high-content screen with induced pluripotent stem cell-derived cardiomyocytes.

Drug-induced cardiotoxicity and hepatotoxicity are major causes of drug attrition. To decrease late-stage drug attrition, pharmaceutical and biotechnology industries need to establish biologically relevant models that use phenotypic screening to detect drug-induced toxicity in vitro. In this study, we sought to rapidly detect patterns of cardiotoxicity using high-content image analysis with deep learning and induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). We screened a library of 1280 bioactive compounds and identified those with potential cardiotoxic liabilities in iPSC-CMs using a single-parameter score based on deep learning. Compounds demonstrating cardiotoxicity in iPSC-CMs included DNA intercalators, ion channel blockers, epidermal growth factor receptor, cyclin-dependent kinase, and multi-kinase inhibitors. We also screened a diverse library of molecules with unknown targets and identified chemical frameworks that show cardiotoxic signal in iPSC-CMs. By using this screening approach during target discovery and lead optimization, we can de-risk early-stage drug discovery. We show that the broad applicability of combining deep learning with iPSC technology is an effective way to interrogate cellular phenotypes and identify drugs that may protect against diseased phenotypes and deleterious mutations.

Complete Mitochondrial Genome Sequence and Identification of a Candidate Gene Responsible for Cytoplasmic Male Sterility in Celery (Apium graveolens L.).

Celery (Apium graveolens L.) is an important leafy vegetable worldwide. The development of F1 hybrids in celery is highly dependent on cytoplasmic male sterility (CMS) because emasculation is difficult. In this study, we first report a celery CMS, which was found in a high-generation inbred line population of the Chinese celery "tanzhixiangqin". Comparative analysis, following sequencing and assembly of the complete mitochondrial genome sequences for this celery CMS line and its maintainer line, revealed that there are 21 unique regions in the celery CMS line and these unique regions contain 15 ORFs. Among these ORFs, only orf768a is a chimeric gene, consisting of 1497 bp sequences of the cox1 gene and 810 bp unidentified sequences located in the unique region, and the predicted protein product of orf768a possesses 11 transmembrane domains. In summary, the results of this study indicate that orf768a is likely to be a strong candidate gene for CMS induction in celery. In addition, orf768a can be a co-segregate marker, which can be used to screen CMS in celery.

Characterization of the Mitochondrial Genome of a Wheat AL-Type Male Sterility Line and the Candidate CMS Gene.

Heterosis utilization is very important in hybrid seed production. An AL-type cytoplasmic male sterile (CMS) line has been used in wheat-hybrid seed production, but its sterility mechanism has not been explored. In the present study, we sequenced and verified the candidate CMS gene in the AL-type sterile line (AL18A) and its maintainer line (AL18B). In the late uni-nucleate stage, the tapetum cells of AL18A showed delayed programmed cell death (PCD) and termination of microspore at the bi-nucleate stage. As compared to AL18B, the AL18A line produced 100% aborted pollens. The mitochondrial genomes of AL18A and AL18B were sequenced using the next generation sequencing such as Hiseq and PacBio. It was found that the mitochondrial genome of AL18A had 99% similarity with that of Triticum timopheevii, AL18B was identical to that of Triticum aestivum cv. Chinese Yumai. Based on transmembrane structure prediction, 12 orfs were selected as candidate CMS genes, including a previously suggested orf256. Only the lines harboring orf279 showed sterility in the transgenic Arabidopsis system, indicating that orf279 is the CMS gene in the AL-type wheat CMS lines. These results provide a theoretical basis and data support to further analyze the mechanism of AL-type cytoplasmic male sterility in wheat.

New PCR-specific markers for pollen fertility restoration QRfp-4R in rye (Secale cereale L.) with Pampa sterilizing cytoplasm.

Pampa cytoplasmic male sterility phenomenon is used extensively in the rye hybrid breeding programs. It relies on sterilizing action of the cytoplasm resulting in non-viable pollen of female lines. The sterilizing effect is problematic for reversion, and efficient restores are needed. The most promising QTL is located on chromosome 4R, but other chromosomes may also code the trait. Advanced recombinant inbred lines formed bi-parental mapping population genotyped with DArTseq markers. Genetic mapping allowed the seven linkage groups to construct with numerous markers and represent all rye chromosomes. Single marker analysis and composite interval mapping were conducted to identify markers linked to the pollen fertility. Association mapping was used to detect additional markers associated with the trait. A highly significant QTL (QRfp-4R) that explained 42.3% of the phenotypic variation was mapped to the distal part of the long arm of the 4R chromosome. The markers localized in the QRfp-4R region achieve R2 association values up to 0.59. The homology of the 43 marker sequences to the loci responsible for fertility restoration in other species and transcription termination factor (mTERF) linked to Rf genes was established. Ten markers were successfully converted into PCR-specific conditions, and their segregation pattern was identical to that of unconverted DArTs.

Transcriptome and MiRNAomics Analyses Identify Genes Associated with Cytoplasmic Male Sterility in Cotton (Gossypium hirsutum L.).

Cytoplasmic male sterility (CMS) is important for large-scale hybrid seed production. Rearrangements in the mitochondrial DNA (mtDNA) for the cotton (Gossypium hirsutum L.) CMS line J4A were responsible for pollen abortion. However, the expression patterns of nuclear genes associated with pollen abortion and the molecular basis of CMS for J4A are unknown, and were the objectives of this study by comparing J4A with the J4B maintainer line. Cytological evaluation of J4A anthers showed that microspore abortion occurs during meiosis preventing pollen development. Changes in enzyme activity of mitochondrial respiratory chain complex IV and mitochondrial respiratory chain complex V and the content of ribosomal protein and ATP during anther abortion were observed for J4A suggesting insufficient synthesis of ATP hindered pollen production. Additionally, levels of sucrose, starch, soluble sugar, and fructose were significantly altered in J4A during the meiosis stage, suggesting reduced sugar metabolism contributed to sterility. Transcriptome and miRNAomics analyses identified 4461 differentially expressed mRNAs (DEGs) and 26 differentially expressed microRNAs (DEMIs). Pathway enrichment analysis indicated that the DEMIs were associated with starch and sugar metabolism. Six deduced target gene regulatory pairs that may participate in CMS were identified, ghi-MIR7484-10/mitogen-activated protein kinase kinase 6 (MAPKK6), ghi-undef-156/agamous-like MADS-box protein AGL19 (AGL19), ghi-MIR171-1-22/SNF1-related protein kinase regulatory subunit gamma-1 and protein trichome birefringence-like 38, and ghi-MIR156-(8/36)/WRKY transcription factor 28 (WRKY28). Overall, a putative CMS mechanism involving mitochondrial dysfunction, the ghi-MIR7484-10/MAPKK6 network, and reduced glucose metabolism was suggested, and ghi-MIR7484-10/MAPKK6 may be related to abnormal microspore meiosis and induction of excessive sucrose accumulation in anthers.

Targeting of CAT and VCAM1 as Novel Therapeutic Targets for DMD Cardiomyopathy.

Duchenne muscular dystrophy (DMD) related cardiomyopathy is the leading cause of early mortality in DMD patients. There is an urgent need to gain a better understanding of the disease molecular pathogenesis and develop effective therapies to prevent the onset of heart failure. In the present study, we used DMD human induced pluripotent stem cells (DMD-hiPSCs) derived cardiomyocytes (CMs) as a platform to explore the active compounds in commonly used Chinese herbal medicine (CHM) herbs. Single CHM herb (DaH, ZK, and CQZ) reduced cell beating rate, decreased cellular ROS accumulation, and improved structure of DMD hiPSC-CMs. Cross-comparison of transcriptomic profiling data and active compound library identified nine active chemicals targeting ROS neutralizing Catalase (CAT) and structural protein vascular cell adhesion molecule 1 (VCAM1). Treatment with Quecetin, Kaempferol, and Vitamin C, targeting CAT, conferred ROS protection and improved contraction; treatment with Hesperidin and Allicin, targeting VCAM1, induced structure enhancement via induction of focal adhesion. Lastly, overexpression of CAT or VCAM1 in DMD hiPSC-CMs reconstituted efficacious effects and conferred increase in cardiomyocyte function. Together, our results provide a new insight in treating DMD cardiomyopathy via targeting of CAT and VCAM1, and serves as an example of translating Bed to Bench back to Bed using a muti-omics approach.

Anthracycline-Induced Cardiotoxicity: Molecular Insights Obtained from Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs).

Anthracyclines are a class of chemotherapy drugs that are highly effective for the treatment of human cancers, but their clinical use is limited by associated dose-dependent cardiotoxicity. The precise mechanisms by which individual anthracycline induces cardiotoxicity are not fully understood. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are emerging as a physiologically relevant model to assess drugs cardiotoxicity. Here, we describe an assay platform by coupling hiPSC-CMs and impedance measurement, which allows real-time monitoring of cardiomyocyte cellular index, beating amplitude, and beating rate. Using this approach, we have performed comparative studies on a panel of four anthracycline drugs (doxorubicin, epirubicin, idarubicin, and daunorubicin) which share a high degree of structural similarity but are associated with distinct cardiotoxicity profiles and maximum cumulative dose limits. Notably, results from our hiPSC-CMs impedance model (dose-dependent responses and EC50 values) agree well with the recommended clinical dose limits for these drugs. Using time-lapse imaging and RNAseq, we found that the differences in anthracycline cardiotoxicity are closely linked to extent of cardiomyocyte uptake and magnitude of activation/inhibition of several cellular pathways such as death receptor signaling, ROS production, and dysregulation of calcium signaling. The results provide molecular insights into anthracycline cardiac interactions and offer a novel assay system to more robustly assess potential cardiotoxicity during drug development.