Proximal telomeric decompaction due to telomere shortening drives FOXC1-dependent myocardial senescence.

Telomeres, TTAGGGn DNA repeat sequences located at the ends of eukaryotic chromosomes, play a pivotal role in aging and are targets of DNA damage response. Although we and others have demonstrated presence of short telomeres in genetic cardiomyopathic and heart failure cardiomyocytes, little is known about the role of telomere lengths in cardiomyocyte. Here, we demonstrate that in heart failure patient cardiomyocytes, telomeres are shortened compared to healthy controls. We generated isogenic human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) with short telomeres (sTL-CMs) and normal telomeres (nTL-CMs) as model. Compared to nTL-CMs, short telomeres result in cardiac dysfunction and expression of senescent markers. Using Hi-C and RNASeq, we observe that short telomeres induced TAD insulation decrease near telomeric ends and this correlated with a transcription upregulation in sTL-CMs. FOXC1, a key transcription factor involved in early cardiogenesis, was upregulated in sTL-CMs and its protein levels were negatively correlated with telomere lengths in heart failure patients. Overexpression of FOXC1 induced hiPSC-CM aging, mitochondrial and contractile dysfunction; knockdown of FOXC1 rescued these phenotypes. Overall, the work presented demonstrate that increased chromatin accessibility due to telomere shortening resulted in the induction of FOXC1-dependent expression network responsible for contractile dysfunction and myocardial senescence.

Attempts to obtain clean biochar from hyperaccumulator through pyrolysis: Removal of heavy metals and transformation of phosphorus.

The sound disposal of the ensuing heavy metal-rich plants can address the aftermath of phytoremediation. In this study, the first attempt was made to obtain heavy metals-free and phosphorus-rich biochar from phytoremediation residue (PR) by pyrolysis, and the effects of chlorinating agent type, chlorine dosage, and pyrolysis residence time on heavy metal removal, phosphorus (P) transformation, and biochar properties were investigated. The results showed that as chlorine dosage and pyrolysis residence time increased, added polyvinyl chloride (PVC) reduced the concentration of Zn in biochar to one-tenth of that in PR by intensified chlorination, where both Zn concentration (2727.50 mg/kg) and its leaching concentration (29.13 mg/L) met the utilization requirements, in which the acid-base property of biochar plays a key role in heavy metal leaching. Meanwhile, more than 90% of P in PR remained in biochar and the bioavailability of P in biochar enhanced with the decomposition of organic P to inorganic P, where the concentration of plant-availability P (Pnac) expanded from 1878.40 mg/kg in PR to 8454.00 mg/kg in biochar. This study demonstrated that heavy metal hyperaccumulator can be converted into heavy metal-free and phosphorus-rich biochar with promising applications, which provides new perspectives for the treatment of such hazardous wastes.

Experimental study on adsorption of PbCl2 and CdCl2 on kaolin modified by leachates from municipal solid waste incineration power plant.

Six kinds of waste liquids produced in the treatment process of leachate in a waste incineration plant were used to improve the adsorption effect of raw kaolin on heavy metal chloride. The capture performances of these modified kaolin on PbCl2 and CdCl2 vapor were investigated in a two-stage fixed bed combustor. The results indicated that the adsorption effects of raw kaolin on PbCl2 and CdCl2 were improved in some experimental groups, main effective component was Na+ in the leachate, but the influences did not change regularly with the increase in the concentration of Na + introduced into kaolin. The adsorbents formed by modifying 10 g kaolin with 21.25 ml leachate 2 were the best adsorbents for PbCl2 and CdCl2. The capture efficiencies of PbCl2 and CdCl2 can reach 95% and 63.88%, with the increase of 36% and 53%, respectively. Using leachate as modifying agent had the same effect as directly using Na+. Adsorptions of PbCl2 and CdCl2 were still mainly chemical adsorptions. After adsorption of PbCl2, the modified kaolin not only generated PbA12Si2O8, but also produced other chemical compounds. The adsorption of CdCl2 by modified kaolin did not generate CdAl2Si2O8, but other chemical reactions occurred to generate CdAl2O4 and Pb8Cd (Si2O7)3.

Granulosa cell mevalonate pathway abnormalities contribute to oocyte meiotic defects and aneuploidy.

With aging, abnormalities during oocyte meiosis become more prevalent. However, the mechanisms of aging-related oocyte aneuploidy are not fully understood. Here we performed Hi-C and SMART-seq of oocytes from young and old mice and reveal decreases in chromosome condensation and disrupted meiosis-associated gene expression in metaphase I oocytes from aged mice. Further transcriptomic analysis showed that meiotic maturation in young oocytes was correlated with robust increases in mevalonate (MVA) pathway gene expression in oocyte-surrounding granulosa cells (GCs), which was largely downregulated in aged GCs. Inhibition of MVA metabolism in GCs by statins resulted in marked meiotic defects and aneuploidy in young cumulus-oocyte complexes. Correspondingly, supplementation with the MVA isoprenoid geranylgeraniol ameliorated oocyte meiotic defects and aneuploidy in aged mice. Mechanically, we showed that geranylgeraniol activated LHR/EGF signaling in aged GCs and enhanced the meiosis-associated gene expression in oocytes. Collectively, we demonstrate that the MVA pathway in GCs is a critical regulator of meiotic maturation and euploidy in oocytes, and age-associated MVA pathway abnormalities contribute to oocyte meiotic defects and aneuploidy.

Genetic polymorphisms and phylogenetic characteristics of Tibeto-Burman-speaking Lahu population from southwest China based on 41 Y-STR loci.

BACKGROUND: Male sex-linked Y-chromosome short tandem repeats (Y-STRs) have been widely used in forensic cases and population genetics research. At present, the forensic-related Y-STR data in the Chinese Lahu population are still poorly understood. AIM: To enrich the available Y-STR data of this Chinese minority population and investigate its phylogenetic relationships with other reported populations. SUBJECTS AND METHODS: The genetic polymorphisms of 41 Y-STR loci were analysed in 299 unrelated healthy Lahu male individuals from Southwest China. Phylogenetic analyses were performed by multidimensional scaling analysis and neighbor-joining phylogenetic tree construction. RESULTS: A total of 379 alleles were observed at the 41 Y-STR loci. The allele frequencies ranged from 0.0033 to 0.9666. The genetic diversity values ranged from 0.0653 to 0.9072. A total of 254 different haplotypes of the 41 Y-STR loci were observed in 299 individuals. The values of haplotype diversity, haplotype match probability, and discrimination capacity were 0.9987, 0.0047, and 0.8495, respectively. The phylogenetic analysis indicated that the Tibeto-Burman-speaking Lahu population showed a close genetic relationship with the Yunnan Yi population. CONCLUSIONS: The haplotype data of the present study can enrich the forensic databases of this Chinese minority population and will be useful for population genetics and forensic DNA application.

Prognostic value of coronary CT angiography in heart failure patients with preserved ejection fraction.

OBJECTIVES: To investigate the prognostic value of coronary CT angiography (CCTA) in heart failure patients with preserved ejection fraction (HFpEF). METHODS: Between January 2009 and December 2013, 6497 participants (mean age 63 ± 9.4 [range 32-86] years; 4111 men) who underwent CCTA and echocardiography were prospectively included. Participants were divided into HFpEF group and without HFpEF group. The primary endpoint was major adverse cardiovascular events (MACEs), including cardiovascular mortality, nonfatal myocardial infarction (MI), or hospitalization for heart failure (HF). RESULTS: Among those participants, 3096 were identified with HFpEF and 3401 were without HFpEF. Higher prevalence of coronary atherosclerosis was observed in HFpEF group than those without (78.3% vs. 64.9%, p < 0.001). During a median of 11.0 [IQR: 9.0-12.0] years follow-up, participants with HFpEF exhibit a heightened risk of MACEs in CAD-RADS = 0, 1-2, and ≥ 3 respectively (p < 0.001 for all). In the risk-adjusted hazard analysis among participants with HFpEF, CAD-RADS = 1-2 increased a 2.5-time risk for non-fatal MI (adjusted HR: 2.5, 95% CI: 1.5 to 4.3, p < 0.001), while CAD-RADS ≥ 3 conferred 3.9-fold and 3.1-fold higher risk for cardiovascular mortality (adjusted HR: 3.9, 95% CI: 2.2 to 7.1, p < 0.001) and hospitalization due to HF (adjusted HR: 3.1, 95% CI: 1.9 to 5.3, p < 0.001) with reference to CAD-RADS = 0 respectively. CONCLUSIONS: Coronary artery disease is common in participants with HFpEF and associated with MACEs. Among those participants, the presence of CAD-RADS = 1-2 increased the risk of nonfatal MI, while CAD-RADS ≥ 3 were correlated with cardiovascular mortality and hospitalization due to HF. KEY POINTS: • Higher median of CACS and higher CAD-RADS categories were observed in the HFpEF group than those without (p < 0.001 for both). • Participants with HFpEF exhibit a heightened risk of MACEs in CAD-RADS = 0, 1-2, and ≥ 3 respectively (p < 0.001 for all). • In the risk-adjusted hazard analysis among participants with HFpEF, CAD-RADS =1-2 increased a 2.5-time risk for non-fatal MI (adjusted HR: 2.5, 95% CI: 1.5 to 4.3, p < 0.001) with reference to CAD-RADS = 0 respectively.

Preparation of Alkali Activated Cementitious Material by Upgraded Fly Ash from MSW Incineration.

Utilization of municipal solid waste incineration fly ash (MSWI-FA) can avoid land occupation and environmental risks of landfill. In this paper, MSWI-FA was used to prepare alkali activated cementitious materials (AACMs) after two-step pretreatment. The ash calcination at 450 °C removed 93% of dioxins. The alkali washing with 0.2 g NaOH/g ash removed 89% of chlorine and retained almost 100% of calcium. The initial setting time of AACMs was too short to detect for 20% of MSWI-FA addition, and the prepared block had extensive cracks and expansion for CaClOH and CaSO4 inside. Alkaline washing pretreatment increased the initial setting time by longer than 3 min with 30% ash addition and eliminated the cracks and expansion. The significance of the factors for compressive strength followed the modulus of alkali activator > silica fume amount > alkaline washing MSWI fly ash (AW-MSWI-FA) amount. When the activator modulus was 1.2, 1.4 and 1.6, the blocks with 30% of AW-MSWI-FA had a compressive strength of up to 36.73, 32.61 and 16.06 MPa, meeting MU15 grade. The leaching test shows that these AACM blocks were not hazardous waste and almost no Zn, Cu, Cd, Pb, Ba, Ni, Be and Ag were released in the leaching solution.

Hydrothermal oxidation degradation of dioxins in fly ash with water-washing and added Ce-Mn catalyst.

A comprehensive analysis of the effects of the temperature, reaction time, liquid-solid ratio (L/S), and initial pH on the hydrothermal degradation of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) (which are both PCDD/Fs) in municipal solid waste incineration (MSWI) fly ash is presented. Consequently, the hydrothermal degradation reaction is catalyzed using Ce-Mn catalyst under low-temperature conditions to study the effect of the catalyst on the degradation efficiency of PCDD/Fs. The experimental results show that temperature is the most critical factor for the reaction. When the hydrothermal oxidation temperature reaches 280 °C (reaction time = 120 min, original pH = 8.5, L/S = 4 mL/g), the toxicity equivalent (I-TEQ) of PCDD/Fs is only 5.4 ng TEQ/kg, and the degradation efficiency reaches 99.71%. Under these conditions, 2,3,4,7,8-P5CDF makes the highest contribution to I-TEQ degradation, reaching 37.4%. There are four main pathways for the reaction of 2,3,4,7,8-P5CDF with hydroxyl radicals. A comparison of the PCDD/F concentrations of different products shows that the addition of 0.5%, 1.0%, and 1.5% of the Ce-Mn catalyst reduces the degradation efficiency by 8.79%, 1.40%, and 0.07%, respectively, which indicates that the addition of a small quantity of Ce-Mn catalyst does not facilitate the degradation of PCDD/Fs. The addition of the catalyst significantly decreases the degradation efficiency of low-chlorinated homologs but has a relatively small effect on that of high-chlorinated homologs. Therefore, it is concluded that Ce-Mn catalysts are more likely to promote resynthesis than degradation of PCDD/Fs.

Population data and phylogenetic structure of 19 STR loci in Chinese Hui ethnic group residing in Yunnan province of China.

Allele frequency distributions and statistical forensic parameters of 19 autosomal STR loci in a sample of 535 unrelated healthy Hui individuals from Yunnan province were estimated. A total of 236 alleles at these loci were identified and their corresponding allele frequencies ranged from 0.000935 to 0.527103. Penta E is the most informative in Hui population, whereas TPOX showed the lowest. All of the STR loci reached the Hardy-Weinberg equilibrium after Bonferroni correction. The combined discrimination power and probability of excluding paternity of the 19 STR loci were 0.999 999 999 999 999 999 999 984 47 16 and 0.999 999 988, respectively. Furthermore, the genetic relationship between the Yunnan Hui population and other 8 different Hui groups or 25 previously investigated groups residing in other areas of China were also estimated based on pairwise genetic distance. These results suggest that the 19 STR loci are highly polymorphic, which is suitable for forensic personal identification and paternity testing.

Stage-resolved Hi-C analyses reveal meiotic chromosome organizational features influencing homolog alignment.

During meiosis, chromosomes exhibit dramatic changes in morphology and intranuclear positioning. How these changes influence homolog pairing, alignment, and recombination remain elusive. Using Hi-C, we systematically mapped 3D genome architecture throughout all meiotic prophase substages during mouse spermatogenesis. Our data uncover two major chromosome organizational features varying along the chromosome axis during early meiotic prophase, when homolog alignment occurs. First, transcriptionally active and inactive genomic regions form alternating domains consisting of shorter and longer chromatin loops, respectively. Second, the force-transmitting LINC complex promotes the alignment of ends of different chromosomes over a range of up to 20% of chromosome length. Both features correlate with the pattern of homolog interactions and the distribution of recombination events. Collectively, our data reveal the influences of transcription and force on meiotic chromosome structure and suggest chromosome organization may provide an infrastructure for the modulation of meiotic recombination in higher eukaryotes.

The SUN1-SPDYA interaction plays an essential role in meiosis prophase I.

Chromosomes pair and synapse with their homologous partners to segregate correctly at the first meiotic division. Association of telomeres with the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex composed of SUN1 and KASH5 enables telomere-led chromosome movements and telomere bouquet formation, facilitating precise pairwise alignment of homologs. Here, we identify a direct interaction between SUN1 and Speedy A (SPDYA) and determine the crystal structure of human SUN1-SPDYA-CDK2 ternary complex. Analysis of meiosis prophase I process in SPDYA-binding-deficient SUN1 mutant mice reveals that the SUN1-SPDYA interaction is required for the telomere-LINC complex connection and the assembly of a ring-shaped telomere supramolecular architecture at the nuclear envelope, which is critical for efficient homologous pairing and synapsis. Overall, our results provide structural insights into meiotic telomere structure that is essential for meiotic prophase I progression.

Clinical Observation on Jiechang Qingre Pills Combined with Conventional Western Medicine Therapy in Improving Dampness-heat Syndrome of Large Intestine at Active Stage of Ulcerative Colitis / 中国实验方剂学杂志

Objective:To observe the clinical efficacy of Jiechang Qingre pills for dampness-heat syndrome of large intestine at active stage of ulcerative colitis （UC） and investigate its effect on inflammatory factors. Method:One hundred and eight patients with active UC were divided into observation group and control group. Both groups were treated with Mesalazine enteric-coated tablets， 2 g/times， 2 times/day， for 2 weeks. If symptoms were poorly controlled， prednisone acetate tablets would be used instead， 0.75 mg·kg^-1·d^-1in 3 times by oral administration. Patients in the observation group took Jiechang Qingre pills， 10 g/time， 3 times/day before meals. Patients in the control group took Jiechang Qingre pills simulated drug， 10 g/time， 3 times/day before meals. The course of treatment was 12 weeks in both groups and the patients were followed up for 3 months. The modified Mayo score was used to evaluate disease activity. Before and after treatment， large intestine dampness-heat syndrome score， inflammatory bowel disease questionnaire （IBDQ）， mucosal histology assessment and scores of major symptoms and intestinal mucosal lesion severity were graded. The incidence of non-reactivity， hormone failure， hormone dependence， and early recurrence were recorded 2 weeks after treatment. Tumor necrosis factor-<italic>α </italic>（TNF-<italic>α</italic>）， interleukin-6（IL-6） and IL-17 levels were measured before and after treatment. Result:The clinical effective rate in the observation group was 94.00% （47/50）， higher than 77.55% （38/49） in the control group （<italic>χ</italic>²=5.514，<italic>P</italic><0.05）. The clinical remission rate was 82.00%（41/50） in the observation group， higher than 61.22% （30/49） in the control group （<italic>χ</italic>²=5.266，<italic>P</italic><0.05）. The endoscopic response rate was 96.00% （48/50） in the observation group， higher than 79.59% （39/49） in the control group （<italic>χ</italic>²=6.251，<italic>P</italic><0.05）. The rate of mucosal healing in the observation group was 90.00% （45/50）， higher than 79.59% （35/49） in the control group （<italic>χ</italic>²=5.503，<italic>P</italic><0.05）. The scores of diarrhea， purulent stool， abdominal pain， tenesmus， hyperemia， edema， erosion and ulcer in the observation group were lower than those in the control group （<italic>P</italic><0.01）. The rate of non-reactivity in the observation group was 16.00% （8/50）， lower than 34.69% （17/49） in the control group（<italic>χ</italic>²=4.581，<italic>P</italic><0.05）. The hormone failure rate in the observation group was 37.50%（3/8）， lower than 64.71%（11/17）in the control group，but the difference was not statistically significant（tested by the exact probaility method）. The hormone dependence rate in the observation group was 12.50%（1/8）， lower than 23.53% （4/17） in the control group，but the difference was not statistically significant（tested by the exact probaility method）. The early recurrence rate in the observation group was 14.00% （7/50）， lower than 32.65%（16/49） in the control group（<italic>χ</italic>²=4.827，<italic>P</italic><0.05）. The scores of Mayo， dampness and heat syndrome and Geboes index in the observation group were lower than those in the control group （<italic>P</italic><0.01）， and the IBDQ scores were significantly higher than those in the control group （<italic>P</italic><0.01）. The TNF-<italic>α， </italic>IL-6 and IL-17 levels of the patients in the observation group were lower than those in the control group （<italic>P</italic><0.01）. Conclusion:Based on the routine treatment of western medicine， Jiechang Qingre pills treatment for the patients with active UC can effectively induce clinical remission， alleviate inflammatory reaction， promote intestinal mucosal healing， improve clinical symptoms， quality of life and the response of treatment. Its clinical efficacy and enteroscopy efficacy are better than western medicine treatment alone， so it is worthy of clinical use.

Effect of Koumine on Proliferation and Apoptosis of Colorectal Cancer Cells via ROS/JNK/FoxO3a Signaling Pathway / 中国实验方剂学杂志

Objective:To explore the effect and underlying mechanism of koumine （Kou） at different concentrations （0， 100， 200， 400 μmol·L^-1） on the proliferation and apoptosis of colorectal cancer HCT-116 cells. Method:After 24 hours of<italic> in vitro</italic> intervention with HCT-116 cells by Kou， cell counting kit-8 （CCK-8） assay was used to detect its effect on cell proliferation. Flow cytometry was used to detect cell cycle， apoptosis， and reactive oxygen species （ROS） expression. Real-time quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of forkhead box O3a （FoxO3a）. Cells were transfected with small interfering ribonucleic acid （siRNA）. Western blot was employed to detect the protein expression of the FoxO3a target gene. Result:Compared with the conditions in the blank group， Kou treatment reduced the proliferation rate of HCT-116 cells （<italic>P</italic><0.05， <italic>P</italic><0.01） in a dose-dependent manner， caused cell cycle arrest in the G₀/G₁ phase， and induced the apoptosis of HCT-116 cells （<italic>P</italic><0.05， <italic>P</italic><0.01）， which was positively correlated with the concentration of Kou. FoxO3a siRNA interference reduced the expression of FoxO3a and its downstream target genes cyclin-dependent kinase inhibitor 1A （p21）， cyclin-dependent kinase inhibitor 1B （p27）， and Bcl-2 interacting mediator of cell death （Bim） （<italic>P</italic><0.01）. Kou treatment induced the activation of c-Jun <italic>N</italic>-terminal kinase （JNK） in HCT116 cells. SP600125 （JNK specific inhibitor） treatment inhibited the Kou-induced FoxO3a activation and the expression of its downstream target genes. <italic>N</italic>-acetyl cysteine （NAC） treatment reduced Kou-induced ROS levels （<italic>P</italic><0.01） and JNK signal activation. The above results were significantly different from those in the blank group （<italic>P</italic><0.01）. Conclusion:Kou can effectively inhibit the proliferation of HCT-116 cells and promote apoptosis， and the mechanism may be related to the regulation of the ROS/JNK/FoxO3a pathway.

Dysregulation of club cell biology in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive, chronic fibrotic lung disease with an irreversible decline of lung function. "Bronchiolization", characterized by ectopic appearance of airway epithelial cells in the alveolar regions, is one of the characteristic features in the IPF lung. Based on the knowledge that club cells are the major epithelial secretory cells in human small airways, and their major secretory product uteroglobin (SCGB1A1) is significantly increased in both serum and epithelial lining fluid of IPF lung, we hypothesize that human airway club cells contribute to the pathogenesis of IPF. By assessing the transcriptomes of the single cells from human lung of control donors and IPF patients, we identified two SCGB1A1+ club cell subpopulations, highly expressing MUC5B, a significant genetic risk factor strongly associated with IPF, and SCGB3A2, a marker heterogeneously expressed in the club cells, respectively. Interestingly, the cellular proportion of SCGB1A1+MUC5B+ club cells was significantly increased in IPF patients, and this club cell subpopulation highly expressed genes related to mucous production and immune cell chemotaxis. In contrast, though the cellular proportion did not change, the molecular phenotype of the SCGB1A1+SCGB3A2high club cell subpopulation was significantly altered in IPF lung, with increased expression of mucins, cytokine and extracellular matrix genes. The single cell transcriptomic analysis reveals the cellular and molecular heterogeneity of club cells, and provide novel insights into the biological functions of club cells in the pathogenesis of IPF.

Cell-specific expression of lung disease risk-related genes in the human small airway epithelium.

BACKGROUND: The human small airway epithelium (SAE) plays a central role in the early events in the pathogenesis of most inherited and acquired lung disorders. Little is known about the molecular phenotypes of the specific cell populations comprising the SAE in humans, and the contribution of SAE specific cell populations to the risk for lung diseases. METHODS: Drop-seq single-cell RNA-sequencing was used to characterize the transcriptome of single cells from human SAE of nonsmokers and smokers by bronchoscopic brushing. RESULTS: Eleven distinct cell populations were identified, including major and rare epithelial cells, and immune/inflammatory cells. There was cell type-specific expression of genes relevant to the risk of the inherited pulmonary disorders, genes associated with risk of chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis and (non-mutated) driver genes for lung cancers. Cigarette smoking significantly altered the cell type-specific transcriptomes and disease risk-related genes. CONCLUSIONS: This data provides new insights into the possible contribution of specific lung cells to the pathogenesis of lung disorders.

Single-Cell Transcriptome Analysis of Mouse Liver Cell-Specific Tropism and Transcriptional Dysregulation Following Intravenous Administration of AAVrh.10 Vectors.

Capitalizing on liver tropism of adeno-associated viral (AAV) vectors, intravenous vector administration is commonly used to genetically modify hepatocytes, a strategy currently in clinical trials for a number of liver-based hereditary disorders. Although hepatocytes are known to exhibit extensive phenotypic heterogeneity influenced by liver zonation and dietary cycle, there is little data available for the tropism capacity, as well as the potential transcriptional dysregulation, of AAV vectors for specific liver cell types. To assess these issues, we employed single-cell RNA sequencing of the mouse liver after intravenous administration of the liver tropic AAVrh.10 vector to characterize cell-specific AAV-mediated transgene expression and transcriptome dysregulation. Wild-type 8-week-old male C57Bl/6 mice under normal feed cycle were randomly divided into three groups and intravenously administered phosphate-buffered saline (PBS), AAVrh.10Null (no transgene), or AAVrh.10mCherry (marker gene). Overall, a total of 46,500 liver cells were sequenced. The single-cell transcriptomic profiles were grouped into three separate clusters of hepatocytes (Ttr-enriched "Hep1," Tat-enriched "Hep2," and Alb-enriched "Hep3") and multiple other cell types. The hepatocyte diversity was driven by glucose and lipid homeostasis signaling. Assessment of the transgene expression demonstrated that AAVrh.10 is primarily Hep1-tropic, with a 10-gene signature positively correlated with AAVrh.10-mediated transgene expression. The transgene expression was less in Hep2 and Hep3 cells with a high receptor tyrosine kinase phenotype. Importantly, AAVrh.10 vector interactions with the liver markedly altered the transcriptional patterns of all cell types, with modified genes enriched in pathways of complement and coagulation cascade, cytochrome P450, peroxisome, antigen processing and presentation, and endoplasmic reticulum protein processing. These observations provide insights into the liver cell-specific consequences of AAV-mediated liver gene transfer, far beyond the well-known organ-specific expression of the vector-delivered transgene.

Exaggerated BMP4 signalling alters human airway basal progenitor cell differentiation to cigarette smoking-related phenotypes.

Airway remodelling in chronic obstructive pulmonary disease (COPD) originates, in part, from smoking-induced changes in airway basal stem/progenitor cells (BCs). Based on the knowledge that bone morphogenetic protein 4 (BMP4) influences epithelial progenitor function in the developing and adult mouse lung, we hypothesised that BMP4 signalling may regulate the biology of adult human airway BCs relevant to COPD.BMP4 signalling components in human airway epithelium were analysed at the mRNA and protein levels, and the differentiation of BCs was assessed using the BC expansion and air-liquid interface models in the absence/presence of BMP4, BMP receptor inhibitor and/or small interfering RNAs against BMP receptors and downstream signalling.The data demonstrate that in cigarette smokers, BMP4 is upregulated in ciliated and intermediate undifferentiated cells, and expression of the BMP4 receptor BMPR1A is enriched in BCs. BMP4 induced BCs to acquire a smoking-related abnormal phenotype in vitro mediated by BMPR1A/Smad signalling, characterised by decreased capacity to differentiate into normal mucociliary epithelium, while generating squamous metaplasia.Exaggerated BMP4 signalling promotes cigarette smoking-relevant airway epithelial remodelling by inducing abnormal phenotypes in human airway BCs. Targeting of BMP4 signalling in airway BCs may represent a novel target to prevent/treat COPD-associated airway disease.

Characterization and improved properties of Glutamine synthetase from Providencia vermicola by site-directed mutagenesis.

In this study, a novel gene for Glutamine synthetase was cloned and characterized for its activities and stabilities from a marine bacterium Providencia vermicola (PveGS). A mutant S54A was generated by site directed mutagenesis, which showed significant increase in the activity and stabilities at a wide range of temperatures. The Km values of PveGS against hydroxylamine, ADP-Na2 and L-Glutamine were 15.7 ± 1.1, (25.2 ± 1.5) × 10-5 and 32.6 ± 1.7 mM, and the kcat were 17.0 ± 0.6, 9.14 ± 0.12 and 30.5 ± 1.0 s-1 respectively. In-silico-analysis revealed that the replacement of Ser at 54th position with Ala increased the catalytic activity of PveGS. Therefore, catalytic efficiency of mutant S54A had increased by 3.1, 0.89 and 2.9-folds towards hydroxylamine, ADP-Na2 and L-Glutamine respectively as compared to wild type. The structure prediction data indicated that the negatively charged pocket becomes enlarged and hydrogen bonding in Ser54 steadily promotes the product release. Interestingly, the residual activity of S54A mutant was increased by 10.7, 3.8 and 3.8 folds at 0, 10 and 50 °C as compared to WT. Structural analysis showed that S54A located on the loop near to the active site improved its flexibility due to the breaking of hydrogen bonds between product and enzyme. This also facilitated the enzyme to increase its cold adaptability as indicated by higher residual activity shown at 0 °C. Thus, replacement of Ala to Ser54 played a pivotal role to enhance the activities and stabilities at a wide range of temperatures.

Enhanced thermostability of halo-tolerant glutaminase from Bacillus licheniformis ATCC 14580 by immobilization onto nano magnetic cellulose sheet and its application in production of glutamic acid.

A halo-tolerant glutaminase gene (BlglsA) was isolated from Bacillus licheniformis. Heterologous expression of BlglsA revealed that it encodes for a 36 kDa protein containing 327 amino acid residues. The purified enzyme showed optimal activity at a pH of 9.5 while 35 °C was found to be the optimum temperature. The enzyme retained about 92 and 97% stability at pH 12 and temperature (40 °C) respectively. Subsequent immobilization of BlglsA on nano magnetic cellulose sheet (NMCS) led to an enhanced tolerance to higher temperature. NMCS-BlglsA showed optimum activity at 45 °C, although it was stable even at 60 °C. NaCl tolerance (≥90% in 0.3 M) was almost similar to BlglsA and NMCS-BlglsA. The metal ions Fe2+ (5 mM) and Mn2+ (2.5 mM) improved the BlglsA relative activity by 61 and 48%, respectively. In contrast, 5 mM Mn2+ was found suitable to enhance the activity of NMCS-BlglsA up to 72%. The production of glutamic acid by NMCS-BlglsA was 1.61 g/l in 48 h. Reusability test of NMCS-BlglsA showed 76 and 35% retention of the actual activity after 4th and 7th cycle, respectively. Such remarkable biochemical properties of NMCS-BlglsA make it an attractive enzyme for food industries.

Ontogeny and Biology of Human Small Airway Epithelial Club Cells.

RATIONALE: Little is known about human club cells, dome-shaped cells with dense cytoplasmic granules and microvilli that represent the major secretory cells of the human small airways (at least sixth-generation bronchi). OBJECTIVES: To define the ontogeny and biology of the human small airway epithelium club cell. METHODS: The small airway epithelium was sampled from the normal human lung by bronchoscopy and brushing. Single-cell transcriptome analysis and air-liquid interface culture were used to assess club cell ontogeny and biology. MEASUREMENTS AND MAIN RESULTS: We identified the club cell population by unbiased clustering using single-cell transcriptome sequencing. Principal component gradient analysis uncovered an ontologic link between KRT5 (keratin 5)+ basal cells and SCGB1A1 (secretoglobin family 1A member 1)+ club cells, a hypothesis verified by demonstrating in vitro that a pure population of human KRT5+ SCGB1A1- small airway epithelial basal cells differentiate into SCGB1A1+KRT5- club cells on air-liquid interface culture. Using SCGB1A1 as the marker of club cells, the single-cell analysis identified novel roles for these cells in host defense, xenobiotic metabolism, antiprotease, physical barrier function, monogenic lung disorders, and receptors for human viruses. CONCLUSIONS: These observations provide novel insights into the molecular phenotype and biologic functions of the human club cell population and identify basal cells as the human progenitor cells for club cells.