Carbon metabolism characteristics of quorum quenching bacteria Rhodococcus sp. BH4 determine the bioaugmentation efficiency under different carbon source conditions.

Carbon sources are critical factors influencing bacterial bioaugmentation, however, the underlying mechanisms, particularly the metabolic characteristics of bioaugmented bacteria remain poorly understood. The bioaugmented bacterium Rhodococcus sp. BH4 secretes the quorum quenching (QQ) enzyme QsdA to disrupt the quorum sensing (QS) in the activated sludge (AS) process, reducing AS yield in-situ. This study investigated the carbon metabolic characteristics of BH4 and explored the effects on bioaugmentation with different influent carbon sources. Because of the absence of glucose-specific phosphoenol phosphotransferase system (PTS), BH4 prefers sodium acetate to glucose. However, the lactones produced during extracellular glucose metabolism enhance BH4 qsdA expression. Moreover, BH4 possess carbon catabolite repression (CCR), acetate inhibits glucose utilization. BH4 microbeads were added to reactors with different carbon sources (R1: sodium acetate; R2: glucose; R3: a mixture of sodium acetate and glucose) for in-situ AS yield reduction. During operation, AS reduction efficiency decreased in the following order: R1 > R3 > R2. R2 and R3 microbeads exhibited similar QQ activity to R1, with less BH4 biomass at 5 d. 13C labeling and Michaelis-Menten equation showed that, due to differences in the competitiveness of carbon sources, R1 BH4 obtained the most carbon, whereas R2 BH4 obtained the least carbon. Moreover, acetate inhibited glucose utilization of R3 BH4. Transcriptome analysis showed that R1 BH4 qsdA expression was the lowest, R2 BH4 was the most serious form of programmed cell death, and the R3 BH4 PTS pathway was inhibited. At 10 d, R1 BH4 biomass and microbead QQ activity were higher than that in R3, and the R2 BH4 lost viability and QQ activity. This study provides new insights into bioaugmentation from the perspectives of carbon source competitiveness, carbon metabolism pathways, and CCR.

Effects and plasma proteomic analysis of GLP-1RA versus CPA/EE, in combination with metformin, on overweight PCOS women: a randomized controlled trial.

PURPOSE: Polycystic ovary syndrome (PCOS) is characterized by reproductive dysfunctions and metabolic disorders. This study aims to compare the therapeutic effectiveness of glucagon-like peptide-1 receptor agonist (GLP-1RA) + Metformin (Met) versus cyproterone acetate/ethinylestradiol (CPA/EE) + Met in overweight PCOS women and identify potential proteomic biomarkers of disease risk in women with PCOS. METHODS: In this prospective, open-label randomized controlled trial, we recruited 60 overweight PCOS women into two groups at a 1:1 ratio to receive CPA/EE (2 mg/day: 2 mg cyproterone acetate and 35-µg ethinylestradiol,) +Met (1500 mg/day) or GLP-1 RA (liraglutide, 1.2-1.8 mg/day) +Met (1500 mg/day) for 12 weeks. The clinical effectiveness and adverse effects were evaluated, followed by plasma proteomic analysis and verification of critical biomarkers by ELISA. RESULTS: Eighty(80%) patients completed the study. Both interventions improved menstrual cycle, polycystic ovaries, LH(luteinizing hormone) and HbA1c(hemoglobin A1c) levels after the 12-week treatment. GLP-1RA + Met was more effective than CPA/EE + Met in reducing body weight, BMI (Body Mass Index), and waist circumference, FBG(fasting blood glucose), AUCI(area under curve of insulin),TC (Total Cholesterol), IL-6(Interleukin-6) and improving insulin sensitivity, and ovulation in overweight women with PCOS, with acceptable short-term side effects. CPA/EE + Met was more effective in improving hyperandrogenemia, including T(total testosterone), LH, LH/FSH(Luteinizing hormone/follicle-stimulating hormone), SHBG(sex hormone-binding globulin) and FAI (free androgen index). By contract, GLP-1RA+Met group only improved LH. Plasma proteomic analysis revealed that the interventions altered proteins involved in reactive oxygen species detoxification (PRDX6, GSTO1, GSTP1, GSTM2), platelet degranulation (FN1), and the immune response (SERPINB9). CONCLUSIONS: Both CPA/EE+Met and GLP-1RA + Met treatment improved reproductive functions in overweight PCOS women. GLP-1RA + Met was more effective than CPA/EE + Met in reducing body weight, BMI, and waist, and improving metabolism, and ovulation in overweight women with PCOS, with acceptable short-term side effects. CPA/EE + Met was more effective in reducing hyperandrogenemia. The novel plasma biomarkers PRDX6, FN1, and SERPINB9, might be indicators and targets for PCOS treatment. TRIAL REGISTRATION CLINICALTIALS. GOV TRIAL NO: NCT03151005. Registered 12 May, 2017, https://clinicaltrials.gov/ct2/show/NCT03151005 .

Light quality regulates plant biomass and fruit quality through a photoreceptor-dependent HY5-LHC/CYCB module in tomato.

Increasing photosynthesis and light capture offers possibilities for improving crop yield and provides a sustainable way to meet the increasing global demand for food. However, the poor light transmittance of transparent plastic films and shade avoidance at high planting density seriously reduce photosynthesis and alter fruit quality in vegetable crops, and therefore it is important to investigate the mechanisms of light signaling regulation of photosynthesis and metabolism in tomato (Solanum lycopersicum). Here, a combination of red, blue, and white (R1W1B0.5) light promoted the accumulation of chlorophyll, carotenoid, and anthocyanin, and enhanced photosynthesis and electron transport rates by increasing the density of active reaction centers and the expression of the genes LIGHT-HARVESTING COMPLEX B (SlLHCB) and A (SlLHCA), resulting in increased plant biomass. In addition, R1W1B0.5 light induced carotenoid accumulation and fruit ripening by decreasing the expression of LYCOPENE ß-CYCLASE (SlCYCB). Disruption of SlCYCB largely induced fruit lycopene accumulation, and reduced chlorophyll content and photosynthesis in leaves under red, blue, and white light. Molecular studies showed that ELONGATED HYPOCOTYL 5 (SlHY5) directly activated SlCYCB, SlLHCB, and SlLHCA expression to enhance chlorophyll accumulation and photosynthesis. Furthermore, R1W1B0.5 light-induced chlorophyll accumulation, photosynthesis, and SlHY5 expression were largely decreased in the slphyb1cry1 mutant. Collectively, R1W1B0.5 light noticeably promoted photosynthesis, biomass, and fruit quality through the photoreceptor (SlPHYB1 and SlCRY1)-SlHY5-SlLHCA/B/SlCYCB module in tomato. Thus, the manipulation of light environments in protected agriculture is a crucial tool to regulate the two vital agronomic traits related to crop production efficiency and fruit nutritional quality in tomato.

N6-Methyladenosine mRNA Modification: From Modification Site Selectivity to Neurological Functions.

The development of various chemical methods has enabled scientists to decipher the distribution features and biological functions of RNA modifications in the past decade. In addition to modifying noncoding RNAs such as tRNAs and rRNAs, N6-methyladenosine (m6A) has been proven to be the most abundant internal chemical modification on mRNAs in eukaryotic cells and is also the most widely studied mRNA modification to date. Extensive studies have repeatedly demonstrated the important functions of m6A in various biological conditions, ranging from embryonic organ development to adult organ function and pathogenesis. Unlike DNA methylation which is relatively stable, the reversible m6A modification on mRNA is highly dynamic and easily influenced by various internal or external factors, such as cell type, developmental stage, nutrient supply, circadian rhythm, and environmental stresses.In this Account, we review our previous findings on the site selectivity mechanisms regulating m6A formation, as well as the physiological roles of m6A modification in cerebellum development and long-term memory consolidation. In our initial efforts to profile m6A in various types of mouse and human cells, we surprisingly found that the sequence motifs surrounding m6A sites were often complementary with the seed sequences of miRNAs. By manipulating the abundance of the miRNA biogenesis enzyme Dicer or individual miRNAs or mutating miRNA sequences, we were able to reveal a new role of nucleus localized miRNAs, which is to guide the m6A methyltransferase METTL3 to bind to mRNAs and to promote m6A formation. As a result, we partially answered the question of why only a small proportion of m6A motifs within an mRNA could have m6A modification at a certain time point. We further explored the functions of m6A modification in regulating brain development and brain functions. We found that cerebellum had the most severe defects when Mettl3 was knocked out in developing mouse embryonic brain and revealed that the underlying mechanisms could be attributed to aberrant mRNA splicing and enhanced cell apoptosis under m6A deficit conditions. On the other hand, knocking out Mettl3 in postnatal hippocampus did not cause morphological defects in the mouse brain but impaired the efficacy of long-term memory consolidation. Under learning stimuli, formation of m6A modifications could be detected on transcripts encoding proteins related to dendrite growth, synapse formation, and other memory related functions. Loss of m6A modifications on these transcripts would result in translation deficiency and reduced protein production, particularly in the translation of early response genes, and therefore would compromise the efficacy of long-term memory consolidation. Interestingly, excessive training sessions or increased training intensity could overcome such m6A deficiency related memory defects, which is likely due to the longer turnover cycle and the cumulative abundance of proteins throughout the training process. In addition to revealing the roles of m6A modification in regulating long-term memory formation, our work also demonstrated an effective method for studying memory formation efficacy. As the lack of an appropriate model for studying memory formation efficacy has been a long-lasting problem in the field of neural science, our hippocampus-specific postnatal m6A knockout model could also be utilized to study other questions related to memory formation efficacy.

Functional heterogeneity of Wnt-responsive and Hedgehog-responsive neural stem cells in the murine adult hippocampus.

Neural stem cells (NSCs) in the adult hippocampus are composed of multiple subpopulations. However, their origin and functional heterogeneity are still unclear. Here, we found that the contribution of murine Wnt-responsive (Axin2+) and Hedgehog-responsive (Gli1+) embryonic neural progenitors to adult NSCs started from early and late postnatal stages, respectively. Axin2+ adult NSCs were intended to actively proliferate, whereas Gli1+ adult NSCs were relatively quiescent and responsive to external stimuli. Moreover, Gli1+ NSC-derived adult-born neurons exhibited more complex dendritic arborization and connectivity than Axin2+ NSC-derived ones. Importantly, genetic cell ablation analysis identified that Axin2+ and Gli1+ adult NSCs were involved in hippocampus-dependent learning, but only Axin2+ adult NSCs were engaged in buffering stress responses and depressive behavior. Together, our study not only defined the heterogeneous multiple origins of adult NSCs but also advanced the concept that different subpopulations of adult NSCs may function differently.

Long-term effect of light rare earth element neodymium on Anammox process.

During the mining of rare earth minerals, the application of neodymium-containing manures, and the treatment of spent neodymium iron boron magnet, the generation of ammonia wastewater containing neodymium is increasing. Thus, the effects of neodymium (Nd(III)) on anaerobic ammonium oxidation (Anammox) were investigated from the aspects of performance, kinetics, statistics, microbial community and sludge morphology, and the recovery strategy of EDTA-2Na wash was discussed. The nitrogen removal efficiency of the Anammox reactor decreased significantly and eventually collapsed at the Nd(III) dosing levels of 20 and 40 mg L-1, respectively. And the toxicity of Nd(III) to AnAOB was determined by the amount internalized into the cells. The EDTA-2Na wash successfully increased the total nitrogen removal rate (TNRR) of Nd(III)-inhibited Anammox to 41.60% of its initial value within 30 days, and the modified Boltzmann model accurately simulated this recovery process. The transient and extended effects of Nd(III), self-recovery, and EDTA-2Na wash on Anammox were effectively assessed using a one-sample t-test. 16S rRNA gene sequencing indicated that Nd(III) remarkably decreased the relative abundance of Planctomycetes and Candidatus Brocadia. The scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) revealed crystal-like neodymium particles on the surface of Anammox sludge. The above-mentioned results demonstrate that the concentration of Nd(III) should be below the toxicity threshold (20 mg L-1) when treating ammonia wastewater containing neodymium by Anammox, and also emphasize the importance of an appropriate recovery strategy.

Acceleration of nitrogen removal performance in a biofilm reactor augmented with Pseudomonas sp. using polycaprolactone as carbon source for treating low carbon to nitrogen wastewater.

Heterotrophic nitrification-aerobic denitrification (HN-AD) process was achieved in a moving bed biofilm reactor after 180-days acclimation using PCL as carbon source for low C/N wastewater treatment. A novel HN-AD strain, JQ-H3, with ability of PCL degradation was augmented to improve nitrogen removal. TN removal efficiencies of 82.31%, 90.05%, and 93.16% were achieved in the augmented reactor (R2), at different HRTs of 24 h, 20 h, and 16 h, while in the control reactor (R1), the TN removal efficiencies were 59.24%, 74.61%, and 76.68%. The effluent COD in R2 was 10.17 mg/L, much lower than that of 42.45 mg/L in R1. Microbial community analysis revealed that JQ-H3 has successfully proliferated with a relative abundance of 4.79%. Relative abundances of functional enzymes of nitrogen cycling remarkably increased due to bioaugmentation based on the analysis of PICRUSt2. This study provides a new approach for enhancing nitrogen removal in low C/N sewage treatment via the HN-AD process.

Electrospinning Preparation, Structure, and Properties of Fe3O4/Tb(acac)3phen/Polystyrene Bifunctional Microfibers.

Compared to single functional materials, multifunctional materials with magnetism and luminescence are more attractive and promising; Thus, it has become an important subject. In our work, bifunctional Fe3O4/Tb(acac)3phen/polystyrene) microfibers with magnetic and luminescent properties (acac: acetylacetone, phen: 1,10-phenanthroline) were synthesized by simple electrospinning process. The doping of Fe3O4 and Tb(acac)3phen made the fiber diameter larger. The surface of pure polystyrene microfibers and microfibers doped only with Fe3O4 nanoparticles were chapped similar to bark, whereas the surface of the microfibers was smoother after doping with Tb(acac)3phen complexes. The luminescent properties of the composite microfibers were systematically studied in contrast to pure Tb(acac)3phen complexes, including excitation and emission spectra, fluorescence dynamics, and the temperature dependence of intensity. Compared with the pure complexes, the thermal activation energy and thermal stability of composite microfiber was significantly improved, and the luminescence of the unit mass of Tb(acac)3phen complexes in composite microfibers was stronger than that in pure Tb(acac)3phen complexes. The magnetic properties of the composite microfibers were also investigated using hysteresis loops, and an interesting experimental phenomenon was found that the saturation magnetization of the composite microfibers gradually increased with the increase in the doping proportion of terbium complexes.

Association Between CYP2B6 Polymorphisms and Efficacy of Clopidogrel in Minor Stroke or Transient Ischemic Attack.

BACKGROUND: CYP2B6 (cytochrome P450 subfamily IIB polypeptide 6), encoded by the CYP2B6 gene, is a critical enzyme involved in clopidogrel metabolism. However, the association between CYP2B6 polymorphisms and the efficacy of clopidogrel in minor stroke or transient ischemic attack for secondary stroke prevention remains unclear. METHODS: Based on CHANCE (Clopidogrel in High-Risk Patients With Acute Nondisabling Cerebrovascular Events) randomized clinical trial of aspirin plus clopidogrel versus aspirin alone, we investigated the role of CYP2B6 polymorphisms and the efficacy of clopidogrel in patients with minor stroke or transient ischemic attack in China from October 2009 to July 2012. A total of 2853 patients were successfully genotyped for CYP2B6-516G>T, rs3745274 and CYP2B6-1456 T>C, rs2054675. The primary efficacy and safety outcomes were new stroke and any bleeding within 90 days. RESULTS: Among the 2853 patients, 32.8% were identified as the carriers of the CYP2B6-516 GT/TT or -1456 TC/CC genotype. The incidences of 90-day new stroke in aspirin plus clopidogrel and aspirin alone groups were 7.1% versus 11.3% among noncarriers, respectively; and 9.7% versus 12.2% among carriers, respectively. The efficacy of aspirin plus clopidogrel versus aspirin alone was not significantly different (P interaction=0.29) in noncarriers (adjusted hazard ratio, 0.61 [95% CI, 0.45-0.83]) compared to carriers (adjusted hazard ratio, 0.80 [95% CI, 0.54-1.18]). The incidence (n=51) of 90-day any bleeding in aspirin plus clopidogrel and aspirin alone groups were 2.2% (21 bleeds) versus 1.9% (18 bleeds) among noncarriers (adjusted hazard ratio, 1.11 [95% CI, 0.59-2.09]) and 1.9% (9 bleeds) versus 0.7% (3 bleeds) among carriers (adjusted hazard ratio, 3.23 [95% CI, 0.86-12.12]). Similar findings were observed during the 1-year follow-up. CONCLUSIONS: In this post hoc analysis of the CHANCE trial, we did not observe a significant difference in the efficacy of aspirin plus clopidogrel compared with aspirin in carriers versus noncarriers of CYP2B6-516 GT/TT or -1456 TC/CC genotype. Our results suggest that both carriers and noncarriers suffering from a minor stroke are likely to benefit from aspirin plus clopidogrel treatment over aspirin monotherapy for secondary prevention. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT00979589.

The Roles of Aerobic Exercise and Folate Supplementation in Hyperhomocysteinemia-Accelerated Atherosclerosis.

Background: Hyperhomocysteinemia (HHcy) is an independent risk factor for atherosclerosis. Effective interventions to reduce HHcy-accelerated atherosclerosis are required. Objectives: This study aimed to investigate the effects of aerobic exercise (AE) and folate (FA) supplementation on plasma homocysteine (Hcy) level and atherosclerosis development in a mouse model. Methods: Six-week-old female apoE-/- mice were grouped into five groups (N = 6-8): HHcy (1.8 g/L DL-homocysteine (DL-Hcy) in drinking water), HHcy + AE (1.8 g/L DL-Hcy and aerobic exercise training on a treadmill), HHcy + FA (1.8 g/L DL-Hcy and 0.006% folate in diet), HHcy + AE + FA (1.8 g/L DL-Hcy, 0.006% folate, and aerobic exercise training on a treadmill), and a control group (regular water and diet). All treatment was sustained for 8 weeks. Triglyceride, cholesterol, lipoprotein, and Hcy levels were determined enzymatically. Plaque and monocyte chemoattractant protein-1 (MCP-1) expression levels in mouse aortic roots were evaluated by immunohistochemistry. Results: Compared to the HHcy group (18.88 ± 6.13 µmol/L), plasma Hcy concentration was significantly reduced in the HHcy + AE (14.79 ± 3.05 µmol/L, p = 0.04), HHcy + FA (9.4 ± 3.85 µmol/L, p < 0.001), and HHcy + AE + FA (9.33 ± 2.21 µmol/L, p < 0.001) groups. Significantly decreased aortic root plaque area and plaque burden were found in the HHcy + AE and HHcy + AE + FA groups compared to those in the HHcy group (both p < 0.05). Plasma MCP-1 level and MCP-1 expression in atherosclerotic lesions were significantly decreased in the HHcy + AE and HHcy + AE + FA groups compared to the HHcy group (all p < 0.05). Conclusions: AE reduced atherosclerosis development in HHcy apoE-/- mice independently of reducing Hcy levels. FA supplementation decreased plasma Hcy levels without attenuating HHcy-accelerated atherosclerosis. AE and FA supplementation have distinct mechanisms in benefiting atherosclerosis.

Tetratricopeptide repeat protein SlREC2 positively regulates cold tolerance in tomato.

Cold stress is a key environmental constraint that dramatically affects the growth, productivity, and quality of tomato (Solanum lycopersicum); however, the underlying molecular mechanisms of cold tolerance remain poorly understood. In this study, we identified REDUCED CHLOROPLAST COVERAGE 2 (SlREC2) encoding a tetratricopeptide repeat protein that positively regulates tomato cold tolerance. Disruption of SlREC2 largely reduced abscisic acid (ABA) levels, photoprotection, and the expression of C-REPEAT BINDING FACTOR (CBF)-pathway genes in tomato plants under cold stress. ABA deficiency in the notabilis (not) mutant, which carries a mutation in 9-CIS-EPOXYCAROTENOID DIOXYGENASE 1 (SlNCED1), strongly inhibited the cold tolerance of SlREC2-silenced plants and empty vector control plants and resulted in a similar phenotype. In addition, foliar application of ABA rescued the cold tolerance of SlREC2-silenced plants, which confirms that SlNCED1-mediated ABA accumulation is required for SlREC2-regulated cold tolerance. Strikingly, SlREC2 physically interacted with ß-RING CAROTENE HYDROXYLASE 1b (SlBCH1b), a key regulatory enzyme in the xanthophyll cycle. Disruption of SlBCH1b severely impaired photoprotection, ABA accumulation, and CBF-pathway gene expression in tomato plants under cold stress. Taken together, this study reveals that SlREC2 interacts with SlBCH1b to enhance cold tolerance in tomato via integration of SlNCED1-mediated ABA accumulation, photoprotection, and the CBF-pathway, thus providing further genetic knowledge for breeding cold-resistant tomato varieties.

Phytopathogenic bacteria utilize host glucose as a signal to stimulate virulence through LuxR homologues.

Chemical signal-mediated biological communication is common within bacteria and between bacteria and their hosts. Many plant-associated bacteria respond to unknown plant compounds to regulate bacterial gene expression. However, the nature of the plant compounds that mediate such interkingdom communication and the underlying mechanisms remain poorly characterized. Xanthomonas campestris pv. campestris (Xcc) causes black rot disease on brassica vegetables. Xcc contains an orphan LuxR regulator (XccR) which senses a plant signal that was validated to be glucose by HPLC-MS. The glucose concentration increases in apoplast fluid after Xcc infection, which is caused by the enhanced activity of plant sugar transporters translocating sugar and cell-wall invertases releasing glucose from sucrose. XccR recruits glucose, but not fructose, sucrose, glucose 6-phosphate, and UDP-glucose, to activate pip expression. Deletion of the bacterial glucose transporter gene sglT impaired pathogen virulence and pip expression. Structural prediction showed that the N-terminal domain of XccR forms an alternative pocket neighbouring the AHL-binding pocket for glucose docking. Substitution of three residues affecting structural stability abolished the ability of XccR to bind to the luxXc box in the pip promoter. Several other XccR homologues from plant-associated bacteria can also form stable complexes with glucose, indicating that glucose may function as a common signal molecule for pathogen-plant interactions. The conservation of a glucose/XccR/pip-like system in plant-associated bacteria suggests that some phytopathogens have evolved the ability to utilize host compounds as virulence signals, indicating that LuxRs mediate an interkingdom signalling circuit.

Upregulated antimicrobial immune response mediated by neutrophils in the development from allergic rhinitis to asthma.

Background: Allergic rhinitis (AR) and asthma are closely related, and AR is regarded as an important risk factor for the onset of asthma. However, the pathogenesis of the development of asthma from AR is still undefined. Objective: The aim of this study was to investigate the mechanisms underlying the development of asthma from AR by comparing the transcriptome features of patients with AR with and without asthma. Methods: Patients with AR with or without asthma caused by weed pollen who presented to the Allergy Clinic of Peking Union Medical College Hospital were recruited for this study. Peripheral blood samples of all the patients were collected during the weed pollen season (September) when the patients had allergic symptoms and outside the pollen season (November) when the patients had no symptoms. Transcriptomic analysis was conducted, and the differentially expressed genes (DEGs) and enriched immune pathways between the patients with AR with asthma (AR-asthma group) and those without asthma (AR group) were identified. In addition, the expression levels of some pivotal differentially expressed RNAs were quantified using quantitative polymerase chain reaction (PCR). Results: During the weed pollen season, the immune-related Gene Ontology (GO) terms with P value < 0.05, enriched by the upregulated genes in the AR-asthma group compared to the AR group included antifungal humoral response, neutrophil-mediated killing of bacterium, antibacterial humoral response, antimicrobial humoral immune response mediated by antimicrobial peptides, and regulation of the T cell receptor signaling pathway. The immune-related GO terms with P values <0.05 enriched by downregulated genes were positive regulation of natural killer cell-mediated cytotoxicity, microglial cell activation, natural killer cell activation, and leukocyte-mediated cytotoxicity. The GO term of antimicrobial humoral immune response mediated by antimicrobial peptides was upregulated both during and outside the pollen season, and the upregulated expression of three DEGs (LTF, PF4, and ELANE) included in this term was verified through quantitative PCR. Conclusions: The activation of the antimicrobial immune response mediated by neutrophils and the depression of cytotoxicity mediated by natural killer cells may play roles in the progression from AR to asthma.

AAV13 Enables Precise Targeting of Local Neural Populations.

As powerful tools for local gene delivery, adeno-associated viruses (AAVs) are widely used for neural circuit studies and therapeutical purposes. However, most of them have the characteristics of large diffusion range and retrograde labeling, which may result in off-target transduction during in vivo application. Here, in order to achieve precise gene delivery, we screened AAV serotypes that have not been commonly used as gene vectors and found that AAV13 can precisely transduce local neurons in the brain, with a smaller diffusion range than AAV2 and rigorous anterograde labeling. Then, AAV13-based single-viral and dual-viral strategies for sparse labeling of local neurons in the brains of C57BL/6 or Cre transgenic mice were developed. Additionally, through the neurobehavioral test in the ventral tegmental area, we demonstrated that AAV13 was validated for functional monitoring by means of carrying Cre recombinase to drive the expression of Cre-dependent calcium-sensitive indicator. In summary, our study provides AAV13-based toolkits for precise local gene delivery, which can be used for in situ small nuclei targeting, sparse labeling and functional monitoring.

Mid-long term forecasting of reservoir inflow using the coupling of time-varying filter-based empirical mode decomposition and gated recurrent unit.

Accurate and reliable runoff forecast is beneficial to watershed planning and management and scientific operation of water resources system. However, due to the comprehensive influence of climatic conditions, geographical environment, and human activities, the runoff series is nonlinear and non-stationary, and there are still great challenges in mid-long term runoff forecasting. In order to improve the prediction accuracy, a novel model TVF-EMD-PE-PSO-GRU (TEPPG) was proposed in this study. Firstly, several intrinsic mode functions (IMFs) were obtained by decomposing the original runoff series by time-varying filter-based empirical mode decomposition (TVF-EMD). Secondly, the permutation entropy (PE) algorithm was used to calculate the complexity of each IMF, and the IMF with similar complexity was combined. Then, the gated recurrent unit (GRU) model based on particle swarm optimization (PSO) was used to predict each IMF after merging. Finally, the prediction results of each IMF were superimposed to obtain the final results. And compared with three models such as TVF-EMD-PSO-GRU, extreme-point symmetric mode decomposition coupled gated recurrent unit and particle swarm optimization (ESMD-PSO-GRU), complete ensemble empirical mode decomposition with adaptive noise coupled gated recurrent unit, and particle swarm optimization (CEEMDAN-PSO-GRU). The monthly and annual runoff forecasting of Tangnaihai hydrological station in the upper reaches of the Yellow River and Cuntan hydrological station in the upstream of the Yangtze River was taken as examples to test the performance of the model. The results show that, compared with the other three models, the TEPPG model had the highest prediction accuracy and was relatively stable in both monthly and annual runoff forecasts. Thus, the proposed method was developed to support the decision-making of water resource system.

Enzymatic Synthesis of Peptide Nanofibers for Self-Delivery of Indomethacin and Tyroservatide in Cancer Therapy.

Non-steroidal anti-inflammatory drugs (NSAIDs) have drawn considerable attention in the field of cancer treatment, yet these drugs display limited potency and selectivity against cancer cells. To address these problems, we designed a peptide-based self-delivery system [Indomethacin-Phe-Phe-Tyr (H2PO3)-Ser-Val, IDM-FFpYSV] that combines an NSAID molecule (indomethacin, or IDM) and a segment of anticancer tripeptide (tyroservatide, or YSV). IDM-FFpYSV is capable of self-assembling in an aqueous solution to afford nanofibrillar hydrogels under the catalysis of alkaline phosphatases (ALPs), which are overexpressed on the plasma membrane of cancer cells. The IDM-FFpYSV + ALP hydrogel displays a continuous release profile of peptide drugs, whereas a solution mixture of pure drugs (IDM-OH + pYSV + ALP) shows burst release of drug moieties. The treatment of IDM-FFpYSV selectively inhibits the proliferation of HeLa cells in vitro, with precise regulations of intracellular targeting proteins (COX-2 and AC-H3). The enhanced potency and selectivity of IDM-FFpYSV are found to be attributed to enhanced cellular uptake of peptide drugs, which involves a caveolae-mediated endocytosis pathway. Furthermore, intravenous administration of the IDM-FFpYSV formulation significantly inhibits the tumor growth in a HeLa-xenografted mouse model, whereas treatment of solution mixtures of pure drugs (IDM-OH + pYSV) fails to do so. Taken together, the study provides a viable strategy to augment anticancer efficacies of self-delivery system through molecular integration of multiple anticancer elements with an enzyme-instructed self-assembly process.

A multi-axis robot-based bioprinting system supporting natural cell function preservation and cardiac tissue fabrication.

Despite the recent advances in artificial tissue and organ engineering, how to generate large size viable and functional complex organs still remains as a grand challenge for regenerative medicine. Three-dimensional bioprinting has demonstrated its advantages as one of the major methods in fabricating simple tissues, yet it still faces difficulties to generate vasculatures and preserve cell functions in complex organ production. Here, we overcome the limitations of conventional bioprinting systems by converting a six degree-of-freedom robotic arm into a bioprinter, therefore enables cell printing on 3D complex-shaped vascular scaffolds from all directions. We also developed an oil bath-based cell printing method to better preserve cell natural functions after printing. Together with a self-designed bioreactor and a repeated print-and-culture strategy, our bioprinting system is capable to generate vascularized, contractible, and long-term survived cardiac tissues. Such bioprinting strategy mimics the in vivo organ development process and presents a promising solution for in vitro fabrication of complex organs.

Intermedin1-53 Inhibits NLRP3 Inflammasome Activation by Targeting IRE1α in Cardiac Fibrosis.

Intermedin (IMD), a paracrine/autocrine peptide, protects against cardiac fibrosis. However, the underlying mechanism remains poorly understood. Previous study reports that activation of nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) inflammasome contributes to cardiac fibrosis. In this study, we aimed to investigate whether IMD mitigated cardiac fibrosis by inhibiting NLRP3. Cardiac fibrosis was induced by angiotensin II (Ang II) infusion for 2 weeks in rats. Western blot, real-time PCR, histological staining, immunofluorescence assay, RNA sequencing, echocardiography, and hemodynamics were used to detect the role and the mechanism of IMD in cardiac fibrosis. Ang II infusion resulted in rat cardiac fibrosis, shown as over-deposition of myocardial interstitial collagen and cardiac dysfunction. Importantly, NLRP3 activation and endoplasmic reticulum stress (ERS) were found in Ang II-treated rat myocardium. Ang II infusion decreased the expression of IMD and increased the expression of the receptor system of IMD in the fibrotic rat myocardium. IMD treatment attenuated the cardiac fibrosis and improved cardiac function. In addition, IMD inhibited the upregulation of NLRP3 markers and ERS markers induced by Ang II. In vitro, IMD knockdown by small interfering RNA significantly promoted the Ang II-induced cardiac fibroblast and NLRP3 activation. Moreover, silencing of inositol requiring enzyme 1 α (IRE1α) blocked the effects of IMD inhibiting fibroblast and NLRP3 activation. Pre-incubation with PKA pathway inhibitor H89 blocked the effects of IMD on the anti-ERS, anti-NLRP3, and anti-fibrotic response. In conclusion, IMD alleviated cardiac fibrosis by inhibiting NLRP3 inflammasome activation through suppressing IRE1α via the cAMP/PKA pathway.

Influence of feeder cells on transcriptomic analysis of pluripotent stem cells.

OBJECTIVES: Human pluripotent stem cells (hPSCs) are of great importance in both scientific research and regenerative medicine. The most classic and widely used culture method for hPSCs is co-culture with feeder cells, usually mouse embryonic fibroblasts. However, whether these feeder cell residues can affect the transcriptomic data analysis of hPSCs, especially gene or miRNA expression quantification, is still largely unknown. METHODS AND RESULTS: In this study, reanalysis of published mRNA-Seq and miRNA-Seq data sets revealed the existence of feeder cell-derived reads in the hPSC transcriptomic samples. We identified potentially influenced human genes and miRNAs due to misalignment of sequencing fragments affected by mouse feeder cells. Furthermore, we developed an optimized miRNA analysis pipeline to avoid quantification bias from different miRNA isoforms in the same family. Finally, by comparing the levels of feeder cell residues in hPSC samples isolated by different methods, we found that fluorescence-activated cell sorting and adhesion methods were more effective in feeder cell removal than the gradient centrifugation method. CONCLUSIONS: Collectively, our results demonstrate that feeder cell residues affect the transcriptomic data analysis of hPSCs. To minimize the impact of feeder cell contamination in hPSC samples, we provide solutions for both data analysis and sample preparation.

Accurate and fast cell marker gene identification with COSG.

Accurate cell classification is the groundwork for downstream analysis of single-cell sequencing data, yet how to identify true marker genes for different cell types still remains a big challenge. Here, we report COSine similarity-based marker Gene identification (COSG) as a cosine similarity-based method for more accurate and scalable marker gene identification. COSG is applicable to single-cell RNA sequencing data, single-cell ATAC sequencing data and spatially resolved transcriptome data. COSG is fast and scalable for ultra-large datasets of million-scale cells. Application on both simulated and real experimental datasets showed that the marker genes or genomic regions identified by COSG have greater cell-type specificity, demonstrating the superior performance of COSG in terms of both accuracy and efficiency as compared with other available methods.