A GMP-compliant manufacturing method for Wharton's jelly-derived mesenchymal stromal cells.

BACKGROUND: Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) hold great therapeutic potential in regenerative medicine. Therefore, it is crucial to establish a Good Manufacturing Practice (GMP)-compliant methodology for the isolation and culture of WJ-MSCs. Through comprehensive research, encompassing laboratory-scale experiments to pilot-scale studies, we aimed to develop standardized protocols ensuring the high yield and quality of WJ-MSCs manufacturing. METHODS: Firstly, optimization of parameters for the enzymatic digestion method used to isolate WJ-MSCs was conducted. These parameters included enzyme concentrations, digestion times, seeding densities, and culture media. Additionally, a comparative analysis between the explant method and the enzymatic digestion method was performed. Subsequently, the consecutive passaging of WJ-MSCs, specifically up to passage 9, was evaluated using the optimized method. Finally, manufacturing processes were developed and scaled up, starting from laboratory-scale flask-based production and progressing to pilot-scale cell factory-based production. Furthermore, a stability study was carried out to assess the storage and use of drug products (DPs). RESULTS: The optimal parameters for the enzymatic digestion method were a concentration of 0.4 PZ U/mL Collagenase NB6 and a digestion time of 3 h, resulting in a higher yield of P0 WJ-MSCs. In addition, a positive correlation between the weight of umbilical cord tissue and the quantities of P0 WJ-MSCs has been observed. Evaluation of different concentrations of human platelet lysate revealed that 2% and 5% concentrations resulted in similar levels of cell expansion. Comparative analysis revealed that the enzymatic digestion method exhibited faster outgrowth of WJ-MSCs compared to the explant method during the initial passage. Passages 2 to 5 exhibited higher viability and proliferation ability throughout consecutive passaging. Moreover, scalable manufacturing processes from the laboratory scale to the pilot scale were successfully developed, ensuring the production of high-quality WJ-MSCs. Multiple freeze-thaw cycles of the DPs led to reduced cell viability and viable cell concentration. Subsequent thawing and dilution of the DPs resulted in a significant decrease in both metrics, especially when stored at 20-27 °C. CONCLUSION: This study offers valuable insights into optimizing the isolation and culture of WJ-MSCs. Our scalable manufacturing processes facilitate the large-scale production of high-quality WJ-MSCs. These findings contribute to the advancement of WJ-MSCs-based therapies in regenerative medicine.

Antipathogenic Activities of Volatile Organic Compounds Produced by Bacillus velezensis LT1 against Sclerotium rolfsii LC1, the Pathogen of Southern Blight in Coptis chinensis.

This study explores the antipathogenic properties of volatile organic compounds (VOCs) produced by Bacillus velezensis LT1, isolated from the rhizosphere soil of Coptis chinensis. The impact of these VOCs on the mycelial growth of Sclerotium rolfsii LC1, the causative agent of southern blight in C. chinensis, was evaluated using a double Petri-dish assay. The biocontrol efficacy of these VOCs was further assessed through leaf inoculation and pot experiments. Antifungal VOCs were collected using headspace solid-phase microextraction (SPME), and their components were identified via gas chromatography-mass spectrometry (GC-MS). The results revealed that the VOCs significantly inhibited the mycelial growth and sclerotia germination of S. rolfsii LC1 and disrupted the morphological integrity of fungal mycelia. Under the influence of these VOCs, genes associated with chitin synthesis were upregulated, while those related to cell wall degrading enzymes were downregulated. Notably, 2-dodecanone and 2-undecanone exhibited inhibition rates of 81.67% and 80.08%, respectively. This research provides a novel approach for the prevention and management of southern blight in C. chinensis, highlighting the potential of microbial VOCs in biocontrol strategies.

Bacillus velezensis LT1: a potential biocontrol agent for southern blight on Coptis chinensis.

Introduction: Southern blight, caused by Sclerotium rolfsii, poses a serious threat to the cultivation of Coptis chinensis, a plant with significant medicinal value. The overreliance on fungicides for controlling this pathogen has led to environmental concerns and resistance issues. There is an urgent need for alternative, sustainable disease management strategies. Methods: In this study, Bacillus velezensis LT1 was isolated from the rhizosphere soil of diseased C. chinensis plants. Its biocontrol efficacy against S. rolfsii LC1 was evaluated through a confrontation assay. The antimicrobial lipopeptides in the fermentation liquid of B. velezensis LT1 were identified using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS). The effects of B. velezensis LT1 on the mycelial morphology of S. rolfsii LC1 were examined using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Results: The confrontation assay indicated that B. velezensis LT1 significantly inhibited the growth of S. rolfsii LC1, with an inhibition efficiency of 78.41%. MALDI-TOF-MS analysis detected the presence of bacillomycin, surfactin, iturin, and fengycin in the fermentation liquid, all known for their antifungal properties. SEM and TEM observations revealed that the mycelial and cellular structures of S. rolfsii LC1 were markedly distorted when exposed to B. velezensis LT1. Discussion: The findings demonstrate that B. velezensis LT1 has considerable potential as a biocontrol agent against S. rolfsii LC1. The identified lipopeptides likely contribute to the antifungal activity, and the morphological damage to S. rolfsii LC1 suggests a mechanism of action. This study underscores the importance of exploring microbial biocontrol agents as a sustainable alternative to chemical fungicides in the management of plant diseases. Further research into the genetic and functional aspects of B. velezensis LT1 could provide deeper insights into its biocontrol mechanisms and facilitate its application in agriculture.

Characteristics of ZjCIPKs and ZjbHLH74-ZjCIPK5 regulated cold tolerance in jujube.

CIPKs are kind of serine/threonine (Ser/Thr) protein kinases which play important roles in response to biotic and abiotic stresses, and in plant growth and development. However, CIPKs in jujube (Ziziphus jujuba Mill.) had limited information, especially regarding their response to cold stress. In the current study, a total of 18 ZjCIPKs were identified in jujube genome which unevenly distributed on seven chromosomes. Conserved motif and gene structural analysis depicted them with conserved DEGLSA and APE motifs and similar structures. Phylogenetic analysis indicated that CIPKs were classified into five subgroups (I-V). In addition, three pairs of ZjCIPKs exhibited tandem duplication while the segmental duplication of ZjCIPKs was not identified. Study on the cis-acting elements indicted that stress or hormone related cis-acting elements were distributed unevenly on ZjCIPKs promoters and most ZjCIPKs were down- or up-regulated by the cold stress. VIGS induced silencing of ZjCIPK5 decreased the cold tolerance of sour jujube. Subcellular location analysis showed ZjCIPK5 located in nucleus. Moreover, transcription factor ZjbHLH74 which was induced at 6 h under cold stress could interact with the promoter of ZjCIPK5 to regulate jujube cold tolerance. These findings provided insights to a molecular basis of CIPK5 in jujube cold tolerance breeding for future.

De Novo RNA Sequencing and Transcriptome Analysis of Sclerotium rolfsii Gene Expression during Sclerotium Development.

Sclerotium rolfsii is a destructive soil-borne fungal pathogen that causes stem rot in cultivated plants. However, little is known about the genetic basis of sclerotium development. In this study, we conducted de novo sequencing of genes from three different stages of S. rolfsii (mycelia, early sclerotium formation, and late sclerotium formation) using Illumina HiSeqTM 4000. We then determined differentially expressed genes (DEGs) across the three stages and annotated gene functions. STEM and weighted gene-co-expression network analysis were used to cluster DEGs with similar expression patterns. Our analysis yielded an average of 25,957,621 clean reads per sample (22,913,500-28,988,848). We identified 8929, 8453, and 3744 DEGs between sclerotium developmental stages 1 versus 2, 1 versus 3, and 2 versus 3, respectively. Additionally, four significantly altered gene expression profiles involved 220 genes related to sclerotium formation, and two modules were positively correlated with early and late sclerotium formation. These results were supported by the outcomes of qPCR and RNA-sequencing conducted on six genes. This is the first study to provide a gene expression map during sclerotial development in S. rolfsii, which can be used to reduce the re-infection ability of this pathogen and provide new insights into the scientific prevention and control of the disease. This study also provides a useful resource for further research on the genomics of S. rolfsii.

The Impact of COVID-19 on Small Businesses in the US: A Longitudinal Study from a Regional Perspective.

Small businesses have suffered disproportionately from the COVID-19 pandemic. We use near-real-time weekly data from the Small Business Pulse Survey (April 26, 2020 - June 17, 2021) to examine the constantly changing impact of COVID-19 on small businesses across the United States. A set of multilevel models for change are adopted to model the trajectories of the various kinds of impact as perceived by business owners (subjective) and those recorded for business operations (objective), providing insights into regional resilience from a small business perspective. The findings reveal spatially uneven and varied trajectories in both the subjectively and the objectively assessed impact of COVID-19 across the U.S., and the different responses to the pandemic shock can be explained by evolving health situations and public policies, as well as by the economic structure and degree of socioeconomic vulnerability in different areas. This study contributes to scholarship on small businesses and regional resilience, as well as identifying policies and practices that build economic resilience and regional development under conditions of global pandemic disruption.

Asymmetric α-Allylation of Glycinate with Switched Chemoselectivity Enabled by Customized Bifunctional Pyridoxal Catalysts.

Owing to the strong nucleophilicity of the NH2 group, free-NH2 glycinates react with MBH acetates to usually deliver N-allylated products even in the absence of catalysts. Without protection of the NH2 group, chiral pyridoxal catalysts bearing an amide side chain at the C3 position of the naphthyl ring switched the chemoselectivity of the glycinates from intrinsic N-allylation to α-C allylation. The reaction formed chiral multisubstituted glutamic acid esters as SN 2'-SN 2' products in good yields with excellent stereoselectivity (up to 86 % yield, >20 : 1 dr, 97 % ee). As compared to pyridoxal catalysts bearing an amide side arm at the C2 position, the pyridoxals in this study have a bigger catalytic cavity to enable effective activation of larger electrophiles, such as MBH acetates and related intermediates. The reaction is proposed to proceed via a cooperative bifunctional catalysis pathway, which accounts for the high level of diastereo- and enantiocontrol of the pyridoxal catalysts.

LncRNA PITPNA-AS1 as a Potential Diagnostic Marker and Therapeutic Target Promotes Hepatocellular Carcinoma Progression via Modulating miR-448/ROCK1 Axis.

Background: Long non-coding RNAs are critical to hepatocellular carcinoma (HCC) developments. LncRNA PITPNA antisense RNA 1 (PITPNA-AS1) is a new regulator in several tumors. However, the mechanism by which PITPNA-AS1 mediates the tumorigenesis of HCC remains unclear. Methods: RT-qPCR was used to detect the level of PITPNA-AS1 in HCC specimens and cells. The biological functions of PITPNA-AS1 were explored by several functional experiments in vivo and in vitro. The binding relationship among PITPNA-AS1, miR-448 and ROCK1 were studied by Luciferase assay and pull-down assays. Results: We found that PITPNA-AS1 expressions were distinctly upregulated in both HCC specimens and cell lines. High PITPNA-AS1 levels were an unfavorable biomarker for patients with HCC. Functionally, knockdown of PITPNA-AS1 suppressed the proliferation, migration and invasion of HCC cells. Mechanistically, PITPNA-AS1 functioned as competing endogenous RNA to increase ROCK1 expressions via sponging miR-448. Conclusion: The newly identified PITPNA-AS/miR-448/ROCK1 axis promoted the oncogenicity of HCC cells. This novel axis is likely to be a promising HCC therapeutic aim.

Emodin Enhances the Chemosensitivity of Endometrial Cancer by Inhibiting ROS-Mediated Cisplatin-resistance.

BACKGROUND: Endometrial cancer is a common cause of death in gynecological malignancies. Cisplatin is a clinically chemotherapeutic agent. However, drug-resistance is the primary cause of treatment failure. OBJECTIVE: Emodin is commonly used clinically to increase the sensitivity of chemotherapeutic agents, yet whether Emodin promotes the role of Cisplatin in the treatment of endometrial cancer has not been studied. METHOD: CCK-8 kit was utilized to determine the growth of two endometrial cancer cell lines, Ishikawa and HEC-IB. The apoptosis level of Ishikawa and HEC-IB cells was detected by Annexin V / propidium iodide double-staining assay. ROS level was detected by DCFH-DA and NADPH oxidase expression. Expressions of drug-resistant genes were examined by real-time PCR and Western blotting. RESULTS: Emodin combined with Cisplatin reduced cell growth and increased the apoptosis of endometrial cancer cells. Co-treatment of Emodin and Cisplatin increased chemosensitivity by inhibiting the expression of drugresistant genes through reducing the ROS levels in endometrial cancer cells. In an endometrial cancer xenograft murine model, the tumor size was reduced and animal survival time was increased by co-treatment of Emodin and Cisplatin. CONCLUSION: This study demonstrates that Emodin enhances the chemosensitivity of Cisplatin on endometrial cancer by inhibiting ROS-mediated expression of drug-resistance genes.

Degradation of lignin in birch sawdust treated by a novel Myrothecium verrucaria coupled with ultrasound assistance.

Combined treatment of a novel fungal endophyte Myrothecium verrucaria coupled with ultrasound assistance was conducted to enhance lignin degradation in birch sawdust. The optimum treatment conditions were confirmed as the materials to liquid ratio 1:20, temperature 30°C, time 4days and pH 7, respectively. The results showed that the combined treatment led to the lignin degradation reaching 67.95±2.14%, while the lignin degradation were 45.50±2.12% and 13.75±0.66% with separate fungal treatment and ultrasound treatment, respectively. Moreover, SEM and FTIR analysis indicated that combined treatment significantly altered surface morphology and chemical structure of birch sawdust. The combined treatment greatly increased lignin removal during short time in mild environment. Therefore, these results demonstrated that the combined treatment of fungal endophyte coupled with ultrasound assistance has the high potential for the removal lignin in lignocellulose.

Production of Laccase by a New Myrothecium verrucaria MD-R-16 Isolated from Pigeon Pea [Cajanus cajan (L.) Millsp.] and its Application on Dye Decolorization.

The present study was conducted to screen a laccase-producing fungal endophyte, optimize fermentation conditions, and evaluate the decolorization ability of the laccase. A new fungal endophyte capable of laccase-producing was firstly isolated from pigeon pea and identified as Myrothecium verrucaria based on a ITS-rRNA sequences analysis. Meanwhile, various fermentation parameters on the laccase production were optimized via response surface methodology (RSM). The optimal fermentation conditions were a fermentation time of five days, temperature 30 °C and pH 6.22. Laccase activity reached 16.52 ± 0.18 U/mL under the above conditions. Furthermore, the laccase showed effective decolorization capability toward synthetic dyes (Congo red, Methyl orange, Methyl red, and Crystal violet) in the presence of the redox mediator ABTS, with more than 70% of dyes decolorizing after 24 h of incubation. Additionally, the activity of laccase was relatively stable with pH (4.5-6.5) and a temperature range of 35-55 °C. Therefore, the high laccase production of the strain and the new fungal laccase could provide a promising alterative approach for industrial and environmental applications.

The level of socioeconomic development of EU countries and the state of ISO 14001 certification.

This study examines the relationship between the level of socioeconomic development of the EU 28 countries and the adoption of International Organization for Standardization (ISO) 14001 environmental management system. First, through a multivariate comparative analysis of the secondary data obtained from the public statistics, a Hellwig's synthetic indicator is created to rank the level of socioeconomic development of the EU 28 countries. Then, using the total number of certificates issued in 2012 and the increase from 2011, this study has found a correlation between the level of national socioeconomic development and the adoption of ISO 14001 system in their businesses. Although there was no relationship between the number of ISO 14001 certificates in 2012 and the level of socioeconomic development at the national level, a weak negative correlation was observed between the increase of certification from 2011 to 2012 and the level of national socioeconomic development. The results suggest a higher interest in ISO 14001 adoption by the firms from the less developed countries than those from the more developed states.

Association of coronary artery calcium with bone mineral density in postmenopausal women.

OBJECTIVES: Atherosclerosis and osteoporosis (OP) are common diseases in elderly individuals and may share common pathogenetic mechanisms. The aim of this study was to investigate the association between bone mineral density (BMD) and coronary artery calcium (CAC) in postmenopausal women. METHODS: In this cross-sectional study, 186 postmenopausal women 50-80 years of age were included. BMD of the spine and femoral neck was measured by dual-energy X-ray absorptiometry. The coronary artery calcium score (CACS) was measured by multidetector computed tomography. RESULTS: The study included postmenopausal women aged 65.6±7.3 years, 109 of whom (58.6%) showed CAC. Thirty-three (17.7%) of the patients were found to have OP in the lumbar spine and 83 (44.6%) had osteopenia, whereas in the femoral neck, 26 patients (14.0%) had OP and 87 patients (46.8%) had osteopenia. The mean CACS was 57.6±108.3 in normal status, 89.7±143.5 in OP, and 156.4±256.9 in osteopenia at the spine (P<0.05). The mean CACS was 43.2±89.9 in normal status, 126.9±180.3 in OP, and 198.2±301.2 in osteopenia at the femoral neck (P<0.05). Multivariable logistic regression analysis showed that BMD was an independent marker for an increased risk of developing CAC in postmenopausal women. The multiple regression model showed that T-scores were the independent predictors of CACS. CONCLUSION: BMD identified on images from dual-energy X-ray absorptiometry were strongly related to multidetector computed tomography measures of CAC. This low-cost, minimal radiation technique used widely for OP screening is a promising marker of generalized coronary atherosclerosis.

Segregated and synchronized vector solutions to linearly coupled systems of Schrödinger equations.

In this paper, we study the following linearly coupled system [Formula: see text], where ε > 0 is a small parameter, Pi (x) are positive potentials, and λij = λji > 0 (i ≠ j) are coupling constants for i, j = 1, …, N. We investigate the effect of potentials to the structure of the solutions. More precisely, we construct multi-spikes solutions concentrating near the local maximum point [Formula: see text] of Pi (x). When [Formula: see text], [Formula: see text], the components have spikes clustering at the same point as ε → 0+. When [Formula: see text], the components have spikes clustering at the different points as ε → 0+.

Does dinitrogen hydrogenation follow different mechanisms for [(eta5-C5Me4H)2Zr]2(mu2,eta2,eta2-N2) and {[PhP(CH2SiMe2NSiMe2CH2)PPh]Zr}2(mu2,eta2,eta2-N2) complexes? A computational study.

The mechanisms of dinitrogen hydrogenation by two different complexes--[(eta(5)-C(5)Me(4)H)(2)Zr](2)(mu(2),eta(2),eta(2)-N(2)), synthesized by Chirik and co-workers [Nature 2004, 427, 527], and {[P(2)N(2)]Zr}(2)(mu(2),eta(2),eta(2)-N(2)), where P(2)N(2) = PhP(CH(2)SiMe(2)NSiMe(2)CH(2))(2)PPh, synthesized by Fryzuk and co-workers [Science 1997, 275, 1445]--are compared with density functional theory calculations. The former complex is experimentally known to be capable of adding more than one H(2) molecule to the side-on coordinated N(2) molecule, while the latter does not add more than one H(2). We have shown that the observed difference in the reactivity of these dizirconium complexes is caused by the fact that the former ligand environment is more rigid than the latter. As a result, the addition of the first H(2) molecule leads to two different products: a non-H-bridged intermediate for the Chirik-type complex and a H-bridged intermediate for the Fryzuk-type complex. The non-H-bridged intermediate requires a smaller energy barrier for the second H(2) addition than the H-bridged intermediate. We have also examined the effect of different numbers of methyl substituents in [(eta(5)-C(5)Me(n)H(5)(-)(n))(2)Zr](2)(mu(2),eta(2),eta(2)-N(2)) for n = 0, 4, and 5 (n = 5 is hypothetical) and [(eta(5)-C(5)H(2)-1,2,4-Me(3))(eta(5)-C(5)Me(5))(2)Zr](2)(mu(2),eta(2),eta(2)-N(2)) and have shown that all complexes of this type would follow a similar H(2) addition mechanism. We have also performed an extensive analysis on the factors (side-on coordination of N(2) to two Zr centers, availability of the frontier orbitals with appropriate symmetry, and inflexibility of the catalyst ligand environment) that are required for successful hydrogenation of the coordinated dinitrogen.

Performance Evaluation of the Three-Layer ONIOM Method: Case Study for a Zwitterionic Peptide.

The performance of the three-layer ONIOM method was systematically investigated by comparing the optimized geometries and calculated deprotonation energy of a zwitterionic peptide molecule, NH3(+)-CH(n)Bu-CO-NH-CH2-CO-NH-CH(n)Bu-COO(-), using all possible combinations of B3LYP/6-31G* as the high-level quantum (HQ), AM1 as the low-level quantum (LQ), and Amber as the MM method. Results show that the three-layer ONIOM(HQ:LQ:MM) method, which includes a medium-level quantum method in the middle system to take into account the electronic effects of the middle layer and to keep the problematic QM-MM boundary away from the action region, is more reliable and more stable than the QM:MM:MM or generic QM/MM method and is the best compromise between accuracy and computational cost.