Selective preparation of lithium carbonate from overhaul slag by high temperature sulfuric acid roasting - Water leaching.

An efficient recycling process is developed to recover valuable materials from overhaul slag and reduce its harm to the ecological environment. The high temperature sulfuric acid roasting - water leaching technology is innovatively proposed to prepare Li2CO3 from overhaul slag. Under roasting conditions, fluorine volatilizes into the flue gas with HF, lithium is transformed into NaLi(SO4), aluminum is firstly transformed into NaAl(SO4)2, and then decomposed into Al2O3, so as to selective extraction of lithium. Under the optimal roasting - leaching conditions, the leaching rate of lithium and aluminum are 95.6% and 0.9%, respectively. Then the processes of impurity removal, and settling lithium are carried out. The Li2CO3 with recovery rate of 72.6% and purity of 98.6% could be obtained under the best settling lithium conditions. Compared with the traditional process, this work has short flow, high controllability, remarkable technical, economic, and environmental benefits. This comprehensive recycling technology is suitable for overhaul slag, and has great practical application potential for the disposal of other hazardous wastes in electrolytic aluminum industry.

Emerging and Fastidious Uropathogens Were Detected by M-PCR with Similar Prevalence and Cell Density in Catheter and Midstream Voided Urine Indicating the Importance of These Microbes in Causing UTIs.

Introduction: This study compared microbial compositions of midstream and catheter urine specimens from patients with suspected complicated urinary tract infections to determine if emerging and fastidious uropathogens are infecting the bladder or are contaminants. Methods: Urine was collected by in-and-out catheter (n = 1000) or midstream voiding (n = 1000) from 2000 adult patients (≥60 years of age) at 17 DispatchHealth sites across 11 states. The two groups were matched by age (mean 81 years), sex (62.1% female, 37.9% male), and ICD-10-CM codes. Microbial detection was performed with multiplex polymerase chain reaction (M-PCR) with a threshold for "positive detection" ≥ 10,000 cells/mL for bacteria or any detection for yeast. Results were divided by sex. Results: In females, 28 of 30 microorganisms/groups were found by both collection methods, while in males 26 of 30 were found by both. There were significant overlaps in the detection and densities of classical uropathogens including Escherichia coli, Enterococcus faecalis, and Klebsiella pneumoniae, as well as emerging uropathogens including Actinotignum schaalii and Aerococcus urinae. In females, detection rates were slightly higher in midstream voided compared to catheter-collected (p = 0.0005) urine samples, while males showed the opposite trend (p < 0.0001). More polymicrobial infections were detected in midstream voided compared to catheter-collected samples (64.4% vs 45.7%, p < 0.0001) in females but the opposite in males (35.6% vs 47.0%, p = 0.002). Discussion: In-and-out catheter-collected and midstream voided urine specimens shared significant similarities in microbial detections by M-PCR, with some differences found for a small subset of organisms and between sexes. Conclusion: Non-invasive midstream voided collection of urine specimens for microbial detection and identification in cases of presumed UTI does not result in significantly more contamination compared to in-and-out catheter-collected specimens. Additionally, organisms long regarded as contaminants should be reconsidered as potential uropathogens.

Improving Patient Outcomes While Reducing Empirical Treatment with Multiplex-Polymerase-Chain-Reaction/Pooled-Antibiotic-Susceptibility-Testing Assay for Complicated and Recurrent Urinary Tract Infections.

This study compared rates of empirical-therapy use and negative patient outcomes between complicated and recurrent urinary tract infection (r/cUTI) cases diagnosed with a multiplex polymerase chain reaction or pooled antibiotic susceptibility testing (M-PCR/P-AST) vs. standard urine culture (SUC). Subjects were 577 symptomatic adults (n = 207 males and n = 370 females) presenting to urology/urogynecology clinics between 03/30/2022 and 05/24/2023. Treatment and outcomes were recorded by the clinician and patient surveys. The M-PCR/P-AST (n = 252) and SUC (n = 146) arms were compared after patient matching for confounding factors. The chi-square and Fisher's exact tests were used to analyze demographics and clinical outcomes between study arms. Reduced empirical-treatment use (28.7% vs. 66.7%), lower composite negative events (34.5% vs. 46.6%, p = 0.018), and fewer individual negative outcomes of UTI-related medical provider visits and UTI-related visits for hospitalization/an urgent care center/an emergency room (p < 0.05) were observed in the M-PCR/P-AST arm compared with the SUC arm. A reduction in UTI symptom recurrence in patients ≥ 60 years old was observed in the M-PCR/P-AST arm (p < 0.05). Study results indicate that use of the M-PCR/P-AST test reduces empirical antibiotic treatment and negative patient outcomes in r/cUTI cases.

A test combining multiplex-PCR with pooled antibiotic susceptibility testing has high correlation with expanded urine culture for detection of live bacteria in urine samples of suspected UTI patients.

We evaluated whether multiplex polymerase chain reaction (M-PCR) detects viable micro-organisms by comparing micro-organism identification with standard urine culture (SUC) and expanded quantitative urine culture (EQUC). Of the 395 organisms detected by M-PCR, EQUC detected 89.1% (p = 0.10), whereas SUC detected 27.3% (p < 0.0001 vs. M-PCR and p < 0.0001 vs EQUC). M-PCR identified 260 nonfastidious bacteria, EQUC detected 96.5% (p = 0.68), whereas SUC detected 41.5% (p < 0.0001). Common nonfastidious bacteria missed by SUC included Escherichia coli (72.5% detected), Klebsiella pneumoniae (66.7% detected), Enterococcus faecalis (34.6% detected) and Enterococcus faecium (0% detected). M-PCR identified 135 fastidious bacteria and EQUC 101 (74.8%, p = 0.01), whereas SUC failed to detect any (0%, p < 0.0001). Clinical samples evaluated using EQUC and M-PCR yielded very similar findings, indicating that most microbes identified by M-PCR represented viable organisms, and validating M-PCR as a diagnostic tool for UTIs.

Quantitative multiplex polymerase chain reaction in copies ml-1 linearly correlates with standard urine culture in colonies ml-1 for urinary tract infection (UTI) pathogens.

Standard urine culture (SUC) is the current standard method for confirmation of a urinary tract infection (UTI). SUC identifies microorganisms in urine samples and semi-quantifies these as colony-forming units (CFUs) ml-1. In contrast, quantitative multiplex polymerase chain reaction (q-MPCR) is a culture-independent assay in which the microbes are quantified by targeting genomic sequences and reported as cells ml-1, calculated from copies ml-1. Using serial dilutions within the 104-105 cells ml-1 range, the usual reporting range of SUC, this study compared the quantification results based on SUC and q-MPCR for four uropathogens with the control hemocytometer counts. The results revealed a linear relationship and a 1:1 correlation between the q-MPCR and SUC results. Additional q-MPCR quantification of 36 uropathogenic non-fastidious and fastidious bacteria and yeast indicated a reproducible linear correlation in a 1:1 manner with the control counts over a range of cell densities (103-106 cells ml-1). The results confirm that the quantifications by q-MPCR in cells ml-1 and by SUC in CFUs ml-1 are comparable and answer to the lingering question of how the results of these two methods correlate. Moreover, q-MPCR provided accurate quantification of various microorganisms over wider cell density ranges without the time required for microbial growth.

A Diagnostic Test Combining Molecular Testing with Phenotypic Pooled Antibiotic Susceptibility Improved the Clinical Outcomes of Patients with Non-E. coli or Polymicrobial Complicated Urinary Tract Infections.

Purpose: Complicated UTIs (cUTIs) cause significant morbidity and healthcare resource utilization and cost. Standard urine culture has limitations in detecting polymicrobial and non-E. coli infections, resulting in the under-diagnosis and under-treatment of cUTIs. In this study, patient-reported outcomes were compared between treated and untreated patients when an advanced diagnostic test combining multiplex-polymerase chain reaction (M-PCR) with a pooled antibiotic susceptibility method (P-AST) was incorporated into the patients' clinical management. Methods: Patients who had symptoms typical of cUTI and positive M-PCR/P-AST test results were recruited from urology clinics. Symptom reduction and clinical cure rates were measured from day 0 through day 14 using the American English Acute Cystitis Symptom Score (ACSS) Questionnaire. Clinical cure was defined based on the sum of the scores of four US Food and Drug Administration (FDA) symptoms and the absence of visible blood in the urine. Results: Of 264 patients with suspected cUTI, 146 (55.4%) had exclusively non-E. coli infections (115 treated and 31 untreated) and 190 (72%) had polymicrobial infections (162 treated and 28 untreated). Treated patients exhibited greater symptom reduction compared to untreated ones on day 14 for those with exclusively non-E. coli organisms (3.18 vs 1.64, p = 0.006) and polymicrobial infections (3.52 vs 1.41, p = 0.002), respectively. A higher percentage of treated patients than of untreated patients achieved clinical cure for polymicrobial infections on day 14 (58.7% vs 36.4%, p = 0.049). Conclusion: Patients with cUTIs treated based on the M-PCR/P-AST diagnostic test had significantly improved symptom reduction and clinical cure rates compared to untreated patients among those with non-E. coli or polymicrobial infections.

Multiplex Polymerase Chain Reaction/Pooled Antibiotic Susceptibility Testing Was Not Associated with Increased Antibiotic Resistance in Management of Complicated Urinary Tract Infections.

Objective: To compare antibiotic resistance results at different time points in patients with urinary tract infections (UTIs), who were either treated based upon a combined multiplex polymerase chain reaction (M-PCR) and pooled antibiotic susceptibility test (P-AST) or were not treated. Methods: The M-PCR/P-AST test utilized here detects 30 UTI pathogens or group of pathogens, 32 antibiotic resistance (ABR) genes, and phenotypic susceptibility to 19 antibiotics. We compared the presence or absence of ABR genes and the number of resistant antibiotics, at baseline (Day 0) and 5-28 days (Day 5-28) after clinical management in the antibiotic-treated (n = 52) and untreated groups (n = 12). Results: Our results demonstrated that higher percentage of patients had a reduction in ABR gene detection in the treated compared to the untreated group (38.5% reduction vs 0%, p = 0.01). Similarly, significantly more patients had reduced numbers of resistant antibiotics, as measured by the phenotypic P-AST component of the test, in the treated than in the untreated group (42.3% reduction vs 8.3%, p = 0.04). Conclusion: Our results with both resistance gene and phenotypic antibiotic susceptibility results demonstrated that treatment based upon rapid and sensitive M-PCR/P-AST resulted in reduction rather than induction of antibiotic resistance in symptomatic patients with suspected complicated UTI (cUTI) in an urology setting, indicating this type of test is valuable in the management of these types of patients. Further studies of the causes of gene reduction, including elimination of ABR gene-carrying bacteria and loss of ABR gene(s), are warranted.

The Diagnosis of Urinary Tract Infections Using a Novel At-home Testing Protocol to Enhance Telemedicine: A Retrospective Analysis.

OBJECTIVE: To retrospectively analyze a novel courier-based home urine collection strategy for patients with symptoms of urinary tract infections (UTIs). This model was developed to provide patient care using telehealth during the coronavirus 2019 pandemic. METHODS: We analyzed data from 2206 patients with symptomatic UTIs to investigate the efficacy of a home urine collection protocol. The primary outcome was the impact of home versus office collection. RESULTS: We analyzed the results of 1112 patient samples collected in-office and 1084 patient samples collected at home. There was no difference in the rate of bacterial identification between females in the office and home collection groups. However, males in the office collection group had a higher rate of bacterial identification (p = .002). The turnaround time was significantly faster in the home collection group than the office collection group (4.08 hours shorter, p < 0.0014). Antibiotic use prior to sample collection was significantly higher in the home collection group for both males (p = .0004) and females (p = .004). Changes in antibiotics were significantly higher in the home collection group than in the office collection group for both males (p = .0009) and females (p = .0006). CONCLUSION: Our home collection protocol is a viable method to provide prompt and reliable outpatient care to urology patients suffering from UTIs. Furthermore, this approach resulted in adequate management and quicker turnaround times. Our findings demonstrate the clinical viability of a decentralized healthcare model to treat UTIs.

Concordance Between Antibiotic Resistance Genes and Susceptibility in Symptomatic Urinary Tract Infections.

PURPOSE: Studies have shown that multiple genes influence antibiotic susceptibility, but the relationship between genotypic and phenotypic antibiotic susceptibility is unclear. We sought to analyze the concordance between the presence of antibiotic resistance (ABR) genes and antibiotic susceptibility results in urine samples collected from patients with symptomatic urinary tract infection (UTI). PATIENTS AND METHODS: Urine samples were collected from patients presenting to 37 geographically disparate urology clinics across the United States from July 2018 to February 2019. Multiplex polymerase chain reaction was used to detect 27 ABR genes. In samples containing at least one culturable organism at a concentration of ≥ 104 cells per mL, pooled antibiotic susceptibility testing (P-AST), which involves simultaneous growing all detected bacteria together in the presence of antibiotic and then measure susceptibility, was performed against 14 antibiotics. The concordance rate between the ABR genes and the P-AST results was generated for the overall group. The concordance rates for each antibiotic between monomicrobial and polymicrobial infection were compared using chi-square test. RESULTS: Results from ABR gene detection and P-AST of urine samples from 1155 patients were included in the concordance analysis. Overall, there was a 60% concordance between the presence or absence of ABR genes and corresponding antimicrobial susceptibility with a range of 49-78% across antibiotic classes. Vancomycin, meropenem, and piperacillin/tazobactam showed significantly lower concordance rates in polymicrobial infections than in monomicrobial infections. CONCLUSION: Given the 40% discordance rate, the detection of ABR genes alone may not provide reliable data to make informed clinical decisions in UTI management. However, when used in conjunction with susceptibility testing, ABR gene data can offer valuable clinical information for antibiotic stewardship.

Analytical Performance of a 15-Gene Prognostic Assay for Early-Stage Non-Small-Cell Lung Carcinoma Using RNA-Stabilized Tissue.

A 15-gene prognostic signature for early-stage, completely resected, non-small-cell lung carcinoma, (which distinguishes between patients with good and poor prognoses) was clinically validated in prior studies. To achieve operational efficiencies, this study was designed to evaluate the assay's performance in RNA-stabilized tissue as an alternative to the fresh-frozen tissue format originally used to develop the assay. The percent concordance between matched tissue formats was 84% (95% Wilson CI, 70%-92%), a level of agreement comparable to the inherent reproducibility of the assay observed within biological replicates of fresh-frozen tissue. Furthermore, the analytical performance of the assay using the RNA-stabilized tissue format was evaluated. When compared to an accredited reference laboratory, the clinical laboratory achieved a concordance of 94% (95% Wilson CI, 81%-98%), and there was no evidence of bias between the laboratories. The lower limit of quantitation for the target RNA concentration was confirmed to be, at most, 12.5 ng/µL. The assay reportable range defined in terms of risk score units was determined to be -4.295 to 4.210. In a large-scale precision study, the assay showed high reproducibility and repeatability. When subjected to a maximal amount of genomic DNA, a potential contaminant, the assay still produced the expected results. The 15-gene signature was confirmed to produce reliable results and, thus, is suitable for its intended use.

In vitro chemoresponse in metachronous pairs of ovarian cancers.

BACKGROUND/AIM: An in vitro chemoresponse assay may aid effective therapy selection in epithelial ovarian cancer (EOC). This study explores changes in chemoresponse between paired primary and recurrent EOC tumors. PATIENTS AND METHODS: RESULTS from metachronous tumors were examined in 242 patients. Changes in in vitro chemoresponse, measured by the area under the dose response curve (AUC) between paired tumors were assessed. RESULTS: A significant increase in AUC was identified in most first-line therapies over time. No significant difference was observed in most recurrent therapies. When the elapsed time between occurrences was <17 months, no difference was observed for any recurrent therapies, and half of first-line therapies exhibited significant increases in AUC. When ≥17 months, all 7 therapies showed significant increases. CONCLUSION: These results suggest an increase in chemoresistance over time, which is more pronounced for first-line therapies. This is consistent with clinical observations and suggests the biologic concordance between assay results and response to chemotherapy.

Analytical performance of a formalin-fixed paraffin-embedded tissue-based 634-probe prognostic assay for predicting outcome of patients with stage II colon cancer.

A formalin-fixed paraffin-embedded tissue-based prognostic assay to assess the risk for recurrence in stage II colon cancer has recently been clinically validated. This study describes the analytical performance and quality control measures of the assay. The reportable range was determined to be [-1.129, 1.414] in risk score units. The accuracy was evaluated with a split sample comparison within the production lab and between the production lab and a reference lab. The concordance between the replicates within the production lab was 79% (95% confidence interval, 64%-91%). There was no evidence of bias, and the concordance was 78% (95% confidence interval, 61%-90%) between the labs. The lab-to-lab concordance was further evaluated by simulating risk scores from the full reportable range. The simulation suggested a higher concordance. The sensitivity study demonstrated that the percentage of tumor tissue did not impact the risk score and that RNA concentration of 9.5 ng/µL was a conservative determination of the analyte lower limit of quantification. From the precision study, the repeatability and reproducibility estimates were 0.1267 and 0.0548 in risk score units, respectively. Furthermore, multifaceted quality control measures were implemented, such as proper tissue processing steps, high-risk and low-risk controls, nontemplate control, and a gene expression-based classifier to evaluate the cDNA amplification kit, a key reagent in the assay. In conclusion, this study demonstrates the strong analytical performance of the assay and further supports its use as an objective standardized prognostic test for stage II colon cancer.

[Basic biological characteristics of mesenchymal stem cells derived from bone marrow and human umbilical cord].

Bone marrow (BM) and umbilical cord (UC) are the major sources of mesenchymal stem cells for therapeutics. This study was aimed to compare the basic biologic characteristics of bone marrow-derived and umbilical cord derived-mesenchymal stem cells (BM-MSC and UC-MSC) and their immunosuppressive capability in vitro. The BM-MSC and UC-MSC were cultured and amplified under same culture condition. The growth kinetics, phenotypic characteristics and immunosuppressive effects of UC-MSC were compared with those of BM-MSC.Gene chip was used to compare the genes differentially expressed between UC-MSC and BM-MSC. The results showed that UC-MSC shared most of the characteristics of BM-MSC, including morphology and immunophenotype. UC-MSC could be ready expanded for 30 passages without visible changes. However, BM-MSC grew slowly, and the mean doubling time increased notably after passage 6. Both UC-MSC and BM-MSC could inhibit phytohemagglutinin-stimulated peripheral blood mononuclear cell proliferation, in which BM-MSC mediated more inhibitory effect. Compared with UC-MSC, BM-MSC expressed more genes associated with immune response. Meanwhile, the categories of up-regulated genes in UC-MSC were concentrated in organ development and growth. It is concluded that the higher proliferation capacity, low human leukocyte antigen-ABC expression and immunosuppression make UC-MSC an excellent alternative to BM-MSC for cell therapy. The differences between BM-MSC and UC-MSC gene expressions can be explained by their ontogeny and different microenvironment in origin tissue. These differences can affect their efficacy in different therapeutic applications.

[Immunomodulatory ability of senile mesenchymal stem cells].

This study was aimed to investigate the immunomodulatory ability of human umbilical cord mesenchymal stem cells (UB-MSC) along with prolonging of culture time and increasing of passages in vitro. Mesenchymal stem cells (MSC) were isolated from human umbilical cord and cultured in vitro. The morphological changes and nucleocytoplasmic ratio of MSC were observed using Giemsa staining. MSC of the 5th passage were selected as control group, and MSC of the 13th passage were taken as senile group. The degree of cell senescence was detected by aging cells in situ test kit. Cell Counting Kit-WST-8 was used to determine the proliferation of lymphocytes in mixed lymphocytes coculture system with different passages of MSC. The expression of immunomodulation-related genes was detected by RT-PCR. The results showed that the length-breadth ratio of MSC increased and nucleocytoplasmic ratio decreased along with the increasing of passages. The senium degree of cells of the 13th passage was higher than that of the 5th passage cells. The capacity of suppressing lymphocyte proliferation of the 13th passage MSC was enhanced, compared with the 5th passage. Moreover, the expression of immunosuppression-related genes of senile MSC increased and the expression of most anti-inflammation associated genes declined as compared with young MSC by RT-PCR. It is concluded that the degree of MSC senescence gradually develops with increasing of culture passage, but the immunosuppressive ability of MSC strengthens with continuous culture.

Adaptation of a chemosensitivity assay to accurately assess pemetrexed in ex vivo cultures of lung cancer.

PURPOSE: Pemetrexed is the only FDA approved treatment for mesothelioma and is a second line agent for treatment of non-small cell lung carcinoma (NSCLC). Pemetrexed is inhibited by folate and its analogs, which are components of many culture media, making it challenging to study pemetrexed in vitro. In order to accurately evaluate pemetrexed's effects in vitro, the protocol for a standard chemosensitivity assay, the ChemoFx drug response marker, had to be modified. EXPERIMENTAL DESIGN: Novel rinse and media change steps were assessed and then added to the assay protocol in order to observe pemetrexed activity. The intraday and interday stability of pemetrexed were also established under the adapted protocol. Then, the modified protocol was used to examine pemetrexed in 65 ex vivo lung cancer specimens. RESULTS: Substituting 5% RPMI + EGF for BEGM allowed pemetrexed to exert its anticancer activity in the ChemoFx DRM. ChemoFx classified 6.2% of the lung specimens as responsive, 9.2% as intermediate responsive and 84.6% as non-responsive to pemetrexed. CONCLUSIONS: Adapting the ChemoFx protocol allowed for the accurate evaluation of pemetrexed anticancer activity in ex vivo lung specimens. ChemoFx evaluation may provide an indication of a patient's clinical response to the drug prior to pemetrexed treatment. Having this information when treatment options are being considered could avoid wasted time, unnecessary costs and needless side effects that are the result of an inappropriate chemotherapy regimen.

The Birt-Hogg-Dubé tumor suppressor Folliculin negatively regulates ribosomal RNA synthesis.

Birt-Hogg-Dubé syndrome (BHD) is a human cancer disorder caused by mutations in the tumor suppressor gene Folliculin (FLCN) with unknown biological functions. Here, we show that the Drosophila homolog of FLCN, dFLCN (a.k.a. dBHD) localizes to the nucleolus and physically interacts with the 19S proteasomal ATPase, Rpt4, a nucleolar resident and known regulator of rRNA transcription. Downregulation of dFLCN resulted in an increase in nucleolar volume and upregulation of rRNA synthesis, whereas dFLCN overexpression reduced rRNA transcription and counteracted the effects of Rpt4 on rRNA production by preventing the association of Rpt4 with the rDNA locus. We further show that human FLCN exhibited evolutionarily conserved function and that Rpt4 knockdown inhibits the growth of FLCN-deficient human renal cancer cells in mouse xenografts. Our study suggests that FLCN functions as a tumor suppressor by negatively regulating rRNA synthesis.

Distinct genes related to drug response identified in ER positive and ER negative breast cancer cell lines.

Breast cancer patients have different responses to chemotherapeutic treatments. Genes associated with drug response can provide insight to understand the mechanisms of drug resistance, identify promising therapeutic opportunities, and facilitate personalized treatment. Estrogen receptor (ER) positive and ER negative breast cancer have distinct clinical behavior and molecular properties. However, to date, few studies have rigorously assessed drug response genes in them. In this study, our goal was to systematically identify genes associated with multidrug response in ER positive and ER negative breast cancer cell lines. We tested 27 human breast cell lines for response to seven chemotherapeutic agents (cyclophosphamide, docetaxel, doxorubicin, epirubicin, fluorouracil, gemcitabine, and paclitaxel). We integrated publicly available gene expression profiles of these cell lines with their in vitro drug response patterns, then applied meta-analysis to identify genes related to multidrug response in ER positive and ER negative cells separately. One hundred eighty-eight genes were identified as related to multidrug response in ER positive and 32 genes in ER negative breast cell lines. Of these, only three genes (DBI, TOP2A, and PMVK) were common to both cell types. TOP2A was positively associated with drug response, and DBI was negatively associated with drug response. Interestingly, PMVK was positively associated with drug response in ER positive cells and negatively in ER negative cells. Functional analysis showed that while cell cycle affects drug response in both ER positive and negative cells, most biological processes that are involved in drug response are distinct. A number of signaling pathways that are uniquely enriched in ER positive cells have complex cross talk with ER signaling, while in ER negative cells, enriched pathways are related to metabolic functions. Taken together, our analysis indicates that distinct mechanisms are involved in multidrug response in ER positive and ER negative breast cells.

[Influence of vascular endothelial growth factor on endothelial components in human bone marrow and umbilical cord mesenchymal stem cells].

This study was aimed to compare the proportion of endothelial cells (EC) in human bone marrow mesenchymal stem cell (BM-MSC) and human umbilical cord mesenchymal stem cells (UC-MSC), and to investigate the influence of vascular endothelial growth factor (VEGF) on proportion of EC in MSC. Flow cytometry was used to detect the proportion of CD34(+)CD133(+) and vWF(+)CD31(+) double positive cells in MSC. Wright's staining was employed to observe the influence of VEGF on morphology of MSC. The expressions of CD34, CD133, CD31, vWF were detected by immunofluorescence. qRT-PCR was performed to detect the influence of VEGF on EC marker genes' expression of MSC. The results showed that there were a small amount of EC and endothelial progenitor cells (EPC) in obtained BM-MSC and UC-MSC. After exposed to VEGF 10 ng/ml for 24 h, aspect ratio of MSC and the proportion of EC increased, while proportion of EPC decreased. Expression of EC related marker genes such as Tie-2 and ecNOS up-regulated, especially in UC-MSC. It is concluded that small amount of EC and EPC exists in cultured BM-MSC and UC-MSC, VEGF can enhance the proportion and function of EC in MSC.

[Changes of biological characteristics and gene expression profile of umbilical cord mesenchymal stem cells during senescence in culture].

This study was purposed to investigate the changes of biological properties and expression patterns of the aging related genes in umbilical cord mesenchymal stem cells (UC-MSC) during in vitro culture. UC-MSC at passage 3 were served as the control cells and those at passage 15 were considered as the aged cells. The biological features of those two kinds of cells including morphology, proliferation activity and phenotypic profile were observed, and the differences of gene expression were analysed by the whole human genome oligo microarray. Several differential genes were selected for further confirmation by quantitative reverse transcription-polymerase chain reaction. The results showed that UC-MSC at passage 15 were larger in size and their proliferation rate was slower compared with those of cells at passage 3, while the positivity of CD44 and CD105 remained unchanged. Compared with UC-MSC at passage 3, relatively aged cells expressed higher levels of genes that are associated with small subunit of ribosome. Further analysis with Gene Ontology functional categories showed that the up-regulated genes were concentrated in those related to steroid biosynthesis, galactose metabolism and the development of autoimmune diseases and degenerative diseases and the down-regulated genes in UC-MSC at passage 15 were concentrated in cytoskeleton molecules, DNA structure binding, mRNA binding and protein function. Functional analysis with Kyoto Encyclopedia of Genes and Genomes functional pathway revealed that the expression of some genes responsible for ribosome composition was elevated while those of associated with extracellular matrix, focal adhesion and cell cycle progression were down-regulated. It is concluded that UC-MSC become senescent due to the declines in metabolism and proliferation activities.

Umbilical cord-derived mesenchymal stem cells retain immunomodulatory and anti-oxidative activities after neural induction.

The immunomodulatory and anti-oxidative activities of differentiated mesenchymal stem cells contribute to their therapeutic efficacy in cell-replacement therapy. Mesenchymal stem cells were isolated from human umbilical cord and induced to differentiate with basic fibroblast growth factor, nerve growth factor, epidermal growth factor, brain-derived neurotrophic factor and forskolin. The mesenchymal stem cells became rounded with long processes and expressed the neural markers, Tuj1, neurofilament 200, microtubule-associated protein-2 and neuron-specific enolase. Nestin expression was significantly reduced after neural induction. The expression of immunoregulatory and anti-oxidative genes was largely unchanged prior to and after neural induction in mesenchymal stem cells. There was no significant difference in the effects of control and induced mesenchymal stem cells on lymphocyte proliferation in co-culture experiments. However, the expression of human leukocyte antigen-G decreased significantly in induced neuron-like cells. These results suggest that growth factor-based methods enable the differentiation of mesenchymal stem cell toward immature neuronal-like cells, which retain their immunomodulatory and anti-oxidative activities.