The membrane-cytoplasmic linker defines activity of FtsH proteases in Pseudomonas aeruginosa clone C.

Pandemic Pseudomonas aeruginosa clone C strains encode two inner-membrane associated ATP-dependent FtsH proteases. PaftsH1 is located on the core genome and supports cell growth and intrinsic antibiotic resistance, whereas PaftsH2, a xenolog acquired through horizontal gene transfer from a distantly related species, is unable to functionally replace PaftsH1. We show that purified PaFtsH2 degrades fewer substrates than PaFtsH1. Replacing the 31-amino acid-extended linker region of PaFtsH2 spanning from the C-terminal end of the transmembrane helix-2 to the first seven highly divergent residues of the cytosolic AAA+ ATPase module with the corresponding region of PaFtsH1 improves hybrid-enzyme substrate processing in vitro and enables PaFtsH2 to substitute for PaFtsH1 in vivo. Electron microscopy indicates that the identity of this linker sequence influences FtsH flexibility. We find membrane-cytoplasmic (MC) linker regions of PaFtsH1 characteristically glycine-rich compared to those from FtsH2. Consequently, introducing three glycines into the membrane-proximal end of PaFtsH2's MC linker is sufficient to elevate its activity in vitro and in vivo. Our findings establish that the efficiency of substrate processing by the two PaFtsH isoforms depends on MC linker identity and suggest that greater linker flexibility and/or length allows FtsH to degrade a wider spectrum of substrates. As PaFtsH2 homologs occur across bacterial phyla, we hypothesize that FtsH2 is a latent enzyme but may recognize specific substrates or is activated in specific contexts or biological niches. The identity of such linkers might thus play a more determinative role in the functionality of and physiological impact by FtsH proteases than previously thought.

Application of the PGT-M strategy using single sperm and/or affected embryos as probands for linkage analysis in males with hereditary tumor syndromes without family history.

Hereditary tumor syndromes have garnered substantial attention due to their adverse effects on both the physical and psychological health of patients, as well as the elevated risk of transmission to subsequent generations. This has prompted a growing interest in exploring preimplantation genetic testing (PGT) as a treatment option to mitigate and eliminate these impacts. Several studies have demonstrated that de novo variants have become a great cause of many hereditary tumor syndromes, which introduce certain difficulties to PGT. In the absence of adequate genetic linkage information (parents and offspring), haplotype construction seems unrealizable. In the study, researchers used single sperm or affected embryos as proband to perform single-nucleotide polymorphism linkage analysis for cases with de novo variants. For complicated variants, the strategy that sperm combined with embryo detection will increase accuracy while avoiding the limitations and potential failures of using a single detection material. The study recruited 11 couples with male de novo carriers, including 3 tumor types and 4 genes. To date, 4 couples have been clinically confirmed as pregnant and three healthy babies have been born. The results of amniocentesis or umbilical cord blood verification were consistent with the results of PGT-M. The study aims to introduce the application of the PGT-M strategy in hereditary tumor syndromes.

Association between ultra-processed foods and risk of cancer: a systematic review and meta-analysis.

Background: Despite increasing evidence that has shown the association of ultra-processed foods (UPFs) with cancer risk, the results remain inconclusive. We, therefore, conducted the meta-analysis to clarify the association by including recently published studies. Methods: A comprehensive search was conducted in PubMed, Embase, and Web of Science to identify all relevant studies from inception to January 2023. To pool data, fixed-effects or random-effects models were used where appropriate. Subgroup analyses, sensitivity analyses, and publication bias tests were performed. Results: A total of 13 studies (4 cohort studies and 9 case-control studies) were included in the analysis, with a total of 625,738 participants. The highest UPFs consumption was associated with increased risk of colorectal cancer (OR = 1.23, 95% CI: 1.10-1.38), colon cancer (OR = 1.25, 95% CI: 1.14-1.36), and breast cancer (OR = 1.10, 95% CI: 1.00-1.20) but not rectal cancer (OR = 1.18, 95% CI: 0.97-1.43) and prostate cancer (OR = 1.03, 95% CI: 0.93-1.12). In addition, the subgroup analyses showed that a positive association between UPFs consumption and colorectal cancer was observed among men (OR = 1.31, 95% CI: 1.15-1.50), whereas no significant association was observed among women (OR = 1.10, 95% CI: 0.94-1.29). Conclusion: The present meta-analysis suggests that high UPFs consumption is associated with a significantly increased risk of certain site-specific cancers, especially the digestive tract and some hormone-related cancers. However, further rigorously designed prospective and experimental studies are needed to better understand causal pathways.

Kinetically and thermodynamically controlled one-pot growth of gold nanoshells with NIR-II absorption for multimodal imaging-guided photothermal therapy.

Since the successful clinical trial of AuroShell for photothermal therapy, there is currently intense interest in developing gold-based core-shell structures with near-infrared (NIR) absorption ranging from NIR-I (650-900 nm) to NIR-II (900-1700 nm). Here, we propose a seed-mediated successive growth approach to produce gold nanoshells on the surface of the nanoscale metal-organic framework (NMOF) of UiO-66-NH2 (UiO = the University of Oslo) in one pot. The key to this strategy is to modulate the proportion of the formaldehyde (reductant) and its regulator / oxidative product of formic acid to harness the particle nucleation and growth rate within the same system. The gold nanoshells propagate through a well-oriented and controllable diffusion growth pattern (points → facets → octahedron), which has not been identified. Most strikingly, the gold nanoshells prepared hereby exhibit an exceedingly broad and strong absorption in NIR-II with a peak beyond 1300 nm and outstanding photothermal conversion efficiency of 74.0%. Owing to such superior performance, these gold nanoshells show promising outcomes in photoacoustic (PA), computed tomography (CT), and photothermal imaging-guided photothermal therapy (PTT) for breast cancer, as demonstrated both in vitro and in vivo.

Fully Reversible Ratiometric Nanosensors for Continuously Quantifying Mitochondrial Glutathione Concentration in Living Cells.

Mitochondrial glutathione (mGSH) is both the cause of the oxidative damage and a mechanism for maintaining the redox homeostasis in mitochondria. To effectively measure mGSH dynamics in living cells, we have developed a new FRET-based nanosensor by immobilizing rhodamine B into dendritic mesoporous silica nanoparticles and installing GSH probes and mitochondria-targeting motifs onto the surface of nanoparticles. The result shows that these nanosensors show efficient FRET and a full reversibility and rapid response (<10 s) to GSH in the range of 0.5-20 mM, due to their unique nanostructure and well-overlapped spectra. The excellent photostability and low cytotoxicity make them an effective means for monitoring mGSH concentration in real time. When the mGSH nanosensors are used for quantitatively measuring mGSH variations under glucose deprivation stimulation in HeLa cells, they successfully prove themselves a useful tool for mitochondrial redox activity studies.

Downregulation of the enhancer of zeste homolog 1 transcriptional factor predicts poor prognosis of triple-negative breast cancer patients.

Background: Triple-negative breast cancer (TNBC) is the most malignant subtype of breast cancer and lacks effective biomarkers. This study seeks to unravel the expression status and the prospective transcriptional mechanisms of EZH1/EZH2 in TNBC tissue samples. Moreover, another objective of this study is to reveal the prognostic molecular signatures for risk stratification in TNBC patients. Methods: To determine the expression status of EZH1/EZH2 in TNBC tissue samples, microarray analysis and immunohistochemistry were performed on in house breast cancer tissue samples. External mRNA expression matrices were used to verify its expression patterns. Furthermore, the prospective transcriptional mechanisms of EZH1/EZH2 in TNBC were explored by performing differential expression analysis, co-expression analysis, and chromatin immunoprecipitation sequencing analysis. Kaplan-Meier survival analysis and univariate Cox regression analysis were utilized to detect the prognostic molecular signatures in TNBC patients. Nomogram and time-dependent receiver operating characteristic curves were plotted to predict the risk stratification ability of the prognostic-signatures-based Cox model. Results: In-house TMAs (66 TNBC vs. 106 non-TNBC) and external gene microarrays, as well as RNA-seq datasets (1,135 TNBC vs. 6,198 non-TNBC) results, confirmed the downregulation of EZH1 at both the protein and mRNA levels (SMD = -0.59 [-0.80, -0.37]), as is opposite to that of EZH2 (SMD = 0.74 [0.40, 1.08]). The upregulated transcriptional target genes of EZH1 were significantly aggregated in the cell cycle pathway, where CCNA2, CCNB1, MAD2L1, and PKMYT1 were determined as key transcriptional targets. Additionally, the downregulated transcriptional targets of EZH2 were enriched in response to the hormone, where ESR1 was identified as the hub gene. The six-signature-based prognostic model produced an impressive performance in this study, with a training AUC of 0.753, 0.981, and 0.977 at 3-, 5-, and 10-year survival probability, respectively. Conclusion: EZH1 downregulation may be a key modulator in the progression of TNBC through negative transcriptional regulation by targeting CCNA2, CCNB1, MAD2L1, and PKMYT1.

Optimal Value for Serum Anti-PLA2R Antibody in Primary Membranous Nephropathy: A Multicenter Observational Study.

INTRODUCTION: Anti-phospholipase A2 receptor antibody (PLA2R-Ab) is highly specific for primary membranous nephropathy (PMN). Here, we compare the diagnostic value of different circulating PLA2R-Ab cutoff titers in multicenter cohorts, with particular focus on determining the optimal cutoff value for Chinese patients. METHODS: In total, 288 patients with PMN and 301 with other nephropathies were recruited retrospectively from five hospitals in China between September 2011 and October 2018. PLA2R-Ab in serum obtained at renal biopsy was determined by enzyme-linked immunosorbent assay. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and receiver operating characteristic (ROC) curve of PLA2R-Ab in diagnosing PMN were assessed. Diagnostic efficiency was evaluated by internal validation. RESULTS: The sensitivity, specificity, PPV, NPV, and Youden index for PMN diagnosis were 71%, 90%, 88%, 75%, and 0.61 at the cutoff of 3.8 RU/mL; 74%, 86%, 84%, 76%, and 0.60 at 2.7 RU/mL; 68%, 92%, 90%, 73%, and 0.60 at 5.2 RU/mL; 64%, 95%, 93%, 72%, and 0.59 at 9.0 RU/mL; 57%, 96%, 94%, 68%, and 0.54 at 14.0 RU/mL; 51%, 97%, 95%, 66%, and 0.49 at 20.0 RU/mL; 47%, 98%, 96%, 64%, and 0.45 at 40.0 RU/mL, respectively. The area under the ROC curve was 0.83. CONCLUSION: By comprehensively considering specificity and sensitivity, we show that 3.8 RU/mL is the optimal cutoff of PLA2R-Ab in Chinese PMN patients, with a sensitivity of 71% and a specificity of 90%. The cutoff values were 5.2 RU/mL and 9.0 RU/mL when the diagnostic specificity was increased to 92% and 95%, respectively.

Immunological Microenvironment Alterations in Follicles of Patients With Autoimmune Thyroiditis.

Autoimmune thyroiditis (AIT) is the most prevalent autoimmune endocrine disease, with a higher incidence in women than in men. Immunological abnormalities may lead to the impairment of ovarian folliculogenesis; however, whether the presence of AIT affects immunological microenvironment in follicles remains controversial. We performed a cross-sectional study including 122 patients, aged 20-40 years, who underwent IVF/ICSI treatment owing to isolated male or tube factor infertility. Patients were divided into AIT and control groups according to clinical presentation, thyroid function, and thyroid autoantibody measurements. Follicular fluid was collected and the distribution of cytokines/chemokines in follicular fluid was measured by flow cytometry using multiplex bead assays between the two groups. Based on differences in levels of intrafollicular chemokines and cytokines between the AIT and control groups, the relevant inflammatory cascade was further demonstrated. Among the 12 chemokines analyzed, three (CXCL9, CXCL10, and CXCL11) showed significantly elevated levels in the follicular fluid of patients with AIT. Among the 11 cytokines detected, compared with those in the control group, significantly higher levels of IFNγ were observed in patients with AIT. IFNγ dose-dependently stimulated the expression and secretion of CXCL9/10/11 in cultured primary granulosa cells. The percentage of CXCR3+ T lymphocytes was significantly elevated in the follicular microenvironment of patients with AIT. We concluded that the IFNγ-CXCL9/10/11-CXCR3+ T lymphocyte inflammatory cascade is activated in the follicular microenvironment of patients with AIT. These findings indicate that a considerable immune imbalance occurred in the follicular microenvironment of patients with AIT.

Serum Fractalkine Levels Were Positively Associated with Disease Severity of Liver Cirrhosis.

OBJECTIVE: The chemokine receptor CX3CR1 and its specific ligand fractalkine (CX3CL1, FKN) has been implicated in modulating inflammatory and fibroproliferative diseases. The current study was performed to investigate the correlation of serum fractalkine levels with disease severity of liver fibrosis/cirrhosis (LC). METHODS: 162 LC patients and 140 healthy controls well enrolled in our study. Serum fractalkine levels were detected using commercial ELISA kit. Liver biopsy specimens were obtained using 16 G disposable needle in LC patients. The Child-Pugh grade was recorded to assess liver function. ROC curve analysis was performed to assess the potential diagnostic power of serum fractalkine with regard to the disease severity of Child-Pugh grade system. Pathological assessment of cirrhotic severity was performed by Laennec staging system. The L3 skeletal muscle index (L3SMI) was applicated to assess the nutrition status. RESULTS: Serum fractalkine levels were significantly higher in LC patients compared with healthy controls. The case group included 50 Child-Pugh A patients, 59 Child-Pugh B patients, and 53 Child-Pugh C patients. Cirrhosis patients with Child-Pugh C had drastically higher serum fractalkine levels compared with those with Child-Pugh B and A. Child-Pugh B patients showed significantly higher serum PACAP concentrations compared with those with Child-Pugh A. ROC curve analysis demonstrated that serum fractalkine may act as a potential indicator for disease progression of LC determined by Child-Pugh classification. Besides, serum fractalkine levels were positively related to ALT and AST concentrations and negatively related to L3SMI. CONCLUSION: Serum fractalkine levels were positively associated with disease severity of LC.

Impact of intravenous dexmedetomidine on postoperative bowel movement recovery after laparoscopic nephrectomy: A consort-prospective, randomized, controlled trial.

BACKGROUND: Postoperative ileus is a frequent postoperative complication, especially after abdominal surgery. Sympathetic excitation is the primary factor for postoperative ileus. Sympathetic activation becomes increased by surgical stress, postoperative pain, and inflammation. Dexmedetomidine (DEX) can inhibit sympathetic nerve activity, inflammation, and pain. AIM: To observe whether DEX promotes bowel movements in patients after laparoscopic nephrectomy. METHODS: One hundred and twenty patients undergoing laparoscopic nephrectomy were assigned to three groups: C (normal saline infusion), D1 (DEX 0.02 µg/kg/h), and D2 (DEX 0.04 µg/kg/h). The primary outcomes were the recorded times to first flatus, defecation, and eating after surgery. The secondary outcomes were postoperative pain, assessed using the numerical rating scale (NRS), adverse effects, and the duration of the postoperative hospital stay. RESULTS: The times to first flatus, defecation, and eating in groups D1 and D2 were significantly shorter than those in group C (P < 0.01). The NRS scores at 8 h and 24 h after surgery were significantly lower in groups D1 and D2 than in group C (P < 0.05). No adverse effects were observed (P > 0.05). CONCLUSION: Postoperative infusion of DEX at 0.04 µg/kg/h facilitates bowel movements in patients undergoing laparoscopic nephrectomy.

Impact of Thyroid Autoimmunity on In Vitro Fertilization/Intracytoplasmic Sperm Injection Outcomes and Fetal Weight.

Several studies have reported the association between thyroid autoimmunity (TAI) and in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) outcomes. However, the findings remain controversial. We performed a large-scale retrospective cohort study to verify the effect of the presence of thyroid antibodies on IVF/ICSI outcomes and fetal growth and to evaluate the association between the types and titers of thyroid antibodies and adverse IVF/ICSI outcomes. A total of 16481 patients with infertility were referred to the Reproductive Center of Peking University Third Hospital for their first IVF/ICSI treatment between January 2018 and June 2019.Patients who sought IVF/ICSI treatment due to tubal or male factors infertility and who achieved fresh embryo transfer were included in our study. Finally, 778 patients with thyroid antibody positivity were selected as the TAI group, and 778 age-matched patients were included in the control group. The number of oocytes retrieved and high-graded embryos and the rates of clinical pregnancy, miscarriage, live birth, and preterm delivery were compared between the TAI and control groups. In addition, subgroup analysis was performed to demonstrate whether different types and titers of thyroid antibodies had different effects on IVF/ICSI outcomes. After adjusting for thyroid function, anti-Müllerian hormone levels, basal follicle stimulating hormone levels, basal estradiol levels and antral follicle count, the number of oocytes retrieved in the TAI group was significantly lower than that in the control group. No significant differences were observed between the two groups in the rates of clinical pregnancy, miscarriage, preterm delivery, live birth, and birth weight in singletons; however, the birth weight in twin pregnancy was significantly lower in the TAI group than in the control group. Subgroup analysis showed no association between the types or titers of thyroid antibodies and adverse IVF/ICSI outcomes. In conclusion, the presence of TAI in patients with infertility did not impair embryo quality or affect pregnancy outcomes, including clinical pregnancy, miscarriage, preterm delivery, and live birth. However, it decreased the number of oocytes retrieved and birth weight in twin pregnancy.

CircCCT3 Acts as a Sponge of miR-613 to Promote Tumor Growth of Pancreatic Cancer Through Regulating VEGFA/VEGFR2 Signaling.

BACKGROUND: Circular RNAs (CircRNAs) have been recently implicated in the progression of pancreatic cancer (PC). AIMS: To investigate the involvement of CircCCT3 in PC and studying its interactions and functioning during the progression of PC in vitro and in vivo, using methods of molecular biology and bioinformatics. STUDY DESIGN: Experimental study. METHODS: The expressions of CircCCT3 and miR-613 in pancreatic carcinoma tissues and cell lines were evaluated by quantitative real-time polymerase chain reaction (PCR). The relationship between clinical pathologic features as well as the survival rate and CircCCT3 expression was analyzed with chi-square test and the Kaplan-Meier method. CCK-8, wound healing, transwell assays, and the fluorescein isothiocyanate- AnnexinV/propidium iodide (FITC-AnnexinV/PI) assay were used to assess cell proliferation, migration, invasion, and apoptosis after CircCCT3 overexpression or downregulation. The Dual- Luciferase reporter assay, RNA immunoprecipitation (RIP), RNA pull-down and fluorescence in situ hybridization (FISH) assays were performed to validate the potential interaction of CircCCT3, miR-613, and vascular endothelial growth factor (VEGFA). The nude mouse xenograft tumor assay was used to detect CircCCT3 effects on pancreatic tumorigenesis in vivo. Western blotting analysis was performed to examine the VEGFA and the vascular endothelial growth factor receptor 2 (VEGFR2) protein expressions following. RESULTS: CircCCT3 expression was significantly increased in PC tissues (3.41 ± 0.57 vs. 1.00 ± 0.10, P < .01) and cell lines (Patu8988 2.57 ± 0.20; SW1990 2.88 ± 0.10; BxPC-3 2.45 ± 0.20; Panc02 2.99 ± 0.10 vs. H6c7 1.00 ± 0.10; all P < .001). CircCCT3 expression was negatively correlated with miR-613 expression. PC patients with high CircCCT3 expression exhibited significantly poorer overall survival rate than those patients with low CircCCT3 expression (P = .013). Moreover, it was found that CircCCT3 promoted cell proliferation, migration, and invasion, and inhibited cell apoptosis in PC cells. The CircCCT3 acted as a sponge for the miR-613 to facilitate VEGFA and VEGFR2 expression. si-CirCCT3 also inhibited tumor growth of PC in nude mice. si-CircCCT3 reduced VEGFA and VEGFR2 expression, whereas overexpression of CircCCT3 increased VEGFA and VEGFR2 expression. CONCLUSION: Increased CircCCT3 suggests a poor prognosis for PC patients and promotes the migration and invasion through targeting VEGFA/VEGFR2 signaling. CircCCT3 may serve as a potential and promising therapeutic target for PC treatment.

Active-Targeting Polymeric Dual Nanosensor for Ratiometrically Measuring Proton and Oxygen Concentrations in Mitochondria.

Dysfunction of mitochondria is closely related to neurodegenerative diseases, heart diseases, cancers, and so on. Because both proton and oxygen participate in vital biochemical reactions occurring in mitochondria such as adenosine triphosphate (ATP) generation, measuring proton and oxygen concentrations in mitochondria is therefore crucial for monitoring mitochondria activities and understanding cellular behavior. For this purpose, we developed a ratiometric fluorescent nanosensor for simultaneously sensing and imaging O2 and pH in mitochondria. The steps are as follows: (1) Styrene was copolymerized with 2-aminoethyl methacrylate hydrochloride to produce amino-functionalized polymer nanoparticles. (2) The reference dye rhodamine B isothiocyanate (RBITC) and oxygen-sensitive dye platinum(II) octaethylporphyrin (PtOEP) were encapsulated into a polymer sphere during polymerization, while the pH indicator fluorescein isothiocyanate (FITC) and mitochondrial-targeting molecule (3-carboxypropyl)triphenylphosphonium bromide (TPP) were further modified on the surface of the nanoparticles. The developed nanosensor shows a narrow distribution of particle size, high sensitivity toward O2 and pH, excellent stability, and low cytotoxicity. These remarkable features of the dual nanosensor render them capable of real-time sensing and imaging of O2 and pH in mitochondria with high spatial resolution. Applying the mitochondrial-targeted dual nanosensor in HeLa cells, we quantitatively measured and imaged mitochondrial proton and oxygen concentration variations after carbonyl cyanide m-chlorophenylhydrazone (CCCP) treatment.

A comprehensive PGT-M strategy for ADPKD patients with de novo PKD1 mutations using affected embryo or gametes as proband.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease characterized by the development of renal cysts and progression to renal failure. Preimplantation genetic testing-monogenic disease (PGT-M) is an alternative option to obtain healthy babies. However, de novo PKD1 mutation of one of the spouses or the absence of a positive family history poses a serious challenge to PGT-M. Here, we described a comprehensive strategy which includes preimplantation genetic testing for aneuploidies (PGT-A) study and monogenic diagnosis study for ADPKD patients bearing de novo mutations. The innovation of our strategy is to use the gamete (polar body or single sperm) as proband for single-nucleotide polymorphism (SNP) linkage analysis to detect an embryo's carrier status. Nine ADPKD couples with either de novo mutation or without a positive family history were recruited and a total of 34 embryos from 13 PGT-M cycles were examined. Within these nine couples, two successfully delivered healthy babies had their genetic status confirmed by amniocentesis. This study provides a creative approach for embryo diagnosis of patients with de novo mutations or patients who lack essential family members for linkage analysis.

Prophylactic administration of fingolimod (FTY720) ameliorated experimental autoimmune myasthenia gravis by reducing the number of dendritic cells, follicular T helper cells and antibody-secreting cells.

Fingolimod (FTY720), a sphingosine 1-phosphate (S1P) receptor antagonist, possesses potent immunomodulatory activity via lymphocyte homing. The effects of FTY720 have been widely studied in various T-cell-mediated autoimmune diseases, while the immunomodulatory effects on experimental autoimmune myasthenia gravis (EAMG), a typical disease model for antibody-mediated autoimmunity, remain elusive. In the present study, FTY720 was administered to EAMG rats as prophylaxis. The clinical scores were recorded every other day, and serum antibodies at different time points were measured by enzyme-linked immunosorbent assay (ELISA). The immune cell subsets in the spleen, bone marrow, circulation, and thymus were determined by flow cytometry. The prophylactic administration alleviated EAMG symptoms by reducing the level of serum antibodies IgG and its isotype IgG2b on days 30 and 46 post immunization, as well as IgG and Ig kappa antibody-secreting cells in the spleen and bone marrow. The mitigated humoral immune response can be attributed to the decreased dendritic cells, follicular T help cells (Tfh) and Tfh subsets (Tfh1, Tfh2, and Tfh17), and T helper cell subsets (Th1, Th2, and Th17) in the spleen. The promotion of lymphocyte homing and inhibition of thymocyte egress contribute to the effects of FTY720 on these effector T cell subsets. Overall, the prophylactic administration of FTY720 ameliorated EAMG partially by regulating humoral immune response，suggesting that FTY720 could be part of a pharmacological strategy for managing myasthenia gravis.

Clinical application of an NGS-based method in the preimplantation genetic testing for Duchenne muscular dystrophy.

PURPOSE: To determine whether next-generation sequencing (NGS) could be used to directly detect different mutations of Duchenne muscular dystrophy (DMD) during preimplantation genetic testing (PGT). METHODS: From Sep. 2016 to Aug. 2018, a total of six couples participated in this study. Four cases carried DMD exon deletions and two carried exon duplications. Trophectoderm cells were biopsied at day 5 or 6 and NGS was used in the genetic testing of the biopsied cells after whole-genome amplification. We developed a new method-DIRected Embryonic Cell Testing of Exon Deletion/Duplication (DIRECTED) to directly detect the single-gene mutation by NGS. Linage analysis based on single-nucleotide polymorphism (SNP) was used to validate the results from DIRECTED. RESULTS: In the four deletion cases, DIRECTED was used to detect DMD exon deletion in 16 biopsied embryos. All DIRECTED results were consistent with linkage analysis, indicating this method was reliable in detecting deletions around 1 Mb. In the two cases carrying exon duplications, no blastocyst was obtained for biopsy. Nonetheless, preliminary experiment results suggested that DIRECTED could also be used for direct detection of exon duplications in embryos. CONCLUSIONS: Exon deletions or duplications in DMD of preimplantation embryos could be detected directly by NGS-based methods during PGT.

Very-low-dose decitabine treatment for patients with intermediate- or high-risk myelodysplastic syndrome: a retrospective analysis of thirteen cases.

Decitabine is a hypomethylating drug that is used to treat myelodysplastic syndrome (MDS) at a recommended dose and schedule (20 mg/m2 per day, for 5 consecutive days). However, due to its relatively high incidence of side effects and its effects on neoplastic cells, many studies have begun to explore the clinical application of a low dose of decitabine for treating MDS. In this retrospective study, we examined the effects of a very-low-dose decitabine schedule for treating MDS. A total of 13 patients diagnosed with de novo MDS received a schedule of intravenous decitabine administration at 6 mg/m2 per day for 7 days, repeated every 4 weeks. The complete response rate was 30.8%, and the overall response rate was 69.2%. In patients with complete remission, the median time to granulocyte recovery greater than 0.5 × 109/L during complete remission (CR) was 15 days. In patients with remission, the median time to granulocyte recovery greater than 0.5 × 109/L was 10.5 days. The 1-year survival rate was 72.7% and the median survival was 28.0 months. In summary, we demonstrated that a very-low-dose decitabine schedule has an appreciable response and survival rate, as well as appreciable tolerance and medical compliance for treating MDS.

Overexpression of miR-221 stimulates proliferation of rat neural stem cell with activating Phosphatase and tensin homolog/protein kinase B signaling pathway.

Neural stem cells (NSCs) are self-renewing, multipotent cells, and remain in our brains throughout life. They could be activated by brain damage and involved in the central nervous system (CNS) repair and motor functional recovery. Previous research demonstrated that miR-221 could regulate proliferation, differentiation, and survival. However, the effect of miR-221 on NSCs remains unknown. In this study, we showed that overexpression of miR-221 inhibited the expression of phosphatase and tensin homolog (PTEN) protein and increased the phosphorylation level of protein kinase B (AKT). More importantly, an AKT-specific inhibitor abolished the effect of miR-221 on the phosphorylation level of AKT. 5-Bromo-2-deoxyUridine (BrdU) incorporation assay and Cyclin D1 expression showed that miR-221 overexpression further promoted the NSCs proliferation. However, knocking down miR-221 inhibited cell proliferation. The AKT-specific inhibitor also blocked the proliferative efficiency of miR-221. These results demonstrated that miR-221 overexpression promoted the proliferation of cultured rat NSCs, for which the PTEN/AKT pathway activation was one possible mechanism. Our research may provide a novel investigating strategy to improve stem cell treatment for CNS diseases.

[Efficacy and mechanism of electroacupuncture on insulin resistant polycystic ovary syndrome].

OBJECTIVE: To explore the therapeutic effect and partial mechanism of electroacupuncture (EA) for patients with insulin resistance (IR) polycystic ovary syndrome (PCOS). METHODS: Seventy patients with IR-PCOS were randomly divided into an EA group (36 cases, 5 cases dropped off) and a medication group (34 cases, 4 cases dropped off). The patients in the medication group were treated with oral administration of metformin hydrochloride, 500 mg each time, twice a day. The patients in the EA group were treated with EA (continuous wave, 2 Hz of frequency) at Zusanli (ST 36), Zhongwan (CV 12), Qihai (CV 6), Yishu (EX-B 3), Shenshu (BL 23), Pishu (BL 20), Ciliao (BL 32) for 30 min, three times a week. One menstrual cycle or 4 weeks were taken as a course of treatment, and 3 continuous courses were given. The follow-up was 3 months. The lipid metabolism indexes of triacylglycerol (TG), total cholesterol (TC), high-density lipoprotein (HDL) and low-density lipoprotein (LDL), homeostasis model assessment-insulin resistance index (HOMA-IR) and testosterone (T) in serum were compared before and after treatment, and the clinical effects of the two groups were evaluated during the follow-up. RESULTS: The total effective rate was 67.7% (21/31) in the EA group and 60.0% (18/30) in the medication group, with no significant difference between the two groups (P>0.05). After treatment, the levels of serum T, HOMA-IR, LDL, TG and TC were decreased significantly in the two groups (P<0.01, P<0.05), and HDL was increased significantly (P<0.01); the levels of TC in the EA group after treatment was lower than that in the medication group (P<0.05). CONCLUSION: EA may adjust some dyslipidemia in patients to correct IR and improve endocrine disorder of PCOS, which had superior/similar effects to metformin.

A risk prediction model for renal damage in a hypertensive Chinese Han population.

Backgroud: While numerous risk factors for renal damage in the hypertensive population have been reported, there is no single prediction model. The purpose of this study was to develop a model to comprehensively evaluate renal damage risk among hypertensive patients. Methods: We analyzed the data of 582 Chinese hypertensive patients from 1 January 2013 to 30 June 2016. Basic patient information was collected along with laboratory test results. According to the albumin-to-creatinine ratio, the subjects were divided into a hypertension with renal damage group and a hypertension without renal damage group. The prediction model was established by logistic regression based on principal component analysis, and the area under the receiver operating characteristic curve was used to evaluate the predictive performance of the model.Results: There are 11 indicators have statistically significant difference between the two groups (P < 0.05); The equation expressed including all 11 risk factors was as follows: Y = (-0.236) - 0.1705 (sex) - 0.0098 (age) - 0.1067 (smoking history) + 0.0303 (drinking history) - 0.3031 (CHD) + 0.1276 (diabetes history) - 0.0596 (CRP level) - 0.0732 (CysC level) + 0.0949 (ß2-MG level) + 0.5407 (blood pressure type) + 0.6470 (RRI). The calculated AUC was 74.4%; The risk in males was much higher than that in females of the same age. However, with increasing age, the male:female risk ratio gradually decreased. Conclusion: Eleven  indicators (including sex, age, smoking history, drinking history, coronary heart disease, diabetes history, C-reactive protein, CystatinC,  ß2-microglobulin protein, blood pressure type, renal artery resistance index)  may be the risk factors of renal damage in hypertension. Our regression equation provides a feasible means of predicting renal damage in Chinese hypertensive populations, and the model showed good predictive power. In addition, estrogen may confer a protective effect on the kidney. Abbreviations: PCA: principal component analysis; SLPs: synthetic latent predictors; CKD: chronic kidney disease; RRI: renal artery resistance index; MLR: multivariate logistic regression; CHD: coronary heart disease; UACR: urine trace albumin/uric creatinine ratio; CysC: CystatinC; TG: Triglyceride; CHO: cholesterol; HDL: high-density lipoprotein cholesterol; LDL: low-density lipoprotein cholesterol; CRP: C-reactive protein; HCY: homocysteine; UA: uric acid; AUC: area under the ROC curve; CVE: cardiovascular events; RFF: renal function related factor; PHF: personal history related factor; CVF: cardiovascular factor; GMF: glucose metabolism factor; IF: inflammatory factor; BPF: blood pressure factor.