In Situ Quantitative Imaging of Plasma Membrane Stiffness in Live Cells Using a Genetically Encoded FRET Sensor.

Cell membrane stiffness is critical for cellular function, with cholesterol and sphingomyelin as pivot contributors. Current methods for measuring membrane stiffness are often invasive, ex situ, and slow in process, prompting the need for innovative techniques. Here, we present a fluorescence resonance energy transfer (FRET)-based protein sensor designed to address these challenges. The sensor consists of two fluorescent units targeting sphingomyelin and cholesterol, connected by a linker that responds to the proximity of these lipids. In rigid membranes, cholesterol and sphingomyelin are in close proximity, leading to an increased FRET signal. We utilized this sensor in combination with confocal microscopy to explore changes in plasma membrane stiffness under various conditions, including differences in osmotic pressure, the presence of reactive oxygen species (ROS) and variations in substrate stiffness. Furthermore, we explored the impact of SARS-CoV-2 on membrane stiffness and the distribution of ACE2 after attachment to the cell membrane. This tool offers substantial potential for future investigations in the field of mechanobiology.

PHPB ameliorates memory deficits and reduces oxidative injury in Alzheimer's disease mouse model by activating Nrf2 signaling pathway.

Alzheimer's disease (AD), a progressive neurodegenerative disorder, is the most common cause of dementia in elderly people and substantially affects patient quality of life. Oxidative stress is considered a key factor in the development of AD. Nrf2 plays a vital role in maintaining redox homeostasis and regulating neuroinflammatory responses in AD. Previous studies show that potassium 2-(1-hydroxypentyl)-benzoate (PHPB) exerts neuroprotective effects against cognitive impairment in a variety of dementia animal models such as APP/PS1 transgenic mice. In this study we investigated whether PHPB ameriorated the progression of AD by reducing oxidative stress (OS) damage. Both 5- and 13-month-old APP/PS1 mice were administered PHPB (100 mg·kg-1·d-1, i.g.) for 10 weeks. After the cognition assessment, the mice were euthanized, and the left hemisphere of the brain was harvested for analyses. We showed that 5-month-old APP/PS1 mice already exhibited impaired performance in the step-down test, and knockdown of Nrf2 gene only slightly increased the impairment, while knockdown of Nrf2 gene in 13-month-old APP/PS1 mice resulted in greatly worse performance. PHPB administration significantly ameliorated the cognition impairments and enhanced antioxidative capacity in APP/PS1 mice. In addition, PHPB administration significantly increased the p-AKT/AKT and p-GSK3ß/GSK3ß ratios and the expression levels of Nrf2, HO-1 and NQO-1 in APP/PS1 mice, but these changes were abolished by knockdown of Nrf2 gene. In SK-N-SH APPwt cells and primary mouse neurons, PHPB (10 µM) significantly increased the p-AKT/AKT and p-GSK3ß/GSK3ß ratios and the level of Nrf2, which were blocked by knockdown of Nrf2 gene. In summary, this study demonstrates that PHPB exerts a protective effect via the Akt/GSK3ß/Nrf2 pathway and it might be a promising neuroprotective agent for the treatment of AD.

Optimizing surgical outcomes for elderly gallstone patients with a high body mass index using enhanced recovery after surgery protocol.

BACKGROUND: Rehabilitation of elderly patients with a high body mass index (BMI) after cholecystectomy carries risks and requires the adoption of effective perioperative management strategies. The enhanced recovery after surgery (ERAS) protocol is a comprehensive treatment approach that facilitates early patient recovery and reduces postoperative complications. AIM: To compare the effectiveness of traditional perioperative management methods with the ERAS protocol in elderly patients with gallbladder stones and a high BMI. METHODS: This retrospective cohort study examined data from 198 elderly patients with a high BMI who underwent cholecystectomy at the Shanghai Fourth People's Hospital from August 2019 to August 2022. Among them, 99 patients were managed using the traditional perioperative care approach (non-ERAS protocol), while the remaining 99 patients were managed using the ERAS protocol. Relevant indicator data were collected for patients preoperatively, intraoperatively, and postoperatively, and surgical outcomes were compared between the two groups. RESULTS: The comparison results between the two groups of patients in terms of age, sex, BMI, underlying diseases, surgical type, and preoperative hospital stay showed no statistically significant differences. However, the ERAS group had a significantly shorter preoperative fasting time than the non-ERAS group (4.0 ± 0.9 h vs 7.6 ± 0.9 h). Regarding intraoperative indicators, there were no significant differences between the two groups of patients. However, in terms of postoperative recovery, the ERAS protocol group exhibited significant advantages over the non-ERAS group, including a shorter hospital stay, lower postoperative pain scores and postoperative hunger scores, and higher satisfaction levels. The readmission rate was lower in the ERAS protocol group than in the non-ERAS group (3.0% vs 8.1%), although the difference was not significant. Furthermore, there were significant differences between the two groups in terms of postoperative nausea and vomiting severity, postoperative abdominal distention at 24 h, and daily life ability scores. CONCLUSION: The findings of this study demonstrate that the ERAS protocol confers significant advantages in postoperative outcomes following cholecystectomy, including reduced readmission rates, decreased postoperative nausea and vomiting, alleviated abdominal distension, and enhanced functional capacity. While the protocol may not exhibit significant improvement in early postoperative symptoms, it does exhibit advantages in long-term postoperative symptoms and recovery. These findings underscore the importance of implementing the ERAS protocol in the postoperative management of cholecystectomy patients, as it contributes to improving patients' recovery and quality of life while reducing health care resource utilization.

Characteristics of Complement Protein Deposition in Proliferative Glomerulonephritis with Monoclonal Immunoglobulin Deposition.

BACKGROUND: Hypocomplementemia and complement co-deposition with monoclonal immunoglobulins in glomeruli are not rare in proliferative glomerulonephritis with monoclonal immunoglobulin deposits (PGNMID). Deposition of monoclonal immunoglobulins in glomeruli has been suggested to activate complement and cause kidney injury. However, the profiles of complement activation in PGNMID and their clinical and pathologic significance need to be clarified. METHODS: Forty-six patients with PGNMID were enrolled. Proteomic analysis of glomeruli using laser microdissection and mass spectrometry was performed for ten patients with PGNMID to determine the composition of glomerular deposits. Kidney deposition of complement components was detected by immunohistochemistry and immunofluorescence. Urinary and plasma levels of complement components were measured by an enzyme-linked immunosorbent assay. Group differences were assessed using t tests or Mann-Whitney U tests depending on the distribution. Correlation analysis was performed using Spearman rank correlation or Pearson correlation. RESULTS: Laser microdissection and mass spectrometry-based proteomic analysis showed that complement components were the most enriched proteins deposited in the glomeruli of patients with PGNMID. Glomerular deposition of C3c, C4d, and C5b-9 was detected in most patients. Levels of urinary and plasma C3a, C5a, soluble C5b-9, C4d, Bb, and C1q as well as urinary mannose-binding lectin were significantly higher in patients with PGNMID compared with healthy controls. The intensity of C3c and C4d deposition in glomeruli correlated with serum creatinine and the percentage of crescents, respectively. Furthermore, levels of urinary complement components correlated positively with serum creatinine, urinary protein excretion, percentage of crescents, and global glomerulosclerosis in kidney biopsies, whereas plasma levels of most complement components did not show a significant correlation with clinicopathologic parameters. In multivariable analysis, a higher level of urinary C4d was identified as an independent risk factor of kidney failure. CONCLUSIONS: The complement system was found to be overactivated in PGNMID, and levels of urinary complements correlated with disease severity. A higher level of urinary C4d was identified as an independent risk factor of kidney failure.

Antibody responses following the surge of SARS-CoV-2 Omicron infection among patients with systemic autoimmune rheumatic diseases.

Objectives: The surge of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant Omicron infections has affected most Chinese residents at the end of 2022, including a number of patients with systemic autoimmune rheumatic diseases (SARDs). Methods: To investigate the antibody level of the Omicron variant in SARD patients after SARS-CoV-2 Omicron infection, we tested BA.5.2 and BF.7 Omicron variant IgG antibody levels using ELISA on blood samples collected from 102 SARD patients and 19 healthy controls (HCs). The type of SARD, demographics, concurrent treatment, doses of SARS-CoV-2 vaccines and outcomes were also recorded. Results: A total of 102 SARD patients (mean age: 40.3 years; 89.2% female), including 60 SLE, 32 RA and 10 other SARDs, were identified. Of these, 87 (85.3%) were infected with SARS-CoV-2. We found that the BA.5.2 and BF.7 antibody levels of infected SARD patients were lower than those of HCs (P < 0.05). Sixty-five (63.7%) patients had at least one dose of a SARS-CoV-2 vaccine. SARD patients with at least two doses of SARS-CoV-2 vaccine had a higher level of BA.5.2 and BF.7 antibodies than the unvaccinated group (P < 0.05). There was no evidence for a significant inhibitory effect of glucocorticoids (GCs) on the BA.5.2 and BF.7 Omicron variant antibody levels in SARD patients. SLE patients using biologic DMARDs had a lower BA.5.2 Omicron variant antibody level than patients using GCs and/or HCQ. Conclusion: These data suggest that patients with SARDs had a lower antibody response than HCs after Omicron infection.

Integrative analysis of platelet-related genes for the prognosis of esophageal cancer.

BACKGROUND: Every year, esophageal cancer is responsible for 509000 deaths and around 572000 new cases worldwide. Although esophageal cancer treatment options have advanced, patients still have a dismal 5-year survival rate. AIM: To investigate the relationship between genes associated to platelets and the prognosis of esophageal cancer. METHODS: We searched differentially expressed genes for changes between 151 tumor tissues and 653 normal, healthy tissues using the "limma" package. To develop a prediction model of platelet-related genes, a univariate Cox regression analysis and least absolute shrinkage and selection operator Cox regression analysis were carried out. Based on a median risk score, patients were divided into high-risk and low-risk categories. A nomogram was created to predict the 1-, 2-, and 3-year overall survival (OS) of esophageal cancer patients using four platelet-related gene signatures, TNM stages, and pathological type. Additionally, the concordance index, receiver operating characteristic curve, and calibration curve were used to validate the nomogram. RESULTS: The prognosis of esophageal cancer was associated to APOOL, EP300, PLA2G6, and VAMP7 according to univariate Cox regression analysis and least absolute shrinkage and selection operator regression analysis. Patients with esophageal cancer at high risk had substantially shorter OS than those with cancer at low risk, according to a Kaplan-Meier analysis (P < 0.05). TNM stage (hazard ratio: 2.187, 95% confidence interval: 1.242-3.852, P = 0.007) in both univariate and multivariate Cox regression and risk score were independently correlated with OS (hazard ratio: 2.451, 95% confidence interval: 1.599-3.756, P < 0.001). CONCLUSION: A survival risk score model and independent prognostic variables for esophageal cancer have been developed using APOOL, EP300, PLA2G6, and VAMP7. OS for esophageal cancer might be predicted using the nomogram based on TNM stage, pathological type, and risk score. The nomogram demonstrated strong predictive ability, as shown by the concordance index, receiver operating characteristic curve, and calibration curve.

Comprehensive analysis of the relationship between cuproptosis-related genes and esophageal cancer prognosis.

BACKGROUND: Esophageal cancer is one of the most common malignant tumors of the digestive system, with a 5-year survival rate of 15% to 50%. Cuproptosis, a unique kind of cell death driven by protein lipoylation, is strongly connected to mitochondrial metabolism. The clinical implications of cuproptosis-related genes in esophageal cancer, however, are mainly unknown. AIM: To identify cuprotosis-related genes that are differentially expressed in esophageal cancer and investigate their prognostic significance. METHODS: With |log fold change| > 1 and false discovery rate < 0.05 as criteria, the Wilcox test was used to evaluate the differentially expressed genes between 151 tumor tissues and 151 normal esophageal tissues. Cuproptosis-related genes were selected to be linked with prognosis using univariate Cox regression analysis. Genes were separated into high- and low- expression groups based on their cutoff value of gene expression, and the correlation between the two groups and overall survival or progression-free survival was investigated using the log-rank test. The C-index, calibration curve, and receiver operator characteristic (ROC) curve were used to assess a nomogram containing clinicopathological characteristics and cuproptosis-related genes. RESULTS: Pyruvate dehydrogenase A1 (PDHA1) was found to be highly correlated with prognosis in univariate Cox regression analysis (hazard ratio = 22.96, 95% confidence interval = 3.09-170.73; P = 0.002). According to Kaplan-Meier survival curves, low expression of PDHA1 was associated with a better prognosis (log-rank P = 0.0007). There was no significant correlation between PDHA1 expression and 22 different types of immune cells. Tumor necrosis factor superfamily member 15 (TNFSF15) (P = 3.2 × 10-6; r = 0.37), TNFRSF14 (P = 8.1 × 10-8; r = 0.42), H long terminal repeat-associating 2 (P = 6.0 × 10-8; r = 0.42) and galectin 9 (P = 3.1 × 10-6; r = 0.37) were all found to be considerably greater in the high PDHA1 expression group, according to an analysis of genes related to 47 immunological checkpoints. The low PDHA1 expression group had significantly lower levels of cluster of differentiation 44 (CD44) (P = 0.00028; R = -0.29), TNFRSF18 (P = 1.2 × 10-5; R = -0.35), programmed cell death 1 ligand 2 (P = 0.0032; R = -0.24), CD86 (P = 0.018; R = -0.19), and CD40 (P = 0.0047; R = -0.23), and the differences were statistically significant. We constructed a prognostic nomogram incorporating pathological type, tumor-node-metastasis stage, and PDHA1 expression, and the C-index, calibration curve, and ROC curve revealed that the nomogram's predictive performance was good. CONCLUSION: Cuproptosis-related genes can be used as a prognostic predictor for esophageal cancer patients, providing novel insights into cancer treatment.

[Spatial Variation and Potential Sources of Microplastics in Rivers in Tongzhou District, Beijing].

Microplastics are emerging contaminants, which can also absorb other contaminants, threatening the health of river ecosystems. However, research on the pollution of microplastics in rivers in northern China is still lacking. In this study, based on the sampling and analysis of water samples in 19 sites in six rivers in Tongzhou district, Beijing, the composition, spatial variation, and potential sources of microplastics were explored. The results showed that all sites were contaminated by microplastics, and the abundance of microplastics in the Xiaozhong River was the highest among all sites (3.50×104 n·m-3), which was 4.04 times that in the Yunchaojian River. The proportion of microplastics with particle sizes smaller than 2000 µm was 90.49%, and microplastics with particle sizes larger than 4000 µm were only found in two out of 19 sampling sites. The microplastics were fiber, film, fragment, and granule shaped. The proportion of fiber microplastics was the highest (90.23%) among all shapes. Most (84.29%) of the microplastics were transparent and blue. Rayon was the most common microplastic in each site, and its proportion in each site was over 66.67%. The proportions of other types of microplastics differed largely among different sites. Spatially, the abundance and types of microplastics in the upper reaches were higher than those in the lower reaches. According to spatial variations in shapes, types, colors, and abundance of microplastics, the potential sources of microplastics were identified. The potential sources of fiber microplastics were washing clothing and using fishing gear and dust-proof nets.

Enhanced Degradation of Juvenile Hormone Promotes Reproductive Diapause in the Predatory Ladybeetle Coccinella Septempunctata.

Improved knowledge on the regulation of reproductive diapause in Coccinella septempunctata, an important predator of aphids, is crucial for improving shelf-life and mass production of the ladybeetles. In many insects, the absence of juvenile hormone (JH) is a central regulator of reproductive diapause. JH is principally degraded by JH esterase (JHE) and JH epoxide hydrolase (JHEH). Previous studies have shown that genes encoding these enzymes were upregulated in early diapause of C. septempunctata, but whether increased JH degradation contributes to the reduction of JH levels and facilitates reproductive diapause remains unknown. Here, we investigate the role of JH and JH degradation genes during reproductive diapause in C. septempunctata females. Applying methoprene, a JH analogue, to the diapause preparation females clearly elevated JH signaling and reversed diapause program, suggesting that a lower level of JH is critical for the induction of reproductive diapause in the ladybeetle. Full-length cDNA sequences of JHE and JHEH were cloned and characterized, and their deduced proteins contain all the conserved active domains and typical motifs as identified in other insects. The expressions of JHE and JHEH were both significantly increased in diapause preparation and remained at a high level for a period throughout diapause, and then decreased after the termination of diapause. Knocking down these JH degradation genes clearly increased the expression levels of JH-inducible genes Krüppel-homolog 1 (Kr-h1) and vitellogenin (Vg), indicating an elevated JH level. Simultaneously, silencing JH degradation genes distinctly reduced diapause-related features and promotes reproduction, indicated by accelerated ovary growth, yolk deposition, and suppressed lipid accumulation. These results indicate that the enhanced JH degradation plays a critical role in regulating reproductive diapause of C. septempunctata.

A novel tumor-specific broad-spectral monoclonal antibody to PL2L60 is highly effective for the treatment of various types of cancers from human and mouse.

There are numerous antibodies used for cancer therapy in clinic, but they are essentially less efficacy than expected. None of them has tumor-specific and broad-spectral properties. PIWIL2-like (PL2L) protein 60 (PL2L60) is a product of alienated activation of PIWIL2 gene, and has been found to be specifically and widely expressed in various types of cancers, including hematopoietic and solid ones. Current study aims to investigate whether a monoclonal antibody (mAb) to PL2L60 has both tumor-specific and broad-spectral properties, which can be used universally to treat various types of cancers. The expression of PL2L60 protein in the cell surface and cytoplasm were determined in a panel of human and mouse tumor cell lines by flow cytometry, immunofluorescent microscopy and Western Blotting. The apoptosis and the cell cycle arrest of the tumor cells treated with mAb KAO3 were evaluated by flow cytometry. The tumorigenesis of the mAb KAO3-pretreated tumor cells was determined by tumor incidence and tumor size, and the efficacy of mAb KAO3 treatment on tumor growth in tumors-bearing mice were kinetically evaluated. Complement-dependent cytotoxicity (CDC) assay was used to determine the capacity of mAb KAO3 to kill tumor cells. Treatment of human or mouse tumor cells from hematopoietic or solid tumors with mAb KAO3 at the time of inoculation efficiently inhibited tumorigenesis in the severe combined immunodeficient (SCID) mice. Moreover, injection of mAb KAO3 into established tumors significantly inhibited their growth, and prolonged survival of the tumor-bearing mice, including lymphoma, breast cancer, lung cancer and cervical cancer. The efficacy of mAb KAO3 treatment is likely associated with its binding to PL2L60 expressed on tumor cell surface, which may lead to cancer cell death through blocking cell cycling and/or activation of complement. In conclusion, we have identified a tumor-specific mAb to PL2L60 (KAO3), which may be used potentially to treat all the types of human cancers including from both hematopoietic and solid ones.

Novel Hybrid Peptide Cathelicidin 2 (1-13)-Thymopentin (TP5) and Its Derived Peptides with Effective Antibacterial, Antibiofilm, and Anti-Adhesion Activities.

The increasing numbers of infections caused by multidrug-resistant (MDR) pathogens highlight the urgent need for new alternatives to conventional antibiotics. Antimicrobial peptides have the potential to be promising alternatives to antibiotics because of their effective bactericidal activity and highly selective toxicity. The present study was conducted to investigate the antibacterial, antibiofilm, and anti-adhesion activities of different CTP peptides (CTP: the original hybrid peptide cathelicidin 2 (1-13)-thymopentin (TP5); CTP-NH2: C-terminal amidated derivative of cathelicidin 2 (1-13)-TP5; CTPQ: glutamine added at the C-terminus of cathelicidin 2 (1-13)-TP5) by determining the minimal inhibitory concentrations (MICs), minimal bactericidal concentrations (MBCs), propidium iodide uptake, and analysis by scanning electron microscopy, transmission electron microscopy, and confocal laser scanning microscopy). The results showed that CTPs had broad-spectrum antibacterial activity against different gram-positive and gram-negative bacteria, with MICs against the tested strains varying from 2 to 64 µg/mL. CTPs at the MBC (2 × MIC 64 µg/mL) showed strong bactericidal effects on a standard methicillin-resistant Staphylococcus aureus strain ATCC 43300 after co-incubation for 6 h through disruption of the bacterial membrane. In addition, CTPs at 2 × MIC also displayed effective inhibition activity of several S. aureus strains with a 40-90% decrease in biofilm formation by killing the bacteria embedded in the biofilms. CTPs had low cytotoxicity on the intestinal porcine epithelial cell line (IPEC-J2) and could significantly decrease the rate of adhesion of S. aureus ATCC 43300 on IPEC-J2 cells. The current study proved that CTPs have effective antibacterial, antibiofilm, and anti-adhesion activities. Overall, this study contributes to our understanding of the possible antibacterial and antibiofilm mechanisms of CTPs, which might be an effective anti-MDR drug candidate.

Pregnancy-associated proliferative glomerulonephritis with monoclonal immunoglobulin deposits.

We report a young woman presented with nephrotic syndrome and normotension during every pregnancy and achieved complete remissions after the deliveries. We thus inferred that her nephrotic syndrome was closely associated with pregnancy. Kidney biopsies were perfromed and showed different histologic patterns: the first biopsy showed a pattern of endocapillary proliferative glomerulonephritis; the second biopsy revealed proliferative glomerulonephritis with monoclonal immunoglobulin deposits (PGNMID) with features of membranous nephropathy. With regard to presentation during the second trimester of pregnancy, achieving complete remission after delivery, and no relapse during the follow-up period, pregnancy associated PGNMID is suggested. To our best knowledge, this is the first reported case of PGNMID associated with pregnancy.

Broadband photomultiplication organic photodetectors.

Broadband photomultiplication organic photodetectors (PMOPDs) can be achieved with a double-layered active layer prepared from IEICO-4F : PBDB-T blend solutions with different weight ratios (1 : 1 or 3 : 100, wt/wt). The response range of the double-layered PMOPDs covers from 310 nm to 930 nm, determined by the photon harvesting range of the IEICO-4F : PBDB-T (1 : 1, wt/wt) layer. The IEICO-4F : PBDB-T (3 : 100, wt/wt) layer was used as a PM layer in the double-layered PMOPDs, achieving external quantum efficiency (EQE) more than 100% based on the work mechanism of trap-assisted hole tunneling injection. The trapped electrons in PBDB-T/IEICO-4F/PBDB-T near the Al electrode will makeinterfacial-band-bending to narrow the injection barrier, resulting in hole-tunneling-injection from the external circuit. The polymer PBDB-T can provide an efficient charge transport channel for the injected hole from the external circuit. The specific detectivity (D*) and responsivity (R) of the double-layered PMOPDs are 1.05 ± 0.03 × 1012 Jones and 0.94 ± 0.03 A W-1 at 810 nm under a -10 V bias, respectively.

Proteomic analysis in different development stages on SP0 generation of rice seeds after space flight.

The space biological effects of plants will drive the development of aerospace science and breeding science. The aim of this study is to reveal changes in the proteome of contemporary plants at different growth and development stages after space flight of rice seeds. We carried the rice seeds (DN416) through the SJ-10 returning satellite and returned to the ground for planting to the three-leaf stage (TLP) and tillering stage (TS) after a 12.5-day orbital flight. We found that the space flight caused the rice germination rate, the TLP plant height, and the number of tillers in the TS decreased by 11.64%, 9.75%, and 9.80%, respectively. In addition, the treatment group ROS and MDA level increased in the TLP and TS. The abundance patterns of proteins in these leaves identified 214 proteins in the TLP and 286 in the TS leaves that were markedly changed. Moreover, our study identified D14 proteins that control plant height and tiller. Our results show that the space environment may affect the downstream signaling mechanism by regulating the level of ROS in the body to achieve a response to the space environment. Meanwhile, the space environment may affect the plant height and tiller of rice by altering the expression of D14 protein and hormone-regulated proteins. Our results reveal changes in the proteome of different growth stages of rice plants, and also reveal the molecular mechanism of space environment regulation of rice plant height and tiller, which provides a new direction for further understanding of space biological effects and space mutation breeding.

The Clinical and Molecular Characteristics of FLT3 Mutations in Chinese De Novo Adolescent and Adult Acute Lymphoblastic Leukemia Patients.

BACKGROUND: Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are frequent in acute myeloid leukemia (AML) and have important prognostic and therapeutic implications. FLT3 aberrations have been detected in a smaller fraction of acute lymphoblastic leukemia (ALL), and their prognostic value is not well established. We therefore assessed the FLT3 mutation in Chinese adolescent and adult ALL patients. PATIENTS AND METHODS: We have examined a cohort of 117 Chinese de novo adolescent and adult ALL patients enrolled between June 2016 and June 2017 from the First Affiliated Hospital of Soochow University. Prognostic factors for the ALL patient population were estimated by the Cox regression method. FLT3 mutation was detected by PCR, and its clinical effect was assessed by Kaplan-Meier curves. Differences in FLT3 mutation rate between subgroups were tested by chi-square test. RESULTS: FLT3 mutations accounted for 6.8% (8/117) in our cohort, including 3 internal tandem duplications (2.6%) and 5 tyrosine kinase domains (4.3%, 3 D835Y mutations, 1 M664I mutation, and 1 I867S mutation), which had no clinical significance on either overall survival (OS) or event-free survival. Alterations in FLT3 occurred more often in early thymic precursor (ETP)-ALL compared to non-ETP T-cell acute lymphoblastic leukemia (P = .028). However, the age at onset (P = .004), initial platelet counts (P = .018), and transplantation status (P = .007) were independent prognostic factors of OS for ALL in multivariate analysis. CONCLUSION: The FLT3 mutation was not common in Chinese ALL patients. Age at onset, platelet counts, and transplantation status rather than the presence of the FLT3 mutation were independent prognostic variables for ALL on OS in our cohort. Despite our small sample size, ETP-ALL may indicate a comparable higher FLT3-mutant rate. Because ETP-ALL has been identified as high-risk subgroup, these data warrant clinical studies with the implementation of FLT3 inhibitors in addition to early allogeneic hematopoietic stem-cell transplantation for FLT3-mutant ETP-ALL.

Involvement of LIMK1/2 in actin assembly during mouse embryo development.

LIMKs (LIMK1 and LIMK2) are serine/threonine protein kinases that involve in various cellular activities such as cell migration, morphogenesis and cytokinesis. However, its roles during mammalian early embryo development are still unclear. In the present study, we disrupted LIMK1/2 activity to explore the functions of LIMK1/2 during mouse early embryo development. We found that p-LIMK1/2 mainly located at the cortex of each blastomeres from 2-cell to 8-cell stage, and p-LIMK1/2 also expressed at morula and blastocyst stage in mouse embryos. Inhibition of LIMK1/2 activity by LIMKi 3 (BMS-5) at the zygote stage caused the failure of embryo early cleavage, and the disruption of LIMK1/2 activity at 8-cell stage caused the defects of embryo compaction and blastocyst formation. Fluorescence staining and intensity analysis results demonstrated that the inhibition of LIMK1/2 activity caused aberrant cortex actin expression and the decrease of phosphorylated cofilin in mouse embryos. Taken together, we identified LIMK1/2 as an important regulator for cofilin phosphorylation and actin assembly during mouse early embryo development.

An unusual intragenic promoter of PIWIL2 contributes to aberrant activation of oncogenic PL2L60.

PIWIL2-like (PL2L) protein 60 (PL2L60), a product of aberrantly activated PIWIL2 gene, is widely expressed in various types of tumors and may promote tumorigenesis. However, the mechanisms underlying the activation of expression of PL2L60 remain unknown. In this study, an intragenic promoter responsible for the activation of PL2L60 within the human PIWIL2 gene has been identified, cloned and characterized. The promoter of PL2L60 is located in the intron 10 of the host gene PIWIL2. Bioinformatic and mutagenic analysis reveals that this intragenic promoter within the sequence of 50 nucleotides contains two closely arranged cis-acting elements specific for the hepatic leukemia factor (HLF) in the positive strand and signal transducer and activator of transcription 3 (STAT3) in the negative strand. Chromatin immunoprecipitation analysis demonstrates that both the HLF and polymerase II (Pol II), a hallmark of active promoters, directly bind to the sequence, although STAT3 does not. Knockdown of HLF and STAT3 alone or both by RNA interference significantly reduced both promoter activity and the PL2L60 protein expression, although there is no additive effect. The expression of PL2L60 proteins was enhanced when host gene Piwil2 was genetically disrupted in a murine cell model. Taken together, we have identified a PL2L60-specific intragenic promoter in the host gene of PIWIL2, which is interdependently activated by HLF and STAT3 through steric interaction. This activation is dependent on cellular milieu rather than the integrity of host gene PIWIL2, highlighting a novel, important mechanism for a cancer-causing gene to be activated during tumorigenesis.

MicroRNA-101 targets von Hippel-Lindau tumor suppressor (VHL) to induce HIF1α mediated apoptosis and cell cycle arrest in normoxia condition.

The activation/inactivation of HIF1α is precisely regulated in an oxygen-dependent manner. HIF1α is essential for hypoxia induced apoptosis and cell cycle arrest. Several recent studies indicated that the expression of miRNAs can be modulated by hypoxia. However, the involvement of miRNAs in the regulation of HIF1α induction remains elusive. In present study, we demonstrated that miR-101 was rapidly and transiently induced after hypoxia in breast cancer cells. Over-expression of miR-101 significantly inhibited cell proliferation in breast cancer cells through increased apoptosis and cell cycle arrest in normoxia condition. This inhibitory phenomenon seems due to miR-101-mediated induction of HIF1α, because we identified that VHL, a negative regulator of HIF1α, is a novel target of miR-101 and over-expression of miR-101 decreased VHL levels and subsequently stabilized HIF1α and induced its downstream target VEGFA. Furthermore, we demonstrated that siRNA-mediated knockdown of VHL or HIF1α overexpression could also induce apoptosis and cell cycle arrest whereas enforced expression of VHL, administration of anti-miR-101 oligos or treatment of 2-MeOE2, an inhibitor of HIF1α, could rescue cells from such inhibition. These results reveal a novel regulatory mechanism of HIF1α induction in normoxia and suggest that miR-101 mediated proliferation inhibition may through HIF1α mediated apoptosis and cell cycle arrest.

Beclin-1-independent autophagy mediates programmed cancer cell death through interplays with endoplasmic reticulum and/or mitochondria in colbat chloride-induced hypoxia.

Autophagy has dual functions in cell survival and death. However, the effects of autophagy on cancer cell survival or death remain controversial. In this study, we show that Autophagy can mediate programmed cell death (PCD) of cancer cells in responding to cobalt chloride (CoCl2)-induced hypoxia in a Beclin-1-independent but autophagy protein 5 (ATG5)-dependent manner. Although ATG5 is not directly induced by CoCl2, its constitutive expression is essential for CoCl2-induced PCD. The ATG5-mediated autophagic PCD requires interplays with endoplasmic reticulum (ER) and/or mitochondria. In this process, ATG5 plays a central role in regulating ER stress protein CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) and mitochondrial protein second mitochondria derived activator of caspases (Smac). Two pathways for autophagic PCD in cancer cells responding to hypoxia have been identified: ATG5/CHOP/Smac pathway and ATG5/Smac pathway, which are probably dependent on the context of cell lines. The former is more potent than the latter for the induction of PCD at the early stage of hypoxia, although the ultimate efficiency of both pathways is comparable. In addition, both pathways may require ATG5-mediated conversion of LC3-I into LC3-II. Therefore, we have defined two autophagy-mediated pathways for the PCD of cancer cells in hypoxia, which are dependent on ATG5, interplayed with ER and mitochondria and tightly regulated by hypoxic status. The findings provide a new evidence that autophagy may inhibit tumor cell proliferation through trigger of PCD, facilitating the development of novel anti-cancer drugs.