Enabling robust blue circularly polarized organic afterglow through self-confining isolated chiral chromophore.

Creating circularly polarized organic afterglow system with elevated triplet energy levels, suppressed non-radiative transitions, and effective chirality, which are three critical prerequisites for achieving blue circularly polarized afterglow, has posed a formidable challenge. Herein, a straightforward approach is unveiled to attain blue circularly polarized afterglow materials by covalently self-confining isolated chiral chromophore within polymer matrix. The formation of robust hydrogen bonds within the polymer matrix confers a distinctly isolated and stabilized molecular state of chiral chromophores, endowing a blue emission band at 414 nm, lifetime of 3.0 s, and luminescent dissymmetry factor of ~ 10-2. Utilizing the synergistic afterglow and chirality energy transfer, full-color circularly polarized afterglow systems are endowed by doping colorful fluorescent molecules into designed blue polymers, empowering versatile applications. This work paves the way for the streamlined design of blue circularly polarized afterglow materials, expanding the horizons of circularly polarized afterglow materials into various domains.

Roflumilast inhibits tumor growth and migration in STK11/LKB1 deficient pancreatic cancer.

Pancreatic cancer is a malignant tumor of the digestive system. It is highly aggressive, easily metastasizes, and extremely difficult to treat. This study aimed to analyze the genes that might regulate pancreatic cancer migration to provide an essential basis for the prognostic assessment of pancreatic cancer and individualized treatment. A CRISPR knockout library directed against 915 murine genes was transfected into TB 32047 cell line to screen which gene loss promoted cell migration. Next-generation sequencing and PinAPL.py- analysis was performed to identify candidate genes. We then assessed the effect of serine/threonine kinase 11 (STK11) knockout on pancreatic cancer by wound-healing assay, chick agnosia (CAM) assay, and orthotopic mouse pancreatic cancer model. We performed RNA sequence and Western blotting for mechanistic studies to identify and verify the pathways. After accelerated Transwell migration screening, STK11 was identified as one of the top candidate genes. Further experiments showed that targeted knockout of STK11 promoted the cell migration and increased liver metastasis in mice. Mechanistic analyses revealed that STK11 knockout influences blood vessel morphogenesis and is closely associated with the enhanced expression of phosphodiesterases (PDEs), especially PDE4D, PDE4B, and PDE10A. PDE4 inhibitor Roflumilast inhibited STK11-KO cell migration and tumor size, further demonstrating that PDEs are essential for STK11-deficient cell migration. Our findings support the adoption of therapeutic strategies, including Roflumilast, for patients with STK11-mutated pancreatic cancer in order to improve treatment efficacy and ultimately prolong survival.

Impact of CRISPR/Cas9-Mediated CD73 Knockout in Pancreatic Cancer.

Pancreatic cancer is among the cancers with the highest mortality rates. Most of the patients are found to have advanced cancer, losing the chance of surgical treatment, and there is an urgent need to find new treatment methods. Targeted therapy for specific genes that play a key role in cancer is now an important means to improve the survival rate of patients. We determined that CD73 is highly expressed in pancreatic cancer by flow cytometry and qRT-PCR assays combined with bioinformatics techniques. Application of CRISPR/Cas9 technology to knockout CD73 in human and murine cell lines, respectively, revealed that CD73 inactivation inhibited cell growth and migration and induced G1 cell cycle arrest. We also found that CD73 deletion inhibited the ERK/STAT3 pathway and activated the E-cadherin pathway. In addition, a CRISPR/Cas9 protein kinase library screen was performed and identified Pbk, Fastk, Cdk19, Adck5, Trim28, and Pfkp as possible genes regulating CD73.

Molecular genetic screening of full-term small for gestational age.

OBJECTIVE: To examine the clinical application of genomic screening in newborns small for gestational age (SGA), hoping to provide an efficient technique for early discovery of neonatal diseases, which is necessary to elevate survival rates and the quality of life in infants. METHODS: Totally 93 full-term SGA newborns were assessed. Dried blood spot (DBS) samples were obtained at 72 h after birth, and tandem mass spectrometry (TMS) and Angel Care genomic screening (GS, using Targeted next generation sequencing) were carried out. RESULTS: All 93 subjects were examined by Angel Care GS and TMS. No children showing inborn errors of metabolism (IEM) were detected by TMS, while 2 pediatric cases (2.15%, 2/93) were confirmed as thyroid dyshormonogenesis 6 (TDH6) by Angel Care GS. Additionally, 45 pediatric cases (48.4%) had one or more variants conferring a carrier status for recessive childhood-onset disorders, with 31 genes and 42 variants associated with 26 diseases. The top three gene-related diseases with carrier status were autosomal recessive deafness (DFNB), abnormal thyroid hormone and Krabbe disease. CONCLUSIONS: SGA is tightly associated with genetic variation. Molecular Genetic Screening allows early detection of congenital hypothyroidism and may be a potent genomic sequencing technique for screening newborns.

Regulatory role of melatonin in Notch1 signaling pathway in cerebral cortex of Aß1-42-induced Alzheimer's disease rat model.

BACKGROUND: Soluble Amyloid-beta (Aß) oligomers are thought to play a key role in the pathogenesis of Alzheimer's disease (AD), which is the most common age-associated neurodegenerative diseases with obvious neuropathological changes and functional decline in both cortical and subcortical regions. Melatonin is ubiquitously distributed and multifunctioning indoleamine. Accumulating studies support that melatonin is potential therapeutic molecule for AD through modulating a broad variety of signaling pathways. In recent years, Notch1 signaling pathway is been known involved in dynamic changes in the cellular architecture and function of adult brain, as well as associated with the pathophysiology of AD and other neurodegenerative disorders. METHODS AND RESULTS: In this study, we performed real-time polymerase chain reaction, immunohistochemistry and western blotting analyses using the cerebral cortical tissues of Aß1-42 oligomers-induced AD rats with or without melatonin treatment. Our results showed that soluble Aß1-42 oligomers decreased the expression of the main components of Notch1 signaling pathway, Notch1, NICD and Hes1 in the cerebral cortex, and melatonin could restore the level of Notch1, NICD and Hes1. CONCLUSION: This observation suggests that targeting of Notch1 signaling might be a promising therapeutic approach for AD and other age-associated neurodegenerative diseases, and melatonin might serve as a potential therapeutic agent for AD and other age-associated neurodegenerative diseases.

An m5C methylation regulator-associated signature predicts prognosis and therapy response in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive digestive malignancy due to frequent late-stage diagnosis, rapid progression and resistance to therapy. With increasing PDAC incidence worldwide, there is an urgent need for new prognostic biomarkers and therapy targets. Recently, RNA methylation has emerged as a new tumorigenic mechanism in different cancers. 5-methylcytosine (m5C) is one of the most frequent RNA modifications and occurs on a variety of RNA species including mRNA, thereby regulating gene expression. Here we investigated the prognostic role of m5C-regulator-associated transcriptional signature in PDAC. We evaluated m5C-regulator status and expression in 239 PDAC samples from publicly available datasets. We used unsupervised consensus clustering analyses to classify PDACs based on m5C-regulator expression. From the resulting signature of differentially expressed genes (DEGs), we selected prognosis-relevant DEGs to stratify patients and build a scoring signature (m5C-score) through LASSO and multivariate Cox regression analyses. The m5C-score represented a highly significant independent prognostic marker. A high m5C-score correlated with poor prognosis in different PDAC cohorts, and was associated with the squamous/basal subtype as well as activated cancer-related pathways including Ras, MAPK and PI3K pathways. Furthermore, the m5C-score correlated with sensitivity to pathway-specific inhibitors of PARP, EGFR, AKT, HER2 and mTOR. Tumors with high m5C-score were characterized by overall immune exclusion, low CD8+ T-cell infiltration, and higher PD-L1 expression. Overall, the m5C-score represented a robust predictor of prognosis and therapy response in PDAC, which was associated with unfavorable molecular subtypes and immune microenvironment.

CDK7 inhibition augments response to multidrug chemotherapy in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a dismal prognosis. Although combined treatment with gemcitabine and albumin-bound paclitaxel has improved the prognosis of PDAC, both intrinsic and acquired chemoresistance remain as severe hurtles towards improved prognosis. Thus, new therapeutic targets and innovative strategies are urgently needed. METHODS: In this study, we used the KPC mouse model-derived PDAC cell line TB32047 to perform kinome-wide CRISPR-Cas9 loss-of-function screening. Next-generation sequencing and MAGeCK-VISPR analysis were performed to identify candidate genes. We then conducted cell viability, clonogenic, and apoptosis assays and evaluated the synergistic therapeutic effects of cyclin-dependent kinase 7 (CDK7) depletion or inhibition with gemcitabine (GEM) and paclitaxel (PTX) in a murine orthotopic pancreatic cancer model. For mechanistic studies, we performed genome enrichment analysis (GSEA) and Western blotting to identify and verify the pathways that render PDAC sensitive to GEM/PTX therapy. RESULTS: We identified several cell cycle checkpoint kinases and DNA damage-related kinases as targets for overcoming chemoresistance. Among them, CDK7 ranked highly in both screenings. We demonstrated that both gene knockout and pharmacological inhibition of CDK7 by THZ1 result in cell cycle arrest, apoptosis induction, and DNA damage at least predominantly through the STAT3-MCL1-CHK1 axis. Furthermore, THZ1 synergized with GEM and PTX in vitro and in vivo, resulting in enhanced antitumor effects. CONCLUSIONS: Our findings support the application of CRISPR-Cas9 screening in identifying novel therapeutic targets and suggest new strategies for overcoming chemoresistance in pancreatic cancer.

Genome-Wide CRISPR Screening Identifies DCK and CCNL1 as Genes That Contribute to Gemcitabine Resistance in Pancreatic Cancer.

Pancreatic cancer is one of the most lethal cancers. Due to the difficulty of early diagnosis, most patients are diagnosed with metastasis or advanced-stage cancer, limiting the possibility of surgical treatment. Therefore, chemotherapy is applied to improve patient outcomes, and gemcitabine has been the primary chemotherapy drug for pancreatic cancer for over a decade. However, drug resistance poses a significant challenge to the efficacy of chemotherapy. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) gene-editing system is a powerful tool, and researchers have developed CRISPR/Cas9 library screening as a means to identify the genes associated with specific phenotype changes. We performed genome-wide CRISPR/Cas9 knockout screening in the mouse pancreatic cancer cell line TB32047 with gemcitabine treatment and identified deoxycytidine kinase (DCK) and cyclin L1 (CCNL1) as the top hits. We knocked out DCK and CCNL1 in the TB32047 and PANC1 cell lines and confirmed that the loss of DCK or CCNL1 enhanced gemcitabine resistance in pancreatic cells. Many researchers have addressed the mechanism of DCK-related gemcitabine resistance; however, no study has focused on CCNL1 and gemcitabine resistance. Therefore, we explored the mechanism of CCNL1-related gemcitabine resistance and found that the loss of CCNL1 activates the ERK/AKT/STAT3 survival pathway, causing cell resistance to gemcitabine treatment.

CRISPR-Cas9 Screen Identifies DYRK1A as a Target for Radiotherapy Sensitization in Pancreatic Cancer.

Although radiation therapy has recently made great advances in cancer treatment, the majority of patients diagnosed with pancreatic cancer (PC) cannot achieve satisfactory outcomes due to intrinsic and acquired radioresistance. Identifying the molecular mechanisms that impair the efficacy of radiotherapy and targeting these pathways are essential to improve the radiation response of PC patients. Our goal is to identify sensitive targets for pancreatic cancer radiotherapy (RT) using the kinome-wide CRISPR-Cas9 loss-of-function screen and enhance the therapeutic effect through the development and application of targeted inhibitors combined with radiotherapy. We transduced pancreatic cancer cells with a protein kinase library; 2D and 3D library cells were irradiated daily with a single dose of up to 2 Gy for 4 weeks for a total of 40 Gy using an X-ray generator. Sufficient DNA was collected for next-generation deep sequencing to identify candidate genes. In this study, we identified several cell cycle checkpoint kinases and DNA damage related kinases in 2D- and 3D-cultivated cells, including DYRK1A, whose loss of function sensitizes cells to radiotherapy. Additionally, we demonstrated that the harmine-targeted suppression of DYRK1A used in conjunction with radiotherapy increases DNA double-strand breaks (DSBs) and impairs homologous repair (HR), resulting in more cancer cell death. Our results support the use of CRISPR-Cas9 screening to identify new therapeutic targets, develop radiosensitizers, and provide novel strategies for overcoming the tolerance of pancreatic cancer to radiotherapy.

Identification of Genes Associated with Liver Metastasis in Pancreatic Cancer Reveals PCSK6 as a Crucial Mediator.

Liver metastasis occurs frequently in patients with pancreatic cancer. We analyzed the molecular profiling in liver metastatic lesions aiming to uncover novel genes responsible for tumor progression. Bioinformatics analysis was applied to identify genes directing liver metastasis. CRISPR/Cas9 technology was used to knock out the candidate gene. Proliferation assays, colony formation assays, cell cycle analysis, migration assays, wound healing assays, Immunofluorescence analysis, and the tumor xenograft model of intrasplenic injection were adopted to evaluate the effects of PCSK6 inactivation on cell growth, migration and liver metastasis. GSEA and Western blot were used to investigate the corresponding signaling pathway. PCSK6 was one of the obtained liver-metastasis-related genes in pancreatic cancer. PCSK6 inactivation inhibited cell growth and cell migration, due to G0/G1 cell cycle arrest and the remodeling of cell-cell junctions or the cell skeleton, respectively. PCSK6 inactivation led to fewer counts and lower outgrowth rates of liver metastatic niches in vivo. The Raf-MEK1/2-ERK1/2 axis was repressed by PCSK6 inactivation. Accordingly, we found PCSK6 inactivation could inhibit cell growth, cell migration, and liver metastasis, and explored the role of the Raf-MEK1/2-ERK1/2 axis in PCSK6 inactivation. PCSK6-targeted therapy might represent a novel approach for combatting liver metastasis in pancreatic cancer.

Pre-clinical Models of Metastasis in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a hostile solid malignancy coupled with an extremely high mortality rate. Metastatic disease is already found in most patients at the time of diagnosis, resulting in a 5-year survival rate below 5%. Improved comprehension of the mechanisms leading to metastasis is pivotal for the development of new targeted therapies. A key field to be improved are modeling strategies applied in assessing cancer progression, since traditional platforms fail in recapitulating the complexity of PDAC. Consequently, there is a compelling demand for new preclinical models that mirror tumor progression incorporating the pressure of the immune system, tumor microenvironment, as well as molecular aspects of PDAC. We suggest the incorporation of 3D organoids derived from genetically engineered mouse models or patients as promising new tools capable to transform PDAC pre-clinical modeling and access new frontiers in personalized medicine.

Metabolic impairment of non-small cell lung cancers by mitochondrial HSPD1 targeting.

BACKGROUND: The identification of novel targets is of paramount importance to develop more effective drugs and improve the treatment of non-small cell lung cancer (NSCLC), the leading cause of cancer-related deaths worldwide. Since cells alter their metabolic rewiring during tumorigenesis and along cancer progression, targeting key metabolic players and metabolism-associated proteins represents a valuable approach with a high therapeutic potential. Metabolic fitness relies on the functionality of heat shock proteins (HSPs), molecular chaperones that facilitate the correct folding of metabolism enzymes and their assembly in macromolecular structures. METHODS: Gene fitness was determined by bioinformatics analysis from available datasets from genetic screenings. HSPD1 expression was evaluated by immunohistochemistry from formalin-fixed paraffin-embedded tissues from NSCLC patients. Real-time proliferation assays with and without cytotoxicity reagents, colony formation assays and cell cycle analyses were used to monitor growth and drug sensitivity of different NSCLC cells in vitro. In vivo growth was monitored with subcutaneous injections in immune-deficient mice. Cell metabolic activity was analyzed through extracellular metabolic flux analysis. Specific knockouts were introduced by CRISPR/Cas9. RESULTS: We show heat shock protein family D member 1 (HSPD1 or HSP60) as a survival gene ubiquitously expressed in NSCLC and associated with poor patients' prognosis. HSPD1 knockdown or its chemical disruption by the small molecule KHS101 induces a drastic breakdown of oxidative phosphorylation, and suppresses cell proliferation both in vitro and in vivo. By combining drug profiling with transcriptomics and through a whole-genome CRISPR/Cas9 screen, we demonstrate that HSPD1-targeted anti-cancer effects are dependent on oxidative phosphorylation and validated molecular determinants of KHS101 sensitivity, in particular, the creatine-transporter SLC6A8 and the subunit of the cytochrome c oxidase complex COX5B. CONCLUSIONS: These results highlight mitochondrial metabolism as an attractive target and HSPD1 as a potential theranostic marker for developing therapies to combat NSCLC.

The pesticide chlorpyrifos promotes obesity by inhibiting diet-induced thermogenesis in brown adipose tissue.

Obesity results from a caloric imbalance between energy intake, absorption and expenditure. In both rodents and humans, diet-induced thermogenesis contributes to energy expenditure and involves the activation of brown adipose tissue (BAT). We hypothesize that environmental toxicants commonly used as food additives or pesticides might reduce BAT thermogenesis through suppression of uncoupling protein 1 (UCP1) and this may contribute to the development of obesity. Using a step-wise screening approach, we discover that the organophosphate insecticide chlorpyrifos suppresses UCP1 and mitochondrial respiration in BAT at concentrations as low as 1 pM. In mice housed at thermoneutrality and fed a high-fat diet, chlorpyrifos impairs BAT mitochondrial function and diet-induced thermogenesis, promoting greater obesity, non-alcoholic fatty liver disease (NAFLD) and insulin resistance. This is associated with reductions in cAMP; activation of p38MAPK and AMPK; protein kinases critical for maintaining UCP1 and mitophagy, respectively in BAT. These data indicate that the commonly used pesticide chlorpyrifos, suppresses diet-induced thermogenesis and the activation of BAT, suggesting its use may contribute to the obesity epidemic.

Crotonaldehyde exposure induces liver dysfunction and mitochondrial energy metabolism disorder in rats.

Crotonaldehyde is a highly toxic pollutant, widely present in tobacco smoke and automobile exhaust. Exposure to crotonaldehyde can cause hepatotoxicity and induce liver tumors in rats; however, the underlying mechanism is unclear. Liver cells contain many mitochondria, which serve to maintain energy levels in the body. We hypothesized that the energy metabolism disorder caused by mitochondrial dysfunction is an important cause of liver injury in rats exposed to crotonaldehyde. To test this, we randomly divided 40 male Wistar rats into four groups, and provided crotonaldehyde at 0, 2.5, 4.5, and 8.5 mg/kg for 90 days by intragastric administration. The results showed that crotonaldehyde exposure caused damage to liver mitochondrial structure, reduced electron-transport chain activity and ATP levels, and interfered with mitochondrial DNA transcription. In response to increased crotonaldehyde exposure, rats exhibited increased reactive oxygen species levels, decreased superoxide dismutase and glutathione activity, and activation of the caspase-mediated apoptosis pathway, as well as elevated levels of alanine aminotransferase, aspartate aminotransferase, total bilirubin, and histopathological damage. Our findings, together with those of previous reports, should help elucidate the underlying mechanism of crotonaldehyde-induced mitochondrial dysfunction and energy metabolism disorder, and provide an important direction for the prevention and clinical intervention of liver diseases caused by crotonaldehyde and aldehydes with similar structures.

[CCL2 promotes angiogenesis of primary rat cardiac microvascular endothelial cells].

The aim of the present study was to investigate the role of chemokine CCL2 in angiogenesis of primary adult rat cardiac microvascular endothelial cells (CMEC). The rat CMECs were isolated and identified through morphology examination and immunostaining with CD31 and factor VIII antibodies. The angiogenesis of CMEC on Matrigel was evaluated at different time points. The expression and secretion of CCL2 during the process of angiogenesis was detected by real-time RT-PCR and ELISA, respectively. The results showed that, the primary rat CMEC was isolated successfully, and the angiogenesis of CMEC was significantly induced after Matrigel treatment for 4 h. The expression of CCL2 and CCR2 were increased during angiogenesis, and the secretion of CCL2 was detected after 2 h of angiogenesis and reached the peak concentration of 1 588.1 pg/mL after 4 h. Either CCL2 blocking antibody or CCR2 antagonist significantly reduced the angiogenesis of CMEC. These results suggest that CCL2 is secreted during the process of angiogenesis of CMEC, and CCL2/CCR2 signaling pathway may play an important role in promoting angiogenesis.

Knockout of DNase1l1l abrogates lens denucleation process and causes cataract in zebrafish.

Removal of nuclei in lens fiber cells is required for organelle-free zone (OFZ) formation during lens development. Defect in degradation of nuclear DNA leads to cataract formation. DNase2ß degrades nuclear DNA of lens fiber cells during lens differentiation in mouse. Hsf4 is the principal heat shock transcription factor in lens and facilitates the lens differentiation. Knockout of Hsf4 in mouse and zebrafish resulted in lens developmental defect that was characterized by retaining of nuclei in lens fiber cells. In previous in vitro studies, we found that Hsf4 promoted DNase2ß expression in human and mouse lens epithelial cells. In this study, it was found that, instead of DNase2ß, DNase1l1l is uniquely expressed in zebrafish lens and was absent in Hsf4-/- zebrafish lens. Using CRISPR-Cas9 technology, a DNase1l1l knockout zebrafish line was constructed, which developed cataract. Deletion of DNase1l1l totally abrogated lens primary and secondary fiber cell denucleation process, whereas had little effect on the clearance of other organelles. The transcriptional regulation of DNase1l1l was dramatically impaired in Hsf4-/- zebrafish lens. Rescue of DNase1l1l mRNA into Hsf4-/- zebrafish embryos alleviated its defect in lens fiber cell denucleation. Our results in vivo demonstrated that DNase1l1l is the primary DNase responsible for nuclear DNA degradation in lens fiber cells, and Hsf4 can transcriptionally activate DNase1l1l expression in zebrafish.

Crotonaldehyde-induced alterations in testicular enzyme function and hormone levels, and apoptosis in the testes of male Wistar rats are associated with oxidative damage.

Crotonaldehyde is a hazardous pollutant present in cigarette smoke and automobile exhausts that is generated by lipid peroxidation, and harmful to reproductive organs. Although we are often exposed to low doses of crotonaldehyde daily, its adverse effects on the reproductive organs have not been fully elucidated. To elucidate them, we administered crotonaldehyde (0, 2.5, 4.5, and 8.5 mg/kg) by gavage for 150 days to male Wister rats, and evaluated its effect on their testicular tissues. Body weight, testis coefficient, sperm count, and motility decreased. Reactive oxygen species and malondialdehyde levels in the 8.5 and 4.5 mg/kg groups significantly increased as antioxidant enzyme activity decreased. Testicular cell apoptosis rate in the exposed groups increased. Testicular enzyme activity and reproductive hormone levels were significantly altered in the 8.5 and 4.5 mg/kg groups. Therefore, long-term exposure to crotonaldehyde may induce oxidative stress, resulting in testicular cell apoptosis, and testicular enzyme and hormone level alteration.

First demonstration of protective effects of purified mushroom polysaccharide-peptides against fatty liver injury and the mechanisms involved.

Fatty liver (FLD) disease is a consequence of metabolic syndrome, which is a health problem worldwide with a phenomenal rise in prevalence. In this study, two hepatoprotective polysaccharide-peptides were extracted from the mushroom Auricularia polytricha followed by chromatographic fractionation of the extract on the ion exchanger DEAE-cellulose and gel filtration on Sephadex-200 to yield two purified fractions: APPI and APPII. The monosaccharide compositions, FT-IR, N-terminal sequences, internal peptide sequences and molecular weights of the two fractions were determined. Furthermore, their hepatoprotective effect on human hepatoma HepG2 cells in vitro and in an animal model of fatty liver disease was evidenced by the findings that APPI and APPII diminished lipid deposit in cells, blood and the liver, increased cellular antioxidant activity and viability, and protected the liver against injury. The mechanistic study revealed that APPI and APPII activated the adiponectin pathway, up-regulated expression of genes controlling free fatty acid (FFA) oxidation, such as AMPK, CPTl, ACOX1 and PPARα genes, enhanced lipid metabolism, preserved hepatic function, promoted the antioxidant defense system and reduced lipid peroxidation. Hence the bioactive compounds of A. polytricha could serve as therapeutic agents in the food and pharmaceutical industries.

TNFAIP8 promotes cisplatin resistance in cervical carcinoma cells by inhibiting cellular apoptosis.

Cervical cancer is the second most prevalent malignant tumor in women worldwide. Failure of successful treatment is most prevalent in patients with the metastatic disease and the chemotherapy refractory disease. Tumor necrosis factor α-induced protein 8 (TNFAIP8) serves as an anti-apoptotic and pro-oncogenic protein, and is associated with cancer progression and poor prognosis in a number of different cancer types. However, the physiological and pathophysiological roles of TNFAIP8 in cervical carcinogenesis and development remain poorly understood. In the present study, it was demonstrated that TNFAIP8 protein expression levels were significantly increased in cervical cancer tissues compared with the non-tumor adjacent tissues using immunohistochemistry. Additionally, it was demonstrated that TNFAIP8 overexpression is associated with cisplatin resistance. Furthermore, depletion of TNFAIP8 impaired HeLa cell proliferation and viability in vitro, improved cisplatin sensitivity, and promoted cisplatin-induced cellular apoptosis and death. Subsequent mechanistic analysis demonstrated that TNFAIP8 silencing promoted caspase-8/-3 activation and p38 phosphorylation in HeLa cells treated with cisplatin, whereas apoptosis regulator B-cell lymphoma-2 expression was inhibited with TNFAIP8-silenced HeLa cells following treatment with cisplatin. These data suggested that TNFAIP8 serves as an anti-apoptotic protein against cisplatin-induced cell death, which eventually leads to chemotherapeutic drug-treatment failure. Therefore, the present data suggested that TNFAIP8 may be a promising therapeutic target for the treatment of cervical cancer.

Establishment of a highly efficient conjugation protocol for Streptomyces kanamyceticus ATCC12853.

Kanamycin B as the secondary metabolite of wild-type Streptomyces kanamyceticus (S. kanamyceticus) ATCC12853 is often used for the synthesis of dibekacin and arbekacin. To construct the strain has the ability for kanamycin B production; the pSET152 derivatives from Escherichia coli ET12567 were introduced to S. kanamyceticus by intergeneric conjugal transfer. In this study, we established a reliable genetic manipulation system for S. kanamyceticus. The key factors of conjugal transfer were evaluated, including donor-to-recipient ratio, heat-shock, and the overlaying time of antibiotics. When spores were used as recipient, the optimal conjugation frequency was up to 6.7 × 10-6 . And mycelia were used as an alternative recipient for conjugation instead of spores; the most suitable donor-to-recipient ratio is 1:1 (107 :107 ). After incubated for only 10-12 hr and overlaid with antibiotics subsequently, the conjugation frequency can reach to 6.2 × 10-5 which is sufficient for gene knockout and other genetic operation. Based on the optimized conjugal transfer condition, kanJ was knocked out successfully. The kanamycin B yield of kanJ-disruption strain can reach to 543.18 ± 42 mg/L while the kanamycin B yield of wild-type strain was only 46.57 ± 12 mg/L. The current work helps improve the content of kanamycin B in the fermentation broth of S. kanamyceticus effectively to ensure the supply for the synthesis of several critical semisynthetic antibiotics.