2D BA2PbI4 Regulating PbI2 Crystallization to Induce Perovskite Growth for Efficient Solar Cells.

Using seeds to control the crystallization of perovskite film is an effective strategy for achieving high-efficiency perovskite solar cells (PSCs). Owing to their excellent environmental stability brought by their long alkyl chain, n-butylammonium (BA) cations are widely used for fabricating efficient and stable PSCs. However, BA-based 2D perovskite is seldom been investigated as a seed. Here, BA2PbI4 is employed to regulate the crystallization of PbI2, acting as nucleation centers. As a result, porous PbI2 film with high crystallinity is obtained, which allows the realization of perovskite film with preferential crystal orientations of (001) and large grain size of over 2 µm. The corresponding PSC achieves a high power conversion efficiency (PCE) of 24.30% and exhibits satisfactory stability, retaining 91.70% of the initial PCE after 300 h of thermal aging at 85°C.

Negative Poisson's ratio of sulfides dominated by strong intralayer electron repulsion.

Geometrical variations in a particular structure or other mechanical factors are often cited as the cause of a negative Poisson's ratio (NPR). These factors are independent of the electronic properties of the materials. This work investigates a class of two-dimensional (2D) sulfides with the chemical formula MX2 (M = Ti, Cr, Mn, Fe, Co, X = S) using first-principles calculations. Among them, monolayered TiS2, CrS2, and MnS2 were found to exhibit a structure-independent NPR. The strong strain response of intra-layer interactions is responsible for this unique phenomenon. This can be traced to the lone pair of electrons of the S atoms and the weak electronegativity of the central atoms in multi-orbital hybridization. Our study provides valuable insights and useful guidelines for designing innovative NPR materials.

Interfacial Electron Potential Well Facilitates the Design of Cobalt Phosphide Heterojunctions for Hydrogen Evolution.

The interfacial electron modulation of electrocatalysts is an effective way to realize efficient hydrogen production, which is of great importance for future renewable energy systems. However, systematic theory-guided design of catalysts in heterojunction coupling is lacking. In this work, a multi-level theoretical calculation is performed to screen optimal candidates to form a heterojunction with CoP (101) surface for electrocatalytic hydrogen production. To overcome the weak adsorption of H+ on CoP (101), rational design of electrons potential well at the heterojunction interface can effectively enhance the hydrogen adsorption. All p-type cobalt-based phosphides are considered potential candidates at the beginning. After screening for conductivity, stability, interface matching screening, and ΔGH* evaluation, the CoP/Co2 P-H system is identified to be able to display optimal hydrogen production performance. To verify the theoretical design, CoP, CoP/Co2 P-H, and CoP/Co2 P-O are synthesized and the electrochemical analysis is carried out. The hydrogen evolution reaction (HER) performance is consistent with the prediction. This work utilizes the electron potential well effect and multi-level screening calculations to design highly efficient heterojunction catalysts, which can provide useful theoretical guidance for the rational design of heterojunction-type catalysts.

Predictive value of NoSAS questionnaire combined with the modified Mallampati grade for hypoxemia during routine sedation for gastrointestinal endoscopy.

BACKGROUND: The incidence of hypoxemia during painless gastrointestinal endoscopy remains a matter of concem. To date, there is no recognized simple method to predict hypoxemia in digestive endoscopic anesthesia. The NoSAS (neck circumference, obesity, snoring, age, sex) questionnaire, an objective and simple assessment scale used to assess obstructive sleep apnea (OSA), combined with the modified Mallampati grade (MMP), may have certain screening value. This combination may allow anesthesiologists to anticipate, manage, and consequently decrease the occurrence of hypoxemia. METHODS: This study was a prospective observational trial. The primary endpoint was the incidence of hypoxaemia defined as pulse oxygen saturation (SpO2) < 95% for 10 s. A total of 2207 patients admitted to our hospital for painless gastrointestinal endoscopy were studied. All patients were measured for age, height, weight, body mass index, neck circumference, snoring, MMP, and other parameters. Patients were divided into hypoxemic and non-hypoxemic groups based on the SpO2. The ROC curve was plotted to evaluate the screening value of the NoSAS questionnaire separately and combined with MMP for hypoxemia. The total NoSAS score was evaluated at cut-off points of 8 and 9. RESULTS: With a NoSAS score ≥ 8 as the critical value for analysis, the sensitivity for hypoxemia was 58.3%, the specificity was 88.4%, and the area under the ROC was 0.734 (P < 0.001, 95% CI: 0.708-0.759). With a NoSAS score ≥ 9 as a critical value, the sensitivity for hypoxemia was 36.50%, the specificity rose to 96.16%, and the area under the ROC was 0.663 (P < 0.001, 95% CI: 0.639-0.688). With the NoSAS Score combined with MMP for analysis, the sensitivity was 78.4%, the specificity was 84%, and the area under the ROC was 0.859 (P < 0.001, 95%CI:0.834-0.883). CONCLUSIONS: As a new screening tool, the NoSAS questionnaire is simple, convenient, and useful for screening hypoxemia. This questionnaire, when paired withMMP, is likely to be helpful for the screening of hypoxemia.

GFPT1 promotes the proliferation of cervical cancer via regulating the ubiquitination and degradation of PTEN.

Cervical cancer demonstrates the fourth incidence and death rate in females worldwide. Glutamine--fructose-6-phosphate transaminase 1 (GFPT1), the first rate-limited enzyme of the hexosamine biosynthesis pathway, has been reported to promote the progression of cancers. However, the prognostic value and roles of GFPT1 in cervical cancer are largely unknown. Transcription expression data for cervical cancer were downloaded from public databases. GFPT1 overexpressed and knockdown cell lines were constructed. Colony formation assays, Edu assays and 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays were used to measure the proliferation capabilities of cervical cancer cells. Western blot, Immunofluorescence and co-immunoprecipitation assays were performed to verify the interaction between GFPT1and Phosphatase and tensin homolog (PTEN). Animal assays were applied to verify the results in vivo. GFPT1 expression was higher in cervical cancer cell lines. The proliferation capabilities of cervical cancer cells were suppressed in GFPT1 knockdown cells and GFPT1 inhibitor L-DON treated cells. And overexpression of GFPT1 promoted cell proliferation. PTEN was up-regulated in GFPT1 knockdown cells and downregulated in GFPT1 overexpression cells. Immunofluorescence and co-immunoprecipitation results showed that GFPT1 was co-localized and interacted with PTEN. GFPT1 promoted the ubiquitination and degradation of PTEN. Silence of PTEN offsets the growth inhibition of cervical cancer caused by GFPT1 knockdown. Animal assays showed that GFPT1 promoted the proliferation of cervical cancer in vivo. Our study revealed that GFPT1 could promote the progression of cervical cancer by regulating PTEN expression. Our study highlights the GFPT1-PTEN regulation as a potential therapy target for cervical cancer. .

The mammalian cytosolic thioredoxin reductase pathway acts via a membrane protein to reduce ER-localised proteins.

Folding of proteins entering the mammalian secretory pathway requires the insertion of the correct disulfides. Disulfide formation involves both an oxidative pathway for their insertion and a reductive pathway to remove incorrectly formed disulfides. Reduction of these disulfides is crucial for correct folding and degradation of misfolded proteins. Previously, we showed that the reductive pathway is driven by NADPH generated in the cytosol. Here, by reconstituting the pathway using purified proteins and ER microsomal membranes, we demonstrate that the thioredoxin reductase system provides the minimal cytosolic components required for reducing proteins within the ER lumen. In particular, saturation of the pathway and its protease sensitivity demonstrates the requirement for a membrane protein to shuttle electrons from the cytosol to the ER. These results provide compelling evidence for the crucial role of the cytosol in regulating ER redox homeostasis, ensuring correct protein folding and facilitating the degradation of misfolded ER proteins.

Downregulation of HSPA12A underlies myotoxicity of local anesthetic agent bupivacaine through inhibiting PGC1α-mediated mitochondrial integrity.

Local anesthetics (LAs) are widely used for intraoperative anesthesia and postoperative analgesia. However, LAs (e.g. Bupivacaine) can evoke myotoxicity that closely associated to mitochondrial damage. PGC1a is a mast co-factor for mitochondrial quality control. We have recently demonstrated that PGC1a can be activated by HSPA12A in hepatocytes, suggesting a possibility that HSPA12A protects from LAs myotoxicity through activating PGC1α-mediated mitochondrial integrity. Here, we reported that HSPA12A was downregulated during Bupivacaine-induced myotoxicity in skeletal muscles of mice in vivo and C2c12 myoblast cultures in vitro. Intriguingly, overexpression of HSPA12A attenuated the Bupivacaine-induced C2c12 cell death. We also noticed that the Bupivacaine-induced decrease of glucose consumption and ATP production was improved by HSPA12A overexpression. Moreover, overexpression of HSPA12A in C2c12 cells attenuated the Bupivacaine-induced decrease of mitochondrial contents and increase of mitochondrial fragmentation. The Bupivacaine-induced reduction of PGC1α expression and nuclear localization was markedly attenuated by HSPA12A overexpression. Importantly, pretreatment with a selective PGC1α inhibitor (SR-18292) abolished the protection of HSPA12A from Bupivacaine-induced death and mitochondrial loss in C2c12 cells. Altogether, the findings indicate that downregulation of HSPA12A underlies myotoxicity of Local anesthetic agent Bupivacaine through inhibiting PGC1α-mediated Mitochondrial Integrity. Thus, HSPA12A might represent a viable strategy for preventing myotoxicity of LAs.

Effect of cobalt phosphide (CoP) vacancies on its hydrogen evolution activity via water splitting: a theoretical study.

Defect engineering plays an important role in improving the performance of catalysts. To clarify the roles of Co and P vacancies in CoP for water splitting, a theoretical study based on density functional theory was carried out in this paper. The geometric and electronic structures, activity and stability of the CoP (101)B surface, CoP (101)B with the Co vacancy (Covac) and the P vacancy (Pvac) are investigated. The results indicate that the CoP (101)B surface with Pvac and Covac can enhance the electron transfer to the surface. The Pvac will upward shift the Co d-band center near the vacancy site, which promotes the adsorption of H on the Co atom. As a result, the bridge Co-Co sites near the vacancy become the active sites for the hydrogen evolution reaction (HER) (ΔGH* = 0.01 eV). The loss of the Co atom also results in an upward shift of its d-band center, which will enhance the H adsorption on the adjacent Co sites. The unevenly distributed electrons due to the presence of vacancies on the surface cause spontaneous dissociation of H2O molecules. Furthermore, the thermodynamic analysis and surface energy find that the CoP (101)B and (101)B facets with Covac and Pvac present good stability. The current work has shed light onto the mechanism of water splitting on the surface of phosphide with vacancies. Our study suggests that engineering vacancies on CoP is a feasible route to improve its catalytic activity.

Protein secondary structure determines the temporal relationship between folding and disulfide formation.

How and when disulfide bonds form in proteins relative to the stage of their folding is a fundamental question in cell biology. Two models describe this relationship: the folded precursor model, in which a nascent structure forms before disulfides do, and the quasi-stochastic model, where disulfides form prior to folding. Here we investigated oxidative folding of three structurally diverse substrates, ß2-microglobulin, prolactin, and the disintegrin domain of ADAM metallopeptidase domain 10 (ADAM10), to understand how these mechanisms apply in a cellular context. We used a eukaryotic cell-free translation system in which we could identify disulfide isomers in stalled translation intermediates to characterize the timing of disulfide formation relative to translocation into the endoplasmic reticulum and the presence of non-native disulfides. Our results indicate that in a domain lacking secondary structure, disulfides form before conformational folding through a process prone to nonnative disulfide formation, whereas in proteins with defined secondary structure, native disulfide formation occurs after partial folding. These findings reveal that the nascent protein structure promotes correct disulfide formation during cotranslational folding.

Upregulated long noncoding RNAs LINC02163 and FEZF1-AS1 exert oncogenic roles in colorectal cancer.

A growing number of evidence has revealed that aberrantly expressed long noncoding RNAs (lncRNAs) are involved in the development of a variety of malignancies, including colorectal cancer (CRC). However, the clinical relevance of most lncRNAs and their potential biological functions in CRC remains poorly understood. The aim of this study was to identify the key lncRNAs related to patient prognosis as well as their biological function and underlying mechanism in CRC. Therefore, five independent datasets containing CRC and normal tissue RNA sequencing, microarray data and the corresponding clinical data from The Cancer Genome Atlas and Gene Expression Omnibus were screened. Hundreds of significantly differentially expressed lncRNAs in CRC were determined, and Kaplan-Meier analyses revealed that some of these lncRNAs were related to the overall survival and progression-free survival of patients with CRC, such as RP11-108K3.2, FOXD3-AS1, H19 and AP001469.9. Among these dysregulated lncRNAs, LINC02163 and FEZF1-AS1 were significantly upregulated in CRC tissues, suggesting that they may have oncogenic roles in CRC. Furthermore, loss of function assays revealed that downregulation of LINC02163 and FEZF1-AS1 impaired CRC cell proliferation. In addition, RNA Immunoprecipitation and Chromatin Immunoprecipitation assays determined that FEZF1-AS1 regulates CRC cell growth via interacting with LSD1 and repressing KLF2 expression. Collectively, hundreds of dysregulated lncRNAs and their associated biological roles identified in this study may provide potentially useful biomarkers and therapeutic targets for CRC.

Penehyclidine mitigates postoperative nausea and vomiting and intraoperative oculocardiac reflex in patients undergoing strabismus surgery: a prospective, randomized, double-blind comparison.

BACKGROUND: Postoperative nausea and vomiting (PONV) is one of the most frequent complications following strabismus surgery. Penehyclidine, an anticholinergic agent, is widely used as premedication. This study investigated the effect of preoperative penehyclidine on PONV in patients undergoing strabismus surgery. METHODS: In this prospective, randomized, double-blind study, patients scheduled for strabismus surgery under general anesthesia were randomly assigned to either penehyclidine (n = 114) or normal saline (n = 104) group. Penehyclidine was administrated immediately after anesthesia induction, and normal saline was substituted as control. PONV was investigated from 0 to 48 h after surgery. Intraoperative oculocardiac reflex (OCR) was also recorded. RESULTS: Compared with normal saline, penehyclidine significantly reduced PONV incidence (30.7% vs. 54.8%, P < 0.01) and mitigated PONV severity as indicated by severity scoring (P < 0.01). Compared with normal saline, penehyclidine also significantly reduced OCR incidence (57.9% vs. 77.9%, P < 0.01) and mitigated OCR severity, as indicated by the requirement for atropine rescue (77.3% vs. 90.1%, P < 0.05) and the maximum decrease of heart rate during OCR (23.1 ± 9.4 bpm vs. 27.3 ± 12.4 bpm, P < 0.05). The recovery course did not differ between groups. CONCLUSIONS: Penehyclidine administrated after anesthesia induction significantly reduced the incidence of PONV and alleviated intraoperative OCR in patients undergoing strabismus surgery. TRIAL REGISTRATION: ClinicalTrials.gov ( NCT04054479 ). Retrospectively registered August 13, 2019.

First-principles investigation of the electronic properties of the Bi2O4(101)/BiVO4(010) heterojunction towards more efficient solar water splitting.

First-principles calculations based on density functional theory were carried out to explore the geometric structure, light absorption, charge separation, over-potential and stability of Bi2O4 (101)/BiVO4 (010) heterojunction. The results show that the formed heterojunction can improve visible light utilization and promote transfer of photo-generated holes from BiVO4 to Bi2O4. Furthermore, the Bi5+ site in the Bi2O4(101) surface is energetically more favorable as the photoanode for the oxygen evolution reaction (OER) than the Bi3+ sites in Bi2O4(101) and BiVO4(010). At the same time, it is also found that the Bi5+ in Bi2O4(101) are more stable than the Bi3+ due to the lower surface energy and stronger bond energy with neighbors. Therefore, forming the Bi2O4/BiVO4 heterojunction can effectively improve the activity and stability of BiVO4 for water splitting reactions.

Comparing the different oxycodone doses of prevent oxycodone for prevention of preventing fentanyl-induced cough during induction of general anaesthesia.

BACKGROUND: Fentanyl-induced cough (FIC) usually occurs after the intravenous administration of fentanyl during general anaesthesia induction. It is a transient condition depending on the fentanyl administration dose and injection speed. Oxycodone can also prevent FIC because it has been proven to treat coughing. This study aimed to evaluate the efficacy of different oxycodone doses to prevent FIC during general anaesthesia induction. METHODS: In a double-blind randomised controlled trial, 210 adult patients who were undergoing elective surgery, classified as American Society of Anaesthesiologists physical status I-II, and aged 20-65 years were randomly assigned into five equally sized groups: Sham group, Group Ⅰ, Group II, Group III and Group IV. Groups Ⅰ-IV were each intravenously injected with oxycodone 0.025, 0.05, 0.075 and 0.100 mg/kg, while an equal volume of normal saline was given instead of oxycodone in the Sham group. Five minutes later, fentanyl 3 µg/kg was intravenously injected within 5 seconds, then, 2 minutes later the other drugs were administered for general anaesthesia induction. The occurrence and severity of coughing were observed within 2 minutes of the fentanyl injection. Vital signs and intensities of coughing were recorded and analysed. RESULTS: Coughing incidences were each 57.1, 50, 42.8, 33.3 and 21.4% in the Sham group and Groups Ⅰ-IV. Significant differences were found in the incidences of coughing between the Sham group and Groups III-IV. No significant differences in FIC incidences have been detected between the Sham group and Groups Ⅰ-II. However, no significant difference in FIC incidence existed between Group III and Group IV. Cough severities in Groups III and IV were significantly lower than in Groups Ⅰ and II (P < .05). No significant differences existed in the hypotension or severe bradycardia incidences during anaesthesia induction among the five groups (P > .05). CONCLUSION: Oxycodone 0.075 mg/kg provided more effective FIC prevention during general anaesthesia induction.

A new self-designed "tongue root holder" device to aid fiberoptic intubation.

OBJECTIVE: In this study, we aimed to assess the feasibility of fiberoptic intubation (FOI), using a new, self-designed, "tongue root holder" device, in combination with the jaw thrust maneuver. METHODS: Three hundred patients undergoing elective surgery requiring orotracheal intubation were enrolled. Patients presented at least one or more risk factors for difficult airway. The patients were randomly allocated at a 1:1 ratio to one of two groups: group L, FOI with tongue root holder, or group C, standard FOI. Orotracheal FOI was performed after commencement of anesthesia. The jaw thrust maneuver was applied in both groups to facilitate advancement of the fiberoptic bronchoscope. The primary endpoint was the feasibility of FOI. The secondary endpoints were number of attempts, time to intubation, and airway clearance at the soft palate and epiglottis levels. RESULTS: The FOI was achieved in all 150 patients in group L, significantly higher than that in group C (100% vs 95.3%; P = 0.015). Less attempts of intubation were made in group L (P = 0.039). Mean time to successful intubation on the first attempt was shorter in group L (P < 0.001). The mean times to view the vocal cord and carina were also shorter in group L (P = 0.011 and P < 0.001, respectively). Airway clearance was better in group L at both the soft palate and the glottis levels (P = 0.010 and P = 0.038, respectively). CONCLUSIONS: This study shows that FOI is feasible with the newly introduced, self-designed, "tongue root holder" device, when combined with the jaw thrust maneuver in patients with risk factors for difficult airway. The device also provides better airway clearance, less intubation attempts, and shorter time to intubation at first attempt. CLINICAL RELEVANCE: Fiberoptic bronchoscope has been the gold standard for routine management of difficult airway. A technique to open the airway is introduced to reduce the incidence rate of upper airway obstruction.

MicroRNA-148b regulates tumor growth of non-small cell lung cancer through targeting MAPK/JNK pathway.

BACKGROUND: MicroRNA-148b (miR-148b) has been detected in various types of tumors, and is generally viewed as a tumor suppressor. Our previous study found the decreased expression of miR-148b in human non small cell lung cancer (NSCLC) specimens and cell lines. However, the underlying mechanisms of miR-148b in regulating tumor progression remain unclear. METHODS: Firstly animal experiments were performed to verify whether miR-148b could inhibit the tumor growth. Then, the underlying mechanisms were studied by transfecting recombinant plasmids containing a miR-148b mimic or a negative control (NC) mimic (shRNA control) into NSCLC cell lines PC14/B and A549 cells. Tumor cells transfected with unpackaged lentiviral vectors was used as blank control. Cell proliferation capabilities were measured by using CCK-8 kit and colony formation assay. Cell cycle arrest was compared to clarify the mechanism underlying the tumor cell proliferation. Annexin V-FITC Apoptosis Detection kit was applied to investigate the effect of miR-148b on cell apoptosis. Furthermore, western blot analysis were performed to study the targeting pathway. RESULTS: We found that over-expression of miR148b could significantly inhibit tumor growth, while knocking down miR148b could obviously promote tumor growth. Further experiment showed that miR-148b inhibited tumor cell proliferation. Besides, over-expression of miR148b decreased the G2/M phase population of the cell cycle by preventing NSCLC cells from entering the mitotic phase and enhanced tumor cell apoptosis. Further western blot analysis indicated that miR148b could inhibit mitogen-activated protein kinase/Jun N-terminal kinase (MAPK/JNK) signaling by decreasing the expression of phosphorylated (p) JNK. CONCLUSIONS: These results demonstrate that miR-148b could inhibit the tumor growth and act as tumor suppressor by inhibiting the proliferation and inducing apoptosis of NSCLC cells by blocking the MAPK/JNK pathway.

Early decrease in postoperative serum albumin predicts severe complications in patients with colorectal cancer after curative laparoscopic surgery.

BACKGROUND: Postoperative severe complications are always associated with prolonged hospital stays, increased economic burdens, and poor prognoses in patients with colorectal cancer (CRC). This present study aimed to investigate potential risk factors including serum albumin (Alb) for severe complications in CRC patients. METHODS: Eligible patients with primary CRC undergoing elective laparoscopic colectomy from July 2015 to July 2017 were included. Postoperative severe complications were defined as grade III and IV according to the Clavien-Dindo classification. ∆Alb was defined as (preoperative Alb - nadir Alb within POD2)/preoperative Alb × 100%. The baseline characteristics, intraoperative data, and laboratory data were obtained from the database for the analysis. Univariate and multivariate logistic regression analyses were utilized for the assessment of the association between risk factors and postoperative severe complications. The predictive value of ∆Alb for postoperative severe complications was evaluated by receiver operating characteristic (ROC) curve analysis. RESULTS: A total of 193 patients were finally included in the analysis data set, of which 38 (19.7%) patients had postoperative severe complications. In the final multivariate logistic regression analysis, ∆Alb was the only independent factor associated with postoperative severe complications (OR 1.66, 95%CI 1.18-2.33, p = 0.003). The area under the curve (AUC) of ∆Alb was 0.916, with the sensitivity and specificity of 0.842 and 0.858 (p < 0.001). CONCLUSIONS: The ∆Alb was an independent risk factor for severe complications in CRC patients after curative laparoscopic surgery.

Autophagy activated by tuberin/mTOR/p70S6K suppression is a protective mechanism against local anaesthetics neurotoxicity.

The local anaesthetics (LAs) are widely used for peripheral nerve blocks, epidural anaesthesia, spinal anaesthesia and pain management. However, exposure to LAs for long duration or at high dosage can provoke potential neuronal damages. Autophagy is an intracellular bulk degradation process for proteins and organelles. However, both the effects of LAs on autophagy in neuronal cells and the effects of autophagy on LAs neurotoxicity are not clear. To answer these questions, both lipid LAs (procaine and tetracaine) and amide LAs (bupivacaine, lidocaine and ropivacaine) were administrated to human neuroblastoma SH-SY5Y cells. Neurotoxicity was evaluated by MTT assay, morphological alterations and median death dosage. Autophagic flux was estimated by autolysosome formation (dual fluorescence LC3 assay), LC3-II generation and p62 protein degradation (immunoblotting). Signalling alterations were examined by immunoblotting analysis. Inhibition of autophagy was achieved by transfection with beclin-1 siRNA. We observed that LAs decreased cell viability in a dose-dependent manner. The neurotoxicity of LAs was tetracaine > bupivacaine > ropivacaine > procaine > lidocaine. LAs increased autophagic flux, as reflected by increases in autolysosome formation and LC3-II generation, and decrease in p62 levels. Moreover, LAs inhibited tuberin/mTOR/p70S6K signalling, a negative regulator of autophagy activation. Most importantly, autophagy inhibition by beclin-1 knockdown exacerbated the LAs-provoked cell damage. Our data suggest that autophagic flux was up-regulated by LAs through inhibition of tuberin/mTOR/p70S6K signalling, and autophagy activation served as a protective mechanism against LAs neurotoxicity. Therefore, autophagy manipulation could be an alternative therapeutic intervention to prevent LAs-induced neuronal damage.

Expression of a chimeric human/salmon calcitonin gene integrated into the Saccharomyces cerevisiae genome using rDNA sequences as recombination sites.

Calcitonin participates in controlling homeostasis of calcium and phosphorus and plays an important role in bone metabolism. The aim of this study was to endow an industrial strain of Saccharomyces cerevisiae with the ability to express chimeric human/salmon calcitonin (hsCT) without the use of antibiotics. To do so, a homologous recombination plasmid pUC18-rDNA2-ura3-P pgk -5hsCT-rDNA1 was constructed, which contains two segments of ribosomal DNA of 1.1 kb (rDNA1) and 1.4 kb (rDNA2), to integrate the heterologous gene into host rDNA. A DNA fragment containing five copies of a chimeric human/salmon calcitonin gene (5hsCT) under the control of the promoter for phosphoglycerate kinase (P pgk ) was constructed to express 5hsCT in S. cerevisiae using ura3 as a selectable auxotrophic marker gene. After digestion by restriction endonuclease HpaI, a linear fragment, rDNA2-ura3-P pgk -5hsCT-rDNA1, was obtained and transformed into the △ura3 mutant of S. cerevisiae by the lithium acetate method. The ura3-P pgk -5hsCT sequence was introduced into the genome at rDNA sites by homologous recombination, and the recombinant strain YS-5hsCT was obtained. Southern blot analysis revealed that the 5hsCT had been integrated successfully into the genome of S. cerevisiae. The results of Western blot and ELISA confirmed that the 5hsCT protein had been expressed in the recombinant strain YS-5hsCT. The expression level reached 2.04 % of total proteins. S. cerevisiae YS-5hsCT decreased serum calcium in mice by oral administration and even 0.01 g lyophilized S. cerevisiae YS-5hsCT/kg decreased serum calcium by 0.498 mM. This work has produced a commercial yeast strain potentially useful for the treatment of osteoporosis.

Application of the Cre/loxP Site-Specific Recombination System for Gene Transformation in Aurantiochytrium limacinum.

The Cre/loxP site-specific recombination system was applied to Aurantiochytrium limacinum to obtain a transformant without the antibiotic resistance marker gene. First, the enhanced green fluorescent protein gene (egfp) and chloramphenicol resistance gene (Cmr), along with the two loxP loci, were integrated into the genome of A. limacinum OUC88 using 18S rDNA sequences as the homologous recombination sites. Then plasmid pSH65, containing a zeocin resistance gene (Bler) was transferred into A. limacinum OUC_CG. After induction with galactose, repeated passage in culture and PCR-based assessment, the pSH65 plasmid was lost and A. limacinum OUC_EG host was shown to no longer have resistance to 100 mg chloramphenicol/L or 5 mg zeocin/L. Through southern blotting and fluorescence detection, egfp was found to be integrated into the genome of A. limacinum OUC_EG, and EGFP was successfully expressed in the cells. The successful application of the Cre/loxP system demonstrates an experimental basis for genetic modification of A. limacinum so as to obtain transformed strains with no antibiotic resistance marker genes.

HSPA12A acts as a scaffolding protein to inhibit cardiac fibroblast activation and cardiac fibrosis.

INTRODUCTION: Cardiac fibrosis is the main driver for adverse remodeling and progressive functional decline in nearly all types of heart disease including myocardial infarction (MI). The activation of cardiac fibroblasts (CF) into myofibroblasts is responsible for cardiac fibrosis. Unfortunately, no ideal approach for controlling CF activation currently exists. OBJECTIVES: This study investigated the role of Heat shock protein A12A (HSPA12A), an atypical member of the HSP70 family, in CF activation and MI-induced cardiac fibrosis. METHODS: Primary CF and Hspa12a knockout mice were used in the experiments. CF activation was indicated by the upregulation of myofibroblast characters including alpha-Smooth muscle actin (αSMA), Collagen, and Fibronectin. Cardiac fibrosis was illustrated by Masson's trichrome and picrosirius staining. Cardiac function was examined using echocardiography. Glycolytic activity was indicated by levels of extracellular lactate and the related protein expression. Protein stability was examined following cycloheximide and MG132 treatment. Protein-protein interaction was examined by immunoprecipitation-immunoblotting analysis. RESULTS: HSPA12A displayed a high expression level in quiescent CF but showed a decreased expression in activated CF, while ablation of HSPA12A in mice promoted CF activation and cardiac fibrosis following MI. HSPA12A overexpression inhibited the activation of primary CF through inhibiting glycolysis, while HSPA12A knockdown showed the opposite effects. Moreover, HSPA12A upregulated the protein expression of transcription factor p53, by which mediated the HSPA12A-induced inhibition of glycolysis and CF activation. Mechanistically, this action of HSPA12A was achieved by acting as a scaffolding protein to bind p53 and ubiquitin specific protease 10 (USP10), thereby promoting the USP10-mediated p53 protein stability and the p53-medicated glycolysis inhibition. CONCLUSION: The present study provided clear evidence that HSPA12A is a novel endogenous inhibitor of CF activation and cardiac fibrosis. Targeting HSPA12A in CF could represent a promising strategy for the management of cardiac fibrosis in patients.