Identification of GnRH-like peptide and its potential signaling pathway involved in the oocyte meiotic maturation in the Chinese mitten crab, Eriocheir sinensis.

Gonadotropin-releasing hormone (GnRH) plays a pivotal role in reproductive regulation in vertebrates. However, GnRH was rarely isolated and its function remains poorly characterized in invertebrates. The existence of GnRH in ecdysozoa has been controversial for a long. Here, we isolated and identified two GnRH-like peptides from brain tissues in Eriocheir sinensis. Immunolocalization showed that the presence of EsGnRH-like peptide in brain, ovary and hepatopancreas. Synthetic EsGnRH-like peptides can induce germinal vesicle breakdown (GVBD) of oocyte. Similar to vertebrates, ovarian transcriptomic analysis revealed a GnRH signaling pathway in the crab, in which most genes exhibited dramatically high expression at GVBD. RNAi knockdown of EsGnRHR suppressed the expression of most genes in the pathway. Co-transfection of the expression plasmid for EsGnRHR with reporter plasmid bearing CRE-luc or SRE-luc response element into 293T cells showed that EsGnRHR transduces its signal via cAMP and Ca2+ signaling transduction pathways. In vitro incubation of the crab oocyte with EsGnRH-like peptide confirmed the cAMP-PKA cascade and Ca2+ mobilization signaling cascade but lack of a PKC cascade. Our data present the first direct evidence of the existence of GnRH-like peptides in the crab and demonstrated its conserved role in the oocyte meiotic maturation as a primitive neurohormone.

Potential role for the germ cell-specific Rad21 in early meiosis of oocyte and spermatocyte in the Chinese mitten crab Eriocheir sinensis.

Rad21/Rec8 family proteins are vital for sister chromatid segregation in mitosis and homologous recombination in meiosis, but no molecular data are available in crustacean species. In this study, a germ cell-specific Rad21 named EsRad21 was identified in the crab Eriocheir sinensis. EsRad21 mRNA has an open reading frame of 2310 base pairs (bp) encoding a 769 amino acids (aa) protein. RT-PCR showed that EsRad21 mRNA was particularly expressed in testis and ovary. The RT-qPCR results further revealed that the EsRad21 mRNA exhibited similar expression pattern in gonads at various developmental stages. EsRad21 mRNA expression level was the highest in testis at early spermatogenesis stage and ovaries at previtellogenesis stage, thereafter decreased significantly at middle spermatogenesis and vitellogenesis, and finally reach the lowest level at late spermatogenesis and vitellogenesis. In situ hybridization (ISH) analysis showed that EsRad21 mRNA was exclusively expressed in germline cells, but not in gonadal somatic cells. Notably, hybridized signal was detected on chromosomes of metaphase spermatocytes. EsRad21 is thus an underlying helpful indicator of the early phases of germ cell development. RNAi knockdown of EsRad21 downregulated the expression of other meiosis-related genes like Smc5-Smc6 and SPO11 and resulted in high mortality of individuals after 24 h post injection of EsRad21 dsRNA. Taken together, our results showed a potential role for EsRad21 in early meiosis of oocytes and spermatocytes in E. sinensis. This is the first report on the molecular characterization of the Rad21 transcript in a crustacean species.

The influence of environmental cognition on green consumption behavior.

With rising consumption and environmental problems, there is an increasing need for green consumption. From a micro perspective, the influence of environmental cognition on consumers' green consumption behaviors and the related mechanisms are examined through multilayer linear analysis and 2010 China General Social Survey (CGSS) microdata with the theory of planned behavior (TPB) as the model framework. The study shows that (1) environmental cognition positively influences attitudes toward green consumption, green consumption subjective norms, and green consumption perceived behavioral control, which leads to increased intentions to engage in green consumption and actual green consumption behaviors. Environmental cognition can either promote the intention toward and lead to green consumption behavior or directly promote green consumption behavior. (2) The more developed a region's economy is, the stronger people's attitudes toward green consumption will be; additionally, the greater the perceived external pressure to engage in green consumption becomes, the greater the likelihood that people will develop the intention to engage in green consumption behavior. Regional environmental quality inhibits green consumption intention to a certain extent. (3) The influence of environmental cognition on green consumption shows regional heterogeneity.

Optimal timing of preoperative intravitreal anti-VEGF injection for proliferative diabetic retinopathy patients.

AIM: To analyze concentrations of vascular endothelial growth factor (VEGF) and fibrosis-related factors in vitreous fluid of proliferative diabetic retinopathy (PDR) patients pre-treated with intravitreal anti-VEGF injections (IVI) at different time periods prior to pars plana vitrectomy (PPV), and their correlation with the degree of vitreoretinal fibrosis and explore the optimal timing of preoperative IVI. METHODS: The prospective case-control study from January 2019 to July 2020 included 31 eyes with PDR-related complications (PDR group) and 21 eyes with non-diabetic ocular disease (control group) requiring PPV. PDR eyes were divided into four groups based on timing of PPV: 3d after IVI (3-day group); 5d after IVI (5-day group); 7 or more days after IVI (≥7-day group); and no IVI. Vitreous fluid samples (0.5-1.0 mL) were collected prior to switching on the infusion before routine 23-G PPV. Concentrations of VEGF, basic fibroblast growth factor (bFGF), periostin (PN), interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α were measured by immunoassay, and concentration differences for each cytokine were compared among the groups. The degree of vitreoretinal fibrosis was graded intraoperatively, and the correlation between the changes in cytokine levels and the severity of vitreoretinal fibrosis was analyzed by univariate ordinal logistic regression analysis. RESULTS: PDR eyes without IVI had significantly higher VEGF, bFGF, PN, and IL-6 concentrations than non-diabetic eyes (all P<0.05), and had a significantly higher concentration of VEGF (P<0.05) and a significantly lower concentration of IL-8 (P<0.05) than PDR eyes with IVI. Statistically significant differences were also observed for concentrations of VEGF, bFGF, PN, IL-6, and IL-8 among 3-day, 5-day, and ≥7-day groups (all P<0.05); meanwhile there was no significant difference in TNF-α among groups (P=0.226). The 5-day group had the lowest concentration of VEGF and the ≥7-day group had the highest concentration of bFGF and PN. The degree of vitreoretinal fibrosis was significantly higher in the ≥7-day group compared to the 3-day (P=0.015) and 5-day group (P=0.039), and vitreoretinal fibrosis correlated significantly with concentrations of bFGF, PN, IL-6, and IL-8 (all P<0.05). Univariate ordinal logistic regression analysis showed that bFGF was an independent risk factor for the severity of vitreoretinal fibrosis in PDR patients pre-treated with IVI. CONCLUSION: The vitreous concentrations of VEGF, bFGF, PN, IL-6, and IL-8 change after pretreatment with IVI before PPV in PDR patients. The degree of vitreoretinal fibrosis is higher in patients with a longer time between IVI treatment and PPV, which may be related to the angio-fibrosis switch. The results suggest that PPV should be performed 5d after IVI administration in PDR patients.

A Special Ancient Bronze Sword and Its Possible Manufacturing Technique from Materials Science Analysis.

In this study, it was found that an ancient bronze sword had special microstructures, i.e., a tin (Sn)-rich layer (Sn: 38.51 wt.%), that was around 0.1-0.3 mm in thickness in the bronze substrate (Sn: 18.57 wt.%). This sword was unearthed from the same Chu tombs of the "Sword of Gou Jian", and dated back to the late Spring and Autumn Period (496 BC-464 BC). The experimental and theoretical analyses revealed that (1) the Sn-rich layer exhibited higher microhardness (around 650 HV) than the sword body (around 300 HV); (2) the Sn-rich layer showed a brittle fracture due to the formation of a large amount of α + δ eutectoid, while the sword body was of good toughness due to a large amount of α-Cu solid solution phase; and (3) theoretical calculations of Sn diffusion in the Cu substrate indicated that this Sn-rich layer could have been formed within several hours or several days if the temperature was above 600 °C. Therefore, this sword was proposed to be a novel kind of composite bronze sword, and the possible manufacturing technique was a surface treatment called "dip or wipe tinning" or tin amalgam, which was widely used in the Bronze Age. Technically, this process possesses more advantages than the well-known two-times casting for making the "double-colour" or bi-metallic composite bronze sword. This research showed that the materials processing level was beyond our expectations for ancient China 2500 years ago.

Impact of Public Environmental Concerns on the Digital Transformation of Heavily Polluting Enterprises.

China is currently facing the arduous tasks of energy conservation, emission reduction and structural transformation, making it of great significance to study the digital transformation of heavily polluting enterprises. As an important informal regulatory system, public environmental concerns affect corporate environmental behavior by increasing external environmental pressure. This study uses the data of listed companies in China's heavily polluting industries from 2012 to 2020 and Baidu Index data to analyze how public environmental concerns affect the digital transformation of heavily polluting enterprises. This study finds that public environmental concerns can significantly promote the digital transformation of heavily polluting enterprises. For non-state-owned, green image and high-tech enterprises, the impact is even more obvious. Furthermore, based on the structural and hierarchical perspective of enterprise digital transformation, we find that public environmental concerns significantly promote digital technology application. This study puts forward some suggestions for government departments to formulate environmental protection regulations, enterprises to fulfill their green responsibilities and the public to participate in environmental governance.

A retrospective study assessing the factors associated with visual outcome in retinal vein occlusion patients after anti-VEGF therapy.

BACKGROUND: Retinal vein occlusion (RVO) is one of the most frequent retinal vascular diseases. In this study, we aimed to investigate the predictive factors of visual outcome for RVO patients who underwent anti-vascular endothelial growth factor (VEGF) therapy. METHODS: RVO patients who underwent anti-VEGF treatment were recruited in this study from January 2018 to June 2020. Clinical data and optical coherence tomography (OCT) parameters were retrospectively reviewed. Best-corrected visual acuity (BCVA) was examined at baseline and after anti-VEGF therapy. Predictive factors associated with visual outcome were assessed by logistic regression model. Treatment-related adverse events were also recorded. RESULTS: The average logMAR BCVA was 0.91 at baseline and 0.70 at final examination (P = 0.003). Among 75 patients, 41 experienced visual improvement were categorized as group A, the remaining 34 patients without improved vision were categorized as group B. Patients in group A demonstrated better visual outcomes, including decreased logMAR BCVA (average logMAR BCVA: 0.53 in group A vs. 0.91 in group B, P < 0.001) and central retinal thickness (CRT) (average CRT: 230.88 µm in group A vs. 404.97 µm in group B, P < 0.001) after anti-VEGF treatment. Multivariable analysis showed that injection frequency (odds ratio [OR], 2.623; 95% confidence interval [CI], [1.282-5.366]), hypertension (odds ratio [OR], 0.189; 95% CI [0.044-0.811]), hyperlipemia (odds ratio [OR], 0.195; 95% CI [0.040-0.941]) and external limiting membrane (ELM) disruption (odds ratio [OR], 0.148; 95% CI [0.032-0.691]) were all significantly associated with the visual outcome of RVO patients who underwent anti-VEGF treatment. In general, anti-VEGF therapy was feasible for all RVO patients, though the response to anti-VEGF was suboptimal in certain patients. Prognostic factors including injection frequency, hypertension, hyperlipemia and ELM disruption may all be useful to provide predictive information of visual outcome of RVO patients in response to anti-VEGF treatment.

Experimental and Simulated Investigations of Thin Polymer Substrates with an Indium Tin Oxide Coating under Fatigue Bending Loadings.

Stress-induced failure is a critical concern that influences the mechanical reliability of an indium tin oxide (ITO) film deposited on a transparently flexible polyethylene terephthalate (PET) substrate. In this study, a cycling bending mechanism was proposed and used to experimentally investigate the influences of compressive and tensile stresses on the mechanical stability of an ITO film deposited on PET substrates. The sheet resistance of the ITO film, optical transmittance of the ITO-coated PET substrates, and failure scheme within the ITO film were measured to evaluate the mechanical stability of the concerned thin films. The results indicated that compressive and tensile stresses generated distinct failure schemes within an ITO film and both led to increased sheet resistance and optical transmittance. In addition, tensile stress increased the sheet resistance of an ITO film more easily than compressive stress did. However, the influences of both compressive and tensile stress on increased optical transmittance were demonstrated to be highly similar. Increasing the thickness of a PET substrate resulted in increased sheet resistance and optical transmittance regardless of the presence of compressive or tensile stress. Moreover, J-Integral, a method based on strain energy, was used to estimate the interfacial adhesion strength of the ITO-PET film through the simulation approach enabled by a finite element analysis.

Characterization of regulatory regions involved in the inducible expression of chiB in Bacillus thuringiensis.

Expression of the chiB gene from Bacillus thuringiensis Bti75 was defined as inducible by the use of transcriptional fusions with the bgaB reporter gene. The transcription start site of the chiB gene was identified as the C base located 132 base pairs upstream of the start codon. Analysis of 5' and 3' deletions of the chiB promoter region revealed that the sequence from position -192 to +36 with respect to the transcription start site was necessary for wild-type levels of inducible expression of the chiB gene. The minimal promoter region for the expression of chiB gene was identified as the sequence from position -100 to +12. Furthermore, a 16-bp sequence (designated dre) downstream of the minimal promoter region of chiB was shown to be required for chitin induction. To confirm the function of this 16-bp sequence, 25 base substitutions were introduced into the dre site. Most of the mutations resulted in constitutive expression, or the efficiency of induction decreased. All mutations identified the dre sequence as a critical site for the inducible expression of chiB. In addition, the dre site was shown to interact with a sequence-specific DNA binding factor of strain Bti75 cultured in the absence of the inducer.

The direct repeat sequence upstream of Bacillus chitinase genes is cis-acting elements that negatively regulate heterologous expression in E. coli.

To explore the influence of the direct repeat sequence (DRS) in Bacillus chitinase genes on heterogonous expression in Escherichia coli, we cloned and sequenced the entire open reading frame (ORF) and upstream sequences of the chitinase B (chiB) and chitinase MY75 (chiMY75) from Bacillus thuringiensis and Bacillus licheniformis. A pair of 8-bp DRS was found upstream of each chi gene. Chi ORFs with a series of truncated DRS were cloned and transformed into E. coli XL-Blue. The activity of the transformants without the DRS were significantly higher in chitinase assays than transformants containing the DRS. SDS-PAGE showed that part and full deletion of the DRS increased chi gene expression by approximately 1.7 and 3.8-fold, respectively. Northern blotting revealed deletion of the DRS regions increased chiB and chiMY75 mRNA expression. Specific binding of DNA-binding factors in the E. coli cell lyaste was observed to both the chiB and chiMY75 promoter regions and DRS elements. This is the first investigation to demonstrate that heterologous expression of Bacillus chi genes in E. coli is negatively regulated by their upstream DRS regions, which act as cis-acting elements.

Construction of a promoter-probe vector for Bacillus thuringiensis: the identification of cis-acting elements of the chiA locus.

The expression and application of Bacillus thuringiensis (Bt) chitinase genes have been extensively investigated. However, little information is available regarding the regulation of chitinase gene expression in Bt. In this study, a shuttle promoter-probe vector was constructed incorporating the thermostable ß-galactosidase gene bgaB of B. stearothermophilus as the reporter for the study of Bt promoters. Using this plasmid, the activity of the chiA gene promoter in Bt was investigated. Deletion analysis of the putative chiA promoter region revealed that the sequence located ~75 bp DNA from positions -116 to -42, with respect to the translation start site, is the core promoter of chiA gene. Furthermore, a site for chitin induction was identified near position -36. This site for negative regulation was indicated downstream of the RNA polymerase binding sites of the promoter of chiA. The expression of chiA started in cell grown for about 6 h and reached the maximum after 60 h of incubation. Induction of chiA expression by chitin was demonstrated by an increase in ß-galactosidase activity of ~2.5-fold.

[Regulation of chitinase genes expression in bacteria].

Chitinases, which can hydrolyze chitin, occur in a wide range of microorganisms including viruses, bacteria, and fungi. The derivatives of chitin are potentially useful in several areas such as food processing, medicines, and biological control in agriculture. Some bacteria can uptake and utilize chitin as carbon source by secreting chitinase. The chitin is degraded into chito-oligosaccharides [(GlcNAc)n] or N-acetylglucosamine (GlcNAc) by chitinases, and then the chitin derivatives are transferred into cells by specific transport systems of bacteria. The intracellular chitin derivatives activate or suppress the transcription of a series of chi genes and affect the amount of chitinase. The expression of chitinase genes are strictly regulated by various regulatory factors and responsive cis-acting elements. The present review will focus on the transport system and the regulation of chitinase genes expression in bacteria.

Identification and characterization of a chitinase-produced bacillus showing significant antifungal activity.

Strain MY75 is a gram-positive, aerobic, endospore-forming bacterium that can secrete high levels of extracellular chitinase (4.645 U/ml) when chitin powder exists as an inducer. This strain was identified as Bacillus licheniformis using the Biolog MicroLog microbial identification system and sequence analysis of 16S rDNA, gyrA and rpoB genes. Strain MY75 has the ability to inhibit the growth of Gibberella saubinetii and Aspergillus niger, two major pathogenic fungi in agriculture, and to restrain their spore germination completely. The chitinase was proved to play an important role in the strain's antifungal activity.

[Study on testing methods of the elastic nature for equivalent material of human skin].

To the specificity of equivalent material of human skin mechanical properties, the static-load pressing method for testing the elastic nature of the equivalent material of human skin is proposed, and the designs experimental set for the test is designed. The result show that the method is correct and feasible.