Site-Directed Mutagenesis: Improving the Acid Resistance and Thermostability of Bacillus velezensis α-Amylase and Its Preliminary Feed Application.

The current study aimed to improve the acid resistance and thermostability of Bacillus velezensis α-amylase through site-directed mutagenesis, with a specific focus on its applicability to the feed industry. Four mutation sites, P546E, H572D, A614E, and K622E, were designed in the C domain of α-amylase, and three mutants, Mut1 (E), Mut2 (ED), and Mut3 (EDEE), were produced. The results showed that the specific activity of Mut3 was 50 U/mg higher than the original α-amylase (Ori) after incubation at 40 °C for 4 h. Compared to Ori, the acid resistance of Mut3 showed a twofold increase in specific activity at pH 2.0. Moreover, the results of preliminary feed hydrolysis were compared between Ori and Mut3 by designing three factors, three levels of orthogonal experiment for enzymatic hydrolysis time, feed quantity, and amount of amylase. It was observed that the enzymatic hydrolysis time and feed quantity showed an extremely significant difference (p < 0.01) in Mut3 compared to Ori. However, the amount of enzyme showed significant (p < 0.05) improvement in the enzymatic hydrolysis in Mut3 as compared to Ori. The study identified Mut3 as a promising candidate for the application of α-amylase in the feed industry.

Glucose-regulated protein 94 facilitates the proliferation of the Bombyx mori nucleopolyhedrovirus via inhibiting apoptosis.

Glucose regulatory protein 94 (GRP94) is an endoplasmic reticulum (ER)-resident member of the heat shock protein 90 (HSP90) family, that plays an important role in secreted protein folding. Bombyx mori nuclear polyhedrosis virus (BmNPV) is one of the main pathogens in sericulture, causing serious economic losses every year. Previous studies showed that HSP90 members promote BmNPV replication in silkworm, but the function of BmGRP94 in BmNPV infection and proliferation is still not understood. In this study, we investigated the interplay between BmGRP94 and BmNPV infection in silkworm. We first identified a single gene of BmGRP94 in the Bombyx mori genome, which encodes a polypeptide with 810 amino acids in length. Spatio-temporal expression profiles showed that BmGRP94 was highly expressed in hemocytes and midgut, and was significantly induced by BmNPV infection. Furthermore, overexpression of BmGRP94 facilitates viral proliferation, while BmGRP94 inhibition evidently decreased BmNPV proliferation in BmN cells and in silkworm midgut. Mechanistically, BmGRP94 inhibition triggers ER stress, as judged by increased expression of PERK/ATF4/ERO1, H2O2 production, and ER calcium efflux, which promotes cell apoptosis to restrict BmNPV replication in silkworm. These results suggest that BmGRP94 plays an important role in facilitating BmNPV proliferation, and provides a potential molecular target for BmNPV prevention.

Characterization of a silkworm UFM1 homolog in regulating Bombyx mori unfolded protein response and nucleopolyhedrovirus replication.

The Ubiquitin (Ub)-like molecules is essential for animal development and the physiopathology of multiple tissues in the vertebrate. Ubiquitin-fold modifier 1 (UFM1) is one of the newly-identified UBL, which is covalently attached to its substrates through the orchestrated action of a dedicated enzymatic cascade. Bombyx mori nuclear polyhedrosis virus (BmNPV) is one of the main pathogens in sericulture, causing serious economic losses every year. However, there are no studies on UFMylation and the effect of UFMylation on BmNPV replication in silkworm. In this study, we identified BmUFM1 in the B. mori genome. Spatio-Temporal expression profiles showed that BmUFM1 expression was highly in hemocytes and response to various pathogenic stimuli. Furthermore, BmUFM1 is involved in the regulation of ER stress induced Unfolded Protein Response (UPR) and knockdown of BmUFM1 inhibited BmNPV replication. Overall, these results suggest that BmUFM1 plays an important role in facilitating BmNPV proliferation in silkworm. Our findings advance the understanding of UFM1's conjugation machinery, and also provides a potentially molecular target for BmNPV prevention and silkworm breeding.

Improving the anti-tumor effect of EGCG in colorectal cancer cells by blocking EGCG-induced YAP activation.

(-)-Epigallocatechin-3-gallate (EGCG) is the primary active ingredient in green tea and has been used for cancer prevention in clinical trials. The anti-tumor effects of EGCG stem from its ability to inhibit the activities of many oncoproteins, such as AKT, VEGFR, STAT3, and mutant p53. However, the clinical efficacy of EGCG is unsatisfactory. How to improve the anti-tumor effects of EGCG is an open question. Here we report that EGCG inhibits the tumor suppressive Hippo signaling pathway and activates downstream YAP in colorectal cancer (CRC) cells. Activation of YAP impedes the anti-tumor effects of EGCG. YAP blockade increases the sensitivity of CRC cells to EGCG treatment.

Uneven Index: A Digital Biomarker to Prompt Demodex Blepharitis Based on Deep Learning.

Purpose: To evaluate ocular surface manifestations and morphological changes in meibomian glands (MGs) based on artificial intelligence (AI) analysis in patients with Demodex blepharitis. Methods: In this retrospective study, 115 subjects were enrolled, including 64 subjects with Demodex blepharitis and 51 subjects without Demodex blepharitis as control group. Morphological indexes were evaluated for height, width, tortuosity, MG density, total variation, and the three types of corrected total variation as Uneven indexes. Results: There were no statistically significant differences in all MGs' average tortuosity and width between the two groups. The average height of all MGs and MG density were significantly lower in the Demodex blepharitis group than control group. The total variation and two types of Uneven indexes were significantly higher in the Demodex blepharitis group than in the control group. Especially the Uneven Index of total variation/MG density had an AUC of 0.822. And the sensitivity and specificity were 59.4% and 92.2%, respectively, at a cut-off value of 3971.667. In addition, Demodex blepharitis was associated with significantly lower meibum quality and expressibility, severe atrophy of MGs, a higher ocular surface disease index (OSDI), and more instability of the tear film. Conclusion: Demodex mites are strongly associated with morphological changes in the MGs and may cause uneven gland atrophy. Therefore, the novel characteristic parameter, the Uneven index, may serve as a digital biomarker to evaluate uneven atrophy of MGs and prompt Demodex blepharitis.

Impact of COVID-19 on stock price crash risk: Evidence from Chinese energy firms.

This paper studies the impact of the outbreak of coronavirus disease 2019 (COVID-19) on the stock price crash risk of energy firms in China. We find that the stock price crash risk of energy firms significantly decreases in the post-COVID-19 period. We also find that firms that engage in more corporate social responsibility (CSR) activities are less exposed to stock price crash risk in the post-COVID-19 period than those that engage in less CSR activities. Finally, we show that the effect of COVID-19 on stock price crash risk is less severe for state-owned enterprises (SOEs) than for non-SOEs in the post-COVID-19 period. Our findings demonstrate China's economic recovery in the post-COVID-19 period and have policy implications for firms to improve their resilience to exogenous shocks.

Production of ß-glucanase and protease from Bacillus velezensis strain isolated from the manure of piglets.

In this study, a strain producing ß-glucanase and protease, identified as Bacillus velezensis Y1, was isolated from the manure of piglet. We attempted to produce ß-glucanase and protease after optimization of various process parameters with the submerged fermentation. The effects of each factor on producing ß-glucanase and protease were as follows: temperature > time > pH > loaded liquid volume. The properties of the ß-glucanase showed that the most suitable reaction temperature was 65 °C and pH was 6.0. However for protease optimum reaction temperature was 50 °C, and pH was 6.0. The amplified PCR fragments of ß-glucanase and protease were 1434 bp containing an open reading frame of 1413 bp encoding a protein with 444 amino acids and 1752 bp containing an open reading frame of 1521 bp encoding a protein with 506 amino acids, respectively. So, the study demonstrated a viable approach of using newly identified B. velezensis Y1 strain for the maximum yield of two industrially important enzymes.

DNA Methylation Is Correlated with Gene Expression during Diapause Termination of Early Embryonic Development in the Silkworm (Bombyx mori).

DNA modification is a naturally occurring DNA modification in prokaryotic and eukaryotic organisms and is involved in several biological processes. Although genome-wide methylation has been studied in many insects, the understanding of global and genomic DNA methylation during insect early embryonic development, is lacking especially for insect diapause. In this study, we analyzed the relationship between DNA methylomes and transcriptomes in diapause-destined eggs compared to diapause-terminated eggs in the silkworm, Bombyx mori (B. mori). The results revealed that methylation was sparse in this species, as previously reported. Moreover, methylation levels in diapause-terminated eggs (HCl-treated) were 0.05% higher than in non-treated eggs, mainly due to the contribution of CG methylation sites. Methylation tends to occur in the coding sequences and promoter regions, especially at transcription initiation sites and short interspersed elements. Additionally, 364 methylome- and transcriptome-associated genes were identified, which showed significant differences in methylation and expression levels in diapause-destined eggs when compared with diapause-terminated eggs, and 74% of methylome and transcriptome associated genes showed both hypermethylation and elevated expression. Most importantly, Kyoto Encyclopaedia of Genes and Genomes (KEGG) analyses showed that methylation may be positively associated with Bombyx mori embryonic development, by regulating cell differentiation, metabolism, apoptosis pathways and phosphorylation. Through analyzing the G2/M phase-specific E3 ubiquitin-protein ligase (G2E3), we speculate that methylation may affect embryo diapause by regulating the cell cycle in Bombyx mori. These findings will help unravel potential linkages between DNA methylation and gene expression during early insect embryonic development and insect diapause.

DNA methyltransferase BmDnmt1 and BmDnmt2 in silkworm (Bombyx mori) and the regulation of silkworm embryonic development.

DNA methylation is one of the most widespread epigenetic marks and has been linked to insect development, especially influencing embryonic development. However, the regulation of DNA methylation in silkworm embryonic development and diapause remain to investigate. In this study, reverse-transcription quantitative polymerase chain reaction was performed to identify the expression level of Bombyx mori DNA methyltransferases (BmDNMTs) 1 and 2 ( BmDnmt1 and BmDnmt2) in different tissues, different embryonic developmental stages, and different strains of the silkworm. The results showed that BmDNMTs were the most highly expressed during embryonic development, especially at early embryonic stages. In particular, the expression of BmDNMTs was significantly upregulated in diapause-terminated eggs by HCl treatment. Moreover, tissue distribution showed that BmDnmt2 was highly expressed in testis and ovary, and BmDnmt1 was highly expressed in testis. This study contributes to understanding the correlation of DNA methylation occurs with embryogenesis and gametogenesis in insect, meanwhile, it provides a research orientation to further analyze the role of DNA methylation in diapause initiation and termination in insect embryonic development.

Ci antagonizes Hippo signaling in the somatic cells of the ovary to drive germline stem cell differentiation.

Many stem cell populations are tightly regulated by their local microenvironment (niche), which comprises distinct types of stromal cells. However, little is known about mechanisms by which niche subgroups coordinately determine the stem cell fate. Here we identify that Yki, the key Hippo pathway component, is essential for escort cell (EC) function in promoting germline differentiation in Drosophila ovary. We found that Hedgehog (Hh) signals emanating primarily from cap cells support the function of ECs, where Cubitus interruptus (Ci), the Hh signaling effector, acts to inhibit Hippo kinase cascade activity. Mechanistically, we found that Ci competitively interacts with Hpo and impairs the Hpo-Wts signaling complex formation, thereby promoting Yki nuclear localization. The actions of Ci ensure effective Yki signaling to antagonize Sd/Tgi/Vg-mediated default repression in ECs. This study uncovers a mechanism explaining how subgroups of niche cells coordinate to determine the stem cell fate via Hh-Hippo signaling crosstalk, and enhances our understanding of mechanistic regulations of the oncogenic Yki/YAP signaling.

N6-methyladenine functions as a potential epigenetic mark in eukaryotes.

N(6)-methyladenine (6mA) is one of the most abundant types of DNA methylation, and plays an important role in bacteria; however, its roles in higher eukaryotes, such as plants, insects, and mammals, have been considered less important. Recent studies highlight that 6mA does indeed occur, and that it plays an important role in eukaryotes, such as worm, fly, and green algae, and thus the regulation of 6mA has emerged as a novel epigenetic mechanism in higher eukaryotes. Despite this intriguing development, a number of important issues regarding its biological roles are yet to be addressed. In this review, we focus on the 5mC and 6mA modifications in terms of their production, distribution, and the erasure of 6mA in higher eukaryotes including mammals. We perform an analysis of the potential functions of 6mA, hence widening understanding of this new epigenetic mark in higher eukaryotes, and suggesting future studies in this field.

Activation of Smurf E3 ligase promoted by smoothened regulates hedgehog signaling through targeting patched turnover.

Hedgehog signaling plays conserved roles in controlling embryonic development; its dysregulation has been implicated in many human diseases including cancers. Hedgehog signaling has an unusual reception system consisting of two transmembrane proteins, Patched receptor and Smoothened signal transducer. Although activation of Smoothened and its downstream signal transduction have been intensively studied, less is known about how Patched receptor is regulated, and particularly how this regulation contributes to appropriate Hedgehog signal transduction. Here we identified a novel role of Smurf E3 ligase in regulating Hedgehog signaling by controlling Patched ubiquitination and turnover. Moreover, we showed that Smurf-mediated Patched ubiquitination depends on Smo activity in wing discs. Mechanistically, we found that Smo interacts with Smurf and promotes it to mediate Patched ubiquitination by targeting the K1261 site in Ptc. The further mathematic modeling analysis reveals that a bidirectional control of activation of Smo involving Smurf and Patched is important for signal-receiving cells to precisely interpret external signals, thereby maintaining Hedgehog signaling reliability. Finally, our data revealed an evolutionarily conserved role of Smurf proteins in controlling Hh signaling by targeting Ptc during development.

The Fused/Smurf complex controls the fate of Drosophila germline stem cells by generating a gradient BMP response.

In the Drosophila ovary, germline stem cells (GSCs) are maintained primarily by bone morphogenetic protein (BMP) ligands produced by the stromal cells of the niche. This signaling represses GSC differentiation by blocking the transcription of the differentiation factor Bam. Remarkably, bam transcription begins only one cell diameter away from the GSC in the daughter cystoblasts (CBs). How this steep gradient of response to BMP signaling is formed has been unclear. Here, we show that Fused (Fu), a serine/threonine kinase that regulates Hedgehog, functions in concert with the E3 ligase Smurf to regulate ubiquitination and proteolysis of the BMP receptor Thickveins in CBs. This regulation generates a steep gradient of BMP activity between GSCs and CBs, allowing for bam expression on CBs and concomitant differentiation. We observed similar roles for Fu during embryonic development in zebrafish and in human cell culture, implying broad conservation of this mechanism.

Systematic identification and characterization of chicken (Gallus gallus) ncRNAs.

Recent studies have demonstrated that non-coding RNAs (ncRNAs) play important roles during development and evolution. Chicken, the first genome-sequenced non-mammalian amniote, possesses unique features for developmental and evolutionary studies. However, apart from microRNAs, information on chicken ncRNAs has mainly been obtained from computational predictions without experimental validation. In the present study, we performed a systematic identification of intermediate size ncRNAs (50-500 nt) by ncRNA library construction and identified 125 chicken ncRNAs. Importantly, through the bioinformatics and expression analysis, we found the chicken ncRNAs has several novel features: (i) comparative genomic analysis against 18 sequenced vertebrate genomes revealed that the majority of the newly identified ncRNA candidates is not conserved and most are potentially bird/chicken specific, suggesting that ncRNAs play roles in lineage/species specification during evolution. (ii) The expression pattern analysis of intronic snoRNAs and their host genes suggested the coordinated expression between snoRNAs and their host genes. (iii) Several spatio-temporal specific expression patterns suggest involvement of ncRNAs in tissue development. Together, these findings provide new clues for future functional study of ncRNAs during development and evolution.