Effects of dietary supplement of ε-polylysine hydrochloride on laying performance, egg quality, serum parameters, organ index, intestinal morphology, gut microbiota and volatile fatty acids in laying hens.

BACKGROUND: ε-polylysine hydrochloride (ε-PLH) is a naturally occurring antimicrobial peptide extensively utilized in the food and medical industries. However, its impact on animal husbandry remains to be further explored. Therefore, the present study aimed to determine the effect of ε-PLH on laying hens' health and laying performance. RESULTS: Dietary supplementation with ε-PLH to the diet significantly increased average egg weight during weeks 1-8. Meanwhile, compared with the control group, supplementation with ε-PLH decreased the feed egg ratio during weeks 9-12 and egg breakage rate during weeks 9-16 ，whereas it increased eggshell strength during weeks 1-4 and 13-16 . The ε-PLH 0.05% group increased yolk percentage during weeks 5-8 and yolk color during weeks 1-4 . Furthermore, ε-PLH supplementation significantly increased the concentrations of total protein, albumin, globulin and reproductive hormones estradiol, as well as decreased interleukin-1 beta and malondialdehyde in the serum. Compared with the control group, supplementation with 0.05% ε-PLH significantly increased the relative abundance of Cyanobacteria and Gastranaerophilales and decreased the abundance of Desulfovibrio and Streptococcus in the cecum microbiota. In addition, ε-PLH 0.1% supplementation also increased acetic acid content in the cecum. CONCLUSION: Dietary supplementation with ε-PLH has a positive impact on both productive performance and egg quality in laying hens. Furthermore, ε-PLH can also relieve inflammation by promoting the immunity and reducing oxidative damage during egg production. ε-PLH has been shown to improve intestinal morphology, gut microbial diversity and intestinal health. © 2023 Society of Chemical Industry.

Porcine promyelocytic leukemia protein isoforms suppress Japanese encephalitis virus replication in PK15 cells.

BACKGROUND: Promyelocytic leukemia protein (PML) is a primary component of PML nuclear bodies (PML-NBs). PML and PML-NBs play critical roles in processes like the cell cycle, DNA damage repair, apoptosis, and the antiviral immune response. Previously, we identified five porcine PML alternative splicing variants and observed an increase in the expression of these PML isoforms following Japanese encephalitis virus (JEV) infection. In this study, we examined the functional roles of these PML isoforms in JEV infection. METHODS: PML isoforms were either knocked down or overexpressed in PK15 cells, after which they were infected with JEV. Subsequently, we analyzed the gene expression of PML isoforms, JEV, and the interferon (IFN)-ß signaling pathway using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. Viral titers were determined through 50% tissue culture infectious dose (TCID50) assays. RESULTS: Our results demonstrated that the knockdown of endogenous PML promoted JEV replication, while the overexpression of PML isoforms 1, 3, 4, and 5 (PML1, PML3, PML4, and PML5) inhibited JEV replication. Further investigation revealed that PML1, PML3, PML4, and PML5 negatively regulated the expression of genes involved in the interferon (IFN)-ß signaling pathway by inhibiting IFN regulatory factor 3 (IRF3) post-JEV infection. CONCLUSIONS: These findings demonstrate that porcine PML isoforms PML1, PML3, PML4, and PML5 negatively regulate IFN-ß and suppress viral replication during JEV infection. The results of this study provide insight into the functional roles of porcine PML isoforms in JEV infection and the regulation of the innate immune response.

High-titer production of staurosporine by heterologous expression and process optimization.

Staurosporine is the most well-known member of the indolocarbazole alkaloid family; it can induce apoptosis of many types of cells as a strong protein kinase inhibitor, and is used as an important lead compound for the synthesis of the antitumor drugs. However, the low fermentation level of the native producer remains the bottleneck of staurosporine production. Herein, integration of multi-copy biosynthetic gene cluster (BGC) in well characterized heterologous host and optimization of the fermentation process were performed to enable high-level production of staurosporine. First, the 22.5 kb staurosporine BGC was captured by CRISPR/Cas9-mediated TAR (transformation-associated recombination) from the native producer (145 mg/L), and then introduced into three heterologous hosts Streptomyces avermitilis (ATCC 31267), Streptomyces lividans TK24 and Streptomyces albus J1074 to evaluate the staurosporine production capacity. The highest yield was achieved in S. albus J1074 (750 mg/L), which was used for further production improvement. Next, we integrated two additional staurosporine BGCs into the chromosome of strain S-STA via two different attB sites (vwb and TG1), leading to a double increase in the production of staurosporine. And finally, optimization of fermentation process by controlling the pH and glucose feeding could improve the yield of staurosporine to 4568 mg/L, which was approximately 30-fold higher than that of the native producer. This is the highest yield ever reported, paving the way for the industrial production of staurosporine. KEYPOINTS: • Streptomyces albus J1074 was the most suitable heterologous host to express the biosynthetic gene cluster of staurosporine. • Amplification of the biosynthetic gene cluster had obvious effect on improving the production of staurosporine. • The highest yield of staurosporine was achieved to 4568 mg/L by stepwise increase strategy.

Enhancement of acarbose production by genetic engineering and fed-batch fermentation strategy in Actinoplanes sp. SIPI12-34.

BACKGROUND: Acarbose, as an alpha-glucosidase inhibitor, is widely used clinically to treat type II diabetes. In its industrial production, Actinoplanes sp. SE50/110 is used as the production strain. Lack of research on its regulatory mechanisms and unexplored gene targets are major obstacles to rational strain design. Here, transcriptome sequencing was applied to uncover more gene targets and rational genetic engineering was performed to increase acarbose production. RESULTS: In this study, with the help of transcriptome information, a TetR family regulator (TetR1) was identified and confirmed to have a positive effect on the synthesis of acarbose by promoting the expression of acbB and acbD. Some genes with low expression levels in the acarbose biosynthesis gene cluster were overexpressed and this resulted in a significant increase in acarbose yield. In addition, the regulation of metabolic pathways was performed to retain more glucose-1-phosphate for acarbose synthesis by weakening the glycogen synthesis pathway and strengthening the glycogen degradation pathway. Eventually, with a combination of multiple strategies and fed-batch fermentation, the yield of acarbose in the engineered strain increased 58% compared to the parent strain, reaching 8.04 g/L, which is the highest fermentation titer reported. CONCLUSIONS: In our research, acarbose production had been effectively and steadily improved through genetic engineering based on transcriptome analysis and fed-batch culture strategy.

Inhibition of Japanese encephalitis virus proliferation by long non-coding RNA SUSAJ1 in PK-15 cells.

BACKGROUND: Japanese encephalitis virus is a mosquito-borne neurotropic flavivirus that causes acute viral encephalitis in humans. Pigs are crucial amplifier host of JEV. Recently, increasing evidence has shown that long non-coding RNAs (lncRNAs) play important roles in virus infection. METHODS: JEV proliferation was evaluated after overexpression or knockdown of lncRNA-SUSAJ1 using western blotting and reverse-transcription polymerase chain reaction (RT-PCR). C-C chemokine receptor type 1 (CCR1) was found to regulate the expression of lncRNA-SUSAJ1 by inhibitors screen. The expression of lncRNA-SUSAJ1 was detected using RT-PCR after overexpression or knockdown of transcription factor SP1. In addition, the enrichments of transcription factor SP1 on the promoter of lncRNA-SUSAJ1 were analyzed by chromatin immunoprecipitation. RESULTS: In this study, we demonstrated that swine lncRNA-SUSAJ1 could suppress JEV proliferation in PK-15 cells. We also found that CCR1 inhibited the expression of lncRNA-SUSAJ1 via the transcription factor SP1. In addition, knockdown of CCR1 could upregulated the expression of SP1 and lncRNA-SUSAJ1, resulting in resistance to JEV proliferation. CONCLUSIONS: These findings illustrate the importance of lncRNAs in virus proliferation, and reveal how this virus regulates lncRNAs in host cells to promote its proliferation.

Coenzyme Q10 ameliorates BPA-induced apoptosis by regulating autophagy-related lysosomal pathways.

Bisphenol A (BPA) is a widely distributed environmental endocrine disruptor. The accumulation of BPA has been proved that produce various toxic effects both on human and animals. However, the strategies to reduce the damage of BPA on the body and related mechanisms remain to be studied. Coenzyme Q10 (CoQ10), as a powerful antioxidant, is ubiquitous in many eukaryotic cells, which can improve the integrity of lysosomal membrane, lysosomal degradation function and promote autophagy. Here, we examined the ability of CoQ10 to alleviate oxidative stress and apoptosis in BPA-induced damages in C2C12 cells, and how to alleviate it. Our results showed that BPA treatment significantly reduced cell viability, increased the number of cell apoptosis and ROS production, decreased mitochondrial membrane potential, and inhibited the gene expression of mitochondria biogenesis. Moreover, we demonstrated that exposure to BPA increased expression levels of autophagy protein (LC3-II, p62), inhibited autophagy flux, and disrupted the acidic pH environment of lysosomes. Importantly, CoQ10 supplementation effectively restored these abnormalities caused by BPA. CoQ10 significantly decreased the apoptotic incidence and ROS levels, improved mitochondrial membrane potential. Moreover, CoQ10 improved lysosome function and enhanced autophagy flux. Taken together, our results indicate that CoQ10 supplementation is a feasible and effective way to promote the level of autophagy by improving lysosomal function, thereby reducing the apoptosis caused by BPA accumulation. This study aims to provide evidence for the role of CoQ10 in repairing BPA-induced cell damage in clinical practice.

Blocking the REDD1/TXNIP axis ameliorates LPS-induced vascular endothelial cell injury through repressing oxidative stress and apoptosis.

The aim of the present study was to investigate the potential role of regulated in development and DNA damage response 1 (REDD1) in LPS-induced vascular endothelial injury by using human umbilical vein endothelial cells (HUVECs). We observed that REDD1 expression was apparently elevated in HUVECs after exposure to LPS. Additionally, elimination of REDD1 strikingly attenuated the secretion of the proinflammatory cytokines TNF-α, IL-6, IL-1ß, and monocyte chemotactic protein-1 and the endothelial cell adhesion markers ICAM-1 and VCAM-1 that was induced by LPS stimulation. Subsequently, knockdown of REDD1 augmented cell viability but ameliorated lactate dehydrogenase release in HUVECs stimulated with LPS. Meanwhile, depletion of REDD1 effectively restricted LPS-induced HUVEC apoptosis, as exemplified by reduced DNA fragmentation, and it also elevated antiapoptotic Bcl-2 protein, concomitant with reduced levels of proapoptotic proteins Bax and cleaved caspase-3. Furthermore, repression of REDD1 remarkably alleviated LPS-triggered intracellular reactive oxygen species generation accompanied by decreased malondialdehyde content and increased the activity of the endogenous antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase. Most important, depletion of REDD1 protected HUVECs against inflammation-mediated apoptosis and oxidative damage partly through thioredoxin-interacting protein (TXNIP). Collectively, these findings indicate that blocking the REDD1/TXNIP axis repressed the inflammation-mediated vascular injury process, which may be closely related to oxidative stress and apoptosis in HUVECs, implying that the REDD1/TXNIP axis may be a new target for preventing the endothelial cell injury process.

Licochalcone A attenuates abdominal aortic aneurysm induced by angiotensin II via regulating the miR-181b/SIRT1/HO-1 signaling.

Licochalcone A (LA), a chalcone derived from liquorice, exhibits multiple biological activities, including anti-oxidation and anti-inflammation. This study aimed to investigate the role and underlying mechanism of LA in the abdominal aortic aneurysm (AAA). AAA model was established by continuous infusion of 1000 ng/kg/min of angiotensin II (AngII) in ApoE -/- mice for 4 weeks. At 7 days before AngII administration, 5 mg/kg/day or 10 mg/kg/day of LA was intraperitoneally administered to mice and continued for 4 weeks. The characteristics and quantification of AAAs were determined in situ. Real-time PCR or western blot was used to measure mRNA or protein levels of matrix metalloproteinase 2 and matrix metalloproteinase 9; pro-inflammatory cytokines tumor necrosis factor-α, interleukin-1ß, and interleukin-6; apoptosis-related proteins Bax, Bcl-2, and active caspase-3; miR-181b; Sirtuin 1 (SIRT1); and heme oxygenase-1 (HO-1). Mouse-aorta-origin vascular smooth muscle (MOVAS) cells were used to confirm the involved pathways in vitro. We found LA administration dose-dependently reduced the incidence of AngII-induced AAA, aneurysm diameter enlargement, elastin degradation, matrix metalloproteinase production, pro-inflammatory cytokines and miR-181b expression, and vascular smooth muscle cell apoptosis. It elevated SIRT1 and HO-1 expression that was suppressed by AngII. AngII enhanced miR-181b but reduced SIRT1 and HO-1 expression in MOVAS cells. In AngII-stimulated MOVAS cells, downregulation of miR-181b significantly upregulated the expression of SIRT1 and HO-1, the effect of which was abrogated by SIRT1 siRNA. Collectively, LA could attenuate AngII-induced AAA by modulating the miR-181b/SIRT1/HO-1 signaling. LA might be a potential medical therapy for small AAA.

microRNA-338-3p promotes ox-LDL-induced endothelial cell injury through targeting BAMBI and activating TGF-ß/Smad pathway.

microRNAs (miRNAs) have been revealed to participate in the pathological process of atherosclerosis (AS). However, the exact role of miR-338-3p, a target miRNA of BMP and activin membrane-bound inhibitor (BAMBI), and its possible molecular mechanism in AS remain unidentified. In this study, we found that BAMBI was significantly decreased, whereas miR-338-3p increased in patients with AS and oxidized low-density lipoprotein (ox-LDL)-induced HUVEC cells. Furthermore, overexpression of miR-338-3p significantly decreased cell viability and elevated cell apoptosis, whereas its inhibition significantly promoted cell viability and inhibited cell apoptosis in ox-LDL-induced HUVEC cells. Moreover, miR-338-3p overexpression increased TGF-ß/Smad pathway activation in ox-LDL-induced HUVEC cells. A dual-luciferase reporter assay confirmed the direct interaction between miR-338-3p and the 3'-untranslated region of BAMBI messenger RNA. Furthermore, the suppression of BAMBI ameliorated the effect of miR-338-3p inhibition against ox-LDL-induced HUVEC cell injury. In conclusion, our study thus suggests that miR-338-3p promoted ox-LDL-induced HUVEC cell injury by targeting BAMBI and activating the TGF-ß/Smad pathway, which may provide a novel and promising therapeutic target for AS.

Lidocaine Potentiates SOCS3 to Attenuate Inflammation in Microglia and Suppress Neuropathic Pain.

Lidocaine is one of the typical local anesthetics that are frequently used in the peripheral nerve blocks and pain management. Emerging evidence have shown that lidocaine may exert anti-inflammatory effect involving neuropathic pain. However, the effect and underlying mechanism of lidocaine in suppressing neuroinflammation in neuropathic pain are incompletely revealed. In this study, effects of lidocaine on the suppressors of cytokine-signaling protein 3 (SOCS3) in microglia are investigated in chronic constriction injury (CCI) rat model and lipopolysaccharide (LPS)-stimulated BV-2 cells. It was shown that intrathecal injection of lidocaine substantially alleviated CCI-induced neuropathic pain, as reflected by the decreased thermal latency and mechanical threshold. Lidocaine reduced the CCI-evoked spinal injury and cell apoptosis. CCI induced an significant increase of IBA1+ microglia accompanied by the increase of inflammatory cytokines IL-6 and IL-1ß, which were suppressed after lidocaine administration. SOCS3 expression in IBA1+ microglia was notably upregulated in response to lidocaine injection, which presented in a similar pattern in LPS-activated BV-2 cells. Furthermore, lidocaine upregulated SOCS3 expression dependent of pCREB, and CREB silencing greatly discounted this effect. The intrathecal injection of lentiviral vectors LV-SOCS3 efficiently alleviated CCI-evoked neuropathic pain and reduced spinal IBA1+ microglia. SOCS3 overexpression contributed to the inhibition of neuroinflammation by decreasing the expression and activation of p38 MAPK and NF-κB stimulated by LPS. Collectively, lidocaine promoted the SOCS3 expression in microglia, in turn leading to suppression of IBA1+ microglia accumulation and p38 MAPK and NF-κB, which may expand our understanding on lidocaine in suppressing neuroinflammation and neuropathic pain.

Inhibition of miR-135b by SP-1 promotes hypoxia-induced vascular endothelial cell injury via HIF-1α.

Myocardial hypoxia-induced endothelial cell apoptosis contributes to cardiac dysfunction, such as myocardial infarction (MI), myocardial ischemia, and heart failure. Thus, it is important to investigate the molecular mechanisms of vascular endothelial cells (VECs) during exposure to hypoxia. SP-1 is an important regulator of cytokines associated with cell functions. We found that SP-1 expression increased in human umbilical vein endothelial cells (HUVECs) exposed to hypoxia by western blot. Then the SP-1 siRNA was transfected into HUVECs under hypoxic condition. MTT assay showed that hypoxia reduced the cell proliferation, but SP-1 siRNA reversed that. Transfection with si-SP-1 also reversed cell apoptosis and reactive oxygen species (ROS) production increased by hypoxia treatment. Moreover, inflammatory phenotype were increased in hypoxia induced HUVECs, including ICAM-1,VCAM-1 levels as well as TNFα, IL-6 and IL-1ß secretion, and the si-SP-1 also reversed this effect of hypoxia. Additionally, si-SP-1 increased expression of miR-135b and reduced expression of hypoxia-inducible factor 1-α (HIF-1α), which is the target gene of miR-135b. To investigate the underlying mechanism of SP-1 on hypoxia induced HUVECs injury, the anti-miR-135b or HIF-1α agonist (CoCl2) were used. Finally, the result indicated that both anti-miR-135b or CoCl2 treatment reversed the effects of SP-1 siRNA under hypoxia. In conclusion, the SP-1/miR-135b/HIF-1α axis may play a critical role in hypoxia-induced vascular endothelial injury. Our study thus provides novel insights into the role of this transcription factor and miRNAs in the pathogenesis of hypoxia-induced cardiac dysfunctions.

Enhancement of FK520 production in Streptomyces hygroscopicus by combining traditional mutagenesis with metabolic engineering.

FK520 (ascomycin), a 23-membered macrolide with immunosuppressive activity, is produced by Streptomyces hygroscopicus. The problem of low yield and high impurities (mainly FK523) limits the industrialized production of FK520. In this study, the FK520 yield was significantly improved by strain mutagenesis and genetic engineering. First, a FK520 high-producing strain SFK-6-33 (2432.2 mg/L) was obtained from SFK-36 (1588.4 mg/L) through ultraviolet radiation mutation coupled with streptomycin resistance screening. The endogenous crotonyl-CoA carboxylase/reductase (FkbS) was found to play an important role in FK520 biosynthesis, identified with CRISPR/dCas9 inhibition system. FkbS was overexpressed in SFK-6-33 to obtain the engineered strain SFK-OfkbS, which produced 2817.0 mg/L of FK520 resulting from an increase in intracellular ethylmalonyl-CoA levels. In addition, the FK520 levels could be further increased with supplementation of crotonic acid in SFK-OfkbS. Overexpression of acetyl-CoA carboxylase (ACCase), used for the synthesis of malonyl-CoA, was also investigated in SFK-6-33, which improved the FK520 yield to 3320.1 mg/L but showed no significant inhibition in FK523 production. To further enhance FK520 production, FkbS and ACCase combinatorial overexpression strain SFK-OASN was constructed; the FK520 production increased by 44.4% to 3511.4 mg/L, and the FK523/FK520 ratio was reduced from 9.6 to 5.6% compared with that in SFK-6-33. Finally, a fed-batch culture was carried out in a 5-L fermenter, and the FK520 yield reached 3913.9 mg/L at 168 h by feeding glycerol, representing the highest FK520 yield reported thus far. These results demonstrated that traditional mutagenesis combined with metabolic engineering was an effective strategy to improve FK520 production.

Genome-wide DNA methylation profiles of porcine ovaries in estrus and proestrus.

DNA methylation is an important epigenetic modification involved in the estrous cycle and the regulation of reproduction. Here, we investigated the genome-wide profiles of DNA methylation in porcine ovaries in proestrus and estrus using methylated DNA immunoprecipitation sequencing. The results showed that DNA methylation was enriched in intergenic and intron regions. The methylation levels of coding regions were higher than those of the 5'- and 3'-flanking regions of genes. There were 4,813 differentially methylated regions (DMRs) of CpG islands in the estrus vs. proestrus ovarian genomes. Additionally, 3,651 differentially methylated genes (DMGs) were identified in pigs in estrus and proestrus. The DMGs were significantly enriched in biological processes and pathways related to reproduction and hormone regulation. We identified 90 DMGs associated with regulating reproduction in pigs. Our findings can serve as resources for DNA methylome research focused on porcine ovaries and further our understanding of epigenetically regulated reproduction in mammals.

DJ-1 Alleviates Angiotensin II-Induced Endothelial Progenitor Cell Damage by Activating the PPARγ/HO-1 Pathway.

There is evidence that angiotensin II (Ang II) may impair the functions of endothelial progenitor cells (EPCs). It was revealed that DJ-1 could resist oxidative stress. In this study, we investigated whether DJ-1 could protect EPCs against Ang II-induced cell damage. The proliferation and migration of EPCs were strongly reduced in the Ang II group and were increased by overexpression of DJ-1. Western blotting indicated that the increased expression of the senescence marker ß-galactosidase and decreased expression of adhesion molecules (ICAM-1, VCAM-1) induced by Ang II were reversed after Ad-DJ-1 transfection. The reduced angiogenic capacity of EPCs caused by Ang II was also improved after Ad-DJ-1 transfection. Moreover, Ang II significantly increased the levels of reactive oxygen species (ROS), malondialdehyde (MDA), and inflammatory cytokines (TNF-α and IL-1ß), reduced the levels of superoxide dismutase (SOD), glutathione (GSH), and these were reversed by Ad-DJ-1 transfection. Expression of peroxisome proliferator-activated receptor-γ (PPARγ) and heme oxygenase (HO-1) was increased by DJ-1. Therefore, HO-1 siRNA were constructed and transfected into EPCs, and the results showed that HO-1 siRNA transfection inhibited the effects of DJ-1 on EPC function. Thus, our study implies that DJ-1 may protect EPCs against Ang II-induced dysfunction by activating the PPARγ/HO-1. J. Cell. Biochem. 119: 392-400, 2018. © 2017 Wiley Periodicals, Inc.

OGG1 regulates the level of symmetric dimethylation of histone H4 arginine-3 by interacting with PRMT5.

OGG1 is the first enzyme in the base excision repair pathway (BER) responsible for repairing 8-oxoguanine DNA lesions. Recent studies found that OGG1 may also be involved in epigenetic regulation. In this study, we focused on the roles of OGG1 in histone modification. First, to study the effects of OGG1 on histone modification, the protein levels of symmetric dimethylation of histone H4 arginine-3 (H4R3me2s) were determined by western blot analysis following the knockdown or overexpression of OGG1. Second, the molecular mechanisms by which OGG1 regulates H4R3me2s were assessed by co-immunoprecipitation (CO-IP) assays in mouse embryonic fibroblast (MEF) wild-type (WT) and Ogg-/- cells. Finally, to verify the regulation of H4R3me2s by OGG1 on specific genes, chromatin immunoprecipitation (CHIP) was performed on MEF WT and Ogg-/- cells. We found that OGG1 affects PRMT5 binding on histone H4 and the formation of H4R3me2s via PRMT5. The methylation level of H4R3me2s was dramatically decreased in MEF Ogg-/- cells compared to WT cells. Knockdown of OGG1 by siRNA led to a decrease in H4R3me2s, while overexpression of OGG1 increased the level of H4R3me2s. OGG1 also interacted with PRMT5 and histone H4, and the interaction between PRMT5 and histone H4 was reduced in MEF Ogg-/- cells. Our data not only illustrate the important roles of OGG1 in histone modification, but also reveal the mechanism by which OGG1 affects PRMT5 binding on H4R3 resulting in the symmetrical dimethylation of histone H4 arginine-3.

Enhancement of A82846B yield and proportion by overexpressing the halogenase gene in Amycolatopsis orientalis SIPI18099.

The glycopeptide antibiotic A82846B (chloroeremomycin) produced by Amycolatopsis orientalis is the precursor of the semi-synthetic antibiotic oritavancin. However, during the industrial production of A82846B, two major impurities, A82846A (63.6%) and A82846C (12%) which are structurally similar to A82846B (24.4%), are also produced. In this study, to improve the ratio of A82846B to A and C, the genes encoding halogenase in A82846B and vancomycin synthesis were integrated into A. orientalis SIPI18099 to test their halogenation ability, respectively. The results indicated that chal from the A82846B biosynthesis pathway was more efficient in reducing A and C factors. Moreover, by increasing the chal copy number, the proportion of A and C were gradually reduced while the titer and proportion of A82846B were improved. In a scaled-up industrial process, the proportion of A and C were decreased to 11.6% and 0.2% in the recombinant strain A.orientalis chal-3 with three gene copies of chal and the titers of A82846B (2.2 g/L) has increased by 2.8-folds compared to 780 mg/L produced by the parental strain, suggesting that the recombinant strain was suitable for the industrial production of A82846B with lower impurities.

Enhanced doxorubicin production by Streptomyces peucetius using a combination of classical strain mutation and medium optimization.

Doxorubicin (DXR), which is produced by Streptomyces peucetius, is an important anthracycline-type antibiotic used for the treatment of various cancers. However, due to the low DXR productivity of wild-type S. peucetius, it is difficult to produce DXR by one-step fermentation. In this study, a DXR-resistance screening method was developed to screen for DXR high-producing mutants. Then, S. peucetius SIPI-11 was treated several times with UV and ARTP (atmospheric and room temperature plasma) to induce mutations. Treated strains were screened by spreading on a DXR-containing plate, isolating a mutant (S. peucetius 33-24) with enhanced DXR yield (570 mg/L vs. 119 mg/L for the original strain). The components of the fermentation medium, including the carbon and nitrogen sources, were optimized to further enhance DXR yield (to 850 mg/L). The pH of the fermentation medium and culture temperature were also optimized for effective DXR production. Finally, DXR production by S. peucetius 33-24 was investigated in flask culture and a fermenter. The yield of DXR was as high as 1100 mg/L in a 5-L fermenter, which is the highest DXR productivity reported thus far, suggesting that S. peucetius 33-24 has the potential to produce DXR by direct fermentation.

miR-124 attenuates Japanese encephalitis virus replication by targeting DNM2.

BACKGROUND: Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes acute viral encephalitis in humans. Pigs are important amplifier hosts of JEV. Emerging evidence indicates that host microRNAs (miRNAs) play key roles in modulating viral infection and pathogenesis. However, mechanistic studies delineating the roles of miRNAs in regulating host-JEV interactions remain scarce. RESULTS: In this study, we demonstrated that miR-124 inhibited JEV replication in porcine kidney epithelial PK15 cells. Furthermore, using bioinformatics tools, we identified dynamin2 (DNM2), a GTPase responsible for vesicle scission, as a target of miR-124. Small interfering RNA (siRNA) depletion studies inicated that dynamin2 was required for efficient JEV replication. We also demonstrated that upregulation of miR-124 expression corresponded to decreased expression of its target, DNM2, in the JEV-infected PK15 cells. CONCLUSIONS: Overall, these results suggest the importance of miR-124 in modulating JEV replication and provide a scientific basis for using cellular miRNAs in anti-JEV therapies.

Goose broodiness is involved in granulosa cell autophagy and homeostatic imbalance of follicular hormones.

Broodiness is observed in most domestic fowls and influences egg production. The goose is one of the most important waterfowls, having strong broody behavior. However, whether autophagy and follicular internal environment play a role in the broodiness behavior of goose is unknown. In this report, we analyzed the follicular internal environment and granulosa cell autophagy of goose follicles. The results show that the contents of hormones, including prolactin (PRL), progesterone (P4), and estradiol (E2), increased in broody goose follicles. Most importantly, the level of granulosa cell autophagy in broody goose follicles was elevated, detected by electron microscopy and western blotting. Also, the expressions of positive regulators of autophagy, including miR-7, miR-29, miR-100, miR-181, PRLR, LC3, p53,Beclin1, Atg9, and Atg12, were up-regulated and the expressions of negative regulators of autophagy, including miR-34b and miR-34c, were down-regulated in broody goose follicles. Our results suggest that goose broodiness is involved in increased granulosa cell autophagy and homeostasis imbalance of internal environment in the follicles. This work contributes to our knowledge of goose broodiness and may influence egg production.