A Cul4 E3 ubiquitin ligase regulates histone hand-off during nucleosome assembly.

Nucleosome assembly following DNA replication and gene transcription is important to maintain genome stability and epigenetic information. Newly synthesized histones H3-H4 first bind histone chaperone Asf1 and are then transferred to other chaperones for nucleosome assembly. However, it is unknown how H3-H4 is transferred from the Asf1-H3-H4 complex to other chaperones because Asf1 binds H3-H4 with high affinity. Here, we show that yeast Rtt101(Mms1) E3 ubiquitin ligase preferentially binds and ubiquitylates new histone H3 acetylated at lysine 56. Inactivation of Rtt101 or mutating H3 lysine residues ubiquitylated by the Rtt101(Mms1) ligase impairs nucleosome assembly and promotes Asf1-H3 interactions. Similar phenotypes occur in human cells in which the ortholog of Rtt101(Mms1), Cul4A(DDB1), is depleted. These results indicate that the transfer of H3-H4 from the Asf1-H3-H4 complex to other histone chaperones is regulated by a conserved E3 ligase and provide evidence for crosstalk between histone acetylation and ubiquitylation in nucleosome assembly.

RPA Interacts with HIRA and Regulates H3.3 Deposition at Gene Regulatory Elements in Mammalian Cells.

The histone chaperone HIRA is involved in depositing histone variant H3.3 into distinct genic regions, including promoters, enhancers, and gene bodies. However, how HIRA deposits H3.3 to these regions remains elusive. Through a short hairpin RNA (shRNA) screening, we identified single-stranded DNA binding protein replication protein A (RPA) as a regulator of the deposition of newly synthesized H3.3 into chromatin. We show that RPA physically interacts with HIRA to form RPA-HIRA-H3.3 complexes, and it co-localizes with HIRA and H3.3 at gene promoters and enhancers. Depletion of RPA1, the largest subunit of the RPA complex, dramatically reduces both HIRA association with chromatin and the deposition of newly synthesized H3.3 at promoters and enhancers and leads to altered transcription at gene promoters. These results support a model whereby RPA, best known for its role in DNA replication and repair, recruits HIRA to promoters and enhancers and regulates deposition of newly synthesized H3.3 to these regulatory elements for gene regulation.

Engineering a non-oxidative glycolysis pathway in escherichia coli for high-level citramalate production.

BACKGROUND: Methyl methacrylate (MMA) is a key precursor of polymethyl methacrylate, extensively used as a transparent thermoplastic in various industries. Conventional MMA production poses health and environmental risks; hence, citramalate serves as an alternative bacterial compound precursor for MMA production. The highest citramalate titer was previously achieved by Escherichia coli BW25113. However, studies on further improving citramalate production through metabolic engineering are limited, and phage contamination is a persistent problem in E. coli fermentation. RESULTS: This study aimed to construct a phage-resistant E. coli BW25113 strain capable of producing high citramalate titers from glucose. First, promoters and heterologous cimA genes were screened, and an effective biosynthetic pathway for citramalate was established by overexpressing MjcimA3.7, a mutated cimA gene from Methanococcus jannaschii, regulated by the BBa_J23100 promoter in E. coli. Subsequently, a phage-resistant E. coli strain was engineered by integrating the Ssp defense system into the genome and mutating key components of the phage infection cycle. Then, the strain was engineered to include the non-oxidative glycolysis pathway while removing the acetate synthesis pathway to enhance the supply of acetyl-CoA. Furthermore, glucose utilization by the strain improved, thereby increasing citramalate production. Ultimately, 110.2 g/L of citramalate was obtained after 80 h fed-batch fermentation. The citramalate yield from glucose and productivity were 0.4 g/g glucose and 1.4 g/(L·h), respectively. CONCLUSION: This is the highest reported citramalate titer and productivity in E. coli without the addition of expensive yeast extract and additional induction in fed-bath fermentation, emphasizing its potential for practical applications in producing citramalate and its derivatives.

Primary neurolymphomatosis with MAG antibody: a case report.

Neurolymphomatosis (NL) is a rare neurologic manifestation of non-Hodgkin lymphoma (NHL) with poor prognosis. Investigations including MRI, PET/CT, nerve biopsy and cerebrospinal fluid (CSF) analysis can aid the diagnosis of NL. In this study, we presented a case of NL with co-existing myelin-associated glycoprotein (MAG) antibody. The patient first presented with symptoms of peripheral neuropathy involving multiple cranial nerves and cauda equina, and later developed obstructive hydrocephalus and deep matter lesions. He also had persistently positive MAG antibody, but did not develop electrophysiologically proven neuropathy and monoclonal immunoglobulin. The final brain biopsy confirmed diffuse large B cell lymphoma.

Targeting deoxynivalenol for degradation by a chimeric manganese peroxidase/glutathione system.

The manganese peroxidase (MnP) can degrade multiple mycotoxins including deoxynivalenol (DON) efficiently; however, the lignin components abundant in foods and feeds were discovered to interfere with DON catalysis. Herein, using MnP from Ceriporiopsis subvermispora (CsMnP) as a model, it was demonstrated that desired catalysis of DON, but not futile reactions with lignin, in the reaction systems containing feeds could be achieved by engineering MnP and supplementing with a boosting reactant. Specifically, two successive strategies (including the fusion of CsMnP to a DON-recognizing ScFv and identification of glutathione as a specific targeting enhancer) were combined to overcome the lignin competition, which together resulted into elevation of the degradation rate from 2.5% to as high as 82.7% in the feeds. The method to construct a targeting MnP and fortify it with an additional enhancer could be similarly applied to catalyze the many other mycotoxins with yet unknown responsive biocatalysts.

USP51 deubiquitylates H2AK13,15ub and regulates DNA damage response.

Dynamic regulation of RNF168-mediated ubiquitylation of histone H2A Lys13,15 (H2AK13,15ub) at DNA double-strand breaks (DSBs) is crucial for preventing aberrant DNA repair and maintaining genome stability. However, it remains unclear which deubiquitylating enzyme (DUB) removes H2AK13,15ub. Here we show that USP51, a previously uncharacterized DUB, deubiquitylates H2AK13,15ub and regulates DNA damage response. USP51 depletion results in increased spontaneous DNA damage foci and elevated levels of H2AK15ub and impairs DNA damage response. USP51 overexpression suppresses the formation of ionizing radiation-induced 53BP1 and BRCA1 but not RNF168 foci, suggesting that USP51 functions downstream from RNF168 in DNA damage response. In vitro, USP51 binds to H2A-H2B directly and deubiquitylates H2AK13,15ub. In cells, USP51 is recruited to chromatin after DNA damage and regulates the dynamic assembly/disassembly of 53BP1 and BRCA1 foci. These results show that USP51 is the DUB for H2AK13,15ub and regulates DNA damage response.

Study on the Rapid Limit Test for Six Sulfonamide Residues in Food Based on the TLC-SERS Method.

Sulfonamides are not only widely applied in clinics but also highly valued in animal husbandry. Recently, it has become common for sulfonamide residues to exceed the standard limits in food, which can affect human health. Current regulations limit these residues. Therefore, we constructed a new limit test method to rapidly determine the levels of sulfonamide residues. Six sulfonamides were detected using the latest method called TLC-SERS, namely, sulfamethasone (A), sulfamethazine (B), sulfadoxine (C), sulfamethoxydiazine (D), sulfamethoxazole (E), and sulfathiazole (F). The optimal conditions for SERS detection were investigated for these six drugs, and the separation effects of different TLC spreaders on them were compared. Then, we successfully established a separation system using dichloromethane-methanol-ammonia in a ratio of 5:1:0.25 (v/v/v), which provided good separation effects on the six drugs. The residues were preliminarily separated via TLC. A silver sol solution was added to the spot on the silica gel G plate at the corresponding specific shift values, and SERS detection was performed. The sample solution was placed on the spot under a 532 nm laser, and the SERS spectrum was collected and analyzed for the six sulfonamides. The results showed obvious variations in the SERS spectrum among the six sulfonamides, with the LODs being 12.5, 6.4, 6.3, 7.1, 18.8, and 6.2 ng/mL from A to F, respectively, and an RSD of <3.0%. Within 48 h, the SERS signal for each sulfonamide drug was kept stable, with an RSD of <3.0%. The detection results of 20 samples using the TLC-SERS method were consistent with those obtained by UPLC-MS/MS. The established TLC-SERS method is simple and fast, providing a useful reference for the rapid detection of residue limits in food.

Identification and Characterization of the Determinants of Copper Resistance in the Acidophilic Fungus Acidomyces richmondensis MEY-1 Using the CRISPR/Cas9 System.

Copper (Cu) homeostasis has not been well documented in filamentous fungi, especially extremophiles. One of the main obstacles impeding their characterization is the lack of a powerful genome-editing tool. In this study, we applied a CRISPR/Cas9 system for efficient targeted gene disruption in the acidophilic fungus Acidomyces richmondensis MEY-1, formerly known as Bispora sp. strain MEY-1. Using this system, we investigated the basis of Cu tolerance in strain MEY-1. This strain has extremely high Cu tolerance among filamentous fungi, and the transcription factor ArAceA (A. richmondensis AceA) has been shown to be involved in this process. The ArAceA deletion mutant (ΔArAceA) exhibits specific growth defects at Cu concentrations of ≥10 mM and is transcriptionally more sensitive to Cu than the wild-type strain. In addition, the putative metallothionein ArCrdA was involved in Cu tolerance only under high Cu concentrations. MEY-1 has no Aspergillus nidulans CrpA homologs, which are targets of AceA-like transcription factors and play a role in Cu tolerance. Instead, we identified the Cu-transporting P-type ATPase ArYgA, homologous to A. nidulans YgA, which was involved in pigmentation rather than Cu tolerance. When the ΔArYgA mutant was grown on medium supplemented with Cu ions, the black color was completely restored. The lack of CrpA homologs in A. richmondensis MEY-1 and its high tolerance to Cu suggest that a novel Cu detoxification mechanism differing from the AceA-CrpA axis exists. IMPORTANCE Filamentous fungi are widely distributed worldwide and play an important ecological role as decomposers. However, the mechanisms of their adaptability to various environments are not fully understood. Various extremely acidophilic filamentous fungi have been isolated from acidic mine drainage (AMD) with extremely low pH and high heavy metal and sulfate concentrations, including A. richmondensis. The lack of genetic engineering tools, particularly genome-editing tools, hinders the study of these acidophilic and heavy metal-resistant fungi at the molecular level. Here, we first applied a CRISPR/Cas9-mediated gene-editing system to A. richmondensis MEY-1. Using this system, we identified and characterized the determinants of Cu resistance in A. richmondensis MEY-1. The conserved roles of the Cu-binding transcription factor ArAceA in Cu tolerance and the Cu-transporting P-type ATPase ArYgA in the Cu-dependent production of pigment were confirmed. Our findings provide insights into the molecular basis of Cu tolerance in the acidophilic fungus A. richmondensis MEY-1. Furthermore, the CRISPR/Cas9 system used here would be a powerful tool for studies of the mechanisms of adaptability of acidophilic fungi to extreme environments.

Development of an efficient protein expression system in the thermophilic fungus Myceliophthora thermophila.

BACKGROUND: Thermophilic fungus Myceliophthora thermophila has been widely used in industrial applications due to its ability to produce various enzymes. However, the lack of an efficient protein expression system has limited its biotechnological applications. RESULTS: In this study, using a laccase gene reporting system, we developed an efficient protein expression system in M. thermophila through the selection of strong constitutive promoters, 5'UTRs and signal peptides. The expression of the laccase was confirmed by enzyme activity assays. The results showed that the Mtpdc promoter (Ppdc) was able to drive high-level expression of the target protein in M. thermophila. Manipulation of the 5'UTR also has significant effects on protein expression and secretion. The best 5'UTR (NCA-7d) was identified. The transformant containing the laccase gene under the Mtpdc promoter, NCA-7d 5'UTR and its own signal peptide with the highest laccase activity (1708 U/L) was obtained. In addition, the expression system was stable and could be used for the production of various proteins, including homologous proteins like MtCbh-1, MtGh5-1, MtLPMO9B, and MtEpl1, as well as a glucoamylase from Trichoderma reesei. CONCLUSIONS: An efficient protein expression system was established in M. thermophila for the production of various proteins. This study provides a valuable tool for protein production in M. thermophila and expands its potential for biotechnological applications.

Recombinant expression of IGF-1 and LR3 IGF-1 fused with xylanase in Pichia pastoris.

Insulin-like growth factor-1 (IGF-1) is a pleiotropic protein hormone and has become an attractive therapeutic target because of its multiple roles in various physiological processes, including growth, development, and metabolism. However, its production is hindered by low heterogenous protein expression levels in various expression systems and hard to meet the needs of clinical and scientific research. Here, we report that human IGF-1 and its analog Long R3 IGF-1 (LR3 IGF-1) are recombinant expressed and produced in the Pichia pastoris (P. pastoris) expression system through being fused with highly expressed xylanase XynCDBFV. Furthermore, purified IGF-1 and LR3 IGF-1 display excellent bioactivity of cell proliferation compared to the standard IGF-1. Moreover, higher heterologous expression levels of the fusion proteins XynCDBFV-IGF-1 and XynCDBFV-LR3 IGF-1 are achieved by fermentation in a 15-L bioreactor, reaching up to about 0.5 g/L XynCDBFV-IGF-1 and 1 g/L XynCDBFV-TEV-LR3 IGF-1. Taken together, high recombinant expression of bioactive IGF-1 and LR3 IGF-1 is acquired with the assistance of xylanase as a fusion partner in P. pastoris, which could be used for both clinical and scientific applications. KEY POINTS: • Human IGF-1 and LR3 IGF-1 are produced in the P. pastoris expression system. • Purified IGF-1 and LR3 IGF-1 show bioactivity comparable to the standard IGF-1. • High heterologous expression of IGF-1 and LR3 IGF-1 is achieved by fermentation in a bioreactor.

Astragaloside IV Alleviates Atorvastatin-Induced Hepatotoxicity via AMPK/SIRT1 Pathway.

INTRODUCTION: Atorvastatin (ATO) is often used to reduce blood lipids and prevent atherosclerosis, but excessive use of ATO will lead to hepatotoxicity. This paper investigated the effects of astragaloside IV (AS IV), which has multiple biological functions, on ATO-induced hepatotoxicity and the underlying mechanism. METHODS: ATO treatment induced a rat model of hepatotoxicity, followed by AS IV treatment. Colorimetric kits were used to detect rat liver function indexes including aspartate aminotransferase (AST), alanine transaminase (ALT), malondialdehyde (MDA), and reduced glutathione (GSH). Reactive oxygen species (ROS) level was determined by 2', 7'-Dichlorodihydrofluorescein diacetate kit. The liver fibrosis and F4/80 expression were detected by Sirius red staining and immunochemistry. Mitochondrial electron transport chain complex I and complex IV activities were examined. The level of mitochondrial membrane potential (MMP) was detected by JC-1 staining. The inflammatory factor levels were detected by quantitative real-time polymerase chain reaction. Western blot detected apoptosis-related proteins and AMPK/SIRT1-related proteins. RESULTS: ATO increased ALT, AST, MDA, and ROS levels and decreased GSH content but was subsequently reversed by AS IV. AS IV alleviated liver tissue damage caused by ATO. AS IV elevated complex I and complex IV activity and promoted MMP levels in ATO rats. ATO promoted inflammatory factor release in SD rats but was then suppressed by AS IV. AS IV inhibited Bax, cleaved caspase-3 but up-regulated Bcl-2 in ATO-induced rats. ATO inhibited SIRT1 expression and AMPK phosphorylation, which was subsequently promoted by AS IV. CONCLUSION: AS IV inhibits ATO-induced hepatotoxicity by activating the AMPK/SIRT1 pathway.

Caspase-1 Regulates the Apoptosis and Pyroptosis Induced by Phthalocyanine Zinc-Mediated Photodynamic Therapy in Breast Cancer MCF-7 Cells.

Photodynamic therapy (PDT) is an innovative and perspective antineoplastic therapy. Tetra-α-(4-carboxyphenoxy) phthalocyanine zinc (TαPcZn)-mediated PDT (TαPcZn-PDT) has shown antitumor activity in some tumor cells, but the manner in which caspase-1 is involved in the regulation of apoptosis and pyroptosis in the TαPcZn-PDT-treated breast cancer MCF-7 cells is unclear. Therefore, effects of TαPcZn-PDT on cytotoxicity, cell viability, apoptosis, pyroptosis, cellular reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm), caspase-1, caspase-3, and nuclear transcription factor-κB (NFκB) in MCF-7 cells was firstly examined in the present study. The findings demonstrated that TαPcZn-PDT resulted in the increase in cytotoxicity and the percentage of apoptotic and pyroptotic cells, the reduction in cell viability and ΔΨm, the production of ROS and the activation of caspase-1, caspase-3 and NFκB in MCF-7 cells. Furthermore, the results also revealed that siRNA-targeting caspase-1 (siRNA-caspase-1) attenuated the effect of TαPcZn-PDT on apoptosis, pyroptosis and the activation of caspase-1, caspase-3 and NFκB in MCF-7 cells. Taken together, we conclude that caspase-1 regulates the apoptosis and pyroptosis induced by TαPcZn-PDT in MCF-7 cells.

Rapid Limit Test of Eight Quinolone Residues in Food Based on TLC-SERS, a New Limit Test Method.

Residual quinolones in food that exceed their maximum residue limit (MRL) are harmful to human health. However, the existing methods used for testing these residues have limitations; so, we developed a new limit test method called TLC-SERS to rapidly determine the levels of residues of the following: enrofloxacin (A), ciprofloxacin (B), ofloxacin (C), fleroxacin (D), sparfloxacin (E), enoxacin (F), gatifloxacin (G), and nadifloxacin (H). The residues ware preliminarily separated via TLC. The tested compounds' position on a thin-layer plate were labeled using their relative Rf under 254 nm ultraviolet light, and an appropriate amount of nanometer silver solution was added to the position. The silver on the plate was irradiated with a 532 nm laser to obtain the SERSs of the compounds. The results show significant differences in the SERS of the eight quinolones: the LODs of H, A, D, E, C, G, F, and B were 9.0, 12.6, 8.9, 19.0, 8.0, 8.7, 19.0, and 12.6 ng/mL, respectively; and the RSD was ≤4.9% for the SERS of each quinolone. The limit test results of 20 samples are consistent with those obtained via UPLC-MS/MS. The results indicate that TLC-SERS is a specific, sensitive, stable, and accurate method, providing a new reference for the rapid limit test of harmful residues in foods.

Enhancing the Thermostability of Phytase to Boiling Point by Evolution-Guided Design.

The good thermostability of enzymes is an important basis for their wide application in industry. In this study, the phytase APPA from Yersinia intermedia was designed by evolution-guided design. Through the collection of homologous sequences in the NCBI database, we obtained a sequence set composed of 5,569 sequences, counted the number and locations of motif N-X-T/S, and selected the sites with high frequency in evolution as candidate sites for experiments. Based on the principle that N-glycosylation modification sites are located on the protein surface, 13 mutants were designed to optimize the number and location of N-glycosylation sites. Through experimental verification, 7 single mutants with improved thermostability were obtained. The best mutant, M14, with equal catalytic efficiency as the wild-type was obtained through combined mutation. The half-life (t1/2) value of mutant M14 was improved from 3.32 min at 65°C to 25 min of at 100°C, allowing it to withstand boiling water treatment, retaining approximately 75% initial activity after a 10-min incubation at 100°C. Differential scanning calorimetry analysis revealed that while the mutants' thermodynamic stability was nearly unchanged, their kinetic stability was greatly improved, and the combined mutant exhibited strong refolding ability. The results of a in vitro digestibility test indicated that the application effect of mutant M14 was about 4.5 times that of wild-type APPA, laying a foundation for its industrial application. IMPORTANCE Due to the harsh reaction conditions of industrial production, the relative instability of enzymes limits their application in industrial production, such as for food, pharmaceuticals, and feed. For example, the pelleting process of feed includes a brief high temperature (80 to 85°C), which requires the enzyme to have excellent thermostability. Therefore, a simple and effective method to improve the thermostability of enzymes has important practical value. In this study, we make full use of the existing homologous sequences (5,569) in the database to statistically analyze the existence frequency of N-X-T/S motifs in this large sequence space to design the phytase APPA with improved thermostability and a high hit rate (~50%). We obtained the best combination mutant, M14, that can tolerate boiling water treatment and greatly improved its kinetic stability without damaging its specific activity. Simultaneously, we proved that its performance improvement is due to its enhanced refolding ability, which comes from N-glycan modification rather than amino acid replacement. Our results provide a feasible and effective method for the modification of enzyme thermostability.

Potential of nobiletin against Alzheimer's disease through inhibiting neuroinflammation.

OBJECTIVE: This study aimed to explore the mechanism of Nobiletin attenuating Alzheimer's disease (AD) by inhibiting neuroinflammation. METHODS: The expression of inflammatory cytokines and HMGB-1 in serum of AD patients were examined. Microglia (MGs) were treated with different doses of Nobiletin before LPS and Nigericin induction. Cell viability and apoptosis were determined by CCK-8 and TUNEL assays, respectively. APP/PS1 mice were gavaged with Nobiletin, and Morris water maze (MWM) was established to record swimming speed, escape latency, the number of platform crossings, and time spent in the platform quadrant. MGs activation in brain tissues was evaluated by immunofluorescence. The expression of pyroptosis-related proteins, inflammatory cytokines, and HMGB-1 was determined in the hippocampus and MGs. RESULTS: The levels of inflammatory cytokines and HMGB-1 were high in serum of AD patients. Treatment with different concentrations of Nobiletin prominently enhanced cell viability and reduced apoptosis and the expression of inflammatory cytokine and pyroptosis-related proteins in LPS + Nigericin-induced MGs. Gavage of different doses of Nobiletin into APP/PS1 mice shortened the escape latency in mice, diminished MGs activation in brain tissues, and remarkably elevated the number of platform crossings and the time spent in the platform quadrant without obvious change in swimming speed, suggesting that Nobiletin improved the spatial learning and memory abilities in APP/PS1 mice. The expression of pyroptosis-related proteins, HMGB-1, and inflammatory cytokines was decreased dramatically by Nobiletin in the hippocampus of APP/PS1 mice. CONCLUSIONS: Nobiletin can inhibit neuroinflammation by inhibiting HMGB-1, pyroptosis-related proteins, and inflammatory cytokines, thus mitigating AD.

Eukaryotic communities in coastal water from Shenzhen in South China.

Eukaryotic microorganisms are ubiquitous in the marine environment, and have a wide variety of ecosystem functions. Shenzhen is one of the most developed cities in South China, but the eukaryotic communities in the water along its coastlines remain poorly understood. The study applied 18S rRNA gene ITS (internal transcribed spacer) sequencing to identify the eukaryotic community from twenty sites of Shenzhen coast water. The alpha-diversity of the samples between these sites were significantly different, and the seawater of eastern coast had higher alpha-diversity compared to that of the western coast. The abundance of Chlorophyta was notably higher in the seawater of western coast, but Picozoa was relatively depleted. Specifically, Cryptocaryon, Pseudovorticella, and Cyclotella were significantly higher in the water of western coast, while Guinardia, Minutocellus, and Amoebophrya were increased in eastern samples. The spatially variations of eukaryotic microorganism community in the seawater of Shenzhen coast were associated with the water quality. The results have important significance for the understanding of coastal eukaryotic community, their interaction network, and build a foundation for future management and protection of coastal water quality.

High-throughput sequencing reveals the spatial distribution variability of microbial community in coastal waters in Shenzhen.

Seashore habitats are located between terrestrial and marine ecosystems, which are a hotspot for anthropogenic impacts. Shenzhen is one of the most developed cities in south China, but the microbial functions of its coastal ecosystems remain poorly understood. The study applied 16S rRNA gene sequencing methods to identify the bacterial community from twenty sites of Shenzhen inshore waters. The microbial structure of the samples between eastern Shenzhen and western Shenzhen seashores is notably different, suggesting the spatial variability. Proteobacteria, Cyanobacteria, Actinobacteria, and Bacteroidetes were dominant phyla in the community, and the relative abundance of Bacteroidetes was significantly higher in eastern seashores. Specifically, samples from western Shenzhen contained much more Prochlorococcus, while Synechococcus was more abundant in eastern samples. Moreover, the metabolism of terpenoids and polyketides, and transport and catabolism were significantly more abundant in eastern samples, while antibiotic-resistant pathways were enriched in western samples. The results have important significance to understand bacterial ecosystem of coastal water and promote water quality management and protection activity in Shenzhen. This study can also help developing an optimal strategy for the green economy development and the policy planning of Guangdong-Hong Kong-Macao Greater Bay Area.

Impacts of anthropogenic disturbances on microbial community of coastal waters in Shenzhen, South China.

During the urbanization, human activities have brought great changes to marine biodiversity and microbial communities of coastal water. Shenzhen is a coastal city that has developed rapidly over the past four decades, but the microbial communities and metabolic potential in offshore water are still not well characterized. Here, 16S rRNA gene V4-V5 sequencing was conducted to determine the microbial components from coastal waters in twenty selected areas of Shenzhen. The results showed a significant difference on the microbial composition between the western and eastern waters. Samples from western coast had more abundant Burkholderiaceae, Sporichthyaceae, Aeromonadaceae, and Methylophilaceae compared to eastern coast, and at the genus level, Candidatus Aquiluna, Aeromonas, Arcobacter, Ottowia and Acidibacter were significantly higher in western waters. There was also a notable difference within the western sample group, suggesting the taxa-compositional heterogeneity. Moreover, analysis of environmental factors and water quality revealed that salinity, pH and dissolved oxygen were relatively decreased in western samples, while total nitrogen, total phosphorus, chemical oxygen demand, and harmful marine vibrio were significantly increased compared to eastern waters. The results suggest the coastal waters pollution is more serious in western Shenzhen than eastern Shenzhen and the microbial communities are altered, which can be associated with anthropogenic disturbances.

Identification of virion-associated transcriptional transactivator (VATT) of SGIV ICP46 promoter and their binding site on promoter.

BACKGROUND: Iridoviruses are large DNA viruses that cause diseases in fish, amphibians and insects. Singapore grouper iridovirus (SGIV) is isolated from cultured grouper and characterized as a ranavirus. ICP46 is defined to be a core gene of the family Iridoviridae and SGIV ICP46 was demonstrated to be an immediate-early (IE) gene associated with cell growth control and could contribute to virus replication in previous research. METHODS: The transcription start site (TSS) and 5'-untranslated region (5'-UTR) of SGIV ICP46 were determined using 5' RACE. The core promoter elements of ICP46s were analyzed by bioinformatics analysis. The core promoter region and the regulation model of SGIV ICP46 promoter were revealed by the construction of serially deleted promoter plasmids, transfections, drug treat and luciferase reporter assays. The identification of virion-associated transcriptional transactivator (VATT) that interact with SGIV ICP46 promoter and their binding site on promoter were performed by electrophoretic mobility shift assays (EMSA), DNA pull-down assays and mass spectrometry (MS). RESULTS: SGIV ICP46 was found to have short 5'-UTR and a presumptive downstream promoter element (DPE), AGACA, which locates at + 36 to + 39 nt downstream of the TSS. The core promoter region of SGIV ICP46 located from - 22 to + 42 nt relative to the TSS. VATTs were involved in the promoter activation of SGIV ICP46 and further identified to be VP12, VP39, VP57 and MCP. A 10-base DNA sequence "ATGGCTTTCG" between the TSS and presumptive DPE was determined to be the binding site of the VATTs. CONCLUSION: Our study showed that four VAATs (VP12, VP39, VP57 and MCP) might bind with the SGIV ICP46 promoter and be involved in the promoter activation. Further, the binding site of the VATTs on promoter was a 10-base DNA sequence between the TSS and presumptive DPE.

H3K9me3 demethylase Kdm4d facilitates the formation of pre-initiative complex and regulates DNA replication.

DNA replication is tightly regulated to occur once and only once per cell cycle. How chromatin, the physiological substrate of DNA replication machinery, regulates DNA replication remains largely unknown. Here we show that histone H3 lysine 9 demethylase Kdm4d regulates DNA replication in eukaryotic cells. Depletion of Kdm4d results in defects in DNA replication, which can be rescued by the expression of H3K9M, a histone H3 mutant transgene that reverses the effect of Kdm4d on H3K9 methylation. Kdm4d interacts with replication proteins, and its recruitment to DNA replication origins depends on the two pre-replicative complex components (origin recognition complex [ORC] and minichromosome maintenance [MCM] complex). Depletion of Kdm4d impairs the recruitment of Cdc45, proliferating cell nuclear antigen (PCNA), and polymerase δ, but not ORC and MCM proteins. These results demonstrate a novel mechanism by which Kdm4d regulates DNA replication by reducing the H3K9me3 level to facilitate formation of pre-initiative complex.