Integrative Analysis of DNA Methylation and Gene Expression Data Identifies Potential Biomarkers and Functional Epigenetic Modules for SARS-CoV-2.

To integrate gene expression and DNA methylation data and find the potential role of DNA methylation in the invasion and replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We first conducted differential expression and methylation analysis between the coronavirus disease of 2019 (COVID-19) and healthy controls. FEM was employed to identify functional epigenetic modules, from which a diagnostic model for COVID-19 was built. SKA1 and WSB1 modules were identified, with SKA1 module enriched in COVID-19 replication and transcription, and WSB1 module related to ubiquitin-protein activity. The differentially expressed or differentially methylated genes in these two modules could be used to distinguish COVID-19 from healthy controls, with AUC reaching 1 and 0.98 for SKA1 and WSB1 modules, respectively. Two epigenetically activated genes (CENPM and KNL1) from the SKA1 module were upregulated in HPV- or HBV-positive tumor samples and were found to be significantly associated with the survival of tumor patients. In conclusion, the identified FEM modules and potential signatures play an essential role in the replication and transcription of coronavirus.

Functional analysis target of rapamycin (TOR) on the Penaeus vannamei in response to acute low temperature stress.

Target of rapamycin (TOR) is an atypical of Ser/Thr protein kinase that plays an important role in many aspects such as cell growth, reproduction, differentiation, cell cycle regulation, autophagy and apoptosis. However, little information is known about the enzyme in crustaceans. Here, a novel TOR was identified from shrimp Penaeus vannamei (PvTOR) and its biological functions were investigated in response low temperature stress. The PvTOR gene encoded a polypeptide of 2464 amino acids with an estimated molecular mass of 279.4 kD and a predicted isoelectronic point (pI) of 7.30. Phylogenetic analysis revealed that PvTOR shared high similarity with other known species. PvTOR mRNA was detected in all the tested tissues and highest transcription in muscle and hepatopancreas. PvTOR transcriptional level was up-regulated significantly at 1.5 h and 3 h, and down-regulated at 12 h and 24 h after low temperature stress. TEM and autophagy indicator system GFP-PvLC3 suggested that low temperature induced autophagy generation. ROS, Ca2+ concentration and apoptosis rate were increased significantly in TOR-knockdown shrimp after low temperature stress. The autophagy associated gene ATG8II/I, PvBeclin-1, PvATG14, apoptosis gene PvPARP, Pvcasp-3, PvBAX and Pvp53 transcripts, and casp-3/8 activity in hemocyte were increased significantly in TOR-knockdown group shrimp at 3 h after low temperature stress. Additionally, THC counts of TOR-knockdown group were significantly higher than the dsGFP group. In summary, these results suggested that PvTOR plays an important role in the adaptation mechanisms of shrimp at low temperature by regulating autophagy and apoptosis.

Identifying the function of LvPI3K during the pathogenic infection of Litopenaeus vannamei by Vibrio alginolyticus.

It is well known that PI3K regulates various processes in mammalian cells by generating a secondary messenger that later activates AKT. However, its innate immune function in crustaceans remains unclear. We report the characterization of Litopenaeus vannamei PI3K (LvPI3K) for investigating how PI3K participates in the innate immunity of crustaceans. Full-length LvPI3K cDNA was 3357 bp long, with a 3222 bp open reading frame (ORF) that encodes a putative protein of 1292 amino acids. The PI3K catalytic domain (PI3Kc) of LvPI3K was found to be rather conserved when the PI3Ks from other species were analyzed. The LvPI3K protein was shown to be localized to the cytoplasm of Drosophila S2 cells, while LvPI3K mRNA was ubiquitously expressed in healthy L. vannamei, with the highest expression found in hemolymph. A dual luciferase reporter gene assay demonstrated that LvPI3K overexpression activated the promoter of antibacterial peptide LvPEN4 in a dose-dependent manner. However, the addition of PDTC, a specific inhibitor of NF-κB, suppressed the LvPI3K-induced LvPEN4 promoter activation. Moreover, Vibrio alginolyticus challenge induced a rapid up-regulation of LvPI3K expression. Further experiments showed that LvPI3K silencing in shrimp challenged with V. alginolyticus significantly increased Vibrio number, ROS production and DNA damage in the hemolymph, as well as significantly decreased total hemocyte count. The mRNA levels of certain molecules related to LvPI3K signaling, such as LvAKT and LvPEN4, also decreased following LvPI3K silencing. Taken together, these results suggest that LvPI3K regulates the downstream signal component LvPEN4 and functions in V. alginolyticus resistance.

Molecular characterization and function analysis of a nucleotide excision repair gene Rad23 from Litopenaeus vannamei after Vibrio alginolyticus challenge.

Nucleotide excision repair (NER) removes many different types of DNA lesions, and NER related host factors are reported to aid recovery steps during viral integration. Here, we report the identification and characterization of a DNA repair gene Rad23 from Litopenaeus vannamei and explore its role in innate immunity of crustaceans. LvRad23 contains a1149 bp open reading frame (ORF) which encodes a 382 amino acids protein with predicted theoretical isoelectric point of 4.21. LvRad23 was ubiquitously expressed in the muscle, eyestalk, gill, stomach, heart, legs, intestine, and hepatopancreas in order from high to low and LvRad23 protein was showed to be located in the cytoplasm of Drosophila S2 cells. The homology analysis showed that it has a high sequence homology with Rad23 protein from Marsupenaeus japonicus. Vibrio alginolyticus challenge induced a remarkable up-regulation of LvRad23 mRNA in hepatopancreas. Knocking down LvRad23can interfere the NER pathway by down regulating the expression of replication protein A (RPA) and proliferating cell nuclear antigen (PCNA). However it didn't cause any significant difference on total hemocyte count (THC) between LvRad23-silenced and non-silenced group.LvRad23-silenced then challenge with V. alginolyticus inducing high level of reactive oxygen species (ROS) and DNA damage in hemolymph. As well as decreased THC, which seriously diminished the innate immune system of L. vannamei. Meanwhile, the NER pathway was reactived by enhancing the expression of LvRad23 and promoting the production of LvPCNA to resist apoptosis and maintain proliferation of hemolymph cells in the later stage. Our results suggest that LvRad23 plays a vital role in shrimp specific immune response to V. alginolytcus through its participation in NER pathway.

Clinical Efficacy of Tacrolimus Ointment + 3% Boric Acid Lotion Joint Chinese Angelica Decoction in Chronic Perianal Eczema.

OBJECTIVE: To unearth the clinical efficacy of tacrolimus ointment + 3% boric acid lotion joint Chinese angelica decoction in chronic perianal eczema. METHODS: Patients with chronic perianal eczema admitted to hospital from June 2018 and June 2019 were retrospectively analyzed. Patients in the control group (n = 38) underwent basic therapy with tacrolimus ointment + 3% boric acid lotion, whereas those in the observation group (n = 38) were given oral Chinese angelica decoction on the basis of the above therapy. Patient's baseline information before therapy and clinical symptoms after therapy were observed and compared, including pruritus ani score, anus drainage and damp score, skin lesion score, skin lesion area score, life quality index score, and IL-2, IL-4, and IgE levels in serum. Overall efficacy in the two groups was also evaluated. RESULTS: No significant differences were found in the baseline information between the observation group and control group before therapy. After therapy, pruritus ani score (P = 0.023), anus drainage and damp score (P = 0.041), skin lesion score (P = 0.025), and skin lesion area score (P = 0.035) of patients in the observation group were remarkably lower than those in the control group. Significantly higher release levels of clinical symptoms of patients in the observation group were indicated. With respect to the control group, the life quality score (P = 0.020) and IgE level in serum (P = 0.003) of patients in the observation group were significantly lower, while IL-4 level in serum was significantly higher (P = 0.129). The therapy in the observation group achieved better clinical efficacy. Overall efficacy in the observation group was markedly favorable with respect to the control group. CONCLUSION: With respect to tacrolimus ointment + 3% boric acid lotion, patients with chronic perianal eczema displayed better clinical efficacy after jointly being treated by Chinese angelica decoction.

SIRT1 Mediates Neuropathic Pain Induced by Sciatic Nerve Chronic Constrictive Injury in the VTA-NAc Pathway.

Background: Mounting evidence has shown that sirtuin 1 (SIRT1), a class III histone deacetylase, alleviated several types of neuropathic pain in the spinal cord and dorsal root ganglion and regulated some aberrant behaviors in the ventral tegmental area (VTA) and the nucleus accumbens (NAc). Methods: In this context, the effect of SIRT1 on neuropathic pain in the VTA-NAc pathway was investigated in the model of chronic constrictive injury (CCI). Results: SIRT1 was localized in the VTA neurons in naive mice. The expression of SIRT1 was decreased in the contralateral VTA of CCI mice. After microinjection of SRT1720 (an activator of SIRT1) in the contralateral VTA of CCI mice, the established thermal hyperalgesia was attenuated. However, it was further exacerbated by EX-527 (an inhibitor of SIRT1). The elevated level of acetyl-histone 3 was reduced by SRT1720 but further elevated by EX-527 in the contralateral VTA of CCI mice. The increased expression of Fos in both VTA and NAc was downregulated by SRT1720 but further upregulated by EX-527 in CCI mice. Conclusions: The discovery of the effect of SIRT1 on neuropathic pain in the VTA represents an important step forward in understanding the analgesic mechanisms of the VTA-NAc pathway.

A Novel Kelch-Like-1 Is Involved in Antioxidant Response by Regulating Antioxidant Enzyme System in Penaeus vannamei.

Heavy metals are typical cumulative pollutants that can enter and poison the human body through the food chain. However, the molecular mechanism of heavy metal-induced oxidative stress is unclear. In this study, we characterize PvKelch-like-1 from P. vannamei and explore its antioxidant roles in immune regulation of crustaceans. PvKelch-like-1 full length contains 2107 nucleotides, consists of a 5' untranslated region (UTR) of 79 bp, a 3' UTR of 180 bp, and a ORF of 1848 encoded 615 amino acids, which contain a BTB, BACK and Kelch motif, putative molecular mass and isoelectric point were 69 KDa and 6.54. PvKelch-like-1 mRNA was ubiquitously expressed in all detected tissue of P. vannamei, and mRNA expression levels were significantly up-regulated from 6 to 24 h after cadmium stress and reached the highest level (3.2-fold) at 12 h in the hepatopancreas. Subcellular localization analysis revealed that PvKelch-like-1 was localized in the nucleus. Silencing PvKelch-like-1 significantly increased reactive oxygen species (ROS) (1.61-fold) production and DNA damage (1.32-fold) in the shrimp hemolymph, and significantly decreased total hemocyte counts (THC) (0.64-fold) at 6 h in hemolymph. Additionally, the antioxidant genes PvCAT (0.43-fold), PvMnSOD (0.72-fold), PvGST (0.31-fold) and PvGPx (0.59-fold) at 6 h were decreased significantly in PvKelch-like-1 silenced shrimp after cadmium stress. Overexpression of PvKelch-like-1 has the opposite results in enzyme activity. The SOD (2.44-fold) and CAT (2.19-fold) activities were significantly increased after overexpressing PvKelch-like-1. These results suggest that PvKelch-like-1 plays a vital role in shrimp innate immune defense by positively regulating the expression of antioxidant enzyme genes to respond to cadmium stress.

The role of delta-1-pyrroline-5-carboxylate dehydrogenase (P5CDh) in the Pacific white shrimp (Litopenaeus vannamei) during biotic and abiotic stress.

Proline (Pro) metabolism is intimately associated with stress adaptation. The catabolism of Pro includes two dehydrogenation reactions catalyzed by proline dehydrogenase (ProDH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDh). P5CDh is a mitochondrial matrix NAD+-dependent dehydrogenase that is critical in preventing P5C-Pro intensive cycling and avoiding ROS production from electron run-off. Little is known about the roles of P5CDh in invertebrates, however. We cloned the P5CDh sequence in the Pacific white shrimp, Litopenaeus vannamei, and found that LvP5CDh is expressed predominantly in pleopod, hepatopancreas and gill. Subcellular localization analysis revealed that LvP5CDh protein was mainly found in the cytoplasm. In addition, overexpressing LvP5CDh in cells reduced ROS formation and inhibited apoptosis induced by LC50 Cd2+. Shrimp were exposed to various stress factors including infection with Vibrio alginolyticus, (½ LC50 and LC50) Cd2+, acid (pH 5.6) and alkali stress (pH 9.3). Both biotic and abiotic stress resulted in increased LvP5CDh expression and Pro accumulation; V. alginolyticus infection, pH 9.3 and LC50 Cd2+ stress apparently stimulated the Glu pathway of Pro synthesis, while pH 5.6 and ½ LC50 Cd2+ stress promoted the Orn pathway of Pro synthesis. Silencing of Lvp53 in shrimp attenuated LvP5CDh expression during Cd2+ stress, but had no effect on LvP5CDh mRNA levels if no Cd2+ stress was imposed. Our study contributes to the functional characterization of LvP5CDh in biotic and abiotic stress and reveals it to protect against ROS generation, damage to the cell, including the mitochondria, and apoptosis. Thus, LvP5CDh plays a critical role in immune defense and antioxidant responses.

LvCdc42 is a potential negative regulator of Lvp53 in Litopenaeus vannamei exposed to Vibrio alginolyticus stress.

As a crucial molecular switch, Cdc42 is a signal regulation hub which is involved in a wide range of cellular processes, including cytokinesis, gene expression, cell cycle progression and apoptosis. It has been reported that this GTPase promotes host defense against fatal infection and plays a vital role in the innate immunity system of mammals. But whether and how Cdc42 participates in innate immunity in invertebrates, such as the shrimp Litopenaeus vannamei, is still unknown. In this study, confocal microscopy analysis showed that LvCdc42 located in both cytoplasm and nucleus of S2 cells depended on its structure. The silencing LvCdc42 induced an increase in the expression of Lvp53 and Lvcaspase-3. When LvCdc42-silenced shrimps were stressed with Vibrio alginolyticus, the expression of Lvp53 and Lvcaspase-3 was markedly up-regulated. Moreover, the increase in the apoptosis rate in hemocytes and in cumulative mortality were in line with Lvp53 mRNA expression. These data suggest that the molecular switch LvCdc42 acts as a negative regulator of Lvp53 and participates in the apoptosis of hemocytes when L. vannamei is challenged with V. alginolyticus.