A novel antimicrobial peptide Scyreptin1-30 from Scylla paramamosain exhibiting potential therapy of Pseudomonas aeruginosa early infection in a mouse burn wound model.

Antimicrobial resistance (AMR) constitutes a significant global threat to human health. In recent years, there has been a concerning surge in infections caused by multidrug-resistant bacteria, highlighting the pressing need to urgently explore novel and effective alternatives to conventional antibiotics. Antimicrobial peptides (AMPs) have emerged as a focal point of research, capturing significant attention as promising antimicrobial agents. In this study, we have identified a novel cationic antimicrobial peptide (AMP) named Scyreptin1-30, derived from the marine invertebrate Scylla paramamosain. The results showed that Scyreptin1-30 exhibits a broad-spectrum antimicrobial activity, demonstrating significant potency against both bacteria and fungi, and even against the clinically isolated multidrug-resistant bacteria Pseudomonas aeruginosa. Moreover, Scyreptin1-30 exhibited rapid bactericidal kinetic. The results of antibacterial mechanism showed that Scyreptin1-30 destroyed the integrity of bacterial membranes, leading to bacterial death and exhibited potent anti-biofilm activity against P. aeruginosa. The activity of Scyreptin1-30 against bacteria had a favorable thermal stability, displayed a certain ion tolerance, and showed no discernible cytotoxicity when assessed against both the mammalian cell line HEK293T and the fish cell lines ZF4. In an In vivo study, Scyreptin1-30 exhibited a remarkably reduction in the bacterial load caused by multidrug-resistant P. aeruginosa at the site of infection, and promoted wound healing in a mouse model of burn infection. This study indicated that Scyreptin1-30 holds promise as an effective antibacterial agent, potentially serving as a topical skin treatment against multidrug-resistant bacterial infections, including those caused by P. aeruginosa.

A protocol of systematic review and meta-analysis of Mozart's Music for drug-resistant epilepsy.

BACKGROUND: This study will aim to assess the effectiveness of Mozart's Music (MM) for the management of patients with drug-resistant epilepsy (DRE). METHODS: In this study, we will search MEDLINE, EMBASE, Cochrane Library, Web of Science, CINAHL, Chinese Scientific Journal Database Information, WANGFANG, and China National Knowledge Infrastructure from their inauguration to March 1, 2020 without language and publication time restrictions. We will also identify other literature resources, such as reference lists of any related reviews. Trial quality will be examined by Cochrane risk of bias tool; reporting bias will be identified by a funnel plot and Egger test; and statistical analysis will be undertaken by RevMan 5.3 software. RESULTS: This study will summarize high quality randomized controlled trials to appraise the effectiveness and safety of MM for the treatment of patients with DRE. CONCLUSIONS: The findings of this study will supply evidence to judge whether MM is effective on DRE at evidence-based medicine level. STUDY REGISTRATION NUMBER: CRD42020170512.

A Multicenter Randomized Controlled Pilot Trial Testing the Efficacy and Safety of Pterygopalatine Fossa Puncture Using One Acupuncture Needle for Moderate-to-Severe Persistent Allergic Rhinitis.

OBJECTIVE: To compare the efficacy and safety of pterygopalatine fossa puncture using one acupuncture needle inserted through the temporal fossa (intervention) and Chinese verum acupuncture (VA) in patients with moderate-to-severe persistent allergic rhinitis. METHODS: The patients were randomized to an intervention group receiving pterygopalatine fossa puncture with one acupuncture needle for 4 weeks (once or twice weekly, 4-8 sessions in total, with a second course performed if required) or to a control group receiving individualized VA for 4 weeks (twice weekly, eight sessions in total). Patients were followed up 4 weeks later. RESULTS: Ninety-six participants were assigned to intervention (n = 48) or VA (n = 48) or VA (P > 0.05 in all cases). Compared with the VA, the time to onset of effect in the intervention group was shorter and the duration of effectiveness was longer. The mean clinical waiting time was significantly shorter in the intervention group than in the control group (6.640 ± 3.035 min and 31.19 ± 10.216 min, respectively). The total number of sessions in the VA group was 384; 7 episodes of subcutaneous bleeding occurred but did not require treatment. The total number of sessions in the intervention group was 185. Two cases of subcutaneous bleeding (one of local hematoma during the intervention and the other one of bruising in the palpebra inferior on the day after intervention) resolved upon withdrawal from the study. CONCLUSIONS: Pterygopalatine fossa puncture using one acupuncture needle resulted in a shorter time to onset of effect, a longer duration of effectiveness, and less clinical waiting time when compared with VA. Though the significant differences for TNSS and TNNSS were shown within intervention and VA groups, there were no differences between the two groups. Although the rate of subcutaneous bleeding was low, these adverse events may influence patient compliance. This trial is registered with ISRCTN21980724.

A CqFerritin protein inhibits white spot syndrome virus infection via regulating iron ions in red claw crayfish Cherax quadricarinatus.

It is well known that iron is an essential element for all living organism. The intracellular iron availability is also important for the host's innate immune response to various pathogens, in which the iron homeostasis can be regulated by ferritin due to its iron storage property. In this study, a full-length cDNA sequence of ferritin (named as CqFerritin) was identified with 1410 bp from red claw crayfish Cherax quadricarinatus, which contained an open reading frame of 513 bp, encoding 170 amino acids with a conserved ferritin domain. Tissue distribution analysis demonstrated that CqFerritin was widely expressed in various tissues with high presence in haemocyte, haematopoietic tissue (Hpt) and heart, while lowest expression in hepatopancreas. In addition, loss-of-function of CqFerritin by gene silencing resulted in significantly higher expression of an envelope protein VP28 of white spot syndrome virus (WSSV) in red claw crayfish Hpt cell cultures, indicating the potential antiviral response of CqFerritin. To further explore the effect on WSSV replication by CqFerritin, recombinant CqFerritin protein (rCqFerritin) was transfected into Hpt cells followed by WSSV infection. Importantly, the replication of WSSV was obviously decreased in Hpt cells if transfected with rCqFerritin protein, suggesting that CqFerritin had clearly negative effect on WSSV infection. Furthermore, intracellular accumulation of iron ions was found to promote the WSSV replication in a dose-dependent manner, illustrating that the iron level regulated by CqFerritin was likely to be vital for WSSV infection in red claw crayfish. Taken together, these data suggest that CqFerritin plays an important role in immune defense against WSSV infection in a crustacean C. quadricarinatus.

[Effect of electroacupuncture on the expression of oligodendrocyte precursor cells in rats with compressed spinal cord injury].

OBJECTIVE: To investigate the effect of electroacupuncture on the expression of oligodendrocyte precursor cells in rats with compressed spinal cord injury (CSCI) and to explore the mechanism of remyelinization. METHODS: Thirty-six SD rats were randomly divided into a control group and three treatment groups with 3 d, 7 d and 14 d of treatment respectively. Acupuncture was given to rats in the treatment groups through jiaji point, double zusanli (ST36), and double taixi (KI3). Electroacupuncture (continuous wave, 2 Hz/1. 5 V, 30 min) was applied for the double zusanli (ST36) and double taixi (KI3). Ethological alterations of the rats were observed with quantitative assessment of neurologic function. The ultrastructure changes of nerve fibers in white matter were determined under electronic microscope. Expressions of NG2 protein, an OPC marker, was observed by Western blot. RESULTS: No significant changes in neurologic function and G-ratio were observed after three days and seven days of electroacupuncture treatment (P>0. 05). However, 14 d of electroacupuncture treatment made a significant change compared to the 7 d treatment group and the control group (P<0. 05). The electronic microscope showed axons with varied degree of swollen, degenerated and lost cell organelle in axoplasm, edema in myelin sheaths, disordered, thickened and even broken layers of myelin sheaths in the rats with CSCI. The rats in the treatment groups had milder swollen axons and more compacted layers of myelin sheaths compared to their controls. Western blot showed that the expression of NG2 was increased with time and the differences among the three treatment groups were statistically significant (P<0. 05). The rats in the treatment groups also had higher expressions of NG2 than their controls at 7 d and 14 d (P<0. 05). CONCLUSION: Electroacupuncture can improve inflammation and edema in the injured nerve fibers and up regulate NG2 expression and remyelination of the injured nerve fibers in rats with CSCI.

Differential gene expression profile of the calanoid copepod, Pseudodiaptomus annandalei, in response to nickel exposure.

To better understand the underlying mechanisms of reactions of copepods exposed to elevated level of nickel, the suppression subtractive hybridization (SSH) was used to elucidate the response of the copepod Pseudodiaptomus annandalei to nickel exposure at the gene level. P. annandale is one of a few copepod species that can be cultured relatively easy under laboratory condition, and it is considered to be a potential model species for toxicity study. In the present study, P. annandalei were exposed to nickel at a concentration of 8.86 mgL(-1) for 24h, after which the RNA was prepared for SSH using unexposed P. annandalei as drivers. A total of 474 clones on the middle scale in the SSH library were sequenced. Among these genes, 129 potential functional genes were recognized based on the BLAST searches in NCBI and Uniprot databases. These genes were then categorized into nine groups in association with different biological processes using AmiGO against the Gene Ontology database. Of the 129 genes, 127 translatable DNA sequences were predicted to be proteins, and the putative amino acid sequences were searched for conserved domains (CD) and proteins using the CD-Search service and BLASTp. Among 129 genes, 119 (92.2%) were annotated to be involved in different biological processes, while 10 genes (7.8%) were classified as an unknown-function gene group. To further confirm the up-regulation of differentially expressed genes, the quantitative real time PCR were performed to test eight randomly selected genes, in which five of them, i.e. α-tubulin, ribosomal protein L13, ferritin, separase and Myohemerythrin-1, exhibited clear up-regulation after nickel exposure. In addition, MnSOD was further studied for the differential expression pattern after nickel exposure and the results showed that MnSOD had a time- and dose-dependent expression pattern in the copepod after nickel exposure. To the best of our knowledge, this is the first attempt to investigate the toxicity effects of nickel on a copepod at molecular level.

Characterization of RAG1 and IgM (mu chain) marking development of the immune system in red-spotted grouper (Epinephelus akaara).

In vertebrates, lymphoid-specific recombinase protein encoded by recombination-activating genes (RAG1/2) plays a key role in V(D)J recombination of the T-cell receptor and B-cell receptor. In this study, both RAG1 and the immunoglobulin M (IgM) mu chain were cloned to characterize their potential role in the immune defense at developmental stages of red-spotted grouper, Epinephelus akaara. The open reading frame (ORF) of E. akaara RAG1 included 2778 nucleotide residues encoding a putative protein of 925 amino acids, while the ORF of the IgM mu chain had 1734 nucleotide residues encoding 578 amino acids including variable (VH) and constant (CH1-CH2-CH3-CH4) regions. E. akaara RAG1 was composed of a zinc-binding dimerization domain (ZDD) with a RING finger and zinc finger A (ZFA) in the non-core region and a nonamer-binding region (NBR), with a zinc finger B (ZFB), the central and C-terminal domains in the core region. Tridimensional models of the ZDD and NBR of E. akaara RAG1 were constructed for the first time in fishes, while a 3D model of the E. akaara IgM mu chain was also clarified. The RAG1 mRNA was only detected in the thymus and kidney of 4-month and 1.5-year old groupers using qPCR, and the RAG1 protein was confirmed using western blotting and immunohistochemistry. The IgM mu mRNA was examined in most tissues except the gonad. RAG1 and IgM mu gene expression were observed at 15 dph (days post-hatching) and 23 dph respectively, and increased to a higher level at 37 dph. In addition, this was the first time that the morphology of the E. akaara thymus was characterized. The oval-shaped thymus of 4-month old fish was clearly seen and there were amounts of T lymphocytes present. The results suggested that the immune action of E. akaara was likely to start to develop around 15 dph to 29 dph. The transcript level of the RAG1 gene and the number of lymphocytes in the thymus between 4-month and 1.5-year old groupers indicated that age-related thymic atrophy also occurs in fishes. The similar functional structures of RAG1 and IgM protein between fish and mammals indicated that teleost species share a similar mechanism of V(D)J recombination with higher vertebrates.

[Evidence of "who knows acupuncture believes the importance of the pressing hand" in ancient literatures and its clinical significance].

According to the records in ancient Chinese literatures such as Lingshu (Miraculous Pivot), Nanjing (The Yellow Emperor's Canon of 81 Difficult Issues), Jinzhen fu (Odes to Golden Needles) and Zhenjiu Dacheng (Compendium of Acupuncture and Moxibustion), the importance of pressing hand has been always stressed. The role of the pressing hand can irreplaceable by the puncturing hand. The touching and pressing with the pressing hand assist on the accurate location of acupoint. The plucking technique may invigorate meridian qi and promote the arrival of qi. Inserting the needle against the finger nail of the pressing hand avoids or alleviates pain to the largest extent. Detecting along meridian and pressing technique main induce qi getting to the affected area. This paper introduces the importance of pressing hand, aiming to suggest the clinical physicians pay more attentions to the effect of the pressing hand in acupuncture practice.

Modulation and Interaction of Immune-Associated Parameters with Antioxidant in the Immunocytes of Crab Scylla paramamosain Challenged with Lipopolysaccharides.

Invertebrates are dependent on cellular and humoral immune defences against microbial infection. Scylla paramamosain is an important commercial species, but the fundamental knowledge on its immune defense related to the antioxidant and immune-associated reactions is still lacking. The study was to differentiate the responses of immune-associated parameters of haemolymph components in S. paramamosain when challenged with bacterial lipopolysaccharides (LPSs). The immunostimulating effects of LPS in crab by triggering various immune parameters (phagocytosis, lysozyme, antibacterial activity, phenoloxidase, and the generation of superoxide and nitric oxide) were investigated. Results showed that the generation of free radicals, phenoloxidase, lysozyme and antibacterial activities was significantly increased through the exposure periods. Conversely, total hemocyte count and lysosomal membrane stability decreased significantly as the exposure period extended to 96 h. The relationship between the antioxidant enzymes and immune reactions due to LPS was highly significant. In addition, ROS production was positively correlated with antioxidant showing immediate response of antioxidant defense to the oxyradicals generated. Overall, the study indicated that nonspecific immune components in hemocytes of crab showed active response to the LPS stimulation, and their responses suggested that many immune-associated parameters could be modulated and interrelated with the influence of antioxidants in crustaceans.

[On problems and thinking of methodology in the inheritance of the esteemed acupuncturists' experiences].

Based on the advantages and disadvantages of the esteemed acupuncturists' experiences, after analyzing the collection work on inheriting the esteemed acupuncturists' experiences, this paper proposes to apply the information technology to build a multimedia platform for the esteemed acupuncturists. Therefore, the precious clinical experiences of the esteemed acupuncturists are preserved. This paper states the potential future of the coming multimedia platform.

Antioxidant enzymes from the crab Scylla paramamosain: gene cloning and gene/protein expression profiles against LPS challenge.

Recent studies revealed that antioxidant enzymes play important roles in antioxidant responses caused by metabolic process or pathogen invasion. Catalase is one of these key enzymes which has been characterized and highly conserved from invertebrates to vertebrates. In the present study, a full-length cDNA sequence of catalase was cloned from the hemocyte suppression subtractive hybridization library of the crab Scylla paramamosain. The Sp-catalase (Sp-CAT) cDNA sequence contained 2551bp with an open reading frame of 1551bp encoding 517 amino acid residues. The conserved catalytic active residues His-71, Asn-144 and Tyr-354 were predicted in the amino acid sequence of Sp-CAT. The deduced Sp-CAT protein had a calculated molecular mass of 59 kDa with an estimated isoelectric point of 6.4. Multiple alignment analysis revealed that the deduced amino acid sequence of Sp-CAT shared high identity (75.4%) with those of other species. The Sp-CAT mRNA transcripts were demonstrated in multiple tissues of normal S. paramamosain. After LPS challenge, the expression level of Sp-CAT gene was increased significantly in hemocyte at 3 and 6 h, and in hepatopancreas at 6 h, respectively, determined by quantitative real-time PCR. Furthermore, the activities of CAT and SOD were also measured in different tissues and serum after LPS challenge. The CAT activity was significantly increased at 3, 6, 24 and 48 h in hemocyte lysate, at 3 h in serum, and at 24 and 48 h in hepatopancreas after LPS challenge. In addition, the SOD activity was significantly induced at 3 and 6 h in hemocyte lysate, 3 and 12 h in serum, 12 and 48 h in hepatopancreas post LPS stimulation, indicating a tissue and time-dependent antioxidant response in the crab. Taken together, these data demonstrated that a strong antioxidant response occurred in the LPS-challenged crab, which might be involved in the protection of host against microbial infections.

Identification of genes differentially expressed in hemocytes of Scylla paramamosain in response to lipopolysaccharide.

Although the crab Scylla paramamosain has been cultured in China for a long time, little knowledge is available on how crabs respond to infection by bacteria. A forward suppression subtractive hybridization (SSH) cDNA library was constructed from their hemocytes and the up-regulated genes were identified in order to isolate differentially expressed genes in S. paramamosain in response to bacterial lipopolysaccharide (LPS). A total of 721 clones on the middle scale in the SSH library were sequenced. Among these genes, 271 potentially functional genes were recognized based on the BLAST searches in NCBI and were categorized into seven groups in association with different biological processes using AmiGO against the Gene Ontology database. Of the 271 genes, 269 translatable DNA sequences were predicted to be proteins, and the putative amino acid sequences were searched for conserved domains and proteins using the CD-Search service and BLASTp. Among 271 genes, 179 (66.1%) were annotated to be involved in different biological processes, while 92 genes (33.9%) were classified as an unknown-function gene group. It was noted that only 18 of the 271 genes (6.6%) had previously been reported in other crustaceans and most of the screened genes showed less similarity to known sequences based on BLASTn results, suggesting that 253 genes were found for the first time in S. paramamosain. Furthermore, two up-regulated genes screened from the SSH library were selected for full-length cDNA sequence cloning and in vivo expression study, including Sp-superoxide dismutase (Sp-Cu-ZnSOD) gene and Sp-serpin gene. The differential expression pattern of the two genes during the time course of LPS challenge was analyzed using real-time PCR. We found that both genes were significantly expressed in LPS-challenged crabs in comparison with control. Taken together, the study primarily provides the data of the up-regulated genes associated with different biological processes in S. paramamosain in response to LPS, by which the interesting genes or proteins potentially involved in the innate immune defense of S. paramamosain will be investigated in future.

Genomic organization and tissue-specific expression analysis of hepcidin-like genes from black porgy (Acanthopagrus schlegelii B).

Hepcidin is an antimicrobial peptide and putative iron regulatory hormone previously described in mice and humans. Dozens of fish hepcidins have been isolated and characterized so far. Here we present seven hepcidin-like cDNA sequences named AS-hepc1-7, amplified from the normal commercially cultured fish (black porgy) by RACE-PCR. Sequence analysis reveals that these seven potential hepcidin peptides have highly conserved sequences with other known hepcidins, but they are different from each other in constitution and characteristics of predicted mature amino acids. Based on the study, it is deduced that AS-hepc1-7 represent different variants of a family of hepcidin genes in black porgy. To understand the organization of these hepcidin-like genes, we sequenced AS-hepc2 DNA, AS-hepc3 DNA, AS-hepc4 DNA, AS-hepc7 DNA and AS-hepc2 upstream region; and all of the four genomic DNAs consisted of two introns and three exons, the same organization as other reported hepcidins. The tissue-specific gene expression of hepcidins in normal black porgy was evaluated using RT-PCR and dot blot approaches. RT-PCR showed that transcripts of hepcidin-like mRNAs were present in each tested tissue of normal juvenile black porgy, including liver, spleen, kidney, heart, brain, stomach, intestine, gill, skin and blood, but abundant hepcidin-like mRNA transcripts were only detected in the liver, kidney, spleen, intestine and stomach by dot blot assay. In addition, using dot blot and Northern blot approach, a significant increase of hepcidin mRNA transcription was observed in the liver within 48 h after immersion in a suspension of live bacteria, which suggested that the expression pattern of hepcidin-like genes in black porgy might be different in the liver from the other tissues as previously reported in several hepcidin studies.