Objective To explore the construction of α-1,3-galactosyltransferase (GGTA1) gene-knockout (GTKO) Diannan miniature pigs and the kidney xenotransplantation from pigs to rhesus macaques, and to assess the effectiveness of GTKO pigs. Methods The GTKO Diannan miniature pigs were constructed using the CRISPR/Cas9 gene-editing system and somatic cell cloning technology. The phenotype of GTKO pigs was verified through polymerase chain reaction, Sanger sequencing and immunofluorescence staining. Flow cytometry was used to detect antigen-antibody (IgM) binding and complement-dependent cytotoxicity. Kidney xenotransplantation was performed from GTKO pigs to rhesus macaques. The humoral immunity, cellular immunity, coagulation and physiological indicators of the recipient monkeys were monitored. The function and pathological changes of the transplanted kidneys were analyzed using ultrasonography, hematoxylin-eosin staining, immunohistochemical staining and immunofluorescence staining. Results Single-guide RNA (sgRNA) targeting exon 4 of the GGTA1 gene in Diannan miniature pigs was designed. The pGL3-GGTA1-sgRNA1-GFP vector was transfected into fetal fibroblasts of Diannan miniature pigs. After puromycin selection, two cell clones, C59# and C89#, were identified as GGTA1 gene-knockout clones. These clones were expanded to form cell lines, which were used as donor cells for somatic cell nuclear transfer. The reconstructed embryos were transferred into the oviducts of trihybrid surrogate sows, resulting in 13 fetal pigs. Among them, fetuses F04 and F11 exhibited biallelic mutations in the GGTA1 gene, and F04 had a normal karyotype. Using this GTKO fetal pig for recloning and transferring the reconstructed embryos into the oviducts of trihybrid surrogate sows, seven surviving piglets were obtained, all of which did not express α-Gal epitope. The binding of IgM from the serum of rhesus monkey 20# to GTKO pig PBMC was reduced, and the survival rate of GTKO pig PBMC in the complement-dependent cytotoxicity assay was higher than that of wild-type pig. GTKO pig kidneys were harvested and perfused until completely white. After the left kidney of the recipient monkey was removed, the pig kidney was heterotopically transplanted. Following vascular anastomosis and blood flow restoration, the pig kidney rapidly turned pink without hyperacute rejection (HAR). Urine appeared in the ureter 6 minutes later, indicating successful kidney transplantation. The right kidney of the recipient was then removed. Seven days after transplantation, the transplanted kidney had good blood flow, the recipient monkey's serum creatinine level was stable, and serum potassium and cystatin C levels were effectively controlled, although they increased 10 days after transplantation. Seven days after transplantation, the levels of white blood cells, lymphocytes, monocytes and eosinophils in the recipient monkey increased, while platelet count and fibrinogen levels decreased. The activated partial thromboplastin time, thrombin time and prothrombin time remained relatively stable but later showed an upward trend. The recipient monkey survived for 10 days. At autopsy, the transplanted kidney was found to be congested, swollen and necrotic, with a small amount of IgG deposition in the renal tissue, and a large amount of IgM, complement C3c and C4d deposition, as well as CD68⁺ macrophage infiltration. Conclusions The kidneys of GTKO Diannan miniature pigs may maintain normal renal function for a certain period in rhesus macaques and effectively overcome HAR, confirming the effectiveness of GTKO pigs for xenotransplantation.

Enterococcus species are Gram-positive cocci normally found in the gut but have emerged as opportunistic pathogens in healthcare settings, with rising multidrug resistance, particularly vancomycin-resistant Enterococci (VRE) posing major therapeutic challenges. This study evaluated the antibacterial and synergistic effects of plant-derived stilbenes from Reynoutria japonica against clinical VRE isolates. Ten clinical Enterococcus faecalis isolates were identified using standard morphological, biochemical, and Gram-staining methods, and antibiotic susceptibility testing. The antibacterial activity of polydatin, resveratroloside, and trans-resveratrol was assessed by agar well diffusion, and evaluation of minimum inhibitory concentrations (MICs) and evaluation of bactericidal activity concentrations (MBCs), with synergistic effects tested in combination with vancomycin.Molecular docking was performed with DapF, vanA, and gelE proteins. All isolates were resistant to vancomycin, ampicillin, imipenem, gentamicin, and ciprofloxacin but sensitive to linezolid. All three stilbenes displayed antibacterial activity. Notably, trans-resveratrol was the most active compound, with a MIC value of 0.156 mM and bactericidal activity (an MBC value equal to the MIC). Furthermore, all compounds exhibited synergistic effects when combined with vancomycin. Phytochemical analysis confirmed these stilbenes as major constituents in the root extract of R. japonica. Molecular docking studies revealed strong binding affinities of the compounds to key virulence and resistance-associated proteins (DapF, VanA, and GelE). These results indicate that transresveratrol and related stilbenes are promising candidates for development as alternative or adjunct therapeutic agents against VRE infections. Additionally, this study substantiates the traditional use of R. japonica as a source of bioactive antimicrobial compounds.

In recent times, the emergence of Clostridium perfringens has posed a significant challenge to public health due to its antibiotic resistance and the formation of biofilms. It is the neuraminidase enzyme that supplies toxin secretion from C. perfringens. Since the sialic acid bond is a target recognition point for bacteria, new molecules are needed to treat infections caused by dangerous pathogens such as C. perfringens.The present work focused on an alternative strategy using compounds from Polygonum cuspidatum Sieb. et Zucc. Nine bioactive compounds derived from this plant emodin, physcion, emodin-1-O-β-D-glucopyranoside, emodin-8-O-β-D-glucopyranoside, physcion-8-O-β-D-glucopyranoside, 2-methoxy-6-acetyl-7-methyl juglone, torachrysone-8-O-β-D-glucoside, polydatin and resveratrol were used as ligands and coupled. The neuraminidase enzyme from C. perfringens was chosen as the target protein. The optimal ligand insertion score and ADMET parameters were determined by employing the Lipinski rules as selection criteria. Emodin-8-O-β-D-glucopyranoside and physcion-8-O-β-D-glucopyranoside exhibited drug-like characteristics in their ability to inhibit neuraminidase, as evidenced by a chelation score of −11.9. A comparison was conducted between emodin-8-O-β-D-glucopyranoside and physcion-8-O-β-D-glucopyranoside, and the positive control quercetin.A comprehensive analysis of the drug-like properties of emodin-8-O-β-D-glucopyranoside and physcion-8-O-β-D-glucopyranoside revealed that exhibited superiority over quercetin across multiple aspects. Quercetin showed a binding affinity of −9.9, while emodin-8-O-β-D-glucopyranoside and physcion-8-O-β-D-glucopyranoside showed a binding affinity of −11.9. The results showed acceptable differential kinetic properties of emodin-8-O-β-D-glucopyranoside and physcion-8-O-β-D-glucopyranoside compared to quercetin. It has been shown to inhibit the neuraminidase enzyme from C. perfringens.

Mullerian agenesis or Mayer-Rokitansky-Kuster-Hauser Syndrome (MRKH) Type-II is a congenital defect in the Mullerian duct that results in the absence of a uterus in women. The aetiology of this syndrome is unknown and has been considered a sporadic genetic disease. MRKH, together with anorectal anomaly, is an extremely rare condition and has only been reported in a few cases without any information on genetic analysis. This study investigated the mutational profile of a girl diagnosed with MRKH and anorectal anomalies with rectovaginal fistula. The whole exome sequencing (WES) trio-genetic analysis of a 5-year-old Malaysian girl diagnosed with MRKH (having anorectal anomaly with rectovaginal fistula) was performed together with her normal parents, using the Ion AmpliSeq Exome RDY kit (ThermoFisher Scientific, USA). Data were analysed using Torrent Suite v.5.0.4 and annotated using ANNOVAR. Single nucleotide polymorphisms (SNPs) with an allele frequency >0.01 were excluded, and the remaining variants were filtered based on de novo mutations, autosomal recessive, and autosomal recessive genetic traits. Related genes were analysed by biological pathway analysis (g:Profiler) and protein-protein interaction (HIPPIE v.2.3, STRING v.11.5, dan GeneMANIA). A total of 36 mutations were identified, and two of them, the LHX5 (p.P358Q), inherited from the father, and CFTR (p.R1158X), inherited from the mother. There were 28 de-novo mutations from 28 genes. All genes were involved in 27 biological processes that connected with 23 interactions, and are likely to cause MRKH syndrome in this patient.

Full thickness skin graft is a simple and reliable method for closure of small facial wound defect. A thorough understanding of how a skin graft heals and how to perform the procedure is essential for successful outcome. We report the use of full thickness skin graft in a wound closure of a facial skin defect caused by Paederus fuscipes, locally known as charlie. An 8-year old boy developed blister and painful swelling over his right cheek following skin contact with charlie. This lesion gradually became extensive, eventually leading to tissue loss and facial wound defect. A full thickness skin grafting was performed with satisfactory functional and excellent aesthetic result.

Mycobacterium tuberculosis is the causative agent of tuberculosis(TB),which is still the leading cause of mortality from a single infectious disease worldwide.The development of novel anti-TB drugs and vaccines is severely hampered by the complicated and time-consuming genetic manipulation techniques for M.tuberculosis.Here,we harnessed an endogenous type Ⅲ-A CRISPR/Cas10 system of M.tuberculosis for efficient gene editing and RNA interference(RNAi).This simple and easy method only needs to transform a single mini-CRISPR array plasmid,thus avoiding the introduction of exogenous protein and minimizing proteotoxicity.We demonstrated that M.tuberculosis genes can be efficiently and specifically knocked in/out by this system as con-firmed by DNA high-throughput sequencing.This system was further applied to single-and multiple-gene RNAi.Moreover,we successfully performed genome-wide RNAi screening to identify M.tuberculosis genes regulating in vitro and intracellular growth.This system can be extensively used for exploring the functional genomics of M.tuberculosis and facilitate the development of novel anti-TB drugs and vaccines.

Hyperinflammation and cytokine storm has been noted as a poor prognostic factor in patients with severe pneumonia related to coronavirus disease 2019 (COVID-19). In COVID-19, pathogenic myeloid cell overactivation is found to be a vital mediator of damage to tissues, hypercoagulability, and the cytokine storm. These cytokines unselectively infiltrate various tissues, such as the lungs and heart, and nervous system. This cytokine storm can hence cause multi-organ dysfunction and life-threatening complications. Mavrilimumab is a monoclonal antibody (mAb) that may be helpful in some cases with COVID-19. During an inflammation, Granulocyte-macrophage colony-stimulating factor (GM-CSF) release is crucial to driving both innate and adaptive immune responses. The GM-CSF immune response is triggered when an antigen attaches to the host cell and induces the signaling pathway. Mavrilimumab antagonizes the action of GM-CSF and decreases the hyperinflammation associated with pneumonia in COVID-19, therefore strengthening the rationale that mavrilimumab when added to the standard protocol of treatment could improve the clinical outcomes in COVID-19 patients, specifically those patients with pneumonia. With this review paper, we aim to demonstrate the inhibitory effect of mavrilimumab on cytokine storms in patients with COVID-19 by reviewing published clinical trials and emphasize the importance of extensive future trials.

Hyperinflammation and cytokine storm has been noted as a poor prognostic factor in patients with severe pneumonia related to coronavirus disease 2019 (COVID-19). In COVID-19, pathogenic myeloid cell overactivation is found to be a vital mediator of damage to tissues, hypercoagulability, and the cytokine storm. These cytokines unselectively infiltrate various tissues, such as the lungs and heart, and nervous system. This cytokine storm can hence cause multi-organ dysfunction and life-threatening complications. Mavrilimumab is a monoclonal antibody (mAb) that may be helpful in some cases with COVID-19. During an inflammation, Granulocyte-macrophage colony-stimulating factor (GM-CSF) release is crucial to driving both innate and adaptive immune responses. The GM-CSF immune response is triggered when an antigen attaches to the host cell and induces the signaling pathway. Mavrilimumab antagonizes the action of GM-CSF and decreases the hyperinflammation associated with pneumonia in COVID-19, therefore strengthening the rationale that mavrilimumab when added to the standard protocol of treatment could improve the clinical outcomes in COVID-19 patients, specifically those patients with pneumonia. With this review paper, we aim to demonstrate the inhibitory effect of mavrilimumab on cytokine storms in patients with COVID-19 by reviewing published clinical trials and emphasize the importance of extensive future trials.

Background and Objectives: There is growing interest in the use of the Level-specific (LS) CE-Chirp® stimulus in auditory brainstem response (ABR) due to its ability to produce prominent ABR waves with robust amplitudes. There are no known studies that investigate the test-retest reliability of the ABR to the LS CE-Chirp® stimulus. The present study aims to investigate the test-retest reliability of the ABR to the LS CE-Chirp® stimulus and compare its reliability with the ABR to standard click stimulus at multiple intensity levels in normal-hearing adults. Subjects and Methods: Eleven normal-hearing adults participated. The ABR test was repeated twice in the same clinical session and conducted again in another session. The ABR was acquired using both the click and LS CE-Chirp® stimuli at 4 presentation levels (80, 60, 40, and 20 dBnHL). Only the right ear was tested using the ipsilateral electrode montage. The reliability of the ABR findings (amplitudes and latencies) to the click and LS CE-Chirp® stimuli within the same clinical session and between the two clinical sessions was calculated using an intra-class correlation coefficient analysis (ICC). Results: The results showed a significant correlation of the ABR findings (amplitude and latencies) to both stimuli within the same session and between the clinical sessions. The ICC values ranged from moderate to excellent. Conclusions The ABR results from both the LS CE-Chirp® and click stimuli were consistent and reliable over the two clinical sessions suggesting that both stimuli can be used for neurological diagnoses with the same reliability.