Multiple morphological abnormalities of the flagella (MMAF) represent a severe form of sperm defects leading to asthenozoospermia and male infertility. In this study, we identified a novel homozygous splicing mutation (c.871-4 ACA>A) in the adenylate kinase 7 (AK7) gene by whole-exome sequencing in infertile individuals. Spermatozoa from affected individuals exhibited typical MMAF characteristics, including coiled, bent, short, absent, and irregular flagella. Transmission electron microscopy analysis showed disorganized axonemal structure and abnormal mitochondrial sheets in sperm flagella. Immunofluorescence staining confirmed the absence of AK7 protein from the patients' spermatozoa, validating the pathogenic nature of the mutation. This study provides direct evidence linking the AK7 gene to MMAF-associated asthenozoospermia in humans, expanding the mutational spectrum of AK7 and enhancing our understanding of the genetic basis of male infertility.
Humans ; Male ; Sperm Tail/ultrastructure* ; Homozygote ; Consanguinity ; Asthenozoospermia/pathology* ; Infertility, Male/genetics* ; Mutation ; Pakistan ; Adenylate Kinase/genetics* ; Adult ; Pedigree ; RNA Splicing ; Exome Sequencing ; Spermatozoa

The syndrome of multiple morphological abnormalities of the sperm flagella (MMAF) is one of the most serious kinds of sperm defects, leading to asthenoteratozoospermia and male infertility. In this study, we use whole-exome sequencing to identify genetic factors that account for male infertility in a patient born from a consanguineous Pakistani couple. A homozygous frameshift mutation (c.1399_1402del; p.Gln468ArgfsTer2) in axonemal dynein light chain domain containing 1 ( AXDND1 ) was identified in the patient. Sanger sequencing data showed that the mutation was cosegregated recessively with male infertility in this family. Papanicolaou staining and scanning electron microscopy analysis of the sperm revealed severely abnormal flagellar morphology in the patient. Immunofluorescence and western blot showed undetectable AXDND1 expression in the sperm of the patient. Transmission electron microscopy analysis showed disorganized sperm axonemal structure in the patient, particularly missing the central pair of microtubules. Immunofluorescence staining showed the absence of sperm-associated antigen 6 (SPAG6) and dynein axonemal light intermediate chain 1 (DNALI1) signals in the sperm flagella of the patient. These findings indicate that AXDND1 is essential for the organization of flagellar axoneme and provide direct evidence that AXDND1 is a MMAF gene in humans, thus expanding the phenotypic spectrum of AXDND1 frameshift mutations.
Humans ; Male ; Sperm Tail/ultrastructure* ; Frameshift Mutation ; Infertility, Male/pathology* ; Pakistan ; Pedigree ; Consanguinity ; Axonemal Dyneins/genetics* ; Adult ; Spermatozoa ; Exome Sequencing

Two new nor-ent-halimane diterpenes and three previously unreported nor-clerodane diterpenes,designated callicain-tides A-E(1-5),were isolated from Callicarpa integerrima.Compounds 1 and 2 feature a distinctive 5/6-membered ring system,while compounds 3-5 are characterized by progressively truncated carbon skeletons,containing 18,17,and 16 carbons,respectively.In addition,four known compounds 6-9 were also identified.Their structures were elucidated using advanced spectroscopic tech-niques,including nuclear magnetic resonance(NMR),high-resolution electrospray ionization mass spectrometry(HR-ESI-MS),ultra-violet(UV),infrared radiation(IR),optical rotatory dispersion(ORD),DP4+analysis and electronic circular dichroism(ECD),sup-ported by quantum chemical calculations.Compounds 1-9 were evaluated for their anti-MRSA activity.Among them,compound 6 demonstrated significant anti-MRSA activity,with a minimum inhibitory concentration(MIC)of 16 μg·mL-1.

Male infertility is a worldwide health issue, affecting 8%-12% of the global population. Oligoasthenoteratozoospermia (OAT) represents a severe type of male infertility, characterized by reduced sperm count and motility and an increased frequency of sperm with aberrant morphology. Using whole-exome sequencing, this study identified a novel missense mutation (c.848C>A, p.A283E) in the coiled-coil domain-containing 34 gene (CCDC34) in a consanguineous Pakistani family. This rare mutation was predicted to be deleterious and to affect the protein stability. Hematoxylin and eosin staining of spermatozoa from the patient with OAT revealed multiple morphological abnormalities of the flagella and transmission electron microscopy indicated axonemal ultrastructural defects with a lack of outer dynein arms. These findings indicated that CCDC34 plays a role in maintaining the axonemal ultrastructure and the assembly or stability of the outer dynein arms, thus expanding the phenotypic spectrum of CCDC34 missense mutations.
Humans ; Male ; Mutation, Missense/genetics* ; Pakistan ; Consanguinity ; Asthenozoospermia/genetics* ; Pedigree ; Infertility, Male/genetics* ; Adult ; Oligospermia/genetics* ; Exome Sequencing ; Axoneme/ultrastructure* ; Spermatozoa/ultrastructure*

Objective To predict the potential mechanism of Punicalagin in the treatment of Inflammatory bowel disease by network pharmacology.Methods The intersection genes of Punicalagin and IBD were obtained from the database,and PPI,GO and KEGG pathways were enriched and analyzed.Punicalagin and the target were verified by molecular docking.C57BL/6J mice were drunk dextran sulfate sodium to establish inflammatory enteritis model,and were given Punicalagin for 7 d of intervention.During the administration,signs of mice in each group were observed,daily disease activity index was calculated;Intestinal permeability test after administration;The colon tissue was stained with hematoxylin eosin to observe the pathological changes and calculate the histological damage score;Detection of tumor necrosis factor(TNF-α),Interleukin-10(IL-10),myeloperoxidase(MPO),chemokine 1(CXCL1)and other cytokines in colon tissue of mice by ELISA.Detection of TNF-α,IL-6,MPO and CXCL1 level in mouse serum by ELISA.CCK8 method was used to determine the effect of Punicalagin on the proliferation activity of caco-2 cells.The levels of cytokines released by caco-2 cells induced by lipopolysaccharide(LPS)were detected by ELISA.Results 14 common targets of Punicalagin and IBD were obtained,including tumor necrosis factor(TNF),arachidonic acid-5-lipoxygenase(ALOX5)and vascular endothelial growth factor A(VEGFA).KEGG enrichment analysis predicted that the treatment of IBD by Punicalagin mainly acted on arachidonic acid signaling pathway,age-rage signaling pathway,VEGR signaling pathway and Ras signaling pathway.Molecular docking showed that Punicalagin had good docking activity with TNF receptor.Compared with the model group,the decreasing range of body mass in Punicalagin group abated(P<0.01);the disease activity index of Punicalagin group decreased significantly(P<0.01);The congestion and edema of colonic mucosa were significantly reduced,and the histological injury score was significantly reduced(P<0.01);The level of TNF-α,IL-1β,MPO,CXCL1,IL-6,IL-18,IFN-γ in colon tissue was significantly decreased(P<0.01);20-300 μmol·L-1 Punicalagin promoted caco-2 cell proliferation and inhibited TNF-α secretion induced by LPS,up-regulation of IL-10 levels.Conclusion Punicalagin inhibits the secretion of TNF-α and other proinflammatory factors,up-regulation of the level of anti-inflammatory factor IL-10,and improvement of colonic inflammatory response in IBD mice.

Aim To investigate the feasibility and mechanism of rhynchophylline in the treatment of in-rhynchophylline flammatory bowel disease (IBD) based on network pharmacology combined with in vivo and in vitro experiments. Methods The target of rhynchophylline-IBD intersection was obtained from the database, and GO and KEGG enrichment analysis were performed. The binding of key target proteins was screened by molecular docking. In vivo the IBD model of mice was induced by sodium dextran sulfate (DSS). After seven days of rhynchophylline intervention, the signs of mice in each group were observed and DAI scores were recorded. The levels of interleukin-1β (3 (IL-1 β), my-eloperoxidase (MPO) and other inflammatory factors in colon tissue of mice were detected by ELISA. The intestinal permeability of each group was detected. In vitro experiments were conducted to establish the inflammatory model of Caco2 cells induced by DSS, and to clarify the regulatory effect of leptosinine on key targets. Results A total of 70 rhynchophylline-IBD intersection targets were screened, and enrichment analysis showed that they were related to the inflammatory prooess, PI3K-Akt and Hippo signaling pathway s. Molecular docking results showed that was most stable in binding with JAK2 and JAK1. In vivo experiment results showed that compared with model group, body weight, colon length and weight of rhynchophylline group significantly increased (P < 0. 05). DAI score, IL-1β, MPO and other inflammatory factors in colon tissue and intestinal permeability significantly decreased (P < 0. 01). In vitro experiment results showed that compared with model group, rhynchophylline group significantly promoted the proliferation of Caco2 cells (P < 0. 05). The levels of IL-6 and NO were significantly reduced (P < 0. 05). Western blot results showed that rhynchophylline could decrease the expressions of JAK2 and JAK1 (P < 0. 05). Conclusion Rhynchophylline may play a role in the treatment of IBD by inhibiting the expression of JAK2 and JAK1 proteins and reducing inflammatory response in body.

Multiple morphological abnormalities of the sperm flagella (MMAF) is a severe form of asthenozoospermia categorized by immotile spermatozoa with abnormal flagella in ejaculate. Whole-exome sequencing (WES) is used to detect pathogenic variants in patients with MMAF. In this study, a novel homozygous frameshift variant (c.6158_6159insT) in dynein axonemal heavy chain 8 (DNAH8) from two infertile brothers with MMAF in a consanguineous Pakistani family was identified by WES. Reverse transcription-polymerase chain reaction (RT-PCR) confirmed DNAH8 mRNA decay in these patients with the DNAH8 mutation. Hematoxylin-eosin staining and transmission electron microscopy revealed highly divergent morphology and ultrastructure of sperm flagella in these patients. Furthermore, an immunofluorescence assay showed the absence of DNAH8 and a reduction in its associated protein DNAH17 in the patients' spermatozoa. Collectively, our study expands the phenotypic spectrum of patients with DNAH8-related MMAF worldwide.
Humans ; Male ; Consanguinity ; Pakistan ; Infertility, Male/metabolism* ; Semen/metabolism* ; Sperm Tail/metabolism* ; Spermatozoa/metabolism* ; Flagella/pathology* ; Mutation

The present study was aimed to evaluate the in-vitro and in-vivo antibacterial effects of the Typha elephantina aqueous extract (TE.AQ), ethanolic extract (TE.ET) and T. elephantina methanolic extract (TE.ME) against eight selected clinical pathogens. The test samples were tested for in-vitro analysis (by disc diffusion method) at different concentrations of 5, 15, 25, 50 and 100 mg/dL against both gram positive and gram-negative strains. The highest potential was observed in TE.ME at a concentration of 100 mg/dL against Pseudomonas aeruginosa exhibiting 19.67 ± 0.577 mm zone of inhibition (ZOI). The same fraction also showed good activity against Staphylococus aureus with ZOI of 17.50 ± 0.70 mm. The TE.ET was found most active against P. aeruginosa and Streptococcus pyogenes having ZOI of 18.53 ± 0.503 and 16.2 ± 1.55 mm respectively at a concentration of 100 mg/dL. The most sensitive bacteria P. aeruginosa was selected for in-vivo study (using poultry chicks) for induction of infection in chicks. The effects of TE.AQ, TE.ET and TE.ME were determined at concentrations of 300 mg/kg body weight based on hematological parameters, liver enzymes and gross pathological findings of lungs and livers. The findings of the in-vivo study in chick’s model showed that treatment of experimental animals with TE.ME significantly restored the hematological parameters, liver enzymes and architecture of lungs and livers. Based on scientific evidence, the current study suggests that TE.ME may serve as a best and new natural antibacterial agent and can be used against infections caused by P. aeruginosa.

The short release half-life of carbon monoxide (CO) is a major obstacle to the effective therapeutic use of carbon monoxide-releasing molecule-2 (CORM-2). The potential of CORM-2-entrapped ultradeformable liposomes (CORM-2-UDLs) to enhance the release half-life of CO and alleviate skin inflammation was investigated in the present study. CORM-2-UDLs were prepared by using soy phosphatidylcholine to form lipid bilayers and Tween 80 as an edge activator. The deformability of CORM-2-UDLs was measured and compared with that of conventional liposomes by passing formulations through a filter device at a constant pressure. The release profile of CO from CORM-2-UDLs was evaluated by myoglobin assay.

The current Zika outbreak is largest of its kind with 1.4 million cases in Brazil alone. World Health Organization declared the current outbreak as the public health emergency of international concerns. The major route of Zika virus transmission is mosquito bites. Sexual transmission and monkey bites are also observed in few cases. There is dire need to evaluate the other routes of transmission like blood transfusion, lactation and contact with body fluids. Zika virus is infecting infants, not only causing microcephaly but also creating number of complications resulting in bad outcomes of pregnancy. In Brazil alone, 4 000 cases of microcephaly have observed during the current outbreak. The incidence of Guillain-Barre (GB) syndrome is also observed during the current Zika virus outbreak. GB syndrome is acute medical condition leading the patients to death due to weakness of respiratory muscles or can cause the life time disability. There is no anti-viral drug or vaccine available for Zika virus. Zika infection can be prevented by using mosquito repellents, mosquito nets, cooling rooms by air conditions and wearing full sleeves or permethrin-treated clothes. The current outbreak of Zika has not only affected the health care but also caused great economic loss. Estimated loss in Latin America and Caribbean is US$3.5 billion. United Nation's sustainable development goal 3.d stresses the strengthening of early warning, risk reduction and management of national and global health risks. The world will keep on facing new challenges in the form of Ebola or Zika; there is strong need to prepare ourselves for any disease outbreak.