Male infertility can result from impaired sperm motility caused by multiple morphological abnormalities of the flagella (MMAF). Distinct projections encircling the central microtubules of the spermatozoal axoneme play pivotal roles in flagellar bending and spermatozoal movement. Mammalian sperm-associated antigen 17 ( SPAG17 ) encodes a conserved axonemal protein of cilia and flagella, forming part of the C1a projection of the central apparatus, with functions related to ciliary/flagellar motility, skeletal growth, and male fertility. This study investigated two novel homozygous SPAG17 mutations (M1: NM_206996.2, c.829+1G>T, p.Asp212_Glu276del; and M2: c.2120del, p.Leu707*) identified in four infertile patients from two consanguineous Pakistani families. These patients displayed the MMAF phenotype confirmed by Papanicolaou staining and scanning electron microscopy assays of spermatozoa. Quantitative real-time polymerase chain reaction (PCR) of patients' spermatozoa also revealed a significant decrease in SPAG17 mRNA expression, and immunofluorescence staining showed the absence of SPAG17 protein signals along the flagella. However, no apparent ciliary-related symptoms or skeletal malformations were observed in the chest X-rays of any of the patients. Transmission electron microscopy of axoneme cross-sections from the patients showed incomplete C1a projection and a higher frequency of missing microtubule doublets 1 and 9 compared with those from fertile controls. Immunofluorescence staining and Western blot analyses of spermatogenesis-associated protein 17 (SPATA17), a component of the C1a projection, and sperm-associated antigen 6 (SPAG6), a marker of the spring layer, revealed disrupted expression of both proteins in the patients' spermatozoa. Altogether, these findings demonstrated that SPAG17 maintains the integrity of spermatozoal flagellar axoneme, expanding the phenotypic spectrum of SPAG17 mutations in humans.
Humans ; Male ; Infertility, Male/pathology* ; Sperm Tail/ultrastructure* ; Homozygote ; Microtubule-Associated Proteins/genetics* ; Axoneme/genetics* ; Spermatozoa/ultrastructure* ; Adult ; Mutation ; Sperm Motility/genetics* ; Pedigree ; Microtubules ; Microtubule Proteins/genetics*

Background: The optimal surgical approach for managing multiple renal arteries in living donor kidney transplantation remains unclear. This study compared outcomes between separate anastomoses (SA) and pantaloon anastomosis (PA). Methods: A retrospective cohort study was conducted from November 2018 to August 2024. In total, 117 patients were included (SA, 67; PA, 50). The primary outcome was arterial complications. Secondary outcomes included renal function, graft and patient survival at 1 year, and intraoperative variables. Statistical analyses utilized the t-test, the Mann-Whitney U-test, and Kaplan-Meier survival curves. Results: The mean warm ischemia time was similar between groups (105.48±35.64 vs.103.92±33.51 min; P=0.811). Cold ischemia time was also comparable (75.90±14.36 vs.80.68±17.85 min; P=0.111). However, anastomosis time was significantly longer in the SA group (39.00 [35.00–43.00] min) compared with the PA group (34.00 [28.50–39.50] min; P<0.001). The immediate postoperative creatinine reduction was slightly greater in the SA group (46.83%±20.68% vs. 39.78%±19.04%; P=0.090). Vascular complications requiring re-exploration were infrequent in both groups (1.5% vs. 2.0%). At 1 year posttransplant, creatinine levels were comparable between the SA (1.20 [0.95–1.45] mg/dL) and PA (1.20 [1.05–1.35] mg/dL) groups (P=0.337). Regression analysis confirmed that postoperative creatinine levels were independently associated with SA (P=0.006). Graft survival was similarly high (97% vs. 98%), as was patient survival (100% vs. 96%). Conclusions Both anastomosis techniques are safe and effective for transplanting kidneys with anatomical anomalies. SA offers surgical efficiency and provides superior early renal recovery.

Aims: The study was aimed to explore the antimicrobial potential of ethanolic leaf extracts of Eucalyptus globulus, Moringa oliefera, Syzygium cumini and Citrus limon against antibiotic-resistant Clostridium perfringens type D (n=5). Methodology and results: Antibiotic resistance pattern of C. perfringens type D isolates against tetracycline, gentamicin, ceftriaxone, amoxicillin and streptomycin was evaluated by disc diffusion method. Well diffusion and micro broth dilution methods were used to determine the anti-bacterial activity, sub-inhibitory concentrations and antibiotic resistance modulating effects of the plant extracts. Ethanolic extract of E. globules was selected to evaluate its modulatory impact and subjected to GC-MS analysis to separate and identify the phytochemicals. The results showed that the isolates were resistant to gentamicin (0 ± 0.00 mm), streptomycin (0 ± 0.00 mm), tetracycline (13.2 ± 2.28 mm) and ceftriaxone (0 ± 0.00 mm) while sensitive to amoxicillin (23.8 ± 1.30 mm) and tetracycline (13.2 ± 2.28 mm). Eucalyptus globulus exhibited the maximum anti-bacterial activity with a zone of inhibition (ZOI) of 14.6 ± 0.54 mm and minimum inhibitory concentration (MIC) (1500 ± 947.85 µg/mL). Other plant extracts (M. oliefera, S. cumini and C. limon) also showed anti-bacterial activity but couldn’t modulate the resistance. The activity of ceftriaxone associated with E. globulus extract was improved with 20.2 ± 0.20 mm ZOI at 78.125 µg/mL sub-inhibitory concentration. Conclusion, significance and impact of study: The study results indicate the possible use of the ethanolic extract of E. globulus alone or in combination with common antibiotics for the treatment of C. perfringens infections in small ruminants.

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

Aims: This paper presents the report on biodiesel and biogas production at a laboratory scale from Scenedesmus strain. Methodology and results: Previously isolated and identified Scenedesmus were grown in 10 Liter flask using BG-11 media at 16 h light and 8 h dark cycle. Oven-dried biomass (20 g) from 16-day-old culture of Scenedesmus was finely grounded and subjected to lipids extraction by chloroform-methanol-NaCl mixture. Microalgal lipids (6 mL) were subjected to transesterification by using NaOH leading to the production of 5 mL biodiesel and 4 mL of glycerin. Biodiesel was rich in methyl esters of linoleic acid, phosphorothioc acid and dodecanoic acid, as shown by gas chromatography-mass spectrometry (GC-MS) analysis. Oven-dried microalgae (2 g) without lipid extraction and leftover biomass (2 g) after lipid extraction were subject to biogas production through anaerobic digestion. Biogas (34, 27 and 19 mL) were recorded respectively in oven-dried whole biomass; lipid extracted biomass and control over a period of 15 days of anaerobic digestion. Conclusion, significance and impact of study It was concluded that water bodies are rich in diverse algae, especially Scenedesmus sp., and this algae can be cultured to produce biodiesel and biogas. But the lipid accumulation potential of microalgae requires special treatment and lipid extraction methods are not up to the mark, which is a major bottleneck in biofuel production from microalgae.
Biofuels ; Scenedesmus--isolation & ; purification

Aims: The study was aimed to isolate and characterize the mycotoxin-producing filamentous Aspergillus parasiticus from the feed samples. The sensitivity pattern of the isolates was assessed against different disinfectants. Methodology and results: Fifty different feed samples were screened for A. parasiticus isolation. Isolates were subjected to macroscopic and microscopic characterization. Polymerase chain reaction was performed to confirm the isolates at the genomic level. Mycotoxin producing potential of the isolates was assessed by thin-layer chromatography (TLC). To quantify the toxins, high performance liquid (HPLC) was employed. The antifungal potential of disinfectants was determined by the well diffusion method followed by minimum inhibitory concentration (MIC) calculation. Out of twenty isolates of A. parasiticus, 11(55%) isolates were observed positive for toxin production. Three toxigenic isolates (AspP2, AspP4 and AspP8) were selected to evaluate their susceptibility against disinfectants by well diffusion method. AspP2 produced maximum (5.90 ng/mL) toxin, followed by AspP4 (3.11 ng/mL) and AspP8 (18.47 ng/mL). Terralin showed maximum fungicidal activity with 29.66 ± 8.08 mm zone of inhibition at 0.42 μg/mL MIC. Hypochlorite and Instru Star showed 99% disinfection with 30, 60 and 90 min contact time (6 mean log reduction) for all A. parasiticus isolates. Alpha Guard inhibited growth after 15 min contact time for all the isolates. Conclusion, significance and impact of study This study provides data indicating the contamination of feed samples with mycotoxin-producing A. parasiticus isolates and their sensitivity against commercially available disinfectants. Use of these disinfectants in appropriate concentrations and time could help prevent the contamination of food, feed and healthcare settings with the fungal species.
Mycotoxins ; Aspergillus

Objectives: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused devastation in over 200 countries. Italy, Spain, and the United States (US) were most severely affected by the first wave of the pandemic. The reasons why some countries were more strongly affected than others remain unknown. We identified the most-affected and lessaffected countries and states and explored environmental, host, and infrastructure risk factors that may explain differences in the SARS-CoV-2 mortality burden. Methods: We identified the top 10 countries/US states with the highest deaths per population until May 2020. For each of these 10 case countries/states, we identified 6 control countries/ states with a similar population size and at least 3 times fewer deaths per population. We extracted data for 30 risk factors from publicly available, trusted sources. We compared case and control countries/states using the non-parametric Wilcoxon rank-sum test, and conducted a secondary cluster analysis to explore the relationship between the number of cases per population and the number of deaths per population using a scalable EM (expectation– maximization) clustering algorithm. Results: Statistically significant differences were found in 16 of 30 investigated risk factors, the most important of which were temperature, neonatal and under-5 mortality rates, the percentage of under-5 deaths due to acute respiratory infections (ARIs) and diarrhea, and tuberculosis incidence (p < 0.05) Conclusion Countries with a higher burden of baseline pediatric mortality rates, higher pediatric mortality from preventable diseases like diarrhea and ARI, and higher tuberculosis incidence had lower rates of coronavirus disease 2019-associated mortality, supporting the hygiene hypothesis.

Multiple morphological abnormalities of the sperm flagella (MMAF) is a specific type of asthenoteratozoospermia, presenting with multiple morphological anomalies in spermatozoa, such as absent, bent, coiled, short, or irregular caliber flagella. Previous genetic studies revealed pathogenic mutations in genes encoding cilia and flagella-associated proteins (CFAPs; e.g., CFAP43, CFAP44, CFAP65, CFAP69, CFAP70, and CFAP251) responsible for the MMAF phenotype in infertile men from different ethnic groups. However, none of them have been identified in infertile Pakistani males with MMAF. In the current study, two Pakistani families with MMAF patients were recruited. Whole-exome sequencing (WES) of patients and their parents was performed. WES analysis reflected novel biallelic loss-of-function mutations in CFAP43 in both families (Family 1: ENST00000357060.3, p.Arg300Lysfs*22 and p.Thr526Serfs*43 in a compound heterozygous state; Family 2: ENST00000357060.3, p.Thr526Serfs*43 in a homozygous state). Sanger sequencing further confirmed that these mutations were segregated recessively in the families with the MMAF phenotype. Semiquantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) was carried out to detect the effect of the mutation on mRNA of the affected gene. Previous research demonstrated that biallelic loss-of-function mutations in CFAP43 accounted for the majority of all CFAP43-mutant MMAF patients. To the best of our knowledge, this is the first study to report CFAP43 biallelic loss-of-function mutations in a Pakistani population with the MMAF phenotype. This study will help researchers and clinicians to understand the genetic etiology of MMAF better.
Adolescent ; Adult ; Humans ; Infertility, Male/epidemiology* ; Loss of Function Mutation/genetics* ; Male ; Microtubule Proteins/genetics* ; Middle Aged ; Pakistan/epidemiology* ; Sperm Tail/physiology*

Objectives: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused devastation in over 200 countries. Italy, Spain, and the United States (US) were most severely affected by the first wave of the pandemic. The reasons why some countries were more strongly affected than others remain unknown. We identified the most-affected and lessaffected countries and states and explored environmental, host, and infrastructure risk factors that may explain differences in the SARS-CoV-2 mortality burden. Methods: We identified the top 10 countries/US states with the highest deaths per population until May 2020. For each of these 10 case countries/states, we identified 6 control countries/ states with a similar population size and at least 3 times fewer deaths per population. We extracted data for 30 risk factors from publicly available, trusted sources. We compared case and control countries/states using the non-parametric Wilcoxon rank-sum test, and conducted a secondary cluster analysis to explore the relationship between the number of cases per population and the number of deaths per population using a scalable EM (expectation– maximization) clustering algorithm. Results: Statistically significant differences were found in 16 of 30 investigated risk factors, the most important of which were temperature, neonatal and under-5 mortality rates, the percentage of under-5 deaths due to acute respiratory infections (ARIs) and diarrhea, and tuberculosis incidence (p < 0.05) Conclusion Countries with a higher burden of baseline pediatric mortality rates, higher pediatric mortality from preventable diseases like diarrhea and ARI, and higher tuberculosis incidence had lower rates of coronavirus disease 2019-associated mortality, supporting the hygiene hypothesis.

Human brucellosis is a neglected zoonotic problem worldwide with a high degree of morbidity in humans and is mostly overlooked due to other febrile conditions. The aim of this study was to evaluate the sero-prevalence and risk factors of human brucellosis among subjects living in Punjab, Pakistan. In this cross-sectional study, human blood samples were collected from seven districts of Punjab, Pakistan. Information regarding personal data, demographic data and potential risk factors was collected through a structured questionnaire. Detection of anti-Brucella antibodies was done through Rose Bengal Plate Test (RBPT) and Enzyme Linked Immunosorbent Assay (ELISA). Descriptive analysis, Chi square test and Odds ratio was applied using STATA software version 12. The sero-prevalence of human brucellosis was 13.13% with significantly higher percentage in males 17.23% and age group 25-40 years 16.50% (P=< 0.001). The demographic factors positively associated with human brucellosis were lack of education (P = 0.003; OR = 1.85) and farming as an occupation (P =<0.001; OR = 2.50) Similarly, among the risk factors studied, keeping animals at home (P =<0.001; OR = 2.03), slaughtering of animals (P =<0.001; OR = 15.87) and consuming raw milk (P =<0.001; OR = 5.42) were the factors strongly connected with human brucellosis. A massive awareness should be given to livestock farmers and individuals directly linked to animals regarding risk factors and transmission of brucellosis. Consumption of unpasteurized milk and its products should be condemned to curtail this neglected disease.