Search

As a precise and versatile tool for genome manipulation, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) platform holds promise for modifying fish traits of interest. With the aim of reducing transgene introgression and controlling reproduction, upscaled disease resistance and reproductive intervention in catfish species have been studied to lower the potential environmental risks of the introgression of escapees as transgenic animals. Taking advantage of the CRISPR/Cas9-mediated system, we succeeded in integrating the cathelicidin gene (As-Cath) from an alligator (Alligator sinensis) into the target luteinizing hormone (lh) locus of channel catfish (Ictalurus punctatus) using two delivery systems assisted by double-stranded DNA (dsDNA) and single-stranded oligodeoxynucleotides (ssODNs), respectively. In this study, high knock in (KI) efficiency (22.38%, 64/286) but low on-target events was achieved using the ssODN strategy, whereas adopting a dsDNA as the donor template led to an efficient on-target KI (10.80%, 23/213). The on-target KI of As-Cath was instrumental in establishing the lh knockout (LH–_As-Cath+) catfish line, which displayed heightened disease resistance and reduced fecundity compared with the wild-type (WT) sibling fish. Furthermore, administration of human chorionic gonadotropin (HCG) and luteinizing hormone-releasing hormone analogue (LHRHa) can restore the reproduction of the transgenic fish line. Overall, we replaced the lh gene with an alligator cathelicidin transgene and then administered hormone therapy to move towards complete reproductive control of disease-resistant transgenic catfish in an environmentally responsible manner. This strategy not only effectively improves consumer-valued traits but also guards against unwanted introgression, providing a breakthrough in aquaculture genetics to confine fish reproduction and prevent the establishment of transgenic or domestic genotypes in the natural environment.

Sterilization and castration have been synonyms for thousands of years. Making an animal sterile meant to render them incapable of producing offspring. Castration or the physical removal of the testes was discovered to be the most simple but reliable method for managing reproduction and sexual behavior in the male. Today, there continues to be global utilization of castration in domestic animals. More than six hundred million pigs are castrated every year, and surgical removal of testes in dogs and cats is a routine practice in veterinary medicine. However, modern biological research has extended the meaning of sterilization to include methods that spare testis removal and involve a variety of options, from chemical castration and immunocastration to various methods of vasectomy. This review begins with the history of sterilization, showing a direct link between its practice in man and animals. Then, it traces the evolution of concepts for inducing sterility, where research has overlapped with basic studies of reproductive hormones and the discovery of testicular toxicants, some of which serve as sterilizing agents in rodent pests. Finally, the most recent efforts to use the immune system and gene editing to block hormonal stimulation of testis function are discussed. As we respond to the crisis of animal overpopulation and strive for better animal welfare, these novel methods provide optimism for replacing surgical castration in some species.

Antimicrobial peptides (AMPs) show promise in enhancing resistance against pathogens. Previously, we integrated two AMP genes, cathelicidin (Cath) from alligator (Alligator mississippiensis or Alligator sinensis) and cecropin (Cec) from moth (Hyalophora cecropia), into the channel catfish (Ictalurus punctatus) genome. This study examines the efficacy of exogenous AMP gene integration in improving bacterial resistance in transgenic channel catfish and assesses the direct and pleiotropic effects of gene replacement/knockout on survival and growth based on insertion site. Transgenic Cath- and Cec-expressing fish exhibited similar or higher survival rates (P ​> ​0.05) compared to controls during the initial culture. Integration of the Cec transgene doubled the survival rate when challenged with Edwardsiella ictaluri, with knock-in (KI) of Cath further increasing bacterial resistance. Coupling Cec KI with mstn knockout (KO) increased survival 3-fold after E. ictaluri infection and growth by 50% at 4 months post-fertilization (mpf). However, random integration of Cec had a minimal effect on disease resistance and did not enhance growth. Random integration of Cath increased survival 2.5-fold and 4-fold against E. ictaluri and Flavobacterium covae, respectively, without affecting growth. Cath KI at the lh locus increased survival 4-fold when challenged with F. covae and reduced growth by 10% (P ​> ​0.05) at 24 mpf, whereas Cath KI coupled with mc4r KO resulted in a 2.5-fold increase in survival following F. covae infection compared with controls, and increased growth by 80% at 3 mpf. Simultaneous KI of Cath and Cec, along with KO of mc4r and mstn, increased survival 4-fold against E. ictaluri, while increasing growth by 50% at 3 mpf. Dual insertion of AMP genes yielded the greatest resistance to disease. These direct and pleiotropic effects may increase comprehension and societal acceptance of genetic engineering in aquaculture.

Channel catfish (Ictalurus punctatus) and blue catfish (Ictalurus furcatus) are two economically important freshwater aquaculture species in the United States, with channel catfish contributing to nearly half of the country’s aquaculture production. While differences in economic traits such as growth rate and disease resistance have been noted, the extent of transcriptomic variance across various tissues between these species remains largely unexplored. The hybridization of female channel catfish with male blue catfish has led to the development of superior hybrid catfish breeds that exhibit enhanced growth rates and improved disease resistance, which dominate more than half of the total US catfish production. While hybrid catfish have significant growth advantages in earthen ponds, channel catfish were reported to grow faster in tank culture environments. In this study, we confirmed channel fish’s superiority in growth over blue catfish in 60-L tanks at 10.8 months of age (30.3 g and 11.6 g in this study, respectively; p < 0.001). In addition, we conducted RNA sequencing experiments and established transcriptomic resources for the heart, liver, intestine, mucus, and muscle of both species. The number of expressed genes varied across tissues, ranging from 5,036 in the muscle to over 20,000 in the mucus. Gene Ontology analysis has revealed the functional specificity of differentially expressed genes within their respective tissues, with significant pathway enrichment in metabolic pathways, immune activity, and stress responses. Noteworthy tissue-specific marker genes, including lrrc10, fabp2, myog, pth1a, hspa9, cyp21a2, agt, and ngtb, have been identified. This transcriptome resource is poised to support future investigations into the molecular mechanisms underlying environment-dependent heterosis and advance genetic breeding efforts of hybrid catfish.

Purpose: The use of deep learning to auto-contour organs at risk (OARs) in gynecologic radiation treatment is well established. Yet, there is limited data investigating the prospective use of auto-contouring in clinical practice. In this study, we assess the accuracy and efficiency of auto-contouring OARs for computed tomography–based brachytherapy treatment planning of gynecologic malignancies. Methods and Materials: An inhouse contouring tool automatically delineated 5 OARs in gynecologic radiation treatment planning: the bladder, small bowel, sigmoid, rectum, and urethra. Accuracy of each auto-contour was evaluated using a 5-point Likert scale: a score of 5 indicated the contour could be used without edits, while a score of 1 indicated the contour was unusable. During scoring, automated contours were edited and subsequently used for treatment planning. Dice similarity coefficient, mean surface distance, 95% Hausdorff distance, Hausdorff distance, and dosimetric changes between original and edited contours were calculated. Contour approval time and total planning time of a prospective auto-contoured (AC) cohort were compared with times from a retrospective manually contoured (MC) cohort. Results: Thirty AC cases from January 2022 to July 2022 and 31 MC cases from July 2021 to January 2022 were included. The mean (±SD) Likert score for each OAR was the following: bladder 4.77 (±0.58), small bowel 3.96 (±0.91), sigmoid colon 3.92 (±0.81), rectum 4.6 (±0.71), and urethra 4.27 (±0.78). No ACs required major edits. All OARs had a mean Dice similarity coefficient > 0.86, mean surface distance < 0.48 mm, 95% Hausdorff distance < 3.2 mm, and Hausdorff distance < 10.32 mm between original and edited contours. There was no significant difference in dose-volume histogram metrics (D2.0 cc/D0.1 cc) between original and edited contours (P values > .05). The average time to plan approval in the AC cohort was 19% less than the MC cohort. (AC vs MC, 117.0 + 18.0 minutes vs 144.9 ± 64.5 minutes, P = .045). Conclusions: Automated contouring is useful and accurate in clinical practice. Auto-contouring OARs streamlines radiation treatment workflows and decreases time required to design and approve gynecologic brachytherapy plans.

Abstract Background Neck injury is a common and often debilitating injury among athletes participating in American football. Limited data exists regarding neck injuries among elite athletes in the National Football League (NFL). To characterize the epidemiology of non-season ending, season-ending, and career-ending neck injuries in the NFL from 2016 through 2021. Methods Athletes who sustained neck injuries were identified using the NFL’s injured reserve (IR) list between the 2016 and 2021 seasons. Demographics and return to sport (RTS) data were collected. Available game footages were reviewed to identify the mechanism of injury (MOI). Injury incidence rates were calculated based on per team play basis. Results During the 6-year study period, 464 players (mean age 26.8 ± 3.2 years) were placed on the injury reserve list due to neck injuries. There were 285 defensive players and 179 offensive players injured (61.4 vs 38.6%, respectively, p

Abstract The cytoplasmic droplet is a conserved dilated area of cytoplasm situated at the neck of the sperm flagellum. Viewed as residual cytoplasm inherited from late spermatids, the cytoplasmic droplet contains numerous saccular elements as its key content. However, the origin of these saccules and the function of the cytoplasmic droplet have long been speculative. Here, we identify the molecular origin of these cytoplasmic droplet components by uncovering a vesicle pathway essential for formation and sequestration of saccules within the cytoplasmic droplet. This process is governed by a transmembrane protein SYPL1 and its interaction with VAMP3. Genetic ablation of SYPL1 in mice reveals that SYPL1 dictates the formation and accumulation of saccular elements in the forming cytoplasmic droplet. Derived from the Golgi, SYPL1 vesicles are critical for segregation of key metabolic enzymes within the forming cytoplasmic droplet of late spermatids and epididymal sperm, which are required for sperm development and male fertility. Our results uncover a mechanism to actively form and segregate saccules within the cytoplasmic droplet to promote sperm fertility.

Abstract The crystal structure of orthorhombic Bovine Pancreatic Ribonuclease A has been determined to 0.85 Å resolution using low temperature, 100 K, synchrotron X-ray data collected at 16000 keV (λ = 0.77 Å). This is the first ultra-high-resolution structure of a native form of Ribonuclease A to be reported. Refinement carried out with anisotropic displacement parameters, stereochemical restraints, inclusion of H atoms in calculated positions, five $${\text{SO}}_{4}^{2 - }$$ SO 4 2 - moieties, eleven ethanol molecules and 293 water molecules, converged with final R values of R1(Free) = 0.129 (4279 reflections) and R1 = 0.112 (85,346 reflections). The refined structure was deposited in the Protein Data Bank as structure 7p4r. Conserved waters, using four high resolution structures, have been investigated. Cluster analysis identified clusters of water molecules that are associated with the active site of Bovine Ribonuclease A. Particular attention has been paid to making detailed comparisons between the present structure and other high quality Bovine Pancreatic Ribonuclease A X-ray crystal structures with special reference to the deposited classic monoclinic structure 3RN3 Howlin et al. (Acta Crystallogr A 45:851–861, 1989). Detailed studies of various aspects of hydrogen bonding and conformation have been carried out with particular reference to active site residues Lys-1, Lys-7, Gln-11, His-12, Lys-41, Asn-44, Thr-45, Lys-66, His-119 and Ser-123. For the two histidine residues in the active site the initial electron density map gives a clear confirmation that the position of His-12 is very similar in the orthorhombic structure to that in 3RN3. In 3RN3 His-119 exhibited poor electron density which was modelled and refined as two distinct sites, A (65%) and B (35%) but with respect to His-119 in the present ultra-high resolution orthorhombic structure there is clear electron density which was modelled and refined as a single conformation distinct from either conformation A or B in 3RN3. Other points of interest include Serine-32 which is disordered at the end of the sidechain in the present orthorhombic form but has been modelled as a single form in 3RN3. Lysine-66: there is density indicating a possible conformation for this residue. However, the density is relatively weak, and the conformation is unclear. Three types of amino acid representation in the ultra-high resolution electron density are examined: (i) sharp with very clearly resolved features, for example Lys-37; (ii) well resolved but clearly divided into two conformations which are well behaved in the refinement, both having high quality geometry, for example Tyr-76; (iii) poor density and difficult or impossible to model, an example is Lys-31 for which density is missing except for Cβ. The side chains of Gln-11, His-12, Lys-41, Thr-45 and His-119 are generally recognised as being closely involved in the enzyme activity. It has also been suggested that Lys-7, Asp-44, Lys-66, Phe-120, Asp-121 and Ser-123 may also have possible roles in this mechanism. A molecular dynamics study on both structures has investigated the conformations of His-119 which was modelled as two conformations in 3RN3 but is observed to have a single clearly defined conformation in the present orthorhombic structure. MD has also been used to investigate Lys-31, Lys-41 and Ser32. The form of the Ribonuclease A enzyme used in both the present study and in 3RN3 (Howlin et al. in Acta Crystallogr A 45:851–861, 1989) includes a sulphate anion which occupies approximately the same location as the $${\text{PO}}_{4}^{2 - }$$ PO 4 2 - phosphate group in protein nucleotide complexes (Borkakoti et al. in J Mol Biol 169:743–755, 1983). The present structure contains 5 $${\text{SO}}_{4}^{2 - }$$ SO 4 2 - groups SO41151–SO41155 two of which, SO41152 and SO41153 are disordered, SO41152 being in the active site, and 11 EtOH molecules, EOH A 201–EOH A 211 all of which have good geometry. H atoms were built into the EtOH molecules geometrically. Illustrations of these features in the present structure are included here. The sulphates are presumably present in the material purchased for use in the present study. 293 water molecules are included in the present structure compared to 134 in 3RN3 (Howlin et al. in Acta Crystallogr A 45:851–861, 1989).

Abstract Reproductive sterilization by surgical gonadectomy is strongly advocated to help manage animal populations, especially domesticated pets, and to prevent reproductive behaviors and diseases. This study explored the use of a single-injection method to induce sterility in female animals as an alternative to surgical ovariohysterectomy. The idea was based on our recent finding that repetitive daily injection of estrogen into neonatal rats disrupted hypothalamic expression of Kisspeptin (KISS1), the neuropeptide that triggers and regulates pulsatile secretion of GnRH. Neonatal female rats were dosed with estradiol benzoate (EB) either by daily injections for 11 days or by subcutaneous implantation of an EB-containing silicone capsule designed to release EB over 2–3 weeks. Rats treated by either method did not exhibit estrous cyclicity, were anovulatory, and became infertile. The EB-treated rats had fewer hypothalamic Kisspeptin neurons, but the GnRH-LH axis remained responsive to Kisspeptin stimulation. Because it would be desirable to use a biodegradable carrier that is also easier to handle, an injectable EB carrier was developed from PLGA microspheres to provide pharmacokinetics comparable to the EB-containing silicone capsule. A single neonatal injection of EB-microspheres at an equivalent dosage resulted in sterility in the female rat. In neonatal female Beagle dogs, implantation of an EB-containing silicone capsule also reduced ovarian follicle development and significantly inhibited KISS1 expression in the hypothalamus. None of the treatments produced any concerning health effects, other than infertility. Therefore, further development of this technology for sterilization in domestic female animals, such as dogs and cats is worthy of investigation.

BackgroundPorphyromonas gingivalis, a major pathogen in periodontitis, produces KGP (Lys-gingipain), a cysteine protease that enhances bacterial virulence by promoting tissue invasion and immune evasion. Recent studies highlight microRNAs' role in viral latency, potentially affecting lytic replication through host mechanisms. Herpes virus (HSV) establishes latency via interactions between microRNA-6 (miRH-6) and the ICP4 transcription factor in neural ganglia. This suggests a potential link between periodontitis and HSV-induced latency. This study aims to identify and validate the insilico inhibitory interaction of P. gingivalis KGP with ICP4 transcripts and correlate the presence of viral latency-associated transcript micro-RNA-6 with periodontitis.MethodsComputational docking analysis was performed to investigate the potential interaction between ICP4 and KGP gingipain. The binding energy and RMSD ligand values were calculated to determine the interaction's strength. Ten patients with recurrent clinical attachment loss despite conventional therapy were included in the clinical study. Subgingival tissue samples were collected post-phase I therapy, and HSV microRNA-6 presence was detected via polymerase chain reaction and confirmed through gel electrophoresis.ResultsComputational docking identified the ICP4-KGP gingipain complex with the lowest binding energy (-288.29 kJ mol^1) and an RMSD ligand of 1.5 Angstroms, indicating strong interaction potential. Gel electrophoresis confirmed miRH-6 presence in all samples.ConclusionThe identification of miRNA-6 in periodontitis patients and the strong interaction potential between P. gingivalis KGP gingipain and ICP4 transcripts indicate a possible link between bacterial virulence factors and viral latency dynamics in periodontal tissues. These results highlight the complex interplay between oral pathogens, viral microRNAs, and host immune responses in periodontitis.

Effects of antimicrobial peptides (AMP) added to diets on aquatic animal health and body function are influenced by multiple factors such as animal species, initial body weight, the dosage of AMP and feeding duration. However, there is limited knowledge on the relationship between these factors and the body function of aquatic animals. Here, we aimed to perform multiple meta-analyses to investigate the effects of dietary AMP on growth performance (feed conversion ratio [FCR], specific growth rate [SGR]), enzyme activity (superoxide dismutase activity [SOD], lysozyme activity [LSA]), disease resistance (cumulative survival rate [CSR], the expression of immune-related genes [GENE]) and the abundance of gut microbiota (MICRO) from a pool of empirical studies. Additionally, the dose-effect model was applied to determine the optimal AMP dose, initial body weight and feeding duration to maximize body function. To conduct the meta-analyses, we included 34 publications that estimated 705 effect sizes across 21 fish, 2 shrimp and 2 shellfish species. The results confirmed that the inclusion of AMP in the diet can significantly improve SGR, SOD, LSA, CSR and GENE and decrease FCR for aquatic animals. Interestingly, our findings implied a slight positive effect of AMP on MICRO albeit with a limited number of studies available on fish gut microbial communities. Although no significant linear or quadratic relationship was predicted by meta-regression, the dose-effect indicated that the optimal AMP doses for FCR, SGR, SOD and LSA were 707.5, 750.0, 1,050.0 and 937.5 mg/kg, respectively. Taken together, fish with an initial body weight of 30 g could be fed with a dose of 600 to 800 mg/kg for 2 mo when AMP-supplemented diets were applied in aquaculture, which can effectively improve body function and health while lowering aquafeed costs. In addition, more studies should address fish gut microbiota to delimitate the influence of dietary AMP on MICRO in the future.

PIWI proteins and their associated piRNAs act to silence transposons and promote gametogenesis. Murine PIWI proteins MIWI, MILI, and MIWI2 have multiple arginine and glycine (RG)-rich motifs at their N-terminal domains. Despite being known as docking sites for the TDRD family proteins, the in vivo regulatory roles for these RG motifs in directing PIWI in piRNA biogenesis and spermatogenesis remain elusive. To investigate the functional significance of RG motifs in mammalian PIWI proteins in vivo, we genetically engineered an arginine to lysine (RK) point mutation of a conserved N-terminal RG motif in MIWI in mice. We show that this tiny MIWI RG motif is indispensable for piRNA biogenesis and male fertility. The RK mutation in the RG motif disrupts MIWI-TDRKH interaction and impairs enrichment of MIWI to the intermitochondrial cement (IMC) for efficient piRNA production. Despite significant overall piRNA level reduction, piRNA trimming and maturation are not affected by the RK mutation. Consequently, MiwiRK mutant mice show chromatoid body malformation, spermatogenic arrest, and male sterility. Surprisingly, LINE1 transposons are effectively silenced in MiwiRK mutant mice, indicating a LINE1-independent cause of germ cell arrest distinctive from Miwi knockout mice. These findings reveal a crucial function of the RG motif in directing PIWI proteins to engage in efficient piRNA production critical for germ cell progression and highlight the functional importance of the PIWI N-terminal motifs in regulating male fertility.

ABSTRACT: Seed germination is a tightly regulated physiological process. Hydrolytic enzymes provide energy that brings physiological, biochemical, and physical changes to the seed during germination. Alpha-galactosidases break down alpha 1-6 linked galactosyl groups in glycoproteins, glycolipids, and oligosaccharides into simple compounds to provide energy during seed germination. Chickpea seed germination characteristics showed that inhibition of alpha-galactosidase using 1-deoxynojirimycin (DNJ), a derivative of deoxygalactonojirimycin, delays germination. To gain insights into the mechanism of inhibition, we modeled the enzyme’s structure and performed in-silico docking of the inhibitor and natural substrates (raffinose and stachyose) to the enzyme. We also validated our model using recombinant chickpea alpha-galactosidase. Our docking studies showed that both the inhibitor and the substrates bind to the same active site pocket in the enzyme but to different amino acid residues, stachyose being a preferred substrate, and DNJ competitively inhibits alpha-galactosidase with a Ki and IC50 of 44.5 µM and 89.13 µM respectively. Delayed germination is a desirable agronomic practice that can be explored for better crop establishment and to prevent pre-harvest sprouting in crops.

Introduction An institution-wide protocol for uncomplicated acute appendicitis was created to improve compliance with best practices between the emergency department (ED), radiology and surgery. Awareness of the protocol was spread with the publication of a smartphone application and communication to clinical leadership. On interim review of quality metrics, poor protocol adherence in diagnostic imaging and antimicrobial stewardship was observed. The authors hypothesised that two further simple interventions would result in more efficient radiographic diagnosis and antimicrobial administration.Materials and methods Surgery residents received targeted in-person education on the appropriate antibiotic choices and diagnostic imaging in the protocol. Signs were placed in the emergency and radiology work areas, immediately adjacent to provider workstations highlighting the preferred imaging for patients with suspected appendicitis and the preferred antibiotic choices for those with proven appendicitis. Protocol adherence was compared before and after each intervention.Results Targeted education was associated with improved antibiotic stewardship within the surgical department from 30% to 91% protocol adherence before/after intervention (p

Background context: Lumbar spine disc pathologies often necessitate removal of the intervertebral disc and implantation of a lumbar interbody fusion (LIF) device. Herein, the objective was to measure use condition forces while simulating intra-operative loading conditions of transforaminal lumbar interbody fusion (TLIF) procedures. Methods: In this work, we developed a transferable method to measure impact forces in a cadaveric intra-operative setting and designed a drop weight benchtop device equipped with force and displacement sensors to replicate cadaveric impact conditions, including number of strikes required for interbody device insertion, peak force, initial slope of the impact waveform, impact duration, and area under the force-time curve (impulse). Following cadaveric testing, a benchtop biomechanical study varying interbody device height, drop height, and drop weight was performed to evaluate the utility of the benchtop device to replicate impact loading conditions which occurred in the cadaveric model. Results: Several benchtop test groups replicated the cadaver group in number of strikes required for device insertion. The average cadaveric peak force, 13.03 kN, was replicated by three benchtop groups: 12 mm device height, 60 cm drop height, 0.75 lb drop weight; 12 mm device height, 60 cm drop height, 1.0 lb drop weight; and 14 mm device height, 60 cm drop height, 0.50 lb drop weight. The initial slope of the impact waveform was replicated best by 12 mm devices. Both the impact duration and impulse were lower in all benchtop groups than in cadaver testing. Conclusions: This work validated the outfitting of an impact force sensor onto an interbody device insertion instrument as a reproducible, quantifiable method for collecting impact loading data in a cadaveric model. This benchtop device and methodology will aid in testing interbody devices relative to anticipated use conditions, enhance interbody device designs, and accelerate surgical technique refinement.

IntroductionPoor mental health, such as post-traumatic stress disorder (PTSD), has been reported after female genital cutting (FGC). However, data documenting adverse consequences of FGC have insufficiently considered confounding factors, such as other traumatising events. Here, we examined the extent to which FGC versus other serious life events disturbed Gambian girls subjected to FGC. We additionally assessed the prevalence of PTSD and the extent to which it was attributed to FGC versus other serious life events.MethodsWe conducted a cross-sectional study with a community-based sample of 12 years-old Gambian girls who had been subjected to FGC (N = 125). Using structured interviews, we assessed serious life events and probable PTSD related to the event that the girls cited as bothering them the most.ResultsMost of the girls reported several serious life events in addition to FGC, such as witnessing violence, experiencing violence or assaults, death of a close relative, and being exposed to natural disasters or serious accidents, for an average 4.5 events per girl. Around one-sixth of the girls (16.8%) stated that FGC was the event that currently bothered them the most, whereas the majority (75.2%) emphasised other experiences. The girls who said they were most troubled by other events reported more impaired daily functioning than those most bothered by FGC. Overall, we found a prevalence of probable PTSD of 19.2%. Of 24 PTSD cases, one was attributed to the experience of FGC, and the remaining 23 were attributed to other events.ConclusionOur findings indicate that FGC is less important than other serious life events in explaining high rates of PTSD in Gambian girls. Associations established in the field between FGC and adverse mental health must be interpreted with caution because girls who have undergone FGC may be severely exposed to other traumatising events.

Background Epstein Barr virus positive (EBV+) immunodeficiency-associated lymphoproliferative disorders (IA-LPD) are heterogeneous diseases with variable treatment strategies that are not well-defined.Case presentation A 68-year-old woman with systemic lupus erythematosus developed EBV+ B-cell polymorphic lymphoproliferative disease (LPD). Positron emission tomography computed tomography (PET/CT) showed a large nasopharyngeal mass, multiple pulmonary lesions, splenomegaly and disseminated lymphadenopathy. Plasma EBV DNA was grossly elevated to 1.5 × 104 IU/mL. There were three paraproteins. Treatment with O-CHOP (obinutuzumab, cyclophosphamide, adriamycin, vincristine, prednisolone) led to undetectable plasma EBV DNA, suggesting eradiation of the EBV-positive malignant clone. However, radiologic abnormalities were still present on PET/CT, and paraprotein persisted. A nasopharyngeal re-biopsy showed infiltration with EBV-negative plasma cells. On treatment with lenalidomide, she finally achieved complete metabolic response. Molecular analysis showed that the EBV+ B-cell LPD and the EBV– plasma cell lesion exhibited identical immunoglobulin gene rearrangements. Next generation sequencing revealed that the EBV+ B-LPD showed mutation in only one gene (TP53), a profile typical of EBV-driven lymphoid neoplasms. However, the EBV– plasma cell lesion showed mutations in five genes (TP53, SF3B1, STAT5B, CD79B and CRKL), suggesting that these mutations instead of EBV infection were the oncogenic driver.Conclusion The presence of both EBV+ and EBV– lesions, which showed different mutational profiles, indicated clonal heterogeneity that might be of biologic and therapeutic significance.

Purpose: The aim of this work was to describe the design and implementation of a more robust workflow for communicating outcomes from a peer-review chart rounds conference. We also provide information regarding cycle times, plan revisions, and other key metrics that we have observed since initial implementation. Methods and Materials: A multidisciplinary team of stakeholders including physicians, physicists, and dosimetrists developed a revised peer-review workflow that addressed key needs to improve the prior process. Consensus terminology was developed to reduce ambiguity regarding the priority of peer-review outcomes and to clarify expectations of the treating physician in response to peer-review outcomes. A custom workflow software tool was developed to facilitate both upstream and downstream processes from the chart rounds conference. The peer-review outcomes of the chart rounds conference and resulting plan changes for the first 18 months of implementation were summarized. Results: In the first 18 months after implementation of the revised processes, 2294 plans were reviewed, and feedback priority levels assigned. Across all cases with feedback, the median time for the treating attending physician to acknowledge conference comments was 1 day and was within 7 calendar days for 89.1% of cases. Conference feedback was acknowledged within 1 day for 74 of 115 (64.3%) cases with level 2 comments and for 18 of 21 (85.7%) cases with level 3 comments (P = .054). Contours were modified in 13 of 116 (11%) cases receiving level 2 feedback and 10 of 21 (48%) cases receiving level 3 feedback (P < .001). The treatment plan was revised in 18 of 116 (16%) cases receiving level 2 feedback and 13 of 21 (61%) cases receiving level 3 feedback (P < .001). Conclusions: We successfully implemented a workflow to improve upstream and downstream processes for a chart rounds conference. Standardizing how peer-review outcomes were communicated and recording physician responses allow for improved ability to monitor conference activities.

Abstract Background There are no previous studies that evaluate the effect of obesity on patients undergoing complex revision thoracolumbar spine surgery. The primary objective was to determine the relationship between obesity and perioperative adverse events (AEs) with patients undergoing complex revision thoracolumbar spine surgery while controlling for psoas muscle index (PMI) as a confounding variable. The secondary objective was to determine the relationship between obesity and 30-day readmission rates, 30-day re-operation rates, rate of discharge to a facility, and post-operative length of stay (LOS). Methods Between May 2016 and February 2020, a retrospective analysis of individuals undergoing complex revision surgery of the thoracolumbar spine was performed at a single institution. Obesity was defined as BMI ≥ 30.0 kg/m2. PMI 10 was used to define complex revision surgery. A multivariable logistic regression model was used to ascertain the effects of low muscle mass, obesity, age, and gender on the likelihood of the occurrence of any AE. Results A total of 114 consecutive patients were included in the study. Fifty-four patients were in the obese cohort and 60 patients in the non-obese cohort. There was not a significant difference in perioperative outcomes of both the obese and non-obese patients. There were 22 obese patients (40.7%) and 33 non-obese patients (55.0%) that experienced any AE (p = 0.130). Multivariable analysis demonstrated that individuals with low muscle mass had a significantly higher likelihood for an AE than individuals with normal or high muscle mass (OR: 7.53, 95% CI: 3.05-18.60). Obesity did not have a significant effect in predicting AEs. Conclusions Obesity is not associated with perioperative AEs, 30-day readmission rates, 30-day re-operation rates, rate of discharge to a facility, or post-operative length of stay (LOS) among patients undergoing complex revision thoracolumbar spine surgery. Level of evidence III

The manchette is a transient and unique structure present in elongating spermatids and required for proper differentiation of the germ cells during spermatogenesis. Previous work indicated that the MEIG1/PACRG complex locates in the manchette and is involved in the transport of cargos, such as SPAG16L, to build the sperm flagellum. Here, using co-immunoprecipitation and pull-down approaches in various cell systems, we established that DNALI1, an axonemal component originally cloned from Chlamydomonas reinhardtii, recruits and stabilizes PACRG and we confirm in vivo, the co-localization of DNALI1 and PACRG in the manchette by immunofluorescence of elongating murine spermatids. We next generated mice with a specific deficiency of DNALI1 in male germ cells, and observed a dramatic reduction of the sperm cells, which results in male infertility. In addition, we observed that the majority of the sperm cells exhibited abnormal morphology including misshapen heads, bent tails, enlarged midpiece, discontinuous accessory structure, emphasizing the importance of DNALI1 in sperm differentiation. Examination of testis histology confirmed impaired spermiogenesis in the mutant mice. Importantly, while testicular levels of MEIG1, PACRG, and SPAG16L proteins were unchanged in the Dnali1 mutant mice, their localization within the manchette was greatly affected, indicating that DNALI1 is required for the formation of the MEIG1/PACRG complex within the manchette. Interestingly, in contrast to MEIG1 and PACRG-deficient mice, the DNALI1-deficient mice also showed impaired sperm spermiation/individualization, suggesting additional functions beyond its involvement in the manchette structure. Overall, our work identifies DNALI1 as a protein required for sperm development.

This study utilizes the Analytic Hierarchy Process (AHP) in the selection of an optimal niche or category of videos for maximizing view count. The main income from videos is derived from RPM, which is a set amount per thousand views. A set of criteria was determined from attributes in the dataset that logically contribute to either the videos’ SEO or trend/popularity. The criteria in question were also determined by commonalities across a vast number of video content platforms, which focus more on the essential attributes of a video. In order to perform pairwise comparison, weights were derived from coefficients generated using Linear Regression. Following the creation of the model, we identify the categories with the highest potential for gaining views. Based on the results, the study may be performed in another time frame to reflect the major shifts in public interest over time. Thus, the importance of its repeatability and degree of usability across datasets from different platforms is emphasized. Doi: 10.28991/HIJ-2022-03-01-07 Full Text: PDF

Abstract Fish is an essential source of high-quality protein for people worldwide. The present study was designed to compare the growth performance among the channel-blue hybrid catfish, channel catfish transgenic for the channel catfish growth hormone (ccGH) cDNA driven by the antifreeze protein promoter from an ocean pout Zoarces americanus (opAFP-ccGH), and non-transgenic channel catfish control. Mean body weight of channel-blue hybrid catfish was 15.80 and 24.06% larger than non-transgenic channel catfish control at 4 and 18 months of age, respectively. However, transgenic opAFP-ccGH channel catfish were 5.52 and 43.41% larger than channel-blue hybrid catfish and 22.19 and 77.91% larger than their controls at 4 and 18 months of age, respectively. Significant differences in mean body weight between the sexes within all genetic types were found. Males were larger than females (P

Background and Aims: A significant factor contributing to poor survival in pancreatic cancer is the often late stage at diagnosis. We sought to develop and validate a risk prediction model to facilitate the distinction between chronic pancreatitis–related vs potential early pancreatic ductal adenocarcinoma (PDAC)-associated changes on pancreatic imaging. Methods: In this retrospective cohort study, patients aged 18–84 years whose abdominal computed tomography/magnetic resonance imaging reports indicated duct dilatation, atrophy, calcification, cyst, or pseudocyst between January 2008 and November 2019 were identified. The outcome of interest is PDAC in 3 years. More than 100 potential predictors were extracted. Random survival forests approach was used to develop and validate risk models. Multivariable Cox proportional hazard model was applied to estimate the effect of the covariates on the risk of PDAC. Results: The cohort consisted of 46,041 (mean age 66.4 years). The 3-year incidence rate was 4.0 (95% confidence interval CI 3.6–4.4)/1000 person-years of follow-up. The final models containing age, weight change, duct dilatation, and either alkaline phosphatase or total bilirubin had good discrimination and calibration (c-indices 0.81). Patients with pancreas duct dilatation and at least another morphological feature in the absence of calcification had the highest risk (adjusted hazard ratio [aHR] = 14.15, 95% CI 8.7–22.6), followed by patients with calcification and duct dilatation (aHR = 7.28, 95% CI 4.09–12.96), and patients with duct dilation only (aHR = 6.22, 95% CI 3.86–10.03), compared with patients with calcifications alone as the reference group. Conclusion: The study characterized the risk of pancreatic cancer among patients with 5 abnormal morphologic findings based on radiology reports and demonstrated the ability of prediction algorithms to provide improved risk stratification of pancreatic cancer in these patients.

Abstract Background Household air pollution (HAP) from cooking with solid fuels has adverse health effects. REACCTING (Research on Emissions, Air quality, Climate, and Cooking Technologies in Northern Ghana) was a randomized cookstove intervention study that aimed to determine the effects of two types of “improved” biomass cookstoves on health using self-reported health symptoms and biomarkers of systemic inflammation from dried blood spots for female adult cooks and children, and anthropometric growth measures for children only. Methods Two hundred rural households were randomized into four different cookstove groups. Surveys and health measurements were conducted at four time points over a two-year period. Chi-square tests were conducted to determine differences in self-reported health outcomes. Linear mixed models were used to assess the effect of the stoves on inflammation biomarkers in adults and children, and to assess the z-score deviance for the anthropometric data for children. Results We find some evidence that two biomarkers of oxidative stress and inflammation, serum amyloid A and C-reactive protein, decreased among adult primary cooks in the intervention groups relative to the control group. We do not find detectable impacts for any of the anthropometry variables or self-reported health. Conclusions Overall, we conclude that the REACCTING intervention did not substantially improve the health outcomes examined here, likely due to continued use of traditional stoves, lack of evidence of particulate matter emissions reductions from “improved” stoves, and mixed results for HAP exposure reductions. Clinical trial registry ClinicalTrials.gov (National Institutes of Health); Trial Registration Number: NCT04633135 ; Date of Registration: 11 November 2020 – Retrospectively registered. URL: https://clinicaltrials.gov/ct2/show/NCT04633135?term=NCT04633135&draw=2&rank=1

The complexity and fragmented nature and the multiple stakeholders in the construction industry often make it difficult to come up with a firm decision regarding sustainable building materials selection. The wrong choice could negatively impact the project objective and performance outcome. This study assessed the critical factor influencing the choice of sustainable building materials (SBM) selection on construction projects in the Southeast geopolitical zone of Nigeria. Data were collected using a well-structured questionnaire, non-probability (purposive and snowball) sampling techniques, and an internet-mediated survey. Data analyses were carried out using the appropriate descriptive and inferential statistical tools and exploratory factor analysis (EFA). The study revealed recycled plastic, natural clay and mud, stone, bricks and tile, cellulose, stray bales, grasses, limestone, and wood timber, are the commonly used sustainable building materials. Also, their level of awareness is high while their adoption is moderate. EFA revealed that the major clusters of determinants of the choice of green building materials are: emissions minimisation, low running cost and reusability, low thermal and energy consumption efficiency, low cost and high health and safety consideration and waste minimisation. The key factors influencing the choice of sustainable building materials selection in construction are: reducing greenhouse gas emissions from buildings, materially embodied energy cost, operating and maintenance costs, non-toxic or low toxic emissions generated by the products/materials, recyclability of the building materials, availability of the technical skills, renewable (reusable) properties, inhibiting the impact of buildings on the environment, safety and health of the occupants, and appearance and aesthetic. It is recommended that consideration be given to these factors in selecting sustainable/green building materials in the designs and specifications of construction projects.

Education has been in a drastic shift from face-to-face classes to distance education. Coping with this educational trend demands innovative practices to provide solutions to varied student conditions. Hence, this study explored the perspectives and experiences of Grade 11 students in the English for Academic and Professional Purposes (EAPP) classes in using mobile devices in the teaching-learning process in one of the public schools in the Department of Education Davao City, Mindanao, Philippines. The study employs a descriptive qualitative approach to conduct the research. Purposive sampling was used in the study that yielded 12 participants. Only Grade 11 students who use mobile devices to learn the lessons in EAPP class are eligible to participate in the study. Thematic analysis was used to analyze the data from focus group discussion and in-depth interviews aided by Quirkos software version 2.4.1. The findings revealed that using mobile technology in the class made learning easy and accessible. It is perceived to be an alternative mode of delivering the lesson. However, the participants also perceived the use of mobile devices in the class as distractive. Hence, guidelines on the responsible use of mobile devices in the classroom must be formulated and implemented. Future researchers might consider adopting a mixed methods research design in exploring this area. This is to balance perspectives with concrete figures that may quantify significant improvement or significant differences in students’ learning.

Introduction Two experiments were designed to investigate the motivational role of the metacognitive self (MCS, meaning self-awareness of biases) and kind of feedback (success vs. failure vs. control group) in willingness to learn. We predict that the condition of failure enhances motivation to learn. Predictions relate to the first experiment and social incentives, not to spatial ones. Material and methods Three hundred ninety-eight participants were individually (in front of a computer with E-Prime) and randomly assigned to experiment 1 of a social task or experiment 2 of a spatial task. Each experiment included three groups: success, failure, and control. The independent variables were metacognitive self (MCS) and type of feedback (success vs. failure vs. control). The dependent variable was the willingness to learn. Logistic regression was applied to investigate the hypothesis that the higher the level of MCS is, the more likely it is that the participants will try to learn. Results As predicted, MCS was positively related to searching for self-diagnostic information in the first experiment. Furthermore, according to expectations, the experiment with a social task showed the main effects of both MCS and type of feedback. The spatial experiment did not reveal significant effects. Conclusions MCS is positively related to motivation to search for self-diagnostic information, and students are more willing to learn in the face of failure. According to expectations, the experiment with a social task substantiated the motivational role of MCS and the role of negative feedback in willingness to learn.

Organisms undergo a variety of characteristic changes as they age, suggesting a substantial commonality in the mechanistic basis of aging. Experiments in model organisms have revealed a variety of cellular systems that impact lifespan, but technical challenges have prevented a comprehensive evaluation of how these components impact the trajectory of aging, and many components likely remain undiscovered. To facilitate the deeper exploration of aging trajectories at a sufficient scale to enable primary screening, we have created the Caenorhabditis elegans Observatory, an automated system for monitoring the behavior of group-housed C. elegans throughout their lifespans. One Observatory consists of a set of computers running custom software to control an incubator containing custom imaging and motion-control hardware. In its standard configuration, the Observatory cycles through trays of standard 6 cm plates, running four assays per day on up to 576 plates per incubator. High-speed image processing captures a range of behavioral metrics, including movement speed and stimulus-induced turning, and a data processing pipeline continuously computes summary statistics. The Observatory software includes a web interface that allows the user to input metadata and view graphs of the trajectory of behavioral aging as the experiment unfolds. Compared to the manual use of a plate-based C. elegans tracker, the Observatory reduces the effort required by close to two orders of magnitude. Within the Observatory, reducing the function of known lifespan genes with RNA interference (RNAi) gives the expected phenotypic changes, including extended motility in daf-2(RNAi) and progeria in hsf-1(RNAi). Lifespans scored manually from worms raised in conventional conditions match those scored from images captured by the Observatory. We have used the Observatory for a small candidate-gene screen and identified an extended youthful vigor phenotype for tank-1(RNAi) and a progeric phenotype for cdc-42(RNAi). By utilizing the Observatory, it is now feasible to conduct whole-genome screens for an aging-trajectory phenotype, thus greatly increasing our ability to discover and analyze new components of the aging program.

The construction industry influences and accelerates national growth and development, but it, however, contributes to unsustainable and eco-unfriendly development which impacts on the economy and environment. To encourage investment in sustainable construction, this study made a case for green building materials (GBM) products and services market in the construction industry of developing countries, through an assessment of the benefits of GBM incorporation in construction projects. The study adopted an internet-mediated questionnaire survey approach and snowball sampling techniques to gathered data from clients, consultants, professionals, and contractors/sub-contractor in the southeast geopolitical zones of Nigeria. Frequency, Percentile, Shapiro-Wilk test, Kaiser–Meyer–Olkin (KMO) and Bartlett’s tests, relative importance index (RII) and Kruskal-Wallis test were used to analyse the collected data. The study revealed that the green construction market is unsaturated and under-tapped. With high awareness and low adoption levels of GBM, it was concluded that the most important benefits of GBM that will trigger demand and supply of GBM products and services are improved daylight and reduced need for artificial lighting, improves the quality of life, Increase employees productivity and reduced absenteeism, improve occupants health and comfort, higher profit and return on investment, leads to construction professionals specialisation, reducing greenhouse gas emission from building, reduces toxicity in the internal environment, low operating and maintenance cost, and creates new job opportunities. The uniqueness of this study lies in the assessment of the views of construction stakeholders' in the five states that make up the South-eastern geo-political zone of Nigeria, regarding the key benefits that could trigger the uptake and incorporation of GBM in construction. Developers and Investors in the industry could invest in green construction since there is a ready and unsaturated market for it.

Follicle-stimulating hormone (fsh) plays an important role in sexual maturation in catfish. Knocking out the fsh gene in the fish zygote should suppress the reproduction of channel catfish (Ictalurus punctatus). In this study, transcription activator-like effector nuclease (TALEN) plasmids targeting the fsh gene were electroporated into fertilized eggs with the standard double electroporation technique. Targeted fsh cleavage efficiency was 63.2% in P1fsh-knockout catfish. Ten of fifteen (66.7%) control pairs spawned, and their eggs had 32.3–74.3% average hatch rates in 2016 and 2017. Without hormone therapy, the spawning rates of P1 mutants ranged from 33.3 to 40.0%, with an average egg hatching rate of 0.75%. After confirmation of the low fertility of P1 mutants in 2016, human chorionic gonadotropin (HCG) hormone therapy improved the spawning rates by 80% for female mutants and 88.9% for male mutants, and the mean hatch rate was 35.0% for F1 embryos, similar to that of the controls (p > 0.05). Polymerase chain reaction (PCR) identification showed no potential TALEN plasmid integration into the P1 channel catfish genome. Neither the P1 nor the F1 mutant fish showed any noticeable changes in in body weight, survival rate, and hatching rate when the reproductive gene was knocked out. F1 families had a mean inheritance rate of 50.3%. The results brought us one step closer to allowing implementation of certain genetic techniques to aquaculture and fisheries management, while essentially eliminating the potential environment risk posed by transgenic, hybrid, and exotic fish as well as domestic fish.

Background and Aim: The periodontal microbiome being complex, this study was aimed to detect and quantify the prevalence of Filifactor alocis in various stages of periodontitis and to evaluate its prospect as a diagnostic marker for periodontal disease. Settings and Design: Sixty subjects were selected (20 healthy controls, 20 with chronic periodontitis, and 20 with aggressive periodontitis) for the study. Materials and Methods: Clinical parameters probing depth and the level of clinical attachment was recorded, subgingival plaque samples were collected. The F. alocis 16srDNA was cloned, sequenced, and used as the standard for real-time quantification of bacterial load using SYBR green chemistry. Statistical Analysis: Clinical, microbiological, and quantitative polymerase chain reaction (PCR) data were analyzed using ANOVA and Pearson's coefficient correlation. Results: (a) Real-time PCR analysis showed the highest average F. alocis count in chronic periodontitis subjects (32,409.85), which was followed by count in healthy controls (3046.15) and the least count in aggressive periodontitis subjects (939.84). The bacterial count was statistically significant at P = 0.005. (b) An intra-group comparison reveals that there was a statistically significant increase in the bacterial count with age and mean probing pocket depth at P = 0.0005. Conclusion: F. alocis population in aggressive periodontitis was lower compared to chronic periodontitis and healthy controls. The F. alocis population surge in healthy controls may be due to geographical variations and the ethnicity of the subjects. A higher population of F. alocis in chronic periodontitis proves its high pathogenic potential to invade the host tissues to aid in further periodontal destruction.

Abstract CRISPR/Cas9-based gene knockout in animal cells, particularly in teleosts, has proven to be very efficient with regards to mutation rates, but the precise insertion of exogenous DNA or gene knock-in via the homology-directed repair (HDR) pathway has seldom been achieved outside of the model organisms. Here, we succeeded in integrating with high efficiency an exogenous alligator cathelicidin gene into a targeted non-coding region of channel catfish (Ictalurus punctatus) chromosome 1 using two different donor templates (synthesized linear dsDNA and cloned plasmid DNA constructs). We also tested two different promoters for driving the gene, zebrafish ubiquitin promoter and common carp β-actin promoter, harboring a 250-bp homologous region flanking both sides of the genomic target locus. Integration rates were found higher in dead fry than in live fingerlings, indicating either off-target effects or pleiotropic effects. Furthermore, low levels of mosaicism were detected in the tissues of P1 individuals harboring the transgene, and high transgene expression was observed in the blood of some P1 fish. This can be an indication of the localization of cathelicidin in neutrophils and macrophage granules as also observed in most antimicrobial peptides. This study marks the first use of CRISPR/Cas9 HDR for gene integration in channel catfish and may contribute to the generation of a more efficient system for precise gene integration in catfish and other aquaculture species, and the development of gene-edited, disease-resistant fish.

The prospect of eradicating malaria continues to be challenging in the face of increasing parasite resistance to antimalarial drugs so that novel antimalarials active against asexual, sexual, and liver-stage malaria parasites are urgently needed. In addition, new antimalarials need to be affordable and available to those most in need and, bearing in mind climate change, should ideally be sustainable. The West African climbing shrub Cryptolepis sanguinolenta is used traditionally for the treatment of malaria; its principal alkaloid, cryptolepine (1), has been shown to have antimalarial properties, and the synthetic analogue 2,7-dibromocryptolepine (2) is of interest as a lead toward new antimalarial agents. Cryptolepine (1) was isolated using a two-step Soxhlet extraction of C. sanguinolenta roots, followed by crystallization (yield 0.8% calculated as a base with respect to the dried roots). Semi-synthetic 7-bromo- (3), 7, 9-dibromo- (4), 7-iodo- (5), and 7, 9-dibromocryptolepine (6) were obtained in excellent yields by reaction of 1 with N-bromo- or N-iodosuccinimide in trifluoroacetic acid as a solvent. All compounds were active against Plasmodia in vitro, but 6 showed the most selective profile with respect to Hep G2 cells: P. falciparum (chloroquine-resistant strain K1), IC50 = 0.25 µM, SI = 113; late stage, gametocytes, IC50 = 2.2 µM, SI = 13; liver stage, P. berghei sporozoites IC50 = 6.13 µM, SI = 4.6. Compounds 3–6 were also active against the emerging zoonotic species P. knowlesi with 5 being the most potent (IC50 = 0.11 µM). In addition, 3–6 potently inhibited T. brucei in vitro at nM concentrations and good selectivity with 6 again being the most selective (IC50 = 59 nM, SI = 478). These compounds were also cytotoxic to wild-type ovarian cancer cells as well as adriamycin-resistant and, except for 5, cisplatin-resistant ovarian cancer cells. In an acute oral toxicity test in mice, 3–6 did not exhibit toxic effects at doses of up to 100 mg/kg/dose × 3 consecutive days. This study demonstrates that C. sanguinolenta may be utilized as a sustainable source of novel compounds that may lead to the development of novel agents for the treatment of malaria, African trypanosomiasis, and cancer.

Fishing is a well-known industry, and there are certain risks of work-related diseases and accidents, occupational hazards and safety issues. This study aimed at examining the determinants of occupational hazards and injuries among fishermen at Tanji fishing site, a major fish-landing site in the Gambia, West Africa. An analytical cross-sectional design was conducted in August to October 2019. Structured questionnaires were administered to fishermen at Tanji fishing site. A simple random sampling method was used to select fishermen in this study. Data entry and processing for preliminary data analysis was done using Stata version 15. Descriptive and bivariate analysis using chi-square/fisher exact test as well as binary logistics regression analysis were used. The adjusted risk ratios (aRRs) and confidence intervals of 95% were calculated. A P -value