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Kari sheep inhabiting the Chitral district of Pakistan show variation in gestation length. In this study, we have analyzed the genetic differences between the three subtypes of Kari sheep (based on variation in gestation length) using microsatellite markers. Kari sheep samples were collected from their breeding tract and were characterized for gestation length and genetic diversity using microsatellite markers. A total of 78 Kari ewes were grouped into three categories based on gestation length (GL), i.e., Kari-S (with a shorter GL), Kari-M (with a medium GL), and Kari-L (with a longer GL). DNA from these samples was used to amplify 31 ovine-specific microsatellite loci through PCR. Of the total 78 Kari specimens, 24 were grouped in Kari-S (GL = 100.7 ± 1.8), 26 were from the Kari-M subtype (GL = 123.1 ± 1.0), and 28 were Kari-L (GL = 143.8 ± 1.5). Microsatellite analysis revealed an association of genotypes at two marker sites (MAF214 and ILSTS5) with variation in GL. A total of 158 alleles were detected across the 22 polymorphic loci with an average of 7.18 alleles per locus. Unique alleles were found in all three subtypes. The highest number of unique alleles was observed in Kari-L (15), followed by Kari-S (10) and Kari-M (8). The results indicated that Kari-S is a genetically distinct subtype (with higher genetic differentiation and distance) from Kari-M and Kari-L. The genetic uniqueness of Kari-S is important for further exploration of the genetic basis for shorter gestation length, and exploitation of their unique values.

We report the genetic analysis of two consanguineous pedigrees of Pakistani ancestry in which two siblings in each family exhibited developmental delay, epilepsy, intellectual disability and aggressive behavior. Whole-genome sequencing was performed in Family 1, and we identified ~80,000 variants located in regions of homozygosity. Of these, 615 variants had a minor allele frequency ≤ 0.001, and 21 variants had CADD scores ≥ 15. Four homozygous exonic variants were identified in both affected siblings: PDZD7 (c.1348_1350delGAG, p.Glu450del), ALG6 (c.1033G>C, p.Glu345Gln), RBM20 (c.1587C>G, p.Ser529Arg), and CNTNAP2 (c.785G>A, p.Gly228Arg). Sanger sequencing revealed co-segregation of the PDZD7, RBM20 , and CNTNAP2 variants with disease in Family 1. Pathogenic variants in PDZD7 and RBM20 are associated with autosomal recessive non-syndromic hearing loss and autosomal dominant dilated cardiomyopathy, respectively, suggesting that these variants are unlikely likely to contribute to the clinical presentation. Gene panel analysis was performed on the two affected siblings in Family 2, and they were found to also be homozygous for the p.Gly228Arg CNTNAP2 variant. Together these families provide a LOD score 2.9 toward p.Gly228Arg CNTNAP2 being a completely penetrant recessive cause of this disease. The clinical presentation of the affected siblings in both families is also consistent with previous reports from individuals with homozygous CNTNAP2 variants where at least one allele was a nonsense variant, frameshift or small deletion. Our data suggests that homozygous CNTNAP2 missense variants can also contribute to disease, thereby expanding the genetic landscape of CNTNAP2 dysfunction., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Badshah, Mattison, Ahmad, Chopra, Johnston, Ahmad, Khan, Sarwar, Cutler, Taylor, Vadlamani, Zwick and Escayg.)

Transmissible spongiform encephalopathies (TSEs) are caused by the accumulation of the abnormal prion protein scrapie (PrP Sc ). Prion protein aggregation, misfolding, and cytotoxicity in the brain are the major causes of neuronal dysfunction and ultimate neurodegeneration in all TSEs. Parkin, an E3 ubiquitin ligase, has been studied extensively in all major protein misfolding aggregating diseases, especially Parkinson's disease and Alzheimer's disease, but the role of parkin in TSEs remains unknown. Here we investigated the role of parkin in a prion disease cell model in which neuroblastoma2a (N2a) cells were treated with prion peptide PrP106-126. We observed a gradual decrease in the soluble parkin level upon treatment with PrP106-126 in a time-dependent manner. Furthermore, endogenous parkin colocalized with FITC-tagged prion fragment106-126. Overexpression of parkin in N2a cells via transfection repressed apoptosis by enhancing autophagy. Parkin-overexpressing cells also showed reductions in apoptotic BAX translocation to the mitochondria and cytochrome c release to the cytosol, which ultimately inhibited activation of proapoptotic caspases. Taken together, our findings reveal a parkin-mediated cytoprotective mechanism against PrP106-126 toxicity, which is a novel potential therapeutic target for treating prion diseases.

Prion infections of the central nervous system (CNS) are characterized by initial reactive gliosis followed by overt neuronal death. Gliosis is likely to be caused initially by the deposition of misfolded, proteinase K-resistant, isoforms (termed PrP Sc ) of the normal cellular prion protein (PrP c ) in the brain. Proinflammatory cytokines and chemokines released by PrP Sc -activated glia and stressed neurons may also contribute directly or indirectly to the disease development by enhancing gliosis and inducing neurotoxicity. Recent studies have illustrated that early neuroinflammation activates nuclear factor of activated T cells (NFAT) in the calcineurin signaling cascade, resulting in nuclear translocation of nuclear factor kappa B (NF-κB) to promote apoptosis. Hence, useful therapeutic approaches to slow down the course of prion disease development should control early inflammatory responses to suppress NFAT signaling. Here we used a hamster model of prion diseases to test, for the first time, the neuroprotective and NFAT-suppressive effect of a second-generation semisynthetic tetracycline derivative, minocycline, versus a calcineurin inhibitor, FK506, with known NFAT suppressive activity. Our results indicate that prolonged treatment with minocycline, starting from the presymptomatic stage of prion disease was more effective than FK506 given either during the presymptomatic or symptomatic stage of prion disease. Specifically, minocycline treatment reduced the expression of the astrocyte activation marker glial fibrillary acidic protein and of the microglial activation marker ionized calcium-binding adapter molecule-1, subsequently reducing the level of proinflammatory cytokines interleukin 1β and tumor necrosis factor-α. We further found that minocycline and FK506 treatment inhibited mitogen-activated protein kinase p38 phosphorylation and NF-κB nuclear translocation in a caspase-dependent manner, and enhanced phosphorylated cyclic adenosine monophosphate response element-binding protein and phosphorylated Bcl2-associated death promoter levels to reduce cognitive impairment and apoptosis. Taken together, our results indicate that minocycline is a better choice for prolonged use in prion diseases and encourage its further clinical development as a possible treatment for this disease.

A captive 8 yr old male bottlenose dolphin Tursiops truncatus succumbed to septicemia with multisystemic inflammation including suppurative enteritis, encephalitis, and pneumonia with chronic pancreatitis. A pure culture of beta-hemolytic, catalase- and oxidase-negative, Gram-positive cocci was isolated from the hilar lymph nodes and pancreas. The isolate was identified by 16S rDNA sequencing as Streptococcus iniae. Histological examination of the digestive system revealed a mixed infection of both bacteria and fungus. Recognized as a pathogen in fish, dolphins, and humans, this is the first report of S. iniae in a dolphin in mainland China. As the number of managed animals in oceanariums is increasing, so is the frequency of contact with fish used as food for marine mammals and humans, highlighting the importance of education and appropriate personal protective protocols to minimize the risk of transmission. An understanding of marine mammal infectious disease organisms is essential to ensuring the health of marine mammals and humans coming into contact with such animals and their food. This study illustrates a systematic clinical, microbiological, and pathological investigation into a septicemic bottlenose dolphin infected with S. iniae. Our findings provide useful information for those involved in the diagnosis and control of infectious diseases in marine mammals and offer insight into an important zoonotic pathogen.

The association between caprine PrP gene polymorphisms and its susceptibility to scrapie has been investigated in current years. As the ORF of the PrP gene is extremely erratic in different breeds of goats, we studied the PrP gene polymorphisms in 80 goats which belong to 11 Pakistani indigenous goat breeds from all provinces of Pakistan. A total of 6 distinct polymorphic sites (one novel) with amino acid substitutions were identified in the PrP gene which includes 126 (A -> G), 304 (G -> T), 379 (A -> G), 414 (C -> T), 428 (A -> G) and 718 (C -> T). The locus c.428 was found highly polymorphic in all breeds as compare to other loci. On the basis of these PrP variants NJ phylogenetic tree was constructed through MEGA6.1 which showed that all goat breeds along with domestic sheep and Mauflon sheep appeared as in one clade and sharing its most recent common ancestors (MRCA) with deer species while Protein analysis has shown that these polymorphisms can lead to varied primary, secondary and tertiary structure of protein. Based on these polymorphic variants, genetic distance, multidimensional scaling plot and principal component analyses revealed the clear picture regarding greater number of substitutions in cattle PrP regions as compared to the small ruminant species. In particular these findings may pinpoint the fundamental control over the scrapie in Capra hircus on genetic basis.

The aggregation of disease-specific misfolded proteins resulting in endoplasmic reticulum stress is associated with early pathological events in many neurodegenerative diseases, and apoptotic signaling is initiated when the stress goes beyond the maximum threshold level of endoplasmic reticulum stress sensors. All eukaryotic cells respond to the accumulation of unfolded proteins in the endoplasmic reticulum (ER) by signaling an adaptive pathway termed as unfolded protein response (UPR). Recently, the focus of research shifted from work on specific proteins as pathogenesis in these neurodegenerative diseases towards a more specific generic pathway known as UPR. ER is a major organelle for protein quality control, and cellular stress disrupts normal functioning of ER. The UPR acts as a protective mechanism during endoplasmic reticulum stress, but persistent long-term stress triggers UPR-mediated apoptotic pathways ultimately leading to cell death. Here in this review, we will briefly summarize the molecular events of endoplasmic reticulum stress-associated UPR signaling pathways and their potential therapeutic role in neurodegenerative diseases.

Dysregulated calcium signaling and accumulation of aberrant proteins causing endoplasmic reticulum stress are the early sign of intra-axonal pathological events in many neurodegenerative diseases, and apoptotic signaling is initiated when the stress goes beyond the maximum threshold level of endoplasmic reticulum. The fate of the cell to undergo apoptosis is controlled by Ca2(+) signaling and dynamics at the level of the endoplasmic reticulum. Endoplasmic reticulum resident inositol 1,4,5-trisphosphate receptors (IP3R) play a pivotal role in cell death signaling by mediating Ca2(+) flux from the endoplasmic reticulum into the cytosol and mitochondria. Hence, many prosurvival and prodeath signaling pathways and proteins affect Ca2(+) signaling by directly targeting IP3R channels, which can happen in an IP3R-isoform-dependent manner. Here, in this review, we summarize the regulatory mechanisms of inositol triphosphate receptors in calcium regulation and initiation of apoptosis during unfolded protein response.

Mycobacterium bovis is the causative agent of tuberculosis in cattle. Infection of macrophages with M. bovis leads to the activation of the "nucleotide binding and oligomerization, leucine-rich repeat and pyrin domains-containing protein 3" (NLRP3) and "absent in melanoma 2" (AIM2) inflammasomes, which in turn triggers release of the proinflammatory cytokine interleukin-1β (IL-1β) that contributes to bacterial clearance and plays a crucial role in the host defense. However, NLRP3 and AIM2 inflammasome activation is influenced by several factors and how IL-1β secretion by M. bovis-infected macrophages is regulated via the inflammasome pathway remains unclear. Here we found that IL-1β secretion and pro-IL-1β protein accumulation were inhibited in THP-1 macrophages upon exposure to the virulent M. bovis Beijing strain in the presence of high K(+) concentrations, cycloheximide (a protein synthesis inhibitor) and PR-619 (a deubiquitinating enzyme inhibitor). Scavenging reactive oxygen species (ROS) induced by N-acetylcysteine reduced IL-1β release independent of the mitochondrial permeability transition. Collectively, our results suggest that IL-1β secretion by M. bovis-infected THP-1 macrophages is reduced by high extracellular K(+) concentration, inhibition of new protein synthesis, deubiquitination, and ROS generation.

Under the "protein-only" hypothesis, prion-based diseases are proposed to result from an infectious agent that is an abnormal isoform of the prion protein in the scrapie form, PrP(Sc). However, since PrP(Sc) is highly insoluble and easily aggregates in vivo, this view appears to be overly simplistic, implying that the presence of PrP(Sc) may indirectly cause neurodegeneration through its intermediate soluble form. We generated a neurotoxic PrP dimer with partial pathogenic characteristics of PrP(Sc) by protein misfolding cyclic amplification in the presence of 1-palmitoyl-2-oleoylphosphatidylglycerol consisting of recombinant hamster PrP (23-231). After intracerebral injection of the PrP dimer, wild-type hamsters developed signs of neurodegeneration. Clinical symptoms, necropsy findings, and histopathological changes were very similar to those of transmissible spongiform encephalopathies. Additional investigation showed that the toxicity is primarily related to cellular apoptosis. All results suggested that we generated a new neurotoxic form of PrP, PrP dimer, which can cause neurodegeneration. Thus, our study introduces a useful model for investigating PrP-linked neurodegenerative mechanisms.

The 37/67-kDa laminin receptor precursor (LRP)/laminin receptor (LR) is a cell surface receptor for cellular prion proteins and misfolded pathological prions. Previous research has shown that blocking or decreasing LRP/LP levels by anti-LRP/LR antibodies or small interfering RNAs (siRNAs) can prolong the incubation phase of experimental prion infection. This study aimed to investigate potential mechanisms contributing to prion resistance/susceptibility by using the rabbit, a species unsusceptible to prion infection, as a model. We investigated the expression level and distribution of LRP/LR in rabbit tissues by real-time polymerase chain reaction and by immunochemical analysis with a monoclonal anti-67 kDa LR antibody. Our results showed LRP/LR mRNA expression in all the tissues examined. Very low LRP/LR expression levels were observed in central nervous system (CNS) tissues, whereas high expression levels were observed in reproductive and digestive tissues, which differed from the expression patterns that have been reported for prion-susceptible animals. The immunochemical staining results were generally consistent with the mRNA findings, although no LR protein was detected in CNS tissues. Our findings provide a basis for further studies on prion resistance in rabbits and other animal species.

Prion diseases, characterized by spongiform degeneration and the accumulation of misfolded and aggregated PrP(Sc) in the central nervous system, are one of fatal neurodegenerative and infectious disorders of humans and animals. In earlier studies, autophagy vacuoles in neurons were frequently observed in neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's diseases as well as prion diseases. Autophagy is a highly conserved homeostatic process by which several cytoplasmic components (proteins or organelles) are sequestered in a double-membrane-bound vesicle termed 'autophagosome' and degraded upon their fusion with lysosome. The pathway of intercellular self-digestion at basal physiological levels is indispensable for maintaining the healthy status of tissues and organs. In case of prion infection, increasing evidence indicates that autophagy has a crucial ability of eliminating pathological PrP(Sc) accumulated within neurons. In contrast, autophagy dysfunction in affected neurons may contribute to the formation of spongiform changes. In this review, we summarized recent findings about the effect of mammalian autophagy in neurodegenerative disorders, particularly in prion diseases. We also summarized the therapeutic potential of some small molecules (such as lithium, rapamycin, Sirtuin 1 and resveratrol) targets to mitigate such diseases on brain function. Furthermore, we discussed the controversial role of autophagy, whether it mediates neuronal toxicity or serves a protective function in neurodegenerative disorders.

Injection drug users (IDUs) are considered as a high risk group to develop hepatitis C due to needle sharing. In this study we have examined 200 injection drug users from various regions of the Khyber Pakhtunkhwa province for the prevalence of active HCV infection and HCV genotypes by Immunochromatographic assays, RT-PCR and Type-specific PCR. Our results indicated that 24% of the IDUs were actively infected with HCV while anti HCV was detected among 31.5% cases. Prevalent HCV genotypes were HCV 2a, 3a, 4 and 1a. Majority of the IDUs were married and had attained primary or middle school education. 95% of the IDUs had a previous history of needle sharing. Our study indicates that the rate of active HCV infection among the IDUs is higher with comparatively more prevalence of the rarely found HCV types in KPK. The predominant mode of HCV transmission turned out to be needle sharing among the IDUs.