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Anaplasmosis, caused by intracellular gram-negative bacteria Anaplasma marginale is one of the most frequently reported tick-borne disease (TBDs) in tropical and sub-tropical countries, including Pakistan. In the present study, a total of 428 cattle blood samples were collected to examine the prevalence and phylogenetic origin of A. marginale in two important livestock regions of Punjab Province in Pakistan, i.e. Lodhran and Dera Ghazi Khan Districts. In addition, association between occurrence of A. marginale in cattle blood and selected epidemiological factors has been also investigated. The presence of A. marginale genetic material was confirmed in 9% of the tested blood samples taken from cattle in Lodhran and in 17% from Dera Ghazi Khan. Prevalence of A. marginale was significantly higher in cattle from Dera Ghazi Khan. All the cattle breeds from both districts were equally susceptible to A. marginale infection. We reported higher prevalence of A. marginale in cattle living indoors or with other dairy animals in Dera Ghazi Khan district. However, no such relationship was observed in the Lodhran district. Sequencing of the msp1b gene shows 96-99% similarity of A. marginale in the study area to those reported from other parts of Pakistan, South Africa, and Israel. We recommend that large scale tick and tick-borne disease control strategies must be implemented in both districts.

Meiosis is pivotal for sexual reproduction and fertility. Meiotic programmed DNA double-strand breaks (DSBs) initiate homologous recombination, ensuring faithful chromosome segregation and generation of gametes. However, few studies have focused on meiotic DSB formation in human reproduction. Here, we report four infertile siblings born to a consanguineous marriage, with three brothers suffering from non-obstructive azoospermia and one sister suffering from unexplained infertility with normal menstrual cycles and normal ovary sizes with follicular activity. An autosomal recessive mutation in TOP6BL was found co-segregating with infertility in this family. Investigation of one male patient revealed failure in programmed meiotic DSB formation and meiotic arrest prior to pachytene stage of prophase I. Mouse models carrying similar mutations to that in patients recapitulated the spermatogenic abnormalities of the patient. Pathogenicity of the mutation in the female patient was supported by observations in mice that meiotic programmed DSBs failed to form in mutant oocytes and oocyte maturation failure due to absence of meiotic recombination. Our study thus illustrates the phenotypical characteristics and the genotype-phenotype correlations of meiotic DSB formation failure in humans.

Background Acromesomelic dysplasia, type Maroteaux (AMDM) is a rare skeletal dysplasia following autosomal recessive mode of inheritance and characterized by abnormal growth plates, short and abnormal bones in the extremities and spine. Objective Present study was designed to report the molecular basis of AMDM in enrolled consanguineous family from Pakistan. Methods A consanguineous family from Vehari District in Pakistan having multiple siblings suffering from AMDM was enrolled in present study. Whole exome sequencing (WES) approach was adopted to identify causative agent of AMDM. Human full length NPR2 gene and sequence with nonsense mutation was amplified by using Myc-tagged pXN vector and transformed in E. coli DH5α cells to confirm mutation. SDS-PAGE and Western blotting were done to confirm the production of truncated protein. Computational three dimensional structure generation through homology modeling approach was done to compare protein structure between patients and controls. Results WES reveled a nonsense mutation (c.613 C>T, p.R205X) in exon 1 of NPR2 gene leading to premature termination codon in mRNA of NPR2 gene resulting in a truncated protein with 204 amino acid residues that was confirmed by SDS-PAGE and Western blotting. Sanger sequencing confirmed that mutation in all subjects and mutation followed Mendalian pattern of inheritance. Multiple sequence alignment by ClustalW revealed that mutated domain of NPR2 is conserved region. Proetin structure comparison revealed a significant structural part of NPR2 was missing in truncated protein as compared to control. Conclusion We are reporting that a novel nonsense mutation (c.613 C>T, p.R205X) in exon 1 of NPR2 gene is causing AMDM in a consanguineous Pakistani family.

Exploring the genetic basis of human infertility is currently under intensive investigation. However, only a handful of genes are validated in animal models as disease-causing genes in infertile men. Thus, to better understand the genetic basis of spermatogenesis in human and to bridge the knowledge gap between human and other animal species, we have constructed FertilityOnline database, which is a resource that integrates the functional genes reported in literature related to spermatogenesis into an existing spermatogenic database, SpermatogenesisOnline 1.0. Additional features like functional annotation and statistical analysis of genetic variants of human genes, are also incorporated into FertilityOnline. By searching this database, users can focus on the top candidate genes associated with infertility and can perform enrichment analysis to instantly refine the number of candidates in a user-friendly web interface. Clinical validation of this database is established by the identification of novel causative mutations in SYCE1 and STAG3 in azoospermia men. In conclusion, FertilityOnline is not only an integrated resource for analysis of spermatogenic genes, but also a useful tool that facilitates to study underlying genetic basis of male infertility.AvailabilityFertilityOnline can be freely accessed at http://mcg.ustc.edu.cn/bsc/spermgenes2.0/index.html.

Spondylo-epimetaphyseal dysplasia Matrilin 3 type (SEMD) is a rare autosomal recessive skeletal dysplasia characterized by short stature, abnormalities in the vertebral bodies and long bones, especially the lower limbs. We enrolled a consanguineous family from Pakistan in which multiple siblings suffered from severe skeletal dysplasia. The six affected subjects ranged in heights from 100 to 136 cm (~-6 standard deviation). Lower limb abnormalities with variable varus and valgus deformities and joint dysplasia were predominant features of the clinical presentation. Whole exome sequencing (WES) followed by Sanger sequencing identified a missense variant, c.542G > A, p.(Arg181Gln) in MATN3 as the genetic cause of the disorder. The variant was homozygous in all affected individuals while the obligate carriers had normal heights with no skeletal symptoms, consistent with a recessive pattern of inheritance. Multiple sequence alignment revealed that MATN3 domain affected by the variant is highly conserved in orthologous proteins. The c.542G > A, p.(Arg181Gln) variant is only the fourth variant in MATN3 causing an autosomal recessive disorder and thus expands the genotypic spectrum.

Infertility is one of the most important and burning issues in present times, as a marked increase in the frequency of infertile cases has been observed all over the world. Chromosomal aneuploidy is among the known factors associated with infertility, and among sex chromosome aneuploidies, 47,XXY and 47,XYY constitute the most common class of chromosome abnormality in human live births. Considerable attention has been given to the somatic abnormalities associated with these conditions, but less is known about their meiotic progression; that is, how sex chromosome imbalance influences the meiotic process. It has been documented that men with the same underlying genetic cause of infertility do not present with uniform pathology, so it is informative to find out how meiotic progression differs in patients with similar chromosomal aneuploidy having different phenotypes. The importance of studying meiotic progression in patients with sex chromosome abnormalities has increased many fold with the introduction of assisted reproductive technologies that have made it possible for infertile men to become biological parents. Hence, exploring the possible consequences of sex chromosome aneuploidy for meiotic chromosome segregation is worthwhile. The objective of this review, in the context of current knowledge, is to discuss problems associated with fertility and progression of meiosis in two relatively common sex chromosome aneuploidies, 47,XXY and 47,XYY, reported in humans.

Anaplasmosis is a tick-borne disease caused by obligate intercellular gram-negative bacteria, Anaplasma (A.) marginale. The present study reports on seasonal prevalence, epidemiology, and phylogeny of A. marginale in three cattle breeds from District Layyah, Southern Punjab, Pakistan. A total of 844 blood samples (Cross = 300, Holstein Friesian = 244, Sahiwal breed = 300) from apparently healthy cattle on seasonal basis were collected along with epidemiological data during May 2018 till April 2019. Polymerase chain reaction generated 265 base-pair amplicon specific for major surface protein-1b encoding gene of A. marginale in 8.6% (73/844) of enrolled cattle. The highest prevalence was observed during autumn (18.3%) followed by summer (9.7%) and winter season (7.1%). Holstein Friesian breed was most susceptible to A. marginale infection (13.1%) followed by Sahiwal (7.6%) and cross breed (6%). Representative amplified partial gene sequences of A. marginale were submitted to GenBank (Accession numbers MK032842 and MK032843). 37/844 (4.3%) Giemsa-stained blood smears were found positive for Anaplasma spp. Small number of ticks including Hyalomma anatolicum, Hyalomma excavatum, Rhipicephalus microplus, Haemaphysalis punctata were identified from cattle but none of them was found PCR positive for the presence of A. marginale. Analysis of epidemiological factors revealed that female cattle and farm with water supply from pool, farms where other dairy animals and dogs were living with cattle and dogs having ticks load on them had significant association with A. marginale prevalence. It was observed that white blood cell, lymphocytes (%), monocytes (%) hematocrit, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration were significantly disturbed in A. marginale-positive than negative cattle.

Babesiosis, theileriosis and anaplasmosis are among the most commonly reported tick-borne diseases in cattle and are associated with significant economic losses. Through the present study the researchers aimed to report the presence of various pathogens that cause babesiosis, theileriosis and anaplasmosis in cattle collected from different provinces in Saudi Arabia and to report their phylogenetic relationship. A total of 362 blood samples of cattle along with ticks that were present on the cattle were collected from four regions (Riyadh, Al-Kharj, Al-Hasa and Al-Qassim) of Saudi Arabia. Blood samples were screened by polymerase chain reaction (PCR) for the presence of various Babesia, Theileria and Anaplasma species by amplification of their 18S rRNA and/or 23S rRNA genes. A total of 541 ticks were collected and identified from the cattle. These included Hyalomma anatolicum, Hyalomma dromedarii, Hyalomma impeltatum, Hyalomma excavatum, Rhipicephalus annulatus and Rhipicephalus turanicus. Regarding tick-borne pathogens, the overall prevalence was 1.9 % (7/362) for Theileria annulata, (2/362) 0.6 % for Theileria and (21/362) 5.8 % for Anaplasma ovis. Four of the cattle were found to be co-infected with more than one pathogen (1.1 %). We did not detect any Babesia species in the blood of the studied cattle. Prevalence of the Theileria and Anaplasma species was highest in cattle that resided in Riyadh, followed by cattle from Al-Hasa and Al-Qassim. Representative amplified partial-gene sequences of T. annulata (GenBank accession numbers MK826137-39) and A. ovis (GenBank acc. no. MK 880224) were submitted to GenBank. The presence of ticks on cattle was found to be associated with a high prevalence of Theileria spp. (P = 0.02) and Anaplasma ovis (P < 0.001). We report novel genotypes of T. annulata and A. ovis from cattle in Saudi Arabia and we recommend that molecular surveys are undertaken throughout the country to address the prevalence and geographical distribution of tick-borne infections for their effective diagnosis and treatment.

Meiosis is an essential step in gametogenesis which is the key process in sexually reproducing organisms as meiotic aberrations may result in infertility. In meiosis, programmed DNA double-strand break (DSB) formation is one of the fundamental processes that are essential for maintaining homolog interactions and correcting segregation of chromosomes. Although the number and distribution of meiotic DSBs are tightly regulated, still abnormalities in DSB formation are known to cause meiotic arrest and infertility. This review is a detailed account of molecular bases of meiotic DSB formation, its evolutionary conservation, and variations in different species. We further reviewed the mutations of DSB formation genes in association with human infertility and also proposed the future directions and strategies about the study of meiotic DSB formation.

Dwarfism or short stature is a condition when human height is less than 147 cm in adult form. We enrolled two families with multiple siblings suffering from dwarfism and Whole exome sequencing (WES) followed by Sanger sequencing approach was used to report genetic base of their dwarfism. Two novel missense mutations, c.2407C > A, p.Pro803Thr in FGFR1 gene in family one and c.1779C > G, p.F539L in FGFR3 gene in second family were identified as the genetic cause of their phenotype. Sanger sequencing confirmed these mutations in all enrolled patients and mutations followed autosomal dominant mode of inheritance. Multiple sequence alignment by Clustal Omega revealed that domains of FGFR1 and FGFR3 containing mutations are highly conserved. Computational 3D structure generations through homology modeling approach revealed no observable differences in FGFR1 and FGFR3 protein structures when compared between control and patients indicating that observed mutations are responsible for functional changes in proteins.

Objectives To examine whether Ephrin type A receptor 2 gene polymorphisms are associated with susceptibility to age-related cataract. METHODS The case-control study was conducted from January to May 2014 in Multan, Pakistan, and comprised patients of age-related cataract enrolled from Nishtar Hospital, Multan, and age-matched healthy controls without any type of cataract from the local population. A questionnaire was used to gather clinical and epidemiological data. Deoxyribonucleic acid was extracted from blood samples, and analysis of rs11260867, rs3568293 and rs7543472 single nucleotide polymorphisms was performed by using tetra amplification-refractory mutation system polymerase chain reaction protocol. Data was analysed using SPSS 17. RESULTS Of the 230 subjects, 129(%) were patients and 101(%) were controls. Among the three polymorphisms analysed, rs7543472 was associated with age-related cataract. Among the epidemiological and clinical factors, age, diabetes, blood pressure, smoking, radiation exposure, steroids usage and use of tranquilisers were associated with age-related cataract (p

International audience; Canine monocytic ehrlichiosis is a tick-borne disease caused by an intracellular alpha-proteobacterium, Ehrlichia canis, which replicates within mononuclear cells in the host. This study was designed to use a polymerase chain reaction (PCR) protocol for the molecular detection of E. canis by the amplification of a portion of its 16S rRNA gene, as well as the effects of this alpha-proteobacterium on the haematological parameters of the sampled dogs and the risk factors associated with E. canis infection. A total of 151 blood samples were collected from dogs of various breeds at three sampling sites (Lahore, Rawalpindi/Islamabad and Multan) in Punjab, Pakistan. Data regarding the epidemiological factors (including age, gender, breed, body temperature, deworming, vaccination, mucous membrane status, hydration status, the presence of haematuria and tick infestation) were collected through a questionnaire at the time of sample collection. A 400 bp DNA fragment of the 16S rRNA gene of E. canis was amplified from 42 dog blood samples (28% of the total), [Lahore (N=24), Rawalpindi/Islamabad (N=13) and Multan (N=05)] through PCR. Data analysis revealed that the character of the animals (age, sex and breed) had no significant association (P>0.05) with the presence of E. canis. Various haematological parameters were also compared, and the results revealed that all of the parameters remained unaffected, except significantly lower white blood cell counts (P=0.004) in E. canis-positive blood samples, as compared with the control group. We concluded that this is the first molecular confirmation of canine infection by E. canis using PCR. Moreover, no specific epidemiological parameter was found associated with the prevalence of E. canis in dogs.

GABAB receptor antagonists are experimentally proved as spatial memory enhancers in mouse models but their role has not been described following hypoxic-ischemic insult. 10-day-old albino mice were subjected to Murine model of hypoxia and ischemia. Following brain damage, mice were fed on normal rodent diet till they were 13 weeks old. At this time point, mice were divided into two groups. Group 1 received saline and group 2 received intraperitoneally CGP 55845 (1 mg/ml solvent/Kg body weight) for 12 days. Behavioural observations were made during rota rod, open field and Morris water maze test along with brain infarct measurement in both CGP 55845 treated and untreated groups. It was observed that application of GABAB receptor antagonist improved the over all motor function in male and female albino mice but effects were more pronounced in males. In open field, CGP 55845-treated female mice showed poor performance. CGP 55845 had no significant effect on learning and memory formation during Morris water maze test and also on brain infract size in both genders following hypoxia ischemia encephalopathy. Effects of CGP 55845 can be further explored in a dose and duration dependent manner to improve the learning and memory in albino mice following neonatal brain damage.

Creatine is known to rescue animals following brain damage. Present study was designed to demonstrate the effect of long term (15 week) supplementation of 2% creatine monohydrate (Cr), following neonatal hypoxic ischemic insult, on learning and memory formation in male albino mouse. Albino mice pups were subjected to right common carotid artery ligation followed by 8% hypoxia for 25 minutes. Following weaning, animals were separated and grouped on the basis of dietry supplementation for 15 weeks followed by a battery of neurological tests including Morris water maze, open field and rota rod. It was observed that HI mice fed on 2% Cr for 15 weeks performed better than their littermates mice on normal rodent diet during water maze (learning and memory) and rotating rod (neuro-muscular coordination and balance) test while the results of open field test remained unaffected. It was also observed that Cr treated animals had a reduced brain infarct volume than untreated but this difference did not reached statistical significance. We have also observed an overall increase in body weight in Cr treated mice during the study. Over all our results are indicating that long term Cr supplementation is beneficial for male albino following hypoxic ischemic insult.

Currently there are no uniform standard treatments for newborn suffering from cerebral hypoxia-ischemia (HI) and to find new and effective strategies for treating the HI injury remains a key direction for future research. Present study was designed to demonstrate that optimal dose (1 or 3%) of creatine monohydrate (Cr) for the treatment of neonatal HI in female albino mice. On postnatal day 10, animals were subjected to left carotid artery ligation followed by 8% hypoxia for 25 minutes. Following weaning on postnatal day 20, mice were divided into three treatments on the basis of diet supplementation (Normal rodent diet, 1% and 3% creatine supplemented diet) for 10 week. A battery of neurological tests (Rota rod, open field and Morris water maze) was used to demonstrate effect of Cr supplementation on neurofunction and infarct size following HI. Open field test results indicated that Cr supplementation had significantly improved locomotory and exploratory behavior in subjects. It was observed that Cr treated mice showed better neuromuscular coordination (rota rod) and improved spatial memory (Morris Water Maze test). A significant affect of creatine supplementation in reducing infarct size was also observed. Post hoc analysis of post hoc multiple comparisons revealed that mice supplemented with 3% Cr for 10 weeks performed better during Morris water maze test while 1% Cr supplementation improved the exploratory behavior and gain in body weight than control group indicating that Cr supplementation has the potential to improve the neurofunction following neonatal brain damage. This article is part of a Special Issue entitled SI: Brain and Memory.

Fanconi anemia (FA) genes play important roles in spermatogenesis. In mice, disruption of Fancm impairs male fertility and testicular integrity, but whether FANCM pathogenic variants (PV) similarly affect fertility in men is unknown. Here we characterize a Pakistani family having three infertile brothers, two manifesting oligoasthenospermia and one exhibiting azoospermia, born to first-cousin parents. A homozygous PV in FANCM (c.1946_1958del, p.P648Lfs*16) was found cosegregating with male infertility. Our objective is to validate that FANCM p.P648Lfs*16 is the PV causing infertility in this family. Exome and Sanger sequencing were used for PV screening. DNA interstrand crosslink (ICL) sensitivity was assessed in lymphocytes from patients. A mouse model carrying a PV nearly equivalent to that in the patients (FancmΔC/ΔC) was generated, followed by functional analysis in spermatogenesis. The loss-of-function FANCM PV increased ICL sensitivity in lymphocytes of patients and FancmΔC/ΔC spermatogonia. Adult FancmΔC/ΔC mice showed spermatogenic failure, with germ cell loss in 50.2% of testicular tubules and round-spermatid maturation arrest in 43.5% of tubules. In addition, neither bone marrow failure nor cancer/tumor was detected in all the patients or adult FancmΔC/ΔC mice. These findings revealed male infertility to be a novel phenotype of human patients with a biallelic FANCM PV.

Disease-associated variants in the human genome are continually being identified using DNA sequencing technologies that are especially effective for Mendelian disorders. Here we sequenced whole genome to high coverage (>30×) of 6 members of a 7-generation family with dwarfism from a consanguineous tribe in Pakistan to determine the causal variant(s). We identified a missense variant rs111033552 (c.2011T>C [p.Ser671Pro]) located in COL10A1 (encodes the alpha chain of type X collagen) as the most likely contributor to the dwarfism. We further confirmed the variant in 22 family members using Sanger sequencing. All affected individuals are heterozygous for the missense mutation rs111033552 and no individual homozygous was observed. Moreover, the mutation was absent in 69,985 individuals representing >150 global populations. Taking advantage of whole-genome sequencing data, we also examined other variant forms, including copy number variation and insertion/deletion, but failed to identify such variants enriched in the affected individuals. Thus rs111033552 had priority for linkage with dwarfism.

Creatine monohydrate (Cr) is a dietary supplement known to improve cognitive functions and has positive therapeutic results under various clinical conditions. The aim of this study was to determine the effect of 2 % Cr supplementation on learning, memory formation, neuromuscular coordination, exploratory and locomotory in male albino mice following hypoxic ischemic insult. At postnatal day, 10 male albino mice pups were subjected to right common carotid artery ligation followed by 8 % hypoxia for 25 min. On postnatal day 20, male mice were separated from the litter and divided into two groups on the basis of special diet supplementation. One group was supplemented with 2 % Cr in diet while the other group was raised on ordinary rodent chow for 8 weeks. Behavioral observations were made during rota rod, open field and Morris water maze test for both treatments. It was observed that supplementation with 2 % Cr for 8 weeks following neonatal brain damage resulted in enhanced muscular strength, neuromuscular coordination and improved body weight. In Morris water maze test, it was observed that Cr supplementation significantly improved mean swimming speed and mice on 2 % Cr diet covered more distance but the spatial memory was not improved significantly following hypoxia ischemia encephalopathy (HIE). Open field parameters and percentage of infarct volume remained unaffected following Cr supplementation. We concluded that 2 % dietary Cr supplementation has a potential to improve the muscle strength and body weight in male albino mice following (HIE) and should be considered for the treatment of neurological ailments.

Purpose Fanconi anemia (FA) genes play important roles in spermatogenesis. In mice, disruption of Fancm impairs male fertility and testicular integrity, but whether FANCM pathogenic variants (PV) similarly affect fertility in men is unknown. Here we characterize a Pakistani family having three infertile brothers, two manifesting oligoasthenospermia and one exhibiting azoospermia, born to first-cousin parents. A homozygous PV in FANCM (c.1946_1958del, p.P648Lfs*16) was found cosegregating with male infertility. Our objective is to validate that FANCM p.P648Lfs*16 is the PV causing infertility in this family. Methods Exome and Sanger sequencing were used for PV screening. DNA interstrand crosslink (ICL) sensitivity was assessed in lymphocytes from patients. A mouse model carrying a PV nearly equivalent to that in the patients (FancmΔC/ΔC) was generated, followed by functional analysis in spermatogenesis. Results The loss-of-function FANCM PV increased ICL sensitivity in lymphocytes of patients and FancmΔC/ΔC spermatogonia. Adult FancmΔC/ΔC mice showed spermatogenic failure, with germ cell loss in 50.2% of testicular tubules and round-spermatid maturation arrest in 43.5% of tubules. In addition, neither bone marrow failure nor cancer/tumor was detected in all the patients or adult FancmΔC/ΔC mice. Conclusion These findings revealed male infertility to be a novel phenotype of human patients with a biallelic FANCM PV.

Androgens are required for normal male sex differentiation and development of male secondary sexual characteristics. Mutations in AR gene are known to cause defects in male sexual differentiation. In current study, we enrolled a 46,XY phenotypically female patient bearing testes in inguinal canal. DNA sequencing of the AR gene detected a missense mutation C.1715A > G (p. Y572C) in exon 2 which is already known to cause complete androgen insensitivity syndrome (CAIS). We focused on the effects of this mutation on the testicular histopathology of the patient. Surface spreading of testicular tissues showed an absence of spermatocytes while H&E staining showed that seminiferous tubules predominantly have only Sertoli cells. This meiotic failure is likely due to the effect of the AR mutation which ultimately leads to Sertoli cell only syndrome. Tubules were stained with SOX9 and AMH which revealed Sertoli cells maturation arrest. Western blot and realtime PCR data showed that patient had higher levels of AMH, SOX9 and inhibin-B in the testis. Therefore, we suggest that the dysfunctioning of AR by mutation enhances AMH expression which ultimately leads to the failure in maturation of Sertoli cells.

The intraleukocytic parasite, Hepatozoon canis, causes the sometimes fatal tick borne disease canine hepatozoonosis. In this study, dogs from Islamabad, Lahore, and Multan Districts of the Punjab region of Pakistan were surveyed to investigate the presence and prevalence of H. canis infection and to determine the effects of the parasite on hematological parameters. Blood samples were collected from 151 domestic dogs (149 pet, 2 stray) of both sexes and varying ages. Data on sex, age, tick infestation, and clinical factors (body temperature, mucous membrane status, and presence of hematuria and vomiting) were collected. Using PCR, 18 dogs (11.9%) were found positive for the presence of H. canis DNA. Partial sequences of the 18S rRNA gene shared 99-100% similarity with the corresponding H. canis isolates. This epidemiological survey revealed higher prevalence of H. canis in Islamabad (11/49, 22.4%) compared to Lahore (3/52, 5.8%) and Multan (4/50, 8%) in Pakistan. No investigated epidemiological or clinical factors were found to be associated with the presence of H. canis (p 0.05) in dogs. H. canis positive dogs exhibited higher minimum inhibitory dilution (p = 0.04), mixed inclusion (p = 0.008) and relative distribution width of red blood cells (p = 0.02), and lower hematocrit (p = 0.03) and mean hemoglobin content (p = 0.03) than did dogs in which H. canis was not detected. We are recommending this PCR-based protocol to the veterinary practitioners for the detection and/or confirmation of H. canis in dogs suspected for hepatozoonosis to improve their health status.

Gastric cancer is the fourth most commonly diagnosed cancer with the second highest mortality rate worldwide. Surgery, chemotherapy and radiation therapy are generally used for the treatment of stomach cancer but only limited clinical response is shown by these therapies and still no effectual therapy for advanced gastric adenocarcinoma patients is available. Therefore, there is a need to identify other therapeutic agents against this life-threatening disease. Plants are considered as one of the most important sources for the development of anticancer drugs. Magnolol, a natural compound possesses anticancer properties. However, effects of Magnolol on human gastric cancer remain unexplored. The effects of Magnolol on the viability of SGC-7901 cells were determined by the MTT assay. Apoptosis, mitochondrial membrane potential and cell cycle were evaluated by flow cytometry. Protein expression of Bcl-2, Bax, caspase-3 and PI3K/Akt was analysed by Western blotting. Magnolol induced morphological changes in SGC-7901 cells and its cytotoxic effects were linked with DNA damage, apoptosis and S-phase arrest in a dose-dependent manner. Magnolol triggered the mitochondrial-mediated apoptosis pathway as shown by an increased ratio of Bax/Bcl-2, dissipation of mitochondrial membrane potential (ΔΨm), and sequential activation of caspase-3 and inhibition of PI3K/Akt. Additionally, Magnolol induced autophagy in SGC-7901 cells at high concentration but was not involved in cell death. Magnolol-induced apoptosis of SGC-7901 cells involves mitochondria and PI3K/Akt-dependent pathways. These findings provide evidence that Magnolol is a promising natural compound for the treatment of gastric cancer and may represent a candidate for in vivo studies of monotherapies or combination antitumor therapies.

OBJECTIVE To find out a correlation between the single nucleotide polymorphisms in cluster of differentiation 28 and cluster of differentiation 40 genes with Graves' disease, if any. METHODS This case-control study was conducted at the Multan Institute of Nuclear Medicine and Radiotherapy, Multan, Pakistan, and comprised blood samples of Graves' disease patients and controls. Various risk factors were also correlated either with the genotype at each single-nucleotide polymorphism or with various combinations of genotypes studied during present investigation. RESULTS Of the 160 samples, there were 80(50%) each from patients and controls. Risk factor analysis revealed that gender (p=0.008), marital status (p

SummaryIn ‘Wake-up call for British psychiatry’ Craddock et al explained how recent attempts to improve psychosocial care for people with mental illness focus on non-specific psychosocial support. This has been at the expense of proper diagnostic assessment and prescription of treatment by psychiatrists aimed at treatment of specific disorders and recovery. They describe a creeping devaluation of psychiatry which is caricatured as narrow, biological, reductionist, oppressive, discriminatory and stigmatising. Some trusts have implemented ‘New Ways of Working for Psychiatrists’ in a way that undermines the central importance of psychiatrists in mental healthcare. Consequently, patients may be treated in secondary care without ever being seen by a psychiatrist. We consider a number of different changes that have interacted in unforeseen ways, with unintended adverse consequences for psychiatric services in England. We aim to continue the debate here.

Endometriosis, a pathological condition in which the endometrium grows outside the uterus, is one of the most common causes of female infertility; it is diagnosed in 25–40% of infertile women. The mechanism by which endometriosis affects the fertility of females remains largely unknown. We examined the ultrastructure of oocytes from patients with minimal or mild endometriosis and control females undergoing in vitro fertilization (IVF) treatment by transmission electron microscopy (TEM) to investigate the physiological significance of oocyte quality for patients with minimal or mild endometriosis. The TEM results revealed that the oocytes from women with minimal or mild endometriosis exhibited abnormal mitochondrial structure and decreased mitochondria mass. Quantitative real time PCR analysis revealed that the mitochondrial DNA copy number was significantly reduced in the oocytes from women with minimal or mild endometriosis compared with those of the control subjects. Our results suggest that decreased oocyte quality because of impaired mitochondrial structure and functions probably an important factor affecting the fertility of endometriosis patients.

To study the effect of CGP 35348 on learning and memory in albino mice following hypoxia ischemia insult, 10 days old albino mice were subjected to right common carotid artery ligation followed by 8% hypoxia for 25 minutes. Following brain damage, mice were fed on normal rodent diet till they were 13 week old. At this time point, mice were divided into two groups. Group 1 received saline and group 2 intrapertoneally CGP 35348 (1 mg/mL solvent/Kg body weight) for 12 days. A battery of tests used to assess long term neurofunction (Morris water maze, Rota rod and open field) along with brain infarct measurement. Overall CGP 35348 has improved the motor function in male and female albino mice but effects were more pronounced in female albino mice. In open field, CGP 35348 treated female albino mice had demonstrated poor exploratory behavior. During Morris water maze test, gender specific effects were observed as CGP 35348 had improved spatial learning and memory and swimming speed in male albino mice but had no effect in female albino mice following hypoxia ischemia encephalopathy (HIE). We concluded that GABAB receptor antagonists CGP 35348 can be used to improve gender based spatial memory.

Background. Present study was designed to report the effect of 2% creatine monohydrate supplementation for 8, 12 and 15 weeks on hematology and serum biochemical profile of male albino mouse following hypoxic ischemic insult on postnatal day 10.Methods. 66 Blood samples (2% creatine monohydrate supplemented (N=34) and unsupplemented (N=32)) were analyzed for various hematological (blood glucose, packed cell volume, total WBC count, total RBC count) and serum biochemical parameters (cholesterol, AST, ALT, HDL, LDL, total protein, triglycerides).Results. ALT had higher concentrations in mice feeding on normal diet for 8 (P>0.01) and 12 weeks (P>0.01) following asphyxia and in 12 weeks treatment without asphyxia (P=0.006) when compared with the creatine supplemented mice. LDL (P=0.011) and cholesterol (P>0.01) had higher concentrations in mice on normal diet for 12 weeks following hypoxia ischemia. Cholesterol (P>0.01) in 12 and glucose (P=0.006) in 15 week treatment group had significantly lower concentrations in creatine supplemented male albino mice when compared with untreated group following hxpoic-ischemic insult.Conclusion. We concluded that creatine supplementation following hypoxic ischemic insult helps in maintain the normal blood chemistry.

Background Oculocutaneous albinism (OCA) is caused by a group of genetically heterogeneous inherited defects that result in the loss of pigmentation in the eyes, skin and hair. Mutations in the TYR, OCA2, TYRP1 and SLC45A2 genes have been shown to cause isolated OCA. No comprehensive analysis has been conducted to study the spectrum of OCA alleles prevailing in Pakistani albino populations. Methods We enrolled 40 large Pakistani families and screened them for OCA genes and a candidate gene, SLC24A5. Protein function effects were evaluated using in silico prediction algorithms and ex vivo studies in human melanocytes. The effects of splice-site mutations were determined using an exon-trapping assay. Results Screening of the TYR gene revealed four known (p.Arg299His, p.Pro406Leu, p.Gly419Arg, p.Arg278*) and three novel mutations (p.Pro21Leu, p.Cys35Arg, p.Tyr411His) in ten families. Ex vivo studies revealed the retention of an EGFP-tagged mutant (p.Pro21Leu, p.Cys35Arg or p.Tyr411His) tyrosinase in the endoplasmic reticulum (ER) at 37°C, but a significant fraction of p.Cys35Arg and p.Tyr411His left the ER in cells grown at a permissive temperature (31°C). Three novel (p.Asp486Tyr, p.Leu527Arg, c.1045-15 T > G) and two known mutations (p.Pro743Leu, p.Ala787Thr) of OCA2 were found in fourteen families. Exon-trapping assays with a construct containing a novel c.1045-15 T > G mutation revealed an error in splicing. No mutation in TYRP1, SLC45A2, and SLC24A5 was found in the remaining 16 families. Clinical evaluation of the families segregating either TYR or OCA2 mutations showed nystagmus, photophobia, and loss of pigmentation in the skin or hair follicles. Most of the affected individuals had grayish-blue colored eyes. Conclusions Our results show that ten and fourteen families harbored mutations in the TYR and OCA2 genes, respectively. Our findings, along with the results of previous studies, indicate that the p.Cys35Arg, p.Arg278* and p.Gly419Arg alleles of TYR and the p.Asp486Tyr and c.1045-15 T > G alleles of OCA2 are the most common causes of OCA in Pakistani families. To the best of our knowledge, this study represents the first documentation of OCA2 alleles in the Pakistani population. A significant proportion of our cohort did not have mutations in known OCA genes. Overall, our study contributes to the development of genetic testing protocols and genetic counseling for OCA in Pakistani families.

Fredrik Svenaeus has applied Heidegger's concept of 'being-in-the-world' to health and illness. Health, Svenaeus contends, is a state of 'homelike being-in-the-world' characterised by being 'balanced' and 'in-tune' with the world. Illness, on the other hand, is a state of 'unhomelike being-in-the-world' characterised by being 'off-balance' and alienated from our own bodies. This paper applies the phenomenological concepts presented by Svenaeus to cases from a study of depression. In doing so, we show that while they can certainly enrich our understanding of depression, they can also reveal a clash between some societal definitions of illness and the individual's definition. Phenomenological analysis may thus cause us to question what we mean, or think should be meant, by the terms 'health' and 'illness'.

To determine the presence of Babesia bovis (B. bovis) in large ruminants in southern Punjab and its effect on hematological and serum biochemical profile of host animals.Blood samples were collected from 144 large ruminants, including 105 cattle and 39 buffaloes, from six districts in southern Punjab including Multan, Layyah, Muzaffar Garh, Bhakar, Bahawalnagar and Vehari. Data on the characteristics of animals and herds were collected through questionnaires. Different blood (hemoglobin, glucose) and serum (ALT, AST, LDH, cholesterol) parameters of calves and cattle were measured and compared between parasite positive and negative samples to demonstrate the effect of B. bovis on the blood and serological profile of infected animals.27 out of 144 animals, from 5 out of 6 sampling districts, produced the 541-bp fragment specific for B. bovis. Age of animals (P=0.02), presence of ticks on animals (P=0.04) and presence of ticks on dogs associated with herds (P=0.5) were among the major risk factors involved in the spread of bovine babesiosis in the study area. ALT concentrations were the only serum biochemical values that significantly varied between parasite positive and negative cattle.: This study has reported for the first time the presence of B. bovis in large ruminant and the results can lead to the prevention of babesiosis in the region to increase the livestock output.

The present study was carried out to determine the prevalence of Theileria annulata in large ruminants in Southern Punjab (Pakistan). Blood samples were collected from 144 large ruminants, consisting of 105 cattle and 39 buffaloes, from six districts of Southern Punjab including Multan, Layyah, Muzaffar Garh, Bhakar, Bahawalnagar, and Vehari. Data on the characteristics of the animals and herds were collected through questionnaires. The age of animals (P = 0.02), presence of ticks on animals (P = 0.02), and presence of ticks on dogs associated with herds (P = 0.05) were among the major risk factors involved in the spread of tropical theileriosis in the study area. Two different parasite detection techniques, PCR amplification and screening of Giemsa-stained slides, were compared, and it was found that PCR amplification is a more sensitive tool (19% parasite detection) as compared to smear scanning (3% parasite detection) for the detection of T. annulata. Twenty eight out of 144 animals produced the 721-bp fragment specific for T. annulata from five out of six sampling districts. Different blood (hemoglobin, glucose) and serum (ALT, AST, LDH, cholesterol) parameters of calves and cattle were measured and compared between parasite-positive and parasite-negative samples to assess the effect of T. annulata on the blood and serological profile of infected animals.

Biotinidase deficiency (BD) is an autosomal recessive disorder of biotin metabolism that causes incomplete recycling of free biotin. The resulting depletion of intracellular biotin leads to impaired activities of biotin-dependent carboxylases. The ensuing clinical phenotype includes progressive neurologic deterioration with epileptic seizures, muscular hypotonia as well as skin eczema. BD may be readily diagnosed by analysing enzyme activity in dried blood spots during newborn screening but typically requires molecular confirmation. More than 100 different mutations in the biotinidase gene have been reported to date. To simplify molecular testing we have developed a rapid and accurate denaturing high pressure liquid chromatography (dHPLC) method of the promoter, 3'UTR, all exons including exon/intron boundaries as a first line screen followed by direct sequencing of the respective PCR products. To validate this method we used DNA from 23 different, newly diagnosed patients with biochemically proven BD from Austria, India, Morocco and Spain. A total of 11 mutations, missense 7, frameshift 3 and 1 nonsense, were screened. Six mutations were novel to this study. All mutations revealed distinct dHPLC pattern thus enabling their accurate detection. This study revealed that dHPLC method is robust, automated, economical and above all highly sensitive for the molecular analysis of biotinidase gene and should be used as a pre-analytical tool followed by sequencing of aberrant heteroduplex forming amplicons.

Folliculogenesis is an important part of ovarian function as it provides the oocytes for female reproductive life. Characterizing genes/proteins involved in folliculogenesis is fundamental for understanding the mechanisms associated with this biological function and to cure the diseases associated with folliculogenesis. A large number of genes/proteins associated with folliculogenesis have been identified from different species. However, no dedicated public resource is currently available for folliculogenesis-related genes/proteins that are validated by experiments. Here, we are reporting a database ‘Follicle Online’ that provides the experimentally validated gene/protein map of the folliculogenesis in a number of species. Follicle Online is a web-based database system for storing and retrieving folliculogenesis-related experimental data. It provides detailed information for 580 genes/proteins (from 23 model organisms, including Homo sapiens, Mus musculus, Rattus norvegicus, Mesocricetus auratus, Bos Taurus, Drosophila and Xenopus laevis) that have been reported to be involved in folliculogenesis, POF (premature ovarian failure) and PCOS (polycystic ovary syndrome). The literature was manually curated from more than 43 000 published articles (till 1 March 2014). The Follicle Online database is implemented in PHP + MySQL + JavaScript and this user-friendly web application provides access to the stored data. In summary, we have developed a centralized database that provides users with comprehensive information about genes/proteins involved in folliculogenesis. This database can be accessed freely and all the stored data can be viewed without any registration. Database URL: http://mcg.ustc.edu.cn/sdap1/follicle/index.php

A 22-month-old boy was referred for further evaluation of muscular hypotonia and mild psychomotor developmental delay. He was born as the second child to healthy, distantly related Caucasian parents following an uneventful pregnancy. The family history was not contributory. Routine neonatal screening for guanidinoacetate methyltransferase (GAMT) deficiency was done in this child, who was found to have a mildly elevated guanidinoacetate (GAA) level in the dry blood filter card on the fifth day of life. When recalled, his urinary GAA levels in a first sample were marginally elevated but were within normal limits in a second specimen. The likelihood of GAMT deficiency was considered to be low, although it is well known that false negative results may occur in neonatal screening programs. His neonatal period and early infancy were uneventful until a mild delay in his motor and speech development was noted at 6 months of age. Extensive workup including metabolic testing and MRI in a peripheral hospital revealed no abnormalities, although his serum …