Characterization of alkaline metalloprotease isolated from halophilic bacterium Bacillus cereus and its applications in various industrial processes.

Microbial proteases are one of the most demanding enzymes for various industries with diverse applications in food, pharmaceutics, and textile industries to name the few. An extracellular alkaline metalloprotease was produced and purified from moderate halophilic bacterial strain, Bacillus cereus TS2, with some unique characteristics required for various industrial applications. The protease was produced in basal medium supplemented with casein and was partially purified by ion exchange chromatography followed by ammonium sulphate precipitation. The alkaline metalloprotease has molecular weight of 35 kDa with specific activity of 535.4 µM/min/mg. It can work at wide range of pH from 3 to 12, while showing optimum activity at pH 10. Similarly, the alkaline metalloprotease is stable till the temperature of 80 °C and works at wide range of temperature from 20 to 90 °C with optimum activity at 60 °C. The turnover rate increases in the presence of NaCl and Co+2 with k cat/KM of 1.42 × 103 and 1.27 × 103 s-1.M-1 respectively, while without NaCl and Co+2 it has a value of 7.58× 102. The alkaline metalloprotease was relatively resistant to thermal and solvent mediated denaturation. Applications revealed that the metalloprotease was efficient to remove hair from goat skin, remove blood stains and degrade milk, thus can be a potential candidate for leather, detergent, and food industry.

Knowledge, Attitudes and Practices Regarding Taeniasis in Pakistan.

Taeniasis is a neglected zoonotic disease responsible for serious health disorders, such as seizures, and may even cause death. Humans are the definitive host for the three species Taenia solium (pork tapeworm), T. saginata (beef tapeworm), and T. asiatica, harboring the adult tapeworm in the small intestine. In this study, a structured questionnaire was circulated to assess the knowledge, attitudes, and practices (KAPs) regarding taeniasis among the rural and urban communities of Rawalpindi and Islamabad, Pakistan. A total of 770 individuals participated in the study. Of the total respondents, 44.4% had little knowledge about the disease and its impact, while the majority (70%) of respondents showed a willingness to participate in elimination campaigns by providing fecal samples. Most respondents kept raw meat separated from clean utensils (81.6%) and checked the internal temperature of meat when cooking it (75.1%). Regression analysis showed a significant association between age and knowledge, especially in the 20-30 years (p < 0.05; OR 0.574) and 30 to 40 years (p < 0.05; OR 0.553) age groups, and being a resident in Rawalpindi (p < 0.05; OR 0.68) and other cities (p < 0.05; OR 2.43), except Islamabad. Income ranges of 31,000-50,000 PKR (p < 0.05; OR: 0.574), 51,000-70,000 PKR (p < 0.05; OR 0.531), and above 70,000 PKR (p < 0.05; OR 0.42) were significantly related to attitude, compared with individuals with incomes of 10,000-30,000 PKR. Income above 70,000 PKR (p < 0.05; OR 0.87) and living in an urban area (p < 0.05; OR 0.616) compared to a rural area were significant with practices. A positive attitude was observed regarding awareness and prevention of the disease. Awareness campaigns and providing health education could be key approaches to manage this disease in the general population of developing countries.

Knowledge, attitudes and practices related to neglected tropical diseases (schistosomiasis and fascioliasis) of public health importance: A cross-sectional study.

Background: Snails play an important role as an intermediate host in various parasitic diseases, which are referred to as snail-borne parasitic diseases (SBPDs). The prevalence of the SBPDs, schistosomiasis and fascioliasis is low in Pakistan compared to other countries. The present study investigated knowledge, attitudes, and practices related to these two SPBDs and risk factors associated with them among the humans living in close contact with livestock and pets from three regions of Pakistan: Punjab, Islamabad and Azad Jammu and Kashmir (AJK). Methods: A cross-sectional survey was conducted using a structured questionnaire to assess participants' knowledge, attitude and practices related to schistosomiasis and fascioliasis during 2021-2022. Results: The majority of the 507 participants who were interviewed had good knowledge of schistosomiasis and fascioliasis: 43% were aware of schistosomiasis and 57% were aware of fascioliasis, but only 25% knew about snails as an intermediate host. Most respondents had a positive attitude toward treatment, prevention and control of the diseases but they did not have any healthcare facilities. Conclusion: This study highlights the importance of the public's awareness for the need to control SBPDs. It also draws attention to the need for health education for risk reduction and the prevention of SBPDs in endemic areas.

Prevalence of different tick species on livestock and associated equines and canine from different agro-ecological zones of Pakistan.

Ticks are ectoparasites that act as vectors for transmission of various pathogens to wild and domesticated animals and pose a serious threat to human health. Because of the hot and humid conditions in different agro-ecological zones of Pakistan, ticks are abundant and parasitize a variety of animals. The aim of this study was to identify different tick species and distribution on different hosts especially livestock, such as sheep, goat, cattle, buffalo, and camel, and livestock associated canines and equines, such as horse, donkey, and dog, across different agro-ecological zones of Pakistan. The ticks samples were collected and morphologically identified at genus and species level using morphological keys under stereomicroscope. A total of 2,846 animals were examined for the tick infestation, and 408 animals were tick-infested. Eleven tick species belonging to 4 genera were identified: Hyalomma anatolicum, Hyalomma scupense, Hyalomma dromedarii, Hyalomma isaaci, Rhipicephalus microplus, Rhipicephalus haemaphysaloides, Rhipicephalus turanicus, Haemaphysalis cornupunctata, Haemaphysalis montgomeryi, Haemaphysalis bispinosa, and Ixodes kashmiricus. The overall tick prevalence was 14.3%; host-wise infestation rate was 12.2% in sheep; 12.6%, goat; 11.7%, buffalo; 11.7%, cattle; 19.6%, camel; 27.4%, donkey; 23.5%, horse; and 24.3%, dog. Tick infestation of different animals differed on the basis of the zones. Camels showed the highest tick infestation rate in zones 1 and 2 (21.4 and 26.7%, respectively), whereas donkeys showed the highest infestation rate in zones 3, 4, 6, and 7 (25, 39.3, 3.3, and 21.4%, respectively). The infestation rates of Hyalomma and Rhipicephalus were the highest in zone 2 (71.4 and 52.9%, respectively). The infestation rate of Hyalomma was the highest (47.4%) in sheep; Haemaphysalis (46.9%), goat; Rhipicephalus (69.7%), buffalo; Rhipicephalus (62.3%), cattle; Hyalomma (70%), camel; Ixodes (60.9%), donkey; Ixodes (75%), horse; and Rhipicephalus (61.1%), dog. This study showed the diversity and infestation rate of different ticks with respect to their hosts and agro-ecological zones of Pakistan. High tick burdens and infestation rates are responsible for the spread of different tick-borne infections, resulting in loss of animal productivity and posing a threat to animal and human health. Understanding different tick species and their distribution across different zones will be helpful for developing efficient control strategies against different tick born infections.

Epidemiological and pathological characteristics of Cutaneous Leishmaniasis from Baluchistan Province of Pakistan.

Cutaneous Leishmaniasis (CL) is considered a neglected tropical disease which in Pakistan can now be considered as a growing public health problem. The exact figures on the magnitude of the disease are lacking both at the national and regional level and only a few health centres are available for diagnosis of CL. The present study was designed to identify the epidemiology of CL infection from August 2018 to December 2019 and to assess clinical aspects of CL in Baluchistan Province of Pakistan. A total of 4072 clinically suspected CL cases were analysed statistically. The highest number of CL cases were reported in May, followed by April, January and then July, February and June and the lowest number of cases were observed in March and November. The highest prevalence rate was found in males where 38% of reported cases were aged 0-9 years. The majority (24.4%) of lesions were found on the hands followed by the face in which cheeks, ears and nose were the effected organs. About 50% of the participants have single lesion while 14% of the participants had two and nearly 3% of the participants have six lesions. The atypical clinical presentations were observed in Baluchistan and common unusual presentations were lupus erythematosus. The study findings suggest that more epidemiological studies and health education campaigns are needed for the population awareness regarding CL in Baluchistan. It is recommended that risk factors should be evaluated to establish control and management strategies to prevent disease at the individual and community level.

Natural Agents-Mediated Targeting of Histone Deacetylases.

In the past few years, basic and clinical scientists have witnessed landmark achievements in many research projects, such as those conducted by the US National Institutes of Health Roadmap Epigenomics Mapping Consortium, the International Human Epigenome Consortium, The Cancer Genome Atlas Network and the International Cancer Genome Consortium, which have provided near-complete resolution of epigenetic landscape in different diseases. Furthermore, genome sequencing of tumors has provided compelling evidence related to frequent existence of mutations in readers, erasers and writers of epigenome in different cancers. Histone acetylation is an intricate mechanism modulated by two opposing sets of enzymes and deeply studied as a key biological phenomenon in 1964 by Vincent Allfrey and colleagues. The research group suggested that this protein modification contributed substantially in transcriptional regulation. Subsequently, histone deacetylases (HDACs), histone acetyltransferases and acetyl-Lys-binding proteins were identified as transcriptional mediators, which further deepened our comprehension regarding biochemical modifications. Overwhelmingly increasing high-impact research is improving our understanding of this molecularly controlled mechanism; moreover, quantification and identification of lysine acetylation by mass spectrometry has added new layers of information. We partition this multi-component review into how both activity and expression of HDAC are targeted using natural agents. We also set spotlight on how oncogenic fusion proteins tactfully utilize HDAC-associated nano-machinery to modulate expression of different genes and how HDAC inhibitors regulate TRAIL-induced apoptosis in cancer cells. HDAC inhibitors have been reported to upregulate expression of TRAIL receptors and protect TRAIL from proteasomal degradation. Deeper understanding of HDAC biology will be useful for stratification and selection of patients who are responders, non-responders and poor-responders for HDACi therapy, and for the rational design of combination studies using HDACi.

Anticancer activity of essential oils: targeting of protein networks in cancer cells.

Cancer is a multifaceted and genomically complex disease and research over decades has gradually and sequentially shown that essential biological mechanisms including cell cycle arrest and apoptosis are deregulated. The benefits of essential oils from different plants have started to gain appreciation as evidenced by data obtained from cancer cell lines and xenografted mice. Encouraging results obtained from preclinical studies have attracted considerable attention and various phytochemicals have entered into clinical trials.

MiR-421, miR-155 and miR-650: emerging trends of regulation of cancer and apoptosis.

It is becoming progressively more understandable that between transcription and translation there lies another versatile regulator that quantitatively controls the expression of mRNAs. Identification of miRNAs as key regulators of wide ranging signaling cascades and modulators of different cell-type and context dependent activities attracted basic and clinical scientists to study modes and mechanisms in details. In line with this approach overwhelmingly increasing in vivo and in vitro studies are deepening our understanding regarding miR-421, mir-155 and miR-650 mediated regulation of cellular activities. We also attempt to provide an overview of long non coding RNAs.

A new autosomal recessive nonsyndromic hearing impairment locus DFNB96 on chromosome 1p36.31-p36.13.

A novel locus for autosomal recessive nonsyndromic hearing impairment (ARNSHI), DFNB96, was mapped to the 1p36.31-p36.13 region. A whole-genome linkage scan was performed using DNA samples from a consanguineous family from Pakistan with ARNSHI. A maximum two-point logarithm of odds (LOD) score of 3.2 was obtained at marker rs8627 (chromosome 1: 8.34 Mb) at θ=0 and a significant maximum multipoint LOD score of 3.8 was achieved at 15 contiguous markers from rs630075 (9.3 Mb) to rs10927583 (15.13 Mb). The 3-unit support interval and the region of homozygosity were both delimited by markers rs3817914 (6.42 Mb) and rs477558 (18.09 Mb) and contained 11.67 Mb. Of the 125 genes within the DFNB96 interval, the previously identified ARNSHI gene for DFNB36, ESPN, and two genes that cause Bartter syndrome, CLCNKA and CLCNKB, were sequenced, but no potentially causal variants were identified.

Novel mutations in the keratin-74 (KRT74) gene underlie autosomal dominant woolly hair/hypotrichosis in Pakistani families.

Autosomal dominant woolly hair (ADWH) is an inherited condition of tightly curled and twisted scalp hair. Recently, a mutation in human keratin-74 (KRT74) gene has been shown to cause this form of hereditary hair disorder. In the present study, we have described two families (A and B) having multiple individuals affected with autosomal dominant form of hair loss disorders. In family A, 10 individuals showed ADWH phenotype while in the family B, 14 individuals showed hypotrichosis of the scalp. Genotyping using polymorphic microsatellite markers showed linkage of both the families to type II keratin gene cluster on the chromosome 12q12-14.1. Mutation analysis of the KRT74 gene identified two novel mutations in the affected individuals of the families. The sequence analysis revealed a splice acceptor site mutation (c.IVS8-1G>A) in family A and a missense variant (c.1444G>A, p.Asp482Asn) in family B. Mutations identified in the present study extend the body of evidence implicating the KRT74 gene in the pathogenesis of autosomal dominant hair loss disorders.

Mapping of a novel autosomal recessive hypotrichosis locus on chromosome 10q11.23­22.3.

Autosomal recessive hypotrichosis is a rare form of human genetic disorder characterized by sparse to absent hair on scalp and rest of the body of affected individuals. Over the past few years at least five autosomal recessive forms of hypotrichosis loci have been mapped on different human chromosomes. In the present study, we report localization of another novel autosomal recessive hypotrichosis locus on human chromosome 10q11.23-22.3 in a four generation consanguineous Pakistani family. All the four patients in the family showed typical features of hereditary hypotrichosis including sparse hair on the scalp and rest of the body. Human genome scan using highly polymorphic microsatellite markers mapped the disease locus to a large region on chromosome 10. This novel locus maps to 29.81 cM (28.5 Mb) region, flanked by markers D10S538 and D10S2327 on chromosome 10q11.23-22.3. A maximum multipoint LOD score of 3.26 was obtained with several markers in this region. DNA sequence analysis of exons and splice-junction sites of four putative candidate genes (P4HA1, ZNF365, ZMYND17, MYST4), located in the linkage interval, were sequenced but were negative for functional sequence variants.

Recurrent mutations in functionally-related EDA and EDAR genes underlie X-linked isolated hypodontia and autosomal recessive hypohidrotic ectodermal dysplasia.

Mutations in three functionally related genes EDA, EDAR and EDARDD have been reported to cause hypohidrotic ectodermal dysplasia (HED), which is characterized by sparse hair, reduced ability to sweat, and hypodontia. In few cases mutations in the EDA gene have been found to result in X-linked recessive isolated hypodontia. In the study, presented here, we have ascertained two large Pakistani families (A and B) with autosomal recessive form of hypohidrotic ectodermal dysplasia and X-linked recessive isolated hypodontia. Genetic mapping showed linkage of family A to EDAR gene on chromosome 2q11-q13 and family B to EDA gene on chromosome Xq12-q13.1. Subsequently, DNA sequencing of the coding regions of EDAR and EDA genes revealed previously described mutations. Sequence analysis identified a four base-pair splice-junction deletion mutation (c.718_721delAAAG) in EDAR gene in family A and a missense mutation (c.T1091C; p.M364T) in EDA gene in family B. Recurrence of mutations in EDAR and EDA genes in unrelated families is evocative of the dispersion of ancestral chromosome in different locality groups through common ancestors.

A novel insertion mutation in the cartilage-derived morphogenetic protein-1 (CDMP1) gene underlies Grebe-type chondrodysplasia in a consanguineous Pakistani family.

BACKGROUND: Grebe-type chondrodysplasia (GCD) is a rare autosomal recessive syndrome characterized by severe acromesomelic limb shortness with non-functional knob like fingers resembling toes. Mutations in the cartilage-derived morphogenetic protein 1 (CDMP1) gene cause Grebe-type chondrodysplasia. METHODS: Genotyping of six members of a Pakistani family with Grebe-type chondrodysplasia, including two affected and four unaffected individuals, was carried out by using polymorphic microsatellite markers, which are closely linked to CDMP1 locus on chromosome 20q11.22. To screen for a mutation in CDMP1 gene, all of its coding exons and splice junction sites were PCR amplified from genomic DNA of affected and unaffected individuals of the family and sequenced directly in an ABI Prism 310 automated DNA sequencer. RESULTS: Genotyping results showed linkage of the family to CDMP1 locus. Sequence analysis of the CDMP1 gene identified a novel four bases insertion mutation (1114insGAGT) in exon 2 of the gene causing frameshift and premature termination of the polypeptide. CONCLUSION: We describe a 4 bp novel insertion mutation in CDMP1 gene in a Pakistani family with Grebe-type chondrodysplasia. Our findings extend the body of evidence that supports the importance of CDMP1 in the development of limbs.