Jehangir Khan

A Comprehensive Entomological, Serological and Molecular Study of 2013 Dengue Outbreak of Swat, Khyber Pakhtunkhwa, Pakistan

Background Aedes aegypti and Aedes albopictus play a fundamental role in transmission of dengue virus to humans. A single infected Aedes mosquito is capable to act as a reservoir/amplifier host for dengue virus and may cause epidemics via horizontal and vertical modes of dengue virus (DENV) transmission. The present and future dengue development can be clarified by understanding the elements which help the dissemination of dengue transmission. The current study deals with molecular surveillance of dengue in addition to ecological and social context of 2013 dengue epidemics in Swat, Pakistan. Methods Herein, we reported dengue vectors surveillance in domestic and peridomistic containers in public and private places in 7 dengue epidemic-prone sites in District Swat, Pakistan from July to November 2013. Using the Flaviviruses genus-specific reverse transcriptase (RT) semi nested-PCR assay, we screened blood samples (N = 500) of dengue positive patients, 150 adult mosquito pools and 25 larval pools. Results The 34 adult and 7 larval mosquito pools were found positive. The adult positive pools comprised 30 pools of Ae. aegypti and 4 pools of Ae. albopictus, while among the 7 larval pools, 5 pools of Ae. aegypti and 2 pools of Ae. albopictus were positive. The detected putative genomes of dengue virus were of DENV-2 (35% in 14 mosquito pools & 39% in serum) and DENV-3 (65% in 27 mosquito pools & 61% in serum). The higher vector density and dengue transmission rate was recorded in July and August (due to favorable conditions for vector growth). About 37% of Ae. aegpti and 34% Ae. albopictus mosquitoes were collected from stagnant water in drums, followed by drinking water tanks (23% & 26%), tires (20% & 18%) and discarded containers (10% & 6%). Among the surveyed areas, Saidu was heavily affected (26%) by dengue followed by Kanju (20% and Landikas (12%). The maximum infection was observed in the age group of <15 (40%) followed by 15–45 (35%) and >45 (25%) years and was more in males (55.3%) as compare to females (44.7%). The increase in vector mosquito density and the subsequent viral transmission was determined by a complex interplay of ecological, biological and social factors. Conclusion The suitable environmental conditions and discriminable role of Aedes through trans-ovarial transmission of DENV is indispensable in the recent geographic increase of dengue in Pakistan. Climate change affects the survival and dispersion of vectors as well as the transmission rates of dengue. Control of Aedes mosquitoes (vectors) and elimination of breeding sources must be emphasized and prioritized. Such actions may not only reduce the risk of dengue transmission during epidemics, but also minimize the chances of dengue viruses establishment in new (non endemic) areas of the region.

Impact of Consanguinity on Health in a Highly Endogamous Population in District Buner,Khyber Pakhtunkhwa, Pakistan

Impact of Consanguinity on Health in a Highly Endogamous Population in District Buner, Khyber Pakhtunkhwa, Pakistan Objective: Consanguineous unions are a deeply rooted social practice among traditional societies in Pakistan. Despite their presumed social advantages, they can result in several genetic disorders. The present study was designed to study the epidemiological trends of consanguineous marriages in district Buner, KPK, Pakistan. Method: A survey from March to September, 2014, was conducted at twelve sampling areas in Tehsil Gaggra of district Buner, Khyber Pakhtunkhwa (KPK), Pakistan. Results: The couples (n=70) participated were 1st cousin (84.3%) and 2nd cousin (15.7%). The minimum age of the couples at the time of marriage was 16 and maximum 30 years, however, maximum marriages were observed at the age of 18year. The offspring were thoroughly observed for the health complications like anemia (28.6%,), obesity (17.1 %), blood pressure (18.6%), diabetes (18.6%), asthma (10%) and respiratory disease (7.1%) respectively. The percentage of malformation found in the live children of consanguineous married couples were mental retardation (28.7%), deafness and speech disorder (23.3%), anemia (14.7%), syndrome (15.5%), dwarfism (3.9%), blindness (8.5%) and skin diseases (5.4%). Among the surveyed areas, Chagharzi has the highest (41.3%) trend of cousin marriages followed by Shalbandi (17.1%) and Bajkata (12.9%). Additionally, the health conditions of the parents (couples) were also observed for the possible health concerns, which were obese (21.4%) and weak (42.8%) while 35.7% were normal. Conclusions: Cousin-marriages are a norm in the population of district Buner. Its effects on inherited disorders deserve comprehensive genetic, education, and premarital genetic counseling programs for awareness and education

Burden of Marble Factories andHealth Risk Assessment of Kidney(renal) Stones Development inDistrict Buner, KhyberPakhtunkhwa, Pakistan

Burden of Marble Factories and Health Risk Assessment of Kidney (renal) Stones Development in District Buner, Khyber Pakhtunkhwa, Pakistan Objective: The marble industry�s disposal, consisting of very fine powder, is becoming one of the environmental problems around the world. The current study was proposed to evaluate health (incidences of kidney stones) risk assessment that might result from marble industries in district Buner. Method: A three months survey was done from August- October 2014 in district Buner, Khyber Pakhtunkhwa (KPK). Different health care centers were visited for sampling purpose. Results: A total of 1000 individuals, exposed to marble effluents (contaminated water), suffering from kidney stones including males and females in a ratio (M: F is 2.33:1) were observed. The individuals having age of 26-40 years suffered 74% infection of urolithiasis followed by 41-60 years (20%), and lowest rate of kidney infections was observed in 15-25 years individuals. The Tehsil Daggar has highest number (390: 65%) of marble industries as compared to Tehsil Gagra (210: 35%). Similarly, the kidney infection rate was high in Daggar (560: 56%) as compared to Gagra (440: 44%). The most common symptom of kidney infection observed was pain (94%) followed by urinary bladder infection (80%) and Anemia (78%).

Correction: The changing epidemiological pattern of Dengue in Swat, Khyber Pakhtunkhwa

[This corrects the article DOI: 10.1371/journal.pone.0195706.].

Assessing Autophagy in Sertoli Cells

Autophagy is an important cellular homeostatic process, it degrades most long-lived proteins and some organelles by lysosome to provide raw materials for the survival of the cells during nutrient or energy deprivation condition. Autophagy is active in Sertoli cells and involved in many cellular processes. However, the precise role of autophagy in Sertoli cells is still largely unknown. Thus, the assessment of autophagy in Sertoli cells should be helpful for investigating the functional roles of autophagy in Sertoli cells. This chapter describes some methods for assessing autophagy in Sertoli cells, including detection of LC3 maturation/aggregation, transmission electron microscopy, half-life assessments of long-lived proteins, immunofluorescence microscopy, and co-localization of autophagy-targeted proteins with autophagy components or lysosomal proteins.

Dengue fever in District Swat: A paradigm shift; changing epidemiology and clinical patterns, Khyber Pakhtunkhwa, Pakistan

Newcastle disease virus (NDV) belongs to the family Paramyxoviridae. It causes Newcastle disease, mainly by the virulent strains of avian paramyxovirus serotype-1 (AMPV-1). This virus represents a huge problem on the world’s economy than any other animal virus, especially in developed countries. ND infection is not obstructed and many studies have attributed the spread of the disease to changes in the genome of the virus and emersion to new strains. We aimed to design a peptide vaccine for NDV particularly for the haemagglutininneuraminidase protein (HN) using computational methods to predict epitopes inducing immune system and can be used later to create a new peptide vaccine could replace conventional vaccines. A total of available 60 virulent strains of HNNDV were retrieved from NCBI for bioinformatics analysis using Immune Epitope Data Base (IEDB) to predict B and T cells Epitopes. We used human MHC class I and II alleles in this study due to the difficulty to determine MHC B complex alleles in Poultry. Then we docked the best predicted CTL epitopes with B-F alleles (BF2*2101 and BF2*0401). Four CTL cell epitopes namely ( 548 ISNTLFGEF 556 , 546 AEISNTLFG 554 , 88 VALESPLAL 96 and 526 YTTSTCFKV 534 ) interacted with MHC class (B-F) I alleles and we suggested them to become universal peptides based vaccine against NDV. We found these CTL epitopes to be T helper epitopes also. The overlapping between MHC class I (B-F) and (B-L) II T cell epitopes suggesting the possibility of antigen presentation to immune cells via both MHC class I and II pathways especially the overlapping between 548 ISNTLFGEF 556 and 546 AEISNTLFG 554 . We considered this study distinctive because no research ever dealt with peptide based vaccine on virulent strains of NDV using in silico approach. Keyword: Newcastle disease virus (NDV), poultry, MHC B complex and vaccine.O third of the human population is infected with parasitic worms. To avoid their elimination, these parasites actively dampen the immune response of their hosts. This immune suppression will also affect immune responses to third party antigens such as vaccines. Accordingly, several studies report a negative correlation between pre-existing helminth infections and response to vaccination in the human population. We use Litomosoides sigmodontis infected mice to further analyze this parasite induced interference with vaccination efficacy. We show that experimental vaccination against the liver stage of Plasmodium berghei elicited reduced numbers of Plasmodium specific CD8+ cytotoxic T lymphocytes in mice with concurrent L. sigmodontis infection. Chronic nematode infection also led to complete suppression of IgG responses to thymus dependent (TD) model antigen vaccinations (DNP-KLH, Ovalbumin). Thereby, the parasite suppressed B-cell function indirectly, via accessory CD4+ T helper cells, as thymus independent vaccination (NIP-Ficoll) was functional and already numbers and frequency of vaccine induced follicular T-helper cells were reduced. Strikingly, a reduced humoral response to TD vaccination was still observed if vaccination was performed more than 16 weeks after clearance of infection. Thus, vaccination may not only fail in helminth infected individuals but also in individuals with a history of previous helminth infections. Helminth induced interference with bystander CD4+T cell activation resulted in a suppressed antigen specific proliferation of Ovalbumin specific TCR transgenic OT-II T cells in vivo, after adoptive transfer into L. sigmodontis infected mice. Using this simplified system, we are currently unraveling the chain of events leading from the presence of L. sigmodontis in the thoracic cavity to the final suppression of T cells specific for a different antigen at a different site.P reproductive and respiratory syndrome virus (PRRSV) is the causative agent of reproductive failure and respiratory disorders in porcine industry. The viral genome consists of eight overlapping open reading frames (ORFs). ORF5 encodes one of the major glycoproteins and is known as an immunologically important structural protein associated with virus neutralization. The ORF5 gene of the Korean PRRSV isolate, CNV-1, was amplified by reverse transcription-polymerase chain reaction (RT-PCR), cloned and sequenced. The nucleotide and amino acid sequences of CNV-1 ORF5 shared 91% and 83% identity, respectively with the American isolate (VR2332 strain) and 57% and 49% identity with the European isolate. For the expression and easy purification of ORF5, the cDNA containing the complete ORF5 sequence selected and fused in frame with sequence encoding glutathione S-transferase (GST) was cloned into a baculovirus transfer vector and transfected into Sf9 cells. The GST-ORF5 fusion protein produced in Sf9 cells was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Sequencing results confirmed that the recombinant baculovirus from Sf9 cells contains the complete ORF5 gene. Further studies in this direction will address whether ORF5 can be a good candidate for a subunit vaccine against PRRSV in Korea and it should be validated safety before using in the field.V preparations based on the extracellular domain of Her1 (Her1-ECD) have demonstrated, both in vitro and in vivo, a potent anti-metastatic effect on EGFR+ Lewis lung carcinoma model, while associated side effects were absent. The Her1-ECD is a glycoprotein with a molecular weight of 105 kDa and has 11 potential sites for N-glycosylation. Currently, in Cuba this vaccine has being evaluated in patients with refractory prostate cancer. Her1-ECD molecule (used for the Cuban clinical trials are obtained from culture supernatant of HEK 293 transfectomes used the culture medium PFHM/PFCHO and is purified using immunoaffinity chromatography. Several studies have demonstrated that glycosylation can affect the protein folding, stability, regulates protein half-life, immunogenecity, biological activity and other functions. In this work, the N-glycosylation of Her1-ECD was preliminarily characterized by SDS-PAGE, glycan differentiation by lectin and normal phase chromatography. Afterwards, the biological activity of the glycosylated and fully deglycosylated Her1-ECD protein was compared. In addition, a comparability study was performed, in order to evaluate the impact of process changes (different culture media) in the physicochemical and biological characteristics of Her1-ECD protein. Such study was carrying out using different techniques such as: SDS-PAGE, SEC-HPLC, isoelectric point, peptide mapping, mass spectrometry, SCX-HPLC, oligosaccharide map, ELISA and flow cytometric. As principal results were obtained that N-glycosylation profile of Her1-ECD is composed of high mannose, hybrid and complex N-glycans types and Her1-ECD glycosylation modifies the humoral immune response, measured as antibody titers, recognition of EGFR in A431 cell line and cell cycle arrest. On the other hand, comparability studies results indicated that media changes has an effect in the degree of sialylation of the protein but does not affect its biological activity (measured as titers of Abs and recognition for A431 cell line).