Countrywide Survey for MERS-Coronavirus Antibodies in Dromedaries and Humans in Pakistan

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a zoonotic pathogen capable of causing severe respiratory disease in humans. Although dromedary camels are considered as a major reservoir host, the MERS-CoV infection dynamics in camels are not fully understood. Through surveillance in Pakistan, nasal (n = 776) and serum (n = 1050)samples were collected from camels between November 2015 and February 2018. Samples were collected from animal markets, free-roaming herds and abattoirs. An in-house ELISA was developed to detect IgG against MERS-CoV. A total of 794 camels were found seropositive for MERS-CoV. Prevalence increased with the age and the highest seroprevalence was recorded in camels aged [ 10 years (81.37%) followed by those aged 3.1–10 years (78.65%) and B 3 years (58.19%).Higher prevalence was observed in female (78.13%) as compared to male (70.70%). Of the camel nasal swabs, 22 were found to be positive by RT-qPCR though with high Ct values. Moreover, 2,409 human serum samples were also collected from four provinces of Pakistan during 2016–2017. Among the sampled population, 840 humans were camel herders.Although we found a high rate of MERS-CoV antibody positive dromedaries (75.62%) in Pakistan, no neutralizing antibodies were detected in humans with and without contact to camels.     

RNAi-mediated silencing of endogenous <Emphasis Type="Italic">Vlnv</Emphasis> gene confers stable reduction of cold-induced sweetening in potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L. cv. Désirée)

Potato tubers must be cold-stored to extend their shelf life and maintain an uninterrupted supply chain for food processors. However, a side-effect of low-temperature storage is manifested in terms of cold-induced sweetening (CIS) of potato tubers, which reduces the processing quality and the commercial value of the end-products. RNA interference (RNAi) technology, whereby transgene-derived small interfering RNAs can trigger the homology-based knockdown of cognate host genes and can initiate gene silencing, has been successfully applied in crop improvement through targeted gene knockout in host plants. In the current study, transgenic potato plants (Solanum tuberosum cv. Désirée) were generated, expressing a 300 bp hairpin loop nucleotide sequence targeting the potato vacuolar invertase gene (VInv), under the constitutive Cauliflower mosaic virus 35S promoter. Tubers collected from transgenic lines showed a significant reduction in reducing sugar content after 180 days of cold storage, without showing any measurable off-target effects on plant morphology and tuberization compared to non-transformed control plants. The cold-stored tubers were further assayed for chip color, which showed a fairly light colored quality in the samples originating from RNAi lines. Together with similar effects seen in previously published experiments involving other potato varieties, the Désirée results described here establish the efficacy of using RNAi for the successful reduction of CIS in potato tubers.     

Time‐lapse microscopy of oxidative stress demonstrates metabolic sensitivity of retinal pericytes under high glucose condition

Hyperglycemia affects retinal vascular cell function, promotes the development and progression of diabetic retinopathy and ultimately causes vision loss. Oxidative stress, reactive oxygen species (ROS) in excess, is a key biomarker for diabetic retinopathy. Using time‐lapse fluorescence microscopy, ROS dynamics was monitored and the metabolic resistivity of retinal endothelial cells (REC) and pericytes (RPC) was compared under metabolic stress conditions including high glucose (HG). In the presence of a mitochondrial stressor, REC exhibited a significant increase in the rate of ROS production compared with RPC. Thus, under normal glucose (NG), REC may utilize oxidative metabolism as the bioenergetic source, while RPC metabolic activity is independent of mitochondrial respiration. In HG condition, the rate of ROS production in RPC was significantly higher, whereas this rate remained unchanged in REC. Thus, under HG condition RPC may preferentially utilize oxidative metabolism, which results in increased rate of ROS production. In contrast, REC use glycolysis as their major bioenergetic source for ATP production, and consequently HG minimally affects their ROS levels. These observations are consistent with our previous studies where we showed HG condition has minimal effect on apoptosis of REC, but results in increased rate of apoptosis in RPC. Collectively, our results suggest that REC and RPC exhibit different metabolic activity preferences under different glucose conditions. Thus, protection of RPC from oxidative stress may provide an early point of intervention in development and progression of diabetic retinopathy.      

Adrenalectomy does not affect the nocturnal peak of fos expression within hypothalamic pro-opiomelanocortin neurons

Circulating glucocorticoid (GC) levels are thought to modulate the basal activity of pro-opiomelanocortin (POMC) neurons within the mediobasal hypothalamus (MBH) of the male rat. In a recent study we demonstrated that Fos-immunoreactivity (Fos-IR) was spontaneously induced throughout the dark phase of the light/dark cycle within a large population of these MBH neurons. Here, we have investigated the effect of adrenalectomy on the nocturnal expression of Fos protein within POMC neurons. To this aim, groups of intact (IN), adrenalectomized (ADX) and sham-operated (sham) rats were killed 7 days after surgery (or no surgery) at times when Fos-IR is known to show either nadir (at light offset) or peak (6 h after light offset) values within MBH POMC neurons. Brains were processed for Fos- and/or POMC-immunohistochemistry. The results showed that, at both times studied, 7-day adrenalectomy did not affect the number of POMC/Fos double-stained neurons within the MBH. The rostro-caudal pattern of distribution of such labeled neurons throughout the MBH of ADX rats was also similar to that of IN or sham rats. The present data demonstrate that the nocturnal induction of Fos within MBH POMC neurons is not controlled via the nychtemeral rhythm of secretion of the adrenal gland. Furthermore, this study shows that basal levels of circulating GC do not alter the nocturnal peak of Fos synthesis within POMC neurons.     

o-Phthalaldehyde activates the Ca(2+) release mechanism from skeletal muscle sarcoplasmic reticulum.

o-Phthalaldehyde (OPA) is a bifunctional reagent that forms an isoindole derivative by reacting with cysteine and lysine residues separated by approximately 0.3 nm. OPA inhibits sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity at low micromolar concentrations and induces Ca(2+) release from actively loaded SR vesicles by activating the ryanodine receptor from fast twitch skeletal muscle. Both ryanodine binding and single-channel activity show a biphasic concentration dependence. At low OPA concentrations (<100 microM), ryanodine binding and single channel activity are stimulated, while at higher concentrations, a time-dependent sequential activation and inhibition of receptor binding is observed. Activation is characterized by a Ca(2+)-independent increase in maximal receptor occupancy. Data are presented to support a model in which Ca(2+) channel and ryanodine binding activity are enhanced due to an intramolecular cross-linking of nearby lysine and nonhyperreactive cysteine residues. OPA complexation with endogenous lysine residue(s) is critical for receptor activation.     

Dream content: Reflections of the emotional and psychological states of earthquake survivors.

This study investigated dream narratives as reflections of the emotional and psychological states of earthquake survivors. Dreams and dreams imagery have reportedly been affected by the emotional and psychological trauma that the dreamer goes through. Dream narratives and dream content ratings used in earlier studies were utilized in this study. Narratives and content ratings were obtained from a sample of 27 volunteer survivors of the October 8, 2005 earthquake in Pakistan (Azad Kashmir area) and 27 volunteer controls from similar demographics. Three independent raters judged the dream narratives and dream content ratings. The judges rating were highly congruent (86.05). Findings revealed that the survivor group had more vivid, unpleasant, horrifying, and hostile dreams compared with the control group. However, there were no significant gender differences. The data suggest that a closer study of dreams can be used to understand the underlying trauma for effective interventions. In addition, interesting emergence of recurrent dreams was seen, which will be independently studied in future. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Identifying people at high risk for developing sleep apnea syndrome (SAS): a cross-sectional study in a Pakistani population

Background  

Obstructive Sleep Apnea (OSA) is associated with many cardiovascular and psychiatric diseases. Day-time sleepiness is a common consequence of sleep apnea and correlates with road-traffic accidents (RTA). Pakistan has a high prevalence of factors which predispose an individual to OSA and death from RTAs are a huge burden. However there is a dearth of prevalence studies in this regard. We aim to understand local relevance of the disease and estimate the prevalence of individuals high-risk for OSA.     

Role of urinary NMP-22 combined with urine cytology in follow-up surveillance of recurring superficial bladder urothelial carcinoma

OBJECTIVE: To determine efficacy and utility of NMP-22 in follow-up of bladder urothelial carcinoma (UC) and compare NMP-22 as a single evaluating test vs combination with cytology. STUDY DESIGN: Ninety-four consecutive urine cytology samples of bladder UC were identified. Patients received follow-up urine cytology, NMP-22 testing and cystoscopy with surgical biopsy. RESULTS: NMP-22 specificity was 100%, sensitivity 45%, positive predictive value (PPV) 100% and negative predictive value (NPV) 87%. NMP-22 showed lower sensitivity for high-grade lesions and higher for low-grade lesions. Cytologic diagnosis had a high inconclusive rate; when regarded as positive, it resulted in 75% sensitivity, 58% specificity, 33% PPV and 89% NPV. NMP-22 correctly classified 60% of false negative cases diagnosed by cytology with low-grade UC and clarified 27 inconclusive cytologic diagnoses. NMP-22 misclassified 9 cases as false negative, all with high-grade UC; all were correctly identified on cytology as true positive. Combined interpretation showed 90% sensitivity, 92% specificity, 75% PPV and 98% NPV. CONCLUSION: NMP-22 complements cytology by its higher sensitivity for low-grade lesions; its values are not affected by bacillus Calmette Guérin therapy changes, which are limiting in cytology. Combined interpretation of NMP-22 and cytology shows promise as an effective, noninvasive method for surveillance of UC.     

Generation of human embryonic stem cell-derived mesoderm and cardiac cells using size-specified aggregates in an oxygen-controlled bioreactor

The ability to generate human pluripotent stem cell-derived cell types at sufficiently high numbers and in a reproducible manner is fundamental for clinical and biopharmaceutical applications. Current experimental methods for the differentiation of pluripotent cells such as human embryonic stem cells (hESC) rely on the generation of heterogeneous aggregates of cells, also called "embryoid bodies" (EBs), in small scale static culture. These protocols are typically (1) not scalable, (2) result in a wide range of EB sizes and (3) expose cells to fluctuations in physicochemical parameters. With the goal of establishing a robust bioprocess we first screened different scalable suspension systems for their ability to support the growth and differentiation of hESCs. Next homogeneity of initial cell aggregates was improved by employing a micro-printing strategy to generate large numbers of size-specified hESC aggregates. Finally, these technologies were integrated into a fully controlled bioreactor system and the impact of oxygen concentration was investigated. Our results demonstrate the beneficial effects of stirred bioreactor culture, aggregate size-control and hypoxia (4% oxygen tension) on both cell growth and cell differentiation towards cardiomyocytes. QRT-PCR data for markers such as Brachyury, LIM domain homeobox gene Isl-1, Troponin T and Myosin Light Chain 2v, as well as immunohistochemistry and functional analysis by response to chronotropic agents, documented the impact of these parameters on cardiac differentiation. This study provides an important foundation towards the robust generation of clinically relevant numbers of hESC derived cells.