Single-molecule fluorescence multiplexing by multi-parameter spectroscopic detection of nanostructured FRET labels.

Multiplexed, real-time fluorescence detection at the single-molecule level can reveal the stoichiometry, dynamics and interactions of multiple molecular species in mixtures and other complex samples. However, fluorescence-based sensing is typically limited to the detection of just 3-4 colours at a time due to low signal-to-noise ratio, high spectral overlap and the need to maintain the chemical compatibility of dyes. Here we engineered a palette of several dozen composite fluorescent labels, called FRETfluors, for multiplexed spectroscopic measurements at the single-molecule level. FRETfluors are compact nanostructures constructed from three chemical components (DNA, Cy3 and Cy5) with tunable spectroscopic properties due to variations in geometry, fluorophore attachment chemistry and DNA sequence. We demonstrate FRETfluor labelling and detection for low-concentration (<100 fM) mixtures of mRNA, dsDNA and proteins using an anti-Brownian electrokinetic trap. In addition to identifying the unique spectroscopic signature of each FRETfluor, this trap differentiates FRETfluors attached to a target from unbound FRETfluors, enabling wash-free sensing. Although usually considered an undesirable complication of fluorescence, here the inherent sensitivity of fluorophores to the local physicochemical environment provides a new design axis complementary to changing the FRET efficiency. As a result, the number of distinguishable FRETfluor labels can be combinatorically increased while chemical compatibility is maintained, expanding prospects for spectroscopic multiplexing at the single-molecule level using a minimal set of chemical building blocks.

Single-molecule fluorescence multiplexing by multi-parameter spectroscopic detection of nanostructured FRET labels.

Multiplexed, real-time fluorescence detection at the single-molecule level is highly desirable to reveal the stoichiometry, dynamics, and interactions of individual molecular species within complex systems. However, traditionally fluorescence sensing is limited to 3-4 concurrently detected labels, due to low signal-to-noise, high spectral overlap between labels, and the need to avoid dissimilar dye chemistries. We have engineered a palette of several dozen fluorescent labels, called FRETfluors, for spectroscopic multiplexing at the single-molecule level. Each FRETfluor is a compact nanostructure formed from the same three chemical building blocks (DNA, Cy3, and Cy5). The composition and dye-dye geometries create a characteristic F\"orster Resonance Energy Transfer (FRET) efficiency for each construct. In addition, we varied the local DNA sequence and attachment chemistry to alter the Cy3 and Cy5 emission properties and thereby shift the emission signatures of an entire series of FRET constructs to new sectors of the multi-parameter detection space. Unique spectroscopic emission of each FRETfluor is therefore conferred by a combination of FRET and this site-specific tuning of individual fluorophore photophysics. We show single-molecule identification of a set of 27 FRETfluors in a sample mixture using a subset of constructs statistically selected to minimize classification errors, measured using an Anti-Brownian ELectrokinetic (ABEL) trap which provides precise multi-parameter spectroscopic measurements. The ABEL trap also enables discrimination between FRETfluors attached to a target (here: mRNA) and unbound FRETfluors, eliminating the need for washes or removal of excess label by purification. We show single-molecule identification of a set of 27 FRETfluors in a sample mixture using a subset of constructs selected to minimize classification errors.

Acquired haemophilia A diagnosed during pregnancy.

Pregnancy-associated haemophilia A is an uncommon, acquired bleeding disorder which usually presents post-partum; very rarely it may present during pregnancy. No consensus guidelines exist on the management of this condition in pregnancy and very few cases have been reported in the literature. Here we describe the case of a woman presenting with acquired haemophilia A during pregnancy and outline the management of her bleeding disorder. We contrast her case with that of two other women, presenting to the same tertiary referral centre, with acquired haemophilia A presenting post-partum. These cases highlight the heterogeneous management of this condition and how it may be successfully managed in pregnancy.

Direct correction of haemoglobin E ß-thalassaemia using base editors.

Haemoglobin E (HbE) ß-thalassaemia causes approximately 50% of all severe thalassaemia worldwide; equating to around 30,000 births per year. HbE ß-thalassaemia is due to a point mutation in codon 26 of the human HBB gene on one allele (GAG; glutamatic acid → AAG; lysine, E26K), and any mutation causing severe ß-thalassaemia on the other. When inherited together in compound heterozygosity these mutations can cause a severe thalassaemic phenotype. However, if only one allele is mutated individuals are carriers for the respective mutation and have an asymptomatic phenotype (ß-thalassaemia trait). Here we describe a base editing strategy which corrects the HbE mutation either to wildtype (WT) or a normal variant haemoglobin (E26G) known as Hb Aubenas and thereby recreates the asymptomatic trait phenotype. We have achieved editing efficiencies in excess of 90% in primary human CD34 + cells. We demonstrate editing of long-term repopulating haematopoietic stem cells (LT-HSCs) using serial xenotransplantation in NSG mice. We have profiled the off-target effects using a combination of circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq) and deep targeted capture and have developed machine-learning based methods to predict functional effects of candidate off-target mutations.

Learned Reconstruction of Protein Folding Trajectories from Noisy Single-Molecule Time Series.

Single-molecule Förster resonance energy transfer (smFRET) is an experimental methodology to track the real-time dynamics of molecules using fluorescent probes to follow one or more intramolecular distances. These distances provide a low-dimensional representation of the full atomistic dynamics. Under mild technical conditions, Takens' Delay Embedding Theorem guarantees that the full three-dimensional atomistic dynamics of a system are diffeomorphic (i.e., related by a smooth and invertible transformation) to a time-delayed embedding of one or more scalar observables. Appealing to these theoretical guarantees, we employ manifold learning, artificial neural networks, and statistical mechanics to learn from molecular simulation training data the a priori unknown transformation between the atomic coordinates and delay-embedded intramolecular distances accessible to smFRET. This learned transformation may then be used to reconstruct atomistic coordinates from smFRET time series data. We term this approach Single-molecule TAkens Reconstruction (STAR). We have previously applied STAR to reconstruct molecular configurations of a C24H50 polymer chain and the mini-protein Chignolin with accuracies better than 0.2 nm from simulated smFRET data under noise free and high time resolution conditions. In the present work, we investigate the role of signal-to-noise ratio, data volume, and time resolution in simulated smFRET data to assess the performance of STAR under conditions more representative of experimental realities. We show that STAR can reconstruct the Chignolin and Villin mini-proteins to accuracies of 0.12 and 0.42 nm, respectively, and place bounds on these conditions for accurate reconstructions. These results demonstrate that it is possible to reconstruct dynamical trajectories of protein folding from time series in noisy, time binned, experimentally measurable observables and lay the foundations for the application of STAR to real experimental data.

Outcomes of Dialysis Among Patients With End-Stage Renal Disease (ESRD).

Background Dialysis-associated morbidity and mortality among end-stage renal disease (ESRD) patients has been increasing, despite the advancement in pharmacological treatment and dialysis technology. The aim of this study was to determine the outcomes of dialysis among ESRD patients presenting at the nephrology department of Jinnah Postgraduate Medical Centre (JPMC).  Methodology This cross-sectional study was conducted during the year 2015-2016, including 105 ESRD patients. Data were collected through a structured questionnaire inquiring about patient's demographics and hemodialysis details. The outcomes in terms of survival and death within one month of dialysis were also recorded. The statistical analysis was carried out using SPSS version 21.0 (IBM Corp, Armonk, NY). Results Gender distribution showed that most of the study patients were males (58.1%). The mean duration of ESRD was 7.65 ± 3.69 months while the mean duration of hemodialysis was 36.5 ± 5.65 hours. Among the comorbid conditions, hypertension (69.5%) and diabetes (64.8%) were the most prevalent, followed by renal stones, chronic pyelonephritis, and chronic nephritis. The outcomes indicated mortality among 16.2% of patients; all deceased ESRD patients had diabetes (p < 0.05). Moreover, the duration of hemodialysis was significantly associated with the outcomes of dialysis (p < 0.05). Conclusion In conclusion, a considerable mortality rate was observed among ESRD patients undergoing hemodialysis. Moreover, patient survival was better with the increased duration of dialysis.

A unified model of human hemoglobin switching through single-cell genome editing.

Key mechanisms of fetal hemoglobin (HbF) regulation and switching have been elucidated through studies of human genetic variation, including mutations in the HBG1/2 promoters, deletions in the ß-globin locus, and variation impacting BCL11A. While this has led to substantial insights, there has not been a unified understanding of how these distinct genetically-nominated elements, as well as other key transcription factors such as ZBTB7A, collectively interact to regulate HbF. A key limitation has been the inability to model specific genetic changes in primary isogenic human hematopoietic cells to uncover how each of these act individually and in aggregate. Here, we describe a single-cell genome editing functional assay that enables specific mutations to be recapitulated individually and in combination, providing insights into how multiple mutation-harboring functional elements collectively contribute to HbF expression. In conjunction with quantitative modeling and chromatin capture analyses, we illustrate how these genetic findings enable a comprehensive understanding of how distinct regulatory mechanisms can synergistically modulate HbF expression.

Frequency of Intradialytic Complications in Patients of End-Stage Renal Disease on Maintenance Hemodialysis.

Introduction Hemodialysis is a vital management option for end-stage renal disease (ESRD) patients. With adequate hemodialysis, patients can have a good quality of life but complications do occur during the session, which can be minor to life-threatening. The objective of this study was to assess the frequency of acute complications during this procedure. Material and methods An observational, cross-sectional study was conducted at Jinnah Postgraduate Medical Centre, Karachi, Pakistan. Patient data were collected about access, comorbid conditions, frequency and duration of hemodialysis, and intradialytic complications. Those with acute kidney injury were excluded. Results There was a total of 94 patients, with a mean age of 45.51±13.29 years, of which 62 (66%) were males and 32 (34%) were females. Diabetes mellitus was the most common cause of ESRD (47.9%, n=45). Patients on twice and thrice-weekly sessions were 51 (54.3%) and 43 (45.7%), respectively. The most common complication was hypotension (28.7%), followed by hypertension (17%), and nausea/vomiting (11.7%). The arteriovenous fistula was the most common access used (75.5%, n=71). Most patients were found to be on hemodialysis for more than five years (51.1%, n=48). Conclusion Blood pressure changes are critical while performing hemodialysis, just like we found hypotension as the most common intradialytic complication in our results, followed by hypertension. Others were fever, muscle cramps, and nausea/vomiting. a prospective follow-up study shall be done to have comparative and long-term results related to the acute and chronic complications of dialysis.

Long-Term Patient-Customized Therapy for a Pathogenic EPO Mutation.

BACKGROUND: Recent advances in genomics have enabled the successful identification of a number of rare pathogenic mutations. Uncovering these mutations is essential as the first step towards devising a cure for the often debilitating and life-limiting diseases arising from them. For many of these mutations targeted agents do not yet exist. Here, we describe the case of a patient who has a novel pathogenic mutation in the erythropoietin (EPO) gene, which is essential for normal erythropoiesis, and who presented with a profound hypoplastic anemia. METHODS: The patient aged 5 months, was started on recombinant erythropoietin, at a standard dose of 500 units (50 U/kg) and subsequently 800 units three time weekly and her blood counts were monitored over 4 years. FINDINGS: A prompt response to the recombinant erythropoietin was found with an increase in hemoglobin levels to 12.8 g/dL and increase in red cell count to 4.89×106/uL. The patient became transfusion independent. The therapy enabled the patient to maintain a hemoglobin level in the normal range without any adverse effects and with no requirement for further blood transfusions. CONCLUSIONS: Patient-customized therapies can be highly effective in the treatment of rare genetic disorders and for many of these disorders effective treatment may already exist in the clinical domain, as described for the patient in this report. FUNDING: This work was supported by the New York Stem Cell Foundation (V.G.S.), a gift from the Lodish Family to Boston Children's Hospital (V.G.S.), and National Institutes of Health Grants R01 DK103794 and R01 HL146500 (V.G.S.).

Uridine-responsive epileptic encephalopathy due to inherited variants in CAD: A Tale of Two Siblings.

We report two siblings with intractable epilepsy, developmental regression, and progressive cerebellar atrophy due to biallelic variants in the gene CAD. For the affected girl, uridine started at age 5 resulted in dramatic improvements in seizure control and development, cessation of cerebellar atrophy, and resolution of hematological abnormalities. Her older brother had a more severe course and only modest response to uridine started at 14 years old. Treatment of this progressive condition via uridine supplementation provides an example of precision diagnosis and treatment using clear outcome measures and biomarkers to monitor efficacy.

Association of Pulmonary Hypertension With End-Stage Renal Disease Among the Obese Population.

Introduction Pulmonary hypertension (PH) is a known complication that occurs in patients of end-stage renal disease (ESRD) that have an arteriovenous fistula (AVF) for hemodialysis (HD). It is defined as pulmonary artery pressure (PAP) of greater than 30 mmHg on echocardiography. The presence of PH in ESRD is an independent risk factor and decreases the survival likelihood among HD patients. Unexplained PH is frequently seen in ESRD following AVF. Obesity can lead to various complications, such as sleep apnea, cardiac complications, pulmonary hypertension, and mortality. Data on the prevalence of coexisting PH and obesity are scarce. Obese patients often have increased albumin excretion rates (AER) that can lead to early renal impairment and an increase in intraglomerular pressure, which may increase the risk of cardiovascular (CV) morbidity and mortality. Therefore, the study aimed to evaluate and compare the associated PH and obesity separately and collectively among ESRD patients. Methods This comparative cross-sectional study was conducted in a tertiary care public sector hospital with the approval of the medical ethics review board committee. The study enrolled all consecutive patients with ESRD as defined by having an estimated glomerular filtration rate (GFR) of <15 mL/min/1.7 3 m2 from April 2017 till March 2019, who presented to our facility. These patients underwent dialysis twice or thrice a week, each session lasting three to four hours approximately. On initial encounter, trans-thoracic echocardiography (TTE) was done by the cardiologist to diagnose pulmonary hypertension. In addition, body mass index (BMI) was calculated for all patients, and the patients were categorized into underweight, normal, overweight, or obese. All patients underwent post-dialysis TTE at one hour or when patients were at the optimal dry weight. Systolic PAP and ejection fraction were measured, and pulmonary hypertension was defined as a PAP of 30 mmHg or greater on TTE. ESRD patients that were diagnosed with PH prior to hemodialysis or had primary PH were excluded from the study. Only ESRD patients developing secondary PH after hemodialysis were included in the study. The chi-square test was used to see the correlation of gender, ambulation status, smoking status, obesity, pulmonary hypertension, body mass index (BMI), and pulmonary hypertension and obesity combined on the final outcome. A p-value of 0.05 was considered significant. Odds ratio (OR) and relative risk (RR) were calculated for pulmonary hypertension and obesity combined, obesity, and pulmonary hypertension in the final outcome. Results The study enrolled 204 patients with a mean age of 46.23 (±20.45 SD) having higher female participation of 108 (52.9%), whereas 96 (47.1%) were males. The average weight of the cohort was 66.78 kg (±22.98 SD) with a mean BMI of 29.91 kg/m2 (±13.29SD), 52 (25.5%) patients were underweight, 40 (19.6%) had a normal BMI, 29 (14.2%) were overweight, and 83 (40.7%) patients were obese. Pulmonary hypertension and obesity combined were observed in 48 (23.5%) of the cases and there was a 4.60 relative risk of death among these individuals, with an odds ratio of 13.35 and a p-value of 0.00. Conclusion The study shows a strong synergistic effect of pulmonary hypertension and obesity towards the final survival outcome in ESRD patients who are on hemodialysis.

Serum Neopterin Is Not Influenced by Age and Blood Group of Transfusion-Transmitted Infection Negative Blood Donors.

Objectives To measure serum neopterin levels in blood donors of the local population and to study its relationship with age and blood group of transfusion-transmitted infection (TTI) negative blood donors. Methodology This cross-sectional study was carried out in the Department of Physiology at Liaquat National Hospital and Medical College (LNMC), Karachi, Pakistan, in collaboration with the Basic Medical Sciences Institute and Jinnah Post Graduate Medical Centre (JPMC). Data were collected from January 2018 to July 2018. A total of 174 blood donors participated in the study, who were selected by using a random sampling technique. They were screened through the standard procedures used for screening at the JPMC blood bank for TTIs as per the World Health Organization recommendations. Serum neopterin was estimated using enzyme-linked immunosorbent assay (ELISA) kits. Data analysis was performed using Statistical Package for Social Sciences (SPSS) version 23 (IBM Corp., Armonk, NY, USA). Chi-square and ANOVA (analysis of variance) were applied, and tests of significance were kept as P < 0.05. Results Neopterin level in the serum of TTI-positive blood donors was 15.1 ± 4.59 nmol/L, which was above the normal range and cutoff value of 10 nmol/L, whereas the neopterin level in the serum of TTI-negative blood donors was 6.1 ± 1.82 nmol/L (P = 0.001). In TTI-negative blood donors, the neopterin levels were within normal limits and were not influenced by age and blood groups (P > 0.05). Conclusions Serum neopterin levels did not report any significant difference in terms of age and blood group of TTI-negative blood donors and were seen to be within normal limits.

Frequency of Risk Factors and Outcome of Hospital-Acquired Acute Kidney Injury.

OBJECTIVE: To determine the frequencies of risk factors and the ultimate outcomes of ccute kidney injury (AKI) among hospitalized patients. MATERIALS AND METHODOLOGY: This prospective, observational study was carried out from September 15, 2018, to March 14, 2019. All admitted patients, both male and female, with AKI, were included. Those with chronic kidney disease (CKD), small size echogenic kidneys (on ultrasonography, performed on admission), and recent history of urological intervention were excluded from the study. All patients were assessed for etiological factors (sepsis, gastroenteritis, surgical, and obstetrical) and outcome (improved, progression to CKD, or expired). RESULTS: Out of a total of 230, most patients were aged between 20-50 years with a mean age of 38.99 ± 7.61 years. Males were 144 (62.61%) and females were 86 (37.39%). About 78 (33.91%) patients were hypertensive while 65 (28.26%) were diabetic. The cause of hospital-acquired AKI was found to be sepsis in most (71.73%, n=165) of the cases, followed by gastroenteritis (10.00%, n=23), surgical (9.56%, n=22), and obstetric (8.69%, n=20) causes. When the outcome was assessed, 10 (4.35%) patients expired, 154 (66.96%) improved completely, while 66 (28.69%) progressed to CKD. CONCLUSION: This study has shown that sepsis is the most common cause of AKI in patients admitted to the hospital. So we recommend that proper steps should be taken to ensure adequate hospital care for avoiding such outcomes in hospitalized patients, and further decrease mortality.

Vitamin D Deficiency in End Stage Renal Disease Patients with Diabetes Mellitus Undergoing Hemodialysis.

OBJECTIVES: To assess the association of hypovitaminosis D with diabetes mellitus (DM) in patients with end stage renal disease (ESRD) undergoing hemodialysis. METHODOLOGY: This cross-sectional study was conducted at the Jinnah Postgraduate Medical Centre between July 2019 and February 2020. Patients with diagnosed ESRD who were on hemodialysis, with or without concomitant DM were registered. Vitamin D levels were categorized according to the severity of the deficiency or excess as 0-10 ng/mL, severely deficient; 11-20 ng/mL, deficient; 21-32 ng/mL; insufficient, 33-49 ng/mL, adequate; 50-65 ng/mL, optimum; and above that as high. Patients were stratified according to the status of DM.  Results: In a total of 80, the mean age was 45.21±12.67 years with 51 (63.75%) males and 29 (36.25%) females. A total of 36 (45%) CKD patients had concomitant diabetes. The median vitamin D levels were 20.25ng/mL. It was found that chronic kidney disease (CKD) patients with concomitant DM had significantly lower levels of vitamin D [15.19±6.83 vs. 30.28±14.22 (p<0.001)]. Out of the 12 patients with a severe deficiency, three-fourths of the population had DM as comorbidity, while in those with 'deficiency', 19 (67.9%) had DM. The majority of the patients without DM had adequate or optimum levels of serum 25-hydroxyvitamin D levels. CONCLUSION: Current study indicated that deficiency of serum vitamin D is associated with concomitant DM in patients with CKD as the majority had a severe deficiency of serum 25(OH)D. Supplemental vitamin D may help correct the deficiency and prevent the associated complications in patients.

Somatic copy number gains of α-synuclein (SNCA) in Parkinson's disease and multiple system atrophy brains.

The α-synuclein protein, encoded by SNCA, has a key role in the pathogenesis of Parkinson's disease and other synucleinopathies. Although usually sporadic, Parkinson's disease can result from inherited copy number variants in SNCA and other genes. We have hypothesized a role of somatic SNCA mutations, leading to mosaicism, in sporadic synucleinopathies. The evidence for mosaicism in healthy and diseased brain is increasing rapidly, with somatic copy number gains of APP reported in Alzheimer's brain. Here we demonstrate somatic SNCA copy number gains in synucleinopathies (Parkinson's disease and multiple system atrophy), focusing on substantia nigra. We selected sporadic cases with relatively young onset or short disease duration, and first excluded high level copy number variant mosaicism by DNA analysis using digital PCR for SNCA, and/or customized array comparative genomic hybridization. To detect low level SNCA copy number variant mosaicism, we used fluorescent in situ hybridization with oligonucleotide custom-designed probes for SNCA, validated on brain and fibroblasts with known copy number variants. We determined SNCA copy number in nigral dopaminergic neurons and other cells in frozen nigra sections from 40 cases with Parkinson's disease and five with multiple system atrophy, and 25 controls, in a blinded fashion. Parkinson's disease cases were significantly more likely than controls to have any SNCA gains in dopaminergic neurons (P = 0.0036), and overall (P = 0.0052). The average proportion of dopaminergic neurons with gains in each nigra was significantly higher in Parkinson's disease than controls (0.78% versus 0.45%; P = 0.017). There was a negative correlation between the proportion of dopaminergic neurons with gains and onset age in Parkinson's disease (P = 0.013), but not with disease duration, or age of death in cases or controls. Cases with tremor at onset were less likely to have gains (P = 0.035). All multiple system atrophy cases had gains, and the highest levels in dopaminergic neurons were in two of these cases (2.76%, 2.48%). We performed selective validation with different probes after dye swapping. All three control probes used showed minimal or no gains (≤0.1% in dopaminergic neurons). We also found occasional SNCA gains in frontal neurons of cases with Parkinson's disease, and the putamen of one multiple system atrophy case. We present evidence of somatic SNCA gains in brain, more commonly in nigral dopaminergic neurons of Parkinson's disease than controls, negatively correlated with onset age, and possibly commonest in some multiple system atrophy cases. Somatic SNCA gains may be a risk factor for sporadic synucleinopathies, or a result of the disease process.10.1093/brain/awy157_video1awy157media15813519976001.

Regulation of Corticosteroidogenic Genes by MicroRNAs.

The loss of normal regulation of corticosteroid secretion is important in the development of cardiovascular disease. We previously showed that microRNAs regulate the terminal stages of corticosteroid biosynthesis. Here, we assess microRNA regulation across the whole corticosteroid pathway. Knockdown of microRNA using Dicer1 siRNA in H295R adrenocortical cells increased levels of CYP11A1, CYP21A1, and CYP17A1 mRNA and the secretion of cortisol, corticosterone, 11-deoxycorticosterone, 18-hydroxycorticosterone, and aldosterone. Bioinformatic analysis of genes involved in corticosteroid biosynthesis or metabolism identified many putative microRNA-binding sites, and some were selected for further study. Manipulation of individual microRNA levels demonstrated a direct effect of miR-125a-5p and miR-125b-5p on CYP11B2 and of miR-320a-3p levels on CYP11A1 and CYP17A1 mRNA. Finally, comparison of microRNA expression profiles from human aldosterone-producing adenoma and normal adrenal tissue showed levels of various microRNAs, including miR-125a-5p to be significantly different. This study demonstrates that corticosteroidogenesis is regulated at multiple points by several microRNAs and that certain of these microRNAs are differentially expressed in tumorous adrenal tissue, which may contribute to dysregulation of corticosteroid secretion. These findings provide new insights into the regulation of corticosteroid production and have implications for understanding the pathology of disease states where abnormal hormone secretion is a feature.

DNA isolation protocol effects on nuclear DNA analysis by microarrays, droplet digital PCR, and whole genome sequencing, and on mitochondrial DNA copy number estimation.

Potential bias introduced during DNA isolation is inadequately explored, although it could have significant impact on downstream analysis. To investigate this in human brain, we isolated DNA from cerebellum and frontal cortex using spin columns under different conditions, and salting-out. We first analysed DNA using array CGH, which revealed a striking wave pattern suggesting primarily GC-rich cerebellar losses, even against matched frontal cortex DNA, with a similar pattern on a SNP array. The aCGH changes varied with the isolation protocol. Droplet digital PCR of two genes also showed protocol-dependent losses. Whole genome sequencing showed GC-dependent variation in coverage with spin column isolation from cerebellum. We also extracted and sequenced DNA from substantia nigra using salting-out and phenol / chloroform. The mtDNA copy number, assessed by reads mapping to the mitochondrial genome, was higher in substantia nigra when using phenol / chloroform. We thus provide evidence for significant method-dependent bias in DNA isolation from human brain, as reported in rat tissues. This may contribute to array "waves", and could affect copy number determination, particularly if mosaicism is being sought, and sequencing coverage. Variations in isolation protocol may also affect apparent mtDNA abundance.

Serum Neopterin: A Potential Marker for Screening Blood Donors.

OBJECTIVE: To determine serum neopterin levels in blood donors of local population and its association with transfusion transmitted infections. STUDY DESIGN: A cross-sectional observational study. PLACE AND DURATION OF STUDY: Department of Physiology, Liaquat National Hospital and Medical College (LNHMC) in collaboration with Basic Medical Sciences Institute (BMSI) and Jinnah Postgraduate Medical Centre (JPMC), Blood Bank, Karachi, Pakistan, from January to June 2015. METHODOLOGY: During this period, a total of 174 blood donors were selected through random sampling technique. All participants fulfilling the inclusion criteria involving apparently healthy blood donors of either gender within the age bracket of 18 - 60 years and consenting to participate were selected. The participants were screened for transfusion transmitted infections as per WHO recommendations through the standard procedures used for screening at the JPMC blood bank. The demographic profile, anthropometric measurements and vitals were recorded for every participant. Serum neopterin was measured using ELISA kits. Data was analysed on SPSS version 21. ANOVA and chi-square tests were applied as tests of significance at a p-value of <0.05. RESULTS: The neopterin content in the sera of disease negative blood donors was 6.23 ±2.19 nmol/l as compared to disease positive blood donors, in whom the neopterin level was increased to 15.10 ±4.93 nmol/l (p =0.001). CONCLUSION: The neopterin assay has the potential to detect a number of transfusion transmissible viral diseases; which may, or may not be revealed by the usually employed battery of routine tests. We conclude that the risk of transfusion transmitted pathogens in our population can be reduced significantly, using neopterin assay as a routine in blood banks.