Search

Background: Objective of the study was to find out the incidence of maternal complications, perinatal mortality andmorbidity in postdated pregnancies. Design of the study was prospective observational study. Methods: Patients who have completed 40 weeks of gestational age, patients who were sure of the date of last menstrualperiod (LMP) along with 1st trimester obstetrics scan were included. Patients not sure of LMP were excluded. Results: There is high fetal and maternal risk associated with postdated pregnancy. Total 100 cases were selected fromantenatal clinic and Labour Room and were divided into two groups. Study group and control group, 50 cases in each. Induction rate is more in postdated pregnancies. Incidence of operative deliveries is 54% in study group. Postpartum haemorrhage and septicaemia are the most common maternal complication in the study group. Perinatal mortality is higher and more NICU admissions were required. Conclusions: Considering this, policy of early intervention should be undertaken in postdated pregnancy to avoidmaternal and perinatal complications.

Background: Maternal weight gain is influenced by several trends in perinatal health that are of greatpublic health concern. Maternal weight gain during the 2nd and 3rd trimesters is an important determinant of fetal growth Objective: To determine the relationship between maternal weight gain and birth weight of baby at term. Methodology : A cross sectional observational study was carried among 50 pregnant women at term wereadmitted in the Department of Obstetrics and Gynaecology, Jawaharlal Nehru Medical College and Hospital, Bhagalpur, Bihar during the period of January 2020 to July 2020. Data were collected pre-designed data collection sheet. Results: This study found maximum (36%) were age group 21-25 years followed by 28% were≤20 years,24% were 26-30 years, 8% were 31-35 years and only 4% were 36-40 years. The average age was 25 years. Among these 50 pregnant women, 2 cases (4%) had BMI

The Oxford MinION, the first commercial nanopore sequencer, is also the first to implement molecule-by-molecule real-time selective sequencing or “Read Until”. As DNA transits a MinION nanopore, real-time pore current data can be accessed and analyzed to provide active feedback to that pore. Fragments of interest are sequenced by default, while DNA deemed non-informative is rejected by reversing the pore bias to eject the strand, providing a novel means of background depletion and/or target enrichment. In contrast to the previously published pattern-matching Read Until approach, our RUBRIC method is the first example of real-time selective sequencing where on-line basecalling enables alignment against conventional nucleic acid references to provide the basis for sequence/reject decisions. We evaluate RUBRIC performance across a range of optimizable parameters, apply it to mixed human/bacteria and CRISPR/Cas9-cut samples, and present a generalized model for estimating real-time selection performance as a function of sample composition and computing configuration.

Introduction:- Vaginal discharge is one of the most common complaint in women attending obstetrics and Gynecology outpatient department. It increases the morbidity in women. AIM : The study is aimed at identifying the impact of the social and demographic variables associated with the complaint of vaginal discharge. Method :This is a hospital based prospective observational study conducted in the obstetries and gynecology outpatient department of Jawaharlal Nehru Medical college , Bhagalpur , Bihar. Pretested questionnaire was done on women with Vaginal discharge . Results : The total number of cases mere 60. The results were vaginal discharge common among 26-35 yr of age . 15.6% among lower socio economic class women , 33.6% among women living with their husband , 27.6% has vaginal discharge who got married before 18 yrs of age. 44% women perceived no specic symptoms. Conclusion : Awarness regarding personal and menstrual hygiene is mandatory among women to lower the incidence of vaginal discharge

Introduction: Nearly 20% of all confirmed pregnancies end in spontaneous abortion. Misoprostol’s use in early pregnancy failure is varied and dose and route are not well established. AIM: To study the efficacy of sublingual misoprostol in causing expulsion of products of conception in early pregnancy failure. Methods: Women with an ultrasound diagnosis of early pregnancy failure, less than 12 weeks gestation were included in the study. Tablet Misoprostol 600 mcg was given six hourly sublingually for 3 doses. All observations were noted and analyzed. Results: Mean gestational age was 7.946+1.2 weeks. Mean induction abortion interval was 18.241+1.2 hours. Women with gestational age six to eight weeks had least mean induction-abortion interval time of 17.38+2.86. Mean dose required was 1560mcg. Efficacy of protocol was 92.85% in achieving complete abortion. Conclusion: The regime had 92.85% efficacy, acceptability (90%) and few side effects. Thus by using a lower dose and appropriate interval between two doses (six hours), the side effects were lessened with high efficacy. Keywords: Efficacy, Missed Abortion, Misoprostol.

The antibiotic resistance crisis has led to renewed interest in phage therapy as an alternative means of treating infection. However, conventional methods for isolating pathogen-specific phage are slow, labor-intensive, and frequently unsuccessful. We have demonstrated that computationally identified prophages carried by near-neighbor bacteria can serve as starting material for production of engineered phages that kill the target pathogen. Our approach and technology platform offer new opportunity for rapid development of phage therapies against most, if not all, bacterial pathogens, a foundational advance for use of phage in treating infectious disease., New therapies are necessary to combat increasingly antibiotic-resistant bacterial pathogens. We have developed a technology platform of computational, molecular biology, and microbiology tools which together enable on-demand production of phages that target virtually any given bacterial isolate. Two complementary computational tools that identify and precisely map prophages and other integrative genetic elements in bacterial genomes are used to identify prophage-laden bacteria that are close relatives of the target strain. Phage genomes are engineered to disable lysogeny, through use of long amplicon PCR and Gibson assembly. Finally, the engineered phage genomes are introduced into host bacteria for phage production. As an initial demonstration, we used this approach to produce a phage cocktail against the opportunistic pathogen Pseudomonas aeruginosa PAO1. Two prophage-laden P. aeruginosa strains closely related to PAO1 were identified, ATCC 39324 and ATCC 27853. Deep sequencing revealed that mitomycin C treatment of these strains induced seven phages that grow on P. aeruginosa PAO1. The most diverse five phages were engineered for nonlysogeny by deleting the integrase gene (int), which is readily identifiable and typically conveniently located at one end of the prophage. The Δint phages, individually and in cocktails, killed P. aeruginosa PAO1 in liquid culture as well as in a waxworm (Galleria mellonella) model of infection. IMPORTANCE The antibiotic resistance crisis has led to renewed interest in phage therapy as an alternative means of treating infection. However, conventional methods for isolating pathogen-specific phage are slow, labor-intensive, and frequently unsuccessful. We have demonstrated that computationally identified prophages carried by near-neighbor bacteria can serve as starting material for production of engineered phages that kill the target pathogen. Our approach and technology platform offer new opportunity for rapid development of phage therapies against most, if not all, bacterial pathogens, a foundational advance for use of phage in treating infectious disease.

New therapies are necessary to combat increasingly antibiotic-resistant bacterial pathogens. We have developed a technology platform of computational, molecular biology, and microbiology tools which together enable on-demand production of phages that target virtually any given bacterial isolate. Two complementary computational tools that identify and precisely map prophages and other integrative genetic elements (IGEs) in bacterial genomes are used to identify prophage-laden bacteria that are close relatives of the target strain. Phage genomes are engineered to disable lysogeny, through use of long amplicon PCR and Gibson assembly. Finally, the engineered phage genomes are introduced into host bacteria for phage production. As an initial demonstration, we used this approach to produce a phage cocktail against the opportunistic pathogen Pseudomonas aeruginosa PAO1. Two prophage-laden P. aeruginosa strains closely related to PAO1 were identified, ATCC 39324 and ATCC 27853. Deep sequencing revealed that mitomycin C treatment of these strains induced seven phages that grow on P. aeruginosa PAO1. The most diverse five of these were engineered for non-lysogeny by deleting the integrase gene (int), which is readily identifiable and typically conveniently located at one end of the prophage. The Δint phages, individually and in cocktails, showed killing of P. aeruginosa PAO1 in vitro as well as in a waxworm (Galleria mellonella) model of infection.SIGNIFICANCE STATEMENTThe antibiotic-resistance crisis in medicine and agriculture has led to renewed interest in phage therapy as an alternative means of treating infection. However, conventional methods for isolating pathogen-specific phage are slow, labor-intensive, and frequently unsuccessful. We have demonstrated that prophages carried by near-neighbor bacteria can serve as starting material for production of engineered phages that kill the target pathogen. Our approach and technology platform offer new opportunity for rapid development of phage therapies against most, if not all, bacterial pathogens, a foundational advance for use of phage in treating infectious disease.

We present the draft genome sequences of three Burkholderia thailandensis strains, E421, E426, and DW503. E421 consists of 90 contigs of 6,639,935 bp and 67.73% GC content. E426 consists of 106 contigs of 6,587,853 bp and 67.73% GC content. DW503 consists of 102 contigs of 6,458,767 bp and 67.64% GC content.

Background: A prospective study was conducted to compare the efficacy of nifedipine against isoxsuprine in preventing preterm labour in third trimester and also to evaluate maternal side effects and neonatal outcome. Methods: This prospective comparative study was conducted at JLNMCH, Bhagalpur, Bihar. 75 antenatal women with gestational age between 28 to 36 weeks wereselected who fulfilled the inclusion criteria for the study, were given nifedipine and were monitored throughout thecourse of treatment. Results: Groups were compared with mean prolongation of delivery, side effects, neonatal outcome, parity, cervicaleffacements. Nifedipine was twice more effective than isoxsuprine hydrochloride as a uterine tocolytic agent whileside effects were comparable with fewer side effects in nifedipine group. Neonatal outcome was better with nifedipinein comparison to isoxsuprine. Conclusions: There is a high incidence of preterm labour in India. The present study found that nifedipine has bettertocolytic efficacy, less side effects and better tolerability as compared to isoxsuprine in third trimester.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Dermal interstitial fluid (ISF) is an underutilized information-rich biofluid potentially useful in health status monitoring applications whose contents remain challenging to characterize. Here, we present a facile microneedle approach for dermal ISF extraction with minimal pain and no blistering for human subjects and rats. Extracted ISF volumes were sufficient for determining transcriptome, and proteome signatures. We noted similar profiles in ISF, serum, and plasma samples, suggesting that ISF can be a proxy for direct blood sampling. Dynamic changes in RNA-seq were recorded in ISF from induced hypoxia conditions. Finally, we report the first isolation and characterization, to our knowledge, of exosomes from dermal ISF. The ISF exosome concentration is 12–13 times more enriched when compared to plasma and serum and represents a previously unexplored biofluid for exosome isolation. This minimally invasive extraction approach can enable mechanistic studies of ISF and demonstrates the potential of ISF for real-time health monitoring applications., Philip Miller et al. present an approach for extracting dermal interstitial fluid (ISF) using an array of hollow microneedles in a cylindrical substrate that minimizes skin compression and tissue damage. They extract larger volumes of ISF suitable for downstream analyses, compared to previous reports.

Emerging sequencing technologies are allowing us to characterize environmental, clinical and laboratory samples with increasing speed and detail, including real-time analysis and interpretation of data. One example of this is being able to rapidly and accurately detect a wide range of pathogenic organisms, both in the clinic and the field. Genomes can have radically different GC content however, such that accurate sequence analysis can be challenging depending upon the technology used. Here, we have characterized the performance of the Oxford MinION nanopore sequencer for detection and evaluation of organisms with a range of genomic nucleotide bias. We have diagnosed the quality of base-calling across individual reads and discovered that the position within the read affects base-calling and quality scores. Finally, we have evaluated the performance of the current state-of-the-art neural network-based MinION basecaller, characterizing its behavior with respect to systemic errors as well as context- and sequence-specific errors. Overall, we present a detailed characterization the capabilities of the MinION in terms of generating high-accuracy sequence data from genomes with a wide range of nucleotide content. This study provides a framework for designing the appropriate experiments that are the likely to lead to accurate and rapid field-forward diagnostics.

Yersinia enterocolitica is typically considered an extracellular pathogen; however, during the course of an infection, a significant number of bacteria are stably maintained within host cell vacuoles. Little is known about this population and the role it plays during an infection. To address this question and to elucidate the spatially and temporally dynamic gene expression patterns of Y. enterocolitica biovar 1B through the course of an in vitro infection, transcriptome sequencing and differential gene expression analysis of bacteria infecting murine macrophage cells were performed under four distinct conditions. Bacteria were first grown in a nutrient-rich medium at 26°C to establish a baseline of gene expression that is unrelated to infection. The transcriptomes of these bacteria were then compared to bacteria grown in a conditioned cell culture medium at 37°C to identify genes that were differentially expressed in response to the increased temperature and medium but not in response to host cells. Infections were then performed, and the transcriptomes of bacteria found on the extracellular surface and intracellular compartments were analyzed individually. The upregulated genes revealed potential roles for a variety of systems in promoting intracellular virulence, including the Ysa type III secretion system, the Yts2 type II secretion system, and the Tad pilus. It was further determined that mutants of each of these systems had decreased virulence while infecting macrophages. Overall, these results reveal the complete set of genes expressed by Y. enterocolitica in response to infection and provide the groundwork for future virulence studies.

Digital microfluidics (DMF) is a powerful technique for sample preparation and analysis for a broad range of biological and chemical applications. In many cases, it is desirable to carry out DMF on an open surface, such that the matrix surrounding the droplets is ambient air. However, the utility of the air-matrix DMF format has been severely limited by problems with droplet evaporation, especially when the droplet-based biochemical reactions require high temperatures for long periods of time. We present a simple solution for managing evaporation in air-matrix DMF: just-in-time replenishment of the reaction volume using droplets of solvent. We demonstrate that this solution enables DMF-mediated execution of several different biochemical reactions (RNA fragmentation, first-strand cDNA synthesis, and PCR) over a range of temperatures (4-95 °C) and incubation times (up to 1 h or more) without use of oil, humidifying chambers, or off-chip heating modules. Reaction volumes and temperatures were maintained roughly constant over the course of each experiment, such that the reaction kinetics and products generated by the air-matrix DMF device were comparable to those of conventional benchscale reactions. This simple yet effective solution for evaporation management is an important advance in developing air-matrix DMF for a wide variety of new, high-impact applications, particularly in the biomedical sciences.

Digital microfluidics (DMF) is a powerful technique for simple and precise manipulation of microscale droplets of fluid. This technique enables processing and analysis of a wide variety of samples and reagents and has proven useful in a broad range of chemical, biological, and medical applications. Handling of "real-world" samples has been a challenge, however, because typically their volumes are greater than those easily accommodated by DMF devices and contain analytes of interest at low concentration. To address this challenge, we have developed a novel "world-to-DMF" interface in which an integrated companion module drives the large-volume sample through a 10 μL droplet region on the DMF device, enabling magnet-mediated recovery of bead-bound analytes onto the device as they pass through the region. To demonstrate its utility, we use this system for extraction of RNA from human whole blood lysates (110-380 μL) and further purification in microscale volumes (5-15 μL) on the DMF device itself. Processing by the system was2-fold faster and consumed 12-fold less reagents, yet produced RNA yields and quality fully comparable to conventional preparations and supporting qRT-PCR and RNA-Seq analyses. The world-to-DMF system is designed for flexibility in accommodating different sample types and volumes, as well as for facile integration of additional modules to enable execution of more complex protocols for sample processing and analysis. As the first technology of its kind, this innovation represents an important step forward for DMF, further enhancing its utility for a wide range of applications.

Here, we present the draft genome sequence of Burkholderia pseudomallei PHLS 6, a virulent clinical strain isolated from a melioidosis patient in Bangladesh in 1960. The draft genome consists of 39 contigs and is 7,322,181 bp long.

Virulence genes on mobile DNAs such as genomic islands (GIs) and plasmids promote bacterial pathogen emergence. Excision is an early step in GI mobilization, producing a circular GI and a deletion site in the chromosome; circular forms are also known for some bacterial insertion sequences (ISs). The recombinant sequence at the junctions of such circles and deletions can be detected sensitively in high-throughput sequencing data, using new computational methods that enable empirical discovery of mobile DNAs. For the rich mobilome of a hospital Klebsiella pneumoniae strain, circularization junctions (CJs) were detected for six GIs and seven IS types. Our methods revealed differential biology of multiple mobile DNAs, imprecision of integrases and transposases, and differential activity among identical IS copies for IS26, ISKpn18 and ISKpn21. Using the resistance of circular dsDNA molecules to exonuclease, internally calibrated with the native plasmids, showed that not all molecules bearing GI CJs were circular. Transpositions were also detected, revealing replicon preference (ISKpn18 prefers a conjugative IncA/C2 plasmid), local action (IS26), regional preferences, selection (against capsule synthesis) and IS polarity inversion. Efficient discovery and global characterization of numerous mobile elements per experiment improves accounting for the new gene combinations that arise in emerging pathogens.

The dual bromodomain protein Brd2 is closely related to the basal transcription factor TAF(II)250, which is essential for cyclin A transactivation and mammalian cell cycle progression. In transgenic mice, constitutive lymphoid expression of Brd2 causes a malignancy most similar to human diffuse large B cell lymphoma. We compare the genome-wide transcriptional expression profiles of these lymphomas with those of proliferating and resting normal B cells. Transgenic tumors reproducibly show differential expression of a large number of genes important for cell cycle control and lymphocyte biology; expression patterns are either tumor-specific or proliferation-specific. Several of their human orthologs have been implicated in human lymphomagenesis. Others correlate with human disease survival time. BRD2 is underexpressed in some subtypes of human lymphoma and these subtypes display a number of similarities to the BRD2-mediated murine tumors. We illustrate with a high degree of detail that cancer is more than rampant cellular proliferation, but involves the additional transcriptional mobilization of many genes, some of them poorly characterized, which show a tumor-specific pattern of gene expression.

Advances in molecular biology, microfluidics, and laboratory automation continue to expand the accessibility and applicability of these methods beyond the confines of conventional, centralized laboratory facilities and into point of use roles in clinical, military, forensic, and field-deployed applications. As a result, there is a growing need to adapt the unit operations of molecular biology (e.g., aliquoting, centrifuging, mixing, and thermal cycling) to compact, portable, low-power, and automation-ready formats. Here we present one such adaptation, the rotary zone thermal cycler (RZTC), a novel wheel-based device capable of cycling up to four different fixed-temperature blocks into contact with a stationary 4-microliter capillary-bound sample to realize 1-3 second transitions with steady state heater power of less than 10 W. We demonstrate the utility of the RZTC for DNA amplification as part of a highly integrated rotary zone PCR (rzPCR) system that uses low-volume valves and syringe-based fluid handling to automate sample loading and unloading, thermal cycling, and between-run cleaning functionalities in a compact, modular form factor. In addition to characterizing the performance of the RZTC and the efficacy of different online cleaning protocols, we present preliminary results for rapid single-plex PCR, multiplex short tandem repeat (STR) amplification, and second strand cDNA synthesis.

Cyclin A is regulated primarily through transcription control during the mammalian cell cycle. A dual mechanism of cyclin A transcriptional repression involves, on the one hand, promoter-bound inhibitory complexes of E2F transcription factors and RB (retinoblastoma) family proteins, and on the other, chromatin-directed histone deacetylase activity that is recruited to the cyclin A promoter early in the cell cycle in association with these RB proteins. This dual regulation maintains transcriptional silence of the cyclin A locus until its transcription is required in S-phase. At that time, RB family members dissociate from E2F proteins and nucleosomal restructuring of the locus takes place, to permit transcriptional activation and resultant S-phase progression to proceed. We have identified a double bromo-domain-containing protein Brd2, which exhibits apparent ‘scaffold’ or transcriptional adapter functions and mediates recruitment of both E2F transcription factors and chromatin-remodelling activity to the cyclin A promoter. We have shown previously that Brd2-containing nuclear, multiprotein complexes contain E2F-1 and -2. In the present study, we show that, in S-phase, they also contain histone H4-directed acetylase activity. Overexpression of Brd2 in fibroblasts accelerates the cell cycle through increased expression of cyclin A and its associated cyclin-dependent kinase activity. Chromatin immunoprecipitation studies show that Brd2 is physically present at the cyclin A promoter and its overexpression promotes increased histone H4 acetylation at the promoter as it becomes transcriptionally active, suggesting a new model for the dual regulation of cyclin A.

In this work, we study the construction of a data gathering tree to maximize the network lifetime, which is defined as the minimum of the lifetime of all the nodes in the sensor network. The problem of finding the optimal data gathering tree is known to be NP-complete [9]. Most works in this area aim to give an heuristic to find a data gathering tree and compare the lifetime obtained with other existing solutions. But it is beyond doubt that best tree with respect to lifetime could be obtained if we can generate all possible spanning trees and choose among them the one giving maximum lifetime. With a sensor network consisting of large number of nodes this approach becomes computationally prohibitive. We have adopted an algorithm to generate all possible spanning trees and modified it to generate only the optimal spanning tree using a branch and bound technique. Here, whenever a partial tree has a node with lifetime less than or equal to the lifetime of a tree generated earlier, that tree is discarded. Simulation results show that the number of complete trees checked by our method is only a small fraction of the total number of possible spanning trees. That way it becomes feasible to find a solution in reasonable time even for large sensor network. We have considered that transmission range of each sensor can be adjusted to be just enough to send a message to its parent. The lifetime obtained by our method is also shown to be better than that obtained by other heuristic algorithm like PEDAP.

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (≥105) in milliliter-scale volumes (≥0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 μl). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

Use of second generation sequencing (SGS) technologies for transcriptional profiling (RNA-Seq) has revolutionized transcriptomics, enabling measurement of RNA abundances with unprecedented specificity and sensitivity and the discovery of novel RNA species. Preparation of RNA-Seq libraries requires conversion of the RNA starting material into cDNA flanked by platform-specific adaptor sequences. Each of the published methods and commercial kits currently available for RNA-Seq library preparation suffers from at least one major drawback, including long processing times, large starting material requirements, uneven coverage, loss of strand information and high cost. We report the development of a new RNA-Seq library preparation technique that produces representative, strand-specific RNA-Seq libraries from small amounts of starting material in a fast, simple and cost-effective manner. Additionally, we have developed a new quantitative PCR-based assay for precisely determining the number of PCR cycles to perform for optimal enrichment of the final library, a key step in all SGS library preparation workflows.

Biological weapons of mass destruction and emerging infectious diseases represent a serious and growing threat to our national security. Effective response to a bioattack or disease outbreak critically depends upon efficient and reliable distinguishing between infected vs healthy individuals, to enable rational use of scarce, invasive, and/or costly countermeasures (diagnostics, therapies, quarantine). Screening based on direct detection of the causative pathogen can be problematic, because culture- and probe-based assays are confounded by unanticipated pathogens (e.g., deeply diverged, engineered), and readily-accessible specimens (e.g., blood) often contain little or no pathogen, particularly at pre-symptomatic stages of disease. Thus, in addition to the pathogen itself, one would like to detect infection-specific host response signatures in the specimen, preferably ones comprised of nucleic acids (NA), which can be recovered and amplified from tiny specimens (e.g., fingerstick draws). Proof-of-concept studies have not been definitive, however, largely due to use of sub-optimal sample preparation and detection technologies. For purposes of pathogen detection, Sandia has developed novel molecular biology methods that enable selective isolation of NA unique to, or shared between, complex samples, followed by identification and quantitation via Second Generation Sequencing (SGS). The central hypothesis of the current study is that variations on this approach willmore » support efficient identification and verification of NA-based host response signatures of infectious disease. To test this hypothesis, we re-engineered Sandia's sophisticated sample preparation pipelines, and developed new SGS data analysis tools and strategies, in order to pioneer use of SGS for identification of host NA correlating with infection. Proof-of-concept studies were carried out using specimens drawn from pathogen-infected non-human primates (NHP). This work provides a strong foundation for large-scale, highly-efficient efforts to identify and verify infection-specific host NA signatures in human populations.« less

Next-generation sequencing (NGS) is emerging as a powerful tool for elucidating genetic information for a wide range of applications. Unfortunately, the surging popularity of NGS has not yet been accompanied by an improvement in automated techniques for preparing formatted sequencing libraries. To address this challenge, we have developed a prototype microfluidic system for preparing sequencer-ready DNA libraries for analysis by Illumina sequencing. Our system combines droplet-based digital microfluidic (DMF) sample handling with peripheral modules to create a fully-integrated, sample-in library-out platform. In this report, we use our automated system to prepare NGS libraries from samples of human and bacterial genomic DNA. E. coli libraries prepared on-device from 5 ng of total DNA yielded excellent sequence coverage over the entire bacterial genome, with >99% alignment to the reference genome, even genome coverage, and good quality scores. Furthermore, we produced a de novo assembly on a previously unsequenced multi-drug resistant Klebsiella pneumoniae strain BAA-2146 (KpnNDM). The new method described here is fast, robust, scalable, and automated. Our device for library preparation will assist in the integration of NGS technology into a wide variety of laboratories, including small research laboratories and clinical laboratories.

Cell membranes are dynamic substrates that achieve a diverse array of functions through multi-scale reconfigurations. We explore the morphological changes that occur upon protein interaction to model membrane systems that induce deformation of their planar structure to yield nanotube assemblies. In the two examples shown in this report we will describe the use of membrane adhesion and particle trajectory to form lipid nanotubes via mechanical stretching, and protein adsorption onto domains and the induction of membrane curvature through steric pressure. Through this work the relationship between membrane bending rigidity, protein affinity, and line tension of phase separated structures were examined and their relationship in biological membranes explored.

We use affinity purification of the double bromodomain protein Brd2 to isolate a multicomponent nuclear complex from cultured cells, and apply mass spectrometry/proteomics methods to identify the participants. We then confirm by immunoblot several transcription co-activators and co-repressors, proteins of the Swi/Snf chromatin remodeling complex, which regulate transcription control of cyclin A. This multiprotein complex is likely to contribute to cell cycle control and play a role in proliferation and cancer.

Transgenic mice with lymphoid-restricted overexpression of the double bromodomain protein bromodomain-containing 2 (Brd2) develop splenic B-cell lymphoma and, upon transplantation, B-cell leukemia with leukemic infiltrates in liver and lung. Brd2 is a nuclear-localized transcription factor kinase that is most closely related to TATA box binding protein–associated factor, 250 kDa (TAFII250) and the Drosophila developmental protein female sterile homeotic. Constitutive expression of BRD2 in the lymphoid compartment increases cyclin A transcription, “priming” transgenic B cells for proliferation. Mice stochastically develop an aggressive B-cell lymphoma with the features of B-1 cells, including CD5 and surface IgM expression. The B-cell lymphoma is monoclonal for immunoglobulin gene rearrangement and is phenotypically stable. The lymphoblasts are very large and express a transcriptome that is similar to human non-Hodgkin lymphomas. Both a wild-type BRD2 transgene and a kinase-null point mutant drive lymphomagenesis; therefore we propose that, rather than kinase activity, Brd2-mediated recruitment of E2 promoter binding factors (E2Fs) and a specific histone acetyltransferase to the cyclinA promoter by both types of transgene is a mechanistic basis for neoplasia. This report is the first to describe a transgenic mouse model for constitutive expression of a protein with more than one bromodomain.

We continue to develop and extend our novel adjuvant therapy approach to lymphoid malignancy. We supplement CHOP with DNA oligonucleotides that mimic the chromosomal telomere, which we call a “T oligo.” These agents are homologous to the 3′ overhang nucleotide sequence of telomeres and have previously been shown to have anti-tumor activity in animal models of malignant melanoma and breast cancer. They are currently being evaluated in melanoma and breast cancer patients. If introduced into human or murine normal, proliferating primary B cells or T cells, T oligo causes transient cell cycle arrest, while exerting no toxicity, but if introduced into human or murine malignant B cells or T cells, the arrest is followed by p53 phosphorylation, p21 message induction and ultimately p53-dependent apoptosis. Other p53-related effector molecules, such as p73, are also likely to be involved. The mechanism immediately suggests a novel method of chemotherapy for leukemia and lymphoma as an adjuvant with CHOP. Furthermore, we have previously shown with in vitro assay and in vivo mouse models of diffuse large B cell lymphoma (DLCL) that T oligo produces a more-than-additive toxicity towards lymphoma cells when combined with vincristine. T oligo alone or in combination with sub-therapeutic doses of CHOP, the standard of care for DLCL, dramatically reduced lymphoma burden in spleen, lung, bone marrow and peritoneum. In combination, which we refer to as T-CHOP, there was a greater reduction in tumor burden than with either therapy alone. We now show in SCID mouse xenograft models of human T cell malignancies, using a Jurkat T cell leukemic line or a MOLT-4 T cell leukemic line that T oligo also works alone to reduce tumor burden dramatically and increase survival. Interestingly, because T oligo-driven apoptosis occurs in p53-null, human lymphoid tumors, even chemotherapy-resistant lymphoid tumors are nevertheless sensitive to T oligo treatment, which may have profound benefit for relapsed leukemia or lymphoma patients.