High-throughput fetal fraction amplification increases analytical performance of noninvasive prenatal screening.

Purpose: The percentage of a maternal cell-free DNA (cfDNA) sample that is fetal-derived (the fetal fraction; FF) is a key driver of the sensitivity and specificity of noninvasive prenatal screening (NIPS). On certain NIPS platforms, >20% of women with high body mass index (and >5% overall) receive a test failure due to low FF (<4%).

Methods: A scalable fetal fraction amplification (FFA) technology was analytically validated on 1264 samples undergoing whole-genome sequencing (WGS)-based NIPS.

Novel MicroRNA Signature to Differentiate Ulcerative Colitis from Crohn Disease: A Genome-Wide Study Using Next Generation Sequencing.

Background: The diagnosis of ulcerative colitis (UC) or Crohn disease (CD) can be challenging given the overlapping features. Knowledge of microRNAs in IBD has expanded recently and supports that microRNAs play an important role. This study aimed to identify novel microRNA biomarkers through comprehensive genome-wide sequencing to distinguish UC from CD.

Fragment Length of Circulating Tumor DNA.

Malignant tumors shed DNA into the circulation. The transient half-life of circulating tumor DNA (ctDNA) may afford the opportunity to diagnose, monitor recurrence, and evaluate response to therapy solely through a non-invasive blood draw. However, detecting ctDNA against the normally occurring background of cell-free DNA derived from healthy cells has proven challenging, particularly in non-metastatic solid tumors.

Digitally guided microdissection aids somatic mutation detection in difficult to dissect tumors.

Molecular genetic testing on formalin fixed, paraffin embedded (FFPE) tumors frequently requires dissection of tumor from tissue sections mounted on glass slides. In a process referred to as "manual macrodissection," the pathologist reviews an H&E stained slide at the light microscope and marks areas for dissection, and then the laboratory performs manual dissection from adjacent sections without the aid of a microscope, using the marked reference H&E slide as a guide. Manual macrodissection may be inadequate for tissue sections with low tumor content.

MicroRNA-146a constrains multiple parameters of intestinal immunity and increases susceptibility to DSS colitis.

Host-microbial interactions within the mammalian intestines must be properly regulated in order to promote host health and limit disease. Because the microbiota provide constant immunological signals to intestinal tissues, a variety of regulatory mechanisms have evolved to ensure proper immune responses to maintain homeostasis. However, many of the genes that comprise these regulatory pathways, including immune-modulating microRNAs (miRNAs), have not yet been identified or studied in the context of intestinal homeostasis.

Novel specific microRNA biomarkers in idiopathic inflammatory bowel disease unrelated to disease activity.

The diagnosis of idiopathic inflammatory bowel disease can be challenging. MicroRNAs (miRNAs) are small, non-coding RNAs that regulate protein synthesis through post-transcriptional suppression. This study is to identify new miRNA markers in inflammatory bowel disease, and to examine whether miRNA biomarkers might assist in the diagnosis of inflammatory bowel disease.

Dynamic linear model for the identification of miRNAs in next-generation sequencing data.

Next-generation sequencing technologies are poised to revolutionize the field of biomedical research. The increased resolution of these data promise to provide a greater understanding of the molecular processes that control the morphology and behavior of a cell. However, the increased amounts of data require innovative statistical procedures that are powerful while still being computationally feasible.

Dicer's helicase domain discriminates dsRNA termini to promote an altered reaction mode.

The role of Dicer's helicase domain is enigmatic, but in vivo it is required for processing certain endogenous siRNA, but not miRNA. By using Caenorhabditis elegans extracts or purified Drosophila Dicer-2 we compared activities of wild-type enzymes and those containing mutations in the helicase domain. We found the helicase domain was essential for cleaving dsRNA with blunt or 5'-overhanging termini, but not those with 3' overhangs, as found on miRNA precursors.

Dicer's helicase domain is required for accumulation of some, but not all, C. elegans endogenous siRNAs.

Years after the discovery that Dicer is a key enzyme in gene silencing, the role of its helicase domain remains enigmatic. Here we show that this domain is critical for accumulation of certain endogenous small interfering RNAs (endo-siRNAs) in Caenorhabditis elegans. The domain is required for the production of the direct products of Dicer, or primary endo-siRNAs, and consequently affects levels of downstream intermediates, the secondary endo-siRNAs.

Genes misregulated in C. elegans deficient in Dicer, RDE-4, or RDE-1 are enriched for innate immunity genes.

We describe the first microarray analysis of a whole animal containing a mutation in the Dicer gene. We used adult Caenorhabditis elegans and, to distinguish among different roles of Dicer, we also performed microarray analyses of animals with mutations in rde-4 and rde-1, which are involved in silencing by siRNA, but not miRNA. Surprisingly, we find that the X chromosome is greatly enriched for genes regulated by Dicer.

Overexpression of groESL in Clostridium acetobutylicum results in increased solvent production and tolerance, prolonged metabolism, and changes in the cell's transcriptional program.

DNA array and Western analyses were used to examine the effects of groESL overexpression and host-plasmid interactions on solvent production in Clostridium acetobutylicum ATCC 824. Strain 824(pGROE1) was created to overexpress the groESL operon genes from a clostridial thiolase promoter. The growth of 824(pGROE1) was inhibited up to 85% less by a butanol challenge than that of the control strain, 824(pSOS95del).

Design of antisense RNA constructs for downregulation of the acetone formation pathway of Clostridium acetobutylicum.

We investigated the effect of antisense RNA (asRNA) structural properties on the downregulation efficacy of enzymes in the acetone-formation pathway (acetoacetate decarboxylase [AADC] and coenzyme A-transferase [CoAT]) of Clostridium acetobutylicum strain ATCC 824. First, we generated three strains, C. acetobutylicum ATCC 824 (pADC38AS), 824(pADC68AS), and 824(pADC100AS), which contain plasmids that produce asRNAs of various lengths against the AADC (adc) transcript.

Northern, morphological, and fermentation analysis of spo0A inactivation and overexpression in Clostridium acetobutylicum ATCC 824.

The Clostridium acetobutylicum ATCC 824 spo0A gene was cloned, and two recombinant strains were generated, an spo0A inactivation strain (SKO1) and an spo0A overexpression strain [824(pMPSOA)]. SKO1 was developed by targeted gene inactivation with a replicative plasmid capable of double-crossover chromosomal integration--a technique never used before with solventogenic clostridia. SKO1 was severely deficient in solvent formation: it produced only 2 mM acetone and 13 mM butanol, compared to the 92 mM acetone and 172 mM butanol produced by the parental strain.

Fermentation characterization and flux analysis of recombinant strains of Clostridium acetobutylicum with an inactivated solR gene.

The effect of solR inactivation on the metabolism of Clostridium acetobutylicum was examined using fermentation characterization and metabolic flux analysis. The solR-inactivated strain (SolRH) of this study had a higher rate of glucose utilization and produced higher solvent concentrations (by 25%, 14%, and 81%, respectively, for butanol, acetone, and ethanol) compared to the wild type. Strain SolRH(pTAAD), carrying a plasmid-encoded copy of the bifunctional alcohol/aldehyde dehydrogenase gene (aad) used in butanol production, produced even higher concentrations of solvents (by 21%, 45%, and 62%, respectively, for butanol, acetone, and ethanol) than strain SolRH.

Metabolic flux analysis elucidates the importance of the acid-formation pathways in regulating solvent production by Clostridium acetobutylicum.

Metabolic flux analysis was used to investigate the roles of the acid formation pathways in Clostridium acetobutylicum. The acid formation pathways were revealed to serve different roles in wildtype fermentations than previously expected. Specifically, enzymes known to catalyze butyrate formation were found to uptake butyrate without concomitant production of acetone.

Characterization of recombinant strains of the Clostridium acetobutylicum butyrate kinase inactivation mutant: need for new phenomenological models for solventogenesis and butanol inhibition?

Two metabolic engineering tools, namely gene inactivation and gene overexpression, were employed to examine the effects of two genetic modifications on the fermentation characteristics of Clostridium acetobutylicum. Inactivation of the butyrate kinase gene (buk) was examined using strain PJC4BK, while the combined effect of buk inactivation and overexpression of the aad gene-encoding the alcohol aldehyde dehydrogense (AAD) used in butanol formation-was examined using strain PJC4BK(pTAAD). The two strains were characterized in controlled pH > or = 5.

Development and characterization of a gene expression reporter system for Clostridium acetobutylicum ATCC 824.

A gene expression reporter system (pHT3) for Clostridium acetobutylicum ATCC 824 was developed by using the lacZ gene from Thermoanaerobacterium thermosulfurogenes EM1 as the reporter gene. In order to test the reporter system, promoters of three key metabolic pathway genes, ptb (coding for phosphotransbutyrylase), thl (coding for thiolase), and adc (coding for acetoacetate decarboxylase), were cloned upstream of the reporter gene in pHT3 in order to construct vectors pHT4, pHT5, and pHTA, respectively. Detection of beta-galactosidase activity in time course studies performed with strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA) demonstrated that the reporter gene produced a functional beta-galactosidase in C.

Expression of Clostridium acetobutylicum ATCC 824 genes in Escherichia coli for acetone production and acetate detoxification.

A synthetic acetone operon (ace4) composed of four Clostridium acetobutylicum ATCC 824 genes (adc, ctfAB, and thl, coding for the acetoacetate decarboxylase, coenzyme A transferase, and thiolase, respectively) under the control of the thl promoter was constructed and was introduced into Escherichia coli on vector pACT. Acetone production demonstrated that ace4 is expressed in E. coli and resulted in the reduction of acetic acid levels in the fermentation broth.

New shuttle vector for cloning in Bacillus stearothermophilus.

Cloning vector plasmid pRP9 was constructed on the basis of the broad host-range plasmid pLM6. pRP9 was a small plasmid (2.9 kb), possessed a convenient polyrestriction site sequence and efficiently transformed Bacillus subtilis, Bacillus stearothermophilus and Escherichia coli.

Expression of cloned homologous fermentative genes in Clostridium acetobutylicum ATCC 824.

We have previously cloned the acetone-formation pathway gene, encoding acetoacetate decarboxylase (adc), and butyrate-formation pathway gene, encoding phosphotransbutyrylase (ptb), of Clostridium acetobutylicum ATCC 824 in Escherichia coli. Here we report their subcloning in Bacillus subtilis and transfer to strain ATCC 824 via electrotransformation, where the corresponding enzyme activities were expressed at elevated levels, using pFNK1, a new B. subtilis/C.

Temperature-induced protein synthesis in Bacillus stearothermophilus NUB36.

Cultures of Bacillus stearothermophilus subjected to a temperature shift-up or shift-down of 15 degrees C within the normal temperature range of growth (45 to 65 degrees C) enter a transient adaptation period before exponential growth at the new temperature. The de novo synthesis of some proteins coincides with the adaptation period.

Cloning and characterization of a glutamine transport operon of Bacillus stearothermophilus NUB36: effect of temperature on regulation of transcription.

We cloned and sequenced a fragment of the Bacillus stearothermophilus NUB36 chromosome that contains two open reading frames (ORFs) whose products were detected only in cells of cultures grown in complex medium at high temperature. The nucleotide sequence of the two ORFs exhibited significant identity to the sequence of the glnQ and glnH loci of the glutamine transport system in enteric bacteria. In addition, growth response to glutamine, sensitivity to the toxic glutamine analog gamma-L-glutamylhydrazide, and glutamine transport assays with parental strain NUB3621 and mutant strain NUB36500, in which the ORF1 coding segment in the chromosome was interrupted with the cat gene, demonstrated that glnQ and glnH encode proteins that are active in the glutamine transport system in B.

Genetic map of the Bacillus stearothermophilus NUB36 chromosome.

A circular genetic map of Bacillus stearothermophilus NUB36 was constructed by transduction with bacteriophage TP-42C and protoplast fusion. Sixty-four genes were tentatively assigned a cognate Bacillus subtilis gene based on growth response to intermediates or end products of metabolism, cross-feeding, accumulation of intermediates, or their relative order in a linkage group. Although the relative position of many genes on the Bacillus stearothermophilus and Bacillus subtilis genetic map appears to be similar, some differences were detected.