A study on early-onset neonatal group B streptococcal infection, Bulgaria, 2007-2011.

This study examines neonatal group B streptococcal (GBS) colonization and its relation to early-onset GBS disease (EOGBSD), based upon the experience of leading obstetrics and gynecology centers in Bulgaria. The objectives of the study were to update neonatal colonization rates and to assess relationships between clinically differentiated cases (culture-proven GBS newborns) and risk factors inherent to the infant and mother, using a computerized file. The neonatal GBS colonization rate ranged from 5.48 to 12.19 per 1000 live births. Maternal-fetal infection (MFI, a provisional clinical diagnosis in culture-proven colonized infants with initial signs of infection that is usually overcome with antibiotic treatment) and/or intrapartum asphyxia (IA) have been demonstrated as the most frequent clinical manifestations, with significant correlations for the primary diagnosis, but not affirmative for the final diagnosis at discharge, resulting from adequate treatment of neonates. MFI and IA were significantly related to prematurity, and reciprocally, prematurity was associated with the risk of MFI, indirectly suggesting that preterm birth or PPROM (preterm premature rupture of membranes, an obstetric indication associated with early labor and delivery, one of the major causes of preterm birth) is a substantial risk factor for EOGBSD. The regression analysis indicated that in the case of a newborn with MFI, a birth weight 593.58 g lower than the birth weight of an infant without this diagnosis might be expected. Testing the inverse relationship, i.e., the way birth weight influences a certain diagnosis (logistic regression) established the presence of a relationship between birth weight categories (degree of prematurity) and the diagnosis of MFI. The proportions and odds ratios, converted into probabilities that a baby would develop MFI, indicate the particularly high risk for newborns with extremely low and very low birth weight: extremely low birth weight (≤1000 g), the probability of developing a MFI is 66%; very low birth weight (1001-1500 g), 81%; low birth weight (the birth weight category including premature and small for gestational age term infants: 1501-2500 g), 40%; normal birth weight (term infants) (>2500 g), 32%. In conclusion, the need to introduce separate categories for early- and late-onset GBS disease in the registration nomenclature of neonatal infectious diseases is highlighted by these results. Drawing up intrapartum antibiotic prophylaxis (IAP) guidelines is also strongly recommended.

An invasive cleavage assay for direct quantitation of specific RNAs.

RNA quantitation is becoming increasingly important in basic, pharmaceutical, and clinical research. For example, quantitation of viral RNAs can predict disease progression and therapeutic efficacy. Likewise, gene expression analysis of diseased versus normal, or untreated versus treated, tissue can identify relevant biological responses or assess the effects of pharmacological agents. As the focus of the Human Genome Project moves toward gene expression analysis, the field will require a flexible RNA analysis technology that can quantitatively monitor multiple forms of alternatively transcribed and/or processed RNAs (refs 3,4). We have applied the principles of invasive cleavage and engineered an improved 5'-nuclease to develop an isothermal, fluorescence resonance energy transfer (FRET)-based signal amplification method for detecting RNA in both total RNA and cell lysate samples. This detection format, termed the RNA invasive cleavage assay, obviates the need for target amplification or additional enzymatic signal enhancement. In this report, we describe the assay and present data demonstrating its capabilities for sensitive (<100 copies per reaction), specific (discrimination of 95% homologous sequences, 1 in > or =20,000), and quantitative (1.2-fold changes in RNA levels) detection of unamplified RNA in both single- and biplex-reaction formats.

RNA template-dependent 5' nuclease activity of Thermus aquaticus and Thermus thermophilus DNA polymerases.

DNA replication and repair require a specific mechanism to join the 3'- and 5'-ends of two strands to maintain DNA continuity. In order to understand the details of this process, we studied the activity of the 5' nucleases with substrates containing an RNA template strand. By comparing the eubacterial and archaeal 5' nucleases, we show that the polymerase domain of the eubacterial enzymes is critical for the activity of the 5' nuclease domain on RNA containing substrates. Analysis of the activity of chimeric enzymes between the DNA polymerases from Thermus aquaticus (TaqPol) and Thermus thermophilus (TthPol) reveals two regions, in the "thumb" and in the "palm" subdomains, critical for RNA-dependent 5' nuclease activity. There are two critical amino acids in those regions that are responsible for the high activity of TthPol on RNA containing substrates. Mutating glycine 418 and glutamic acid 507 of TaqPol to lysine and glutamine, respectively, increases its RNA-dependent 5' nuclease activity 4-10-fold. Furthermore, the RNA-dependent DNA polymerase activity is controlled by a completely different region of TaqPol and TthPol, and mutations in this region do not affect the 5' nuclease activity. The results presented here suggest a novel substrate binding mode of the eubacterial DNA polymerase enzymes, called a 5' nuclease mode, that is distinct from the polymerizing and editing modes described previously. The application of the enzymes with improved RNA-dependent 5' nuclease activity for RNA detection using the invasive signal amplification assay is discussed.

Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes.

Flap endonucleases (FENs) isolated from archaea are shown to recognize and cleave a structure formed when two overlapping oligonucleotides hybridize to a target DNA strand. The downstream oligonucleotide probe is cleaved, and the precise site of cleavage is dependent on the amount of overlap with the upstream oligonucleotide. We have demonstrated that use of thermostable archaeal FENs allows the reaction to be performed at temperatures that promote probe turnover without the need for temperature cycling. The resulting amplification of the cleavage signal enables the detection of specific DNA targets at sub-attomole levels within complex mixtures. Moreover, we provide evidence that this cleavage is sufficiently specific to enable discrimination of single-base differences and can differentiate homozygotes from heterozygotes in single-copy genes in genomic DNA.

Role of Ca2+ in the early stages of murine adipocyte differentiation as evidenced by calcium mobilizing agents.

We have investigated the effect of the endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin and the calcium ionophore A23187 on the differentiation of 3T3-L1 preadipocytes. Treatment of 3T3-L1 preadipocytes with either agent resulted in a dose-dependent inhibition of adipocyte differentiation. The cells accumulated neither fat droplets nor the adipocyte-specific mRNAs encoding stearoyl-CoA desaturase 1 (SCD1) and adipocyte-P2 (aP2). These late markers of differentiation were specifically affected, because thapsigargin and A23187 did not inhibit the expression of beta-tubulin mRNA. No inhibition of differentiation or the expression of the mRNAs occurred when the drugs were added either prior to or 2 days after the initiation of differentiation. Thapsigargin and A23187 were also shown to dramatically block cell proliferation and DNA replication, which occur early in differentiation. Furthermore, during the first 48 h, thapsigargin and A23187 mediated an elevated and prolonged expression of the immediate-early gene corresponding to c-myc, and altered intracellular levels of calcium. Our results suggests that changes in intracellular calcium levels elicited by thapsigargin and A23187 prevent differentiation of 3T3-L1 preadipocytes into adipocytes by blocking the postconfluent mitotic phase of the differentiation process and also by mediating c-myc gene expression.

A model cell line to study regulation of stearoyl-CoA desaturase gene 1 expression by insulin and polyunsaturated fatty acids.

Insulin and polyunsaturated fatty acids (PUFAs) regulate the expression of SCD1 gene in mouse liver. Accordingly, we examined the insulin and PUFA regulation of SCD1 gene expression in H2.35 cells. The levels of SCD1 mRNA in H2.35 cells increased at the restrictive temperature of 39 degrees C, when the glucose-containing medium was supplemented with insulin. The insulin-stimulated expression of SCD1 mRNA was significantly blunted when the induction medium was supplemented with linolenic acid (18:2n-3) and arachidonic acid (20:4n-6). Stearic acid (18:0n-9) and oleic acid (18:1n-9) were without dramatic effects. The effect of insulin and PUFAs on a transfected SCD1 fusion gene (SCD1.CAT4.3) was also examined in H2.35 cells. Whereas insulin stimulated SCD1.CAT4.3 expression, arachidonic acid significantly decreased SCD1.CAT4.3 activity. These studies suggest that insulin and PUFAs regulate SCD1 gene transcription via regulatory DNA sequences flanking the 5' end of the gene.

A novel cis-acting element required for lipopolysaccharide-induced transcription of the murine interleukin-1 beta gene.

Regulatory elements important for transcription of the murine interleukin-1 beta (IL-1 beta) gene lie within a DNase I-hypersensitive region located > 2,000 bp upstream from the transcription start site. We have identified within this region a novel positive regulatory element that is required for activation of an IL-1 beta promoter-chloramphenicol acetyltransferase (CAT) fusion gene in the murine macrophage line RAW264.7. Electrophoretic mobility shift analysis of the 3' portion (-2315 to -2106) of the hypersensitive region revealed at least two nuclear factor binding sites, one of which is located between positions -2285 and -2256. Competitive inhibition studies localized the binding site to a 15-bp sequence between -2285 and -2271. Nuclear factor binding was lost by mutation of the 6-bp sequence from -2280 to -2275. The specific retarded complex formed with RAW264.7 nuclear extract was not detected under similar conditions with nuclear extracts from RLM-11, a murine T-cell line which does not express IL-1 beta RNA. Mutation of the 6-bp sequence (-2280 to -2275) in the chimeric IL-1 beta promoter -4093 +I CAT plasmid virtually eliminated the activation of this reporter gene by lipopolysaccharide (LPS) in transfected RAW264.7 cells. Multimerization of the 15-bp sequence containing the core wild-type 6-bp sequence 5' of minimal homologous or heterologous promoters in CAT reporter plasmids resulted in significant enhancement of CAT expression compared with parallel constructs containing the mutant 6-bp core sequence. This element was LPS independent and position and orientation dependent. The multimerized 15-bp sequence did not enhance expression in RLM-11 cells. Methylation interference revealed contact residues from -2281 to -2271, CCAAAAAGGAA. Because a search of the NIH TFD data bank with the 11-bp binding site sequence found no homology to known nuclear factor binding sites, we have designated this sequence the IL1 beta -upstream nuclear factor 1 (IL1 beta -UNF1) target. UV cross-linking and sodium dodecyl sulfate-polyacrylamide electrophoresis identified an IL1 beta -UNF1-specific binding factor approximately 85 to 90 kDa in size.

Upstream NFIL-6-like site located within a DNase I hypersensitivity region mediates LPS-induced transcription of the murine interleukin-1 beta gene.

To define the cis-acting elements that regulate LPS-stimulated IL-1 beta gene transcription, we analyzed the murine IL-1 beta gene by digestion with DNase I. At least two hypersensitive sites were located between 2200 and 2600 bp upstream of the transcription start site in mononuclear phagocytes, but not in an IL-1 nonproducing immature T cell line. Specific DNA sequences required for LPS induction of IL-1 beta gene expression were identified within the DNase I hypersensitive (DH) region using transfection of reporter constructs that contained portions of the IL-1 beta 5'-flanking region. Two specific DNA sequences were targets for nuclear factor binding as assessed with use of electrophoretic mobility shift analysis (EMSA). One site contained a consensus sequence for NFIL-6 binding. Base substitutions within this NFIL-6 site resulted in virtual elimination of LPS-induced IL-1 beta gene transcription. Introduction of multimers of the NFIL-6-like sequence immediately 5' to homologous or heterologous promoters conferred LPS-induced transcription, indicating that this NFIL-6-like consensus site was a transcriptional activator. Anti-C/EBP beta (NFIL-6) and anti-C/EBP delta (NFIL-6 beta) Abs identified both of these proteins in complexes formed between the NFIL-6-like element and mononuclear cell nuclear extracts. C/EBP delta (NFIL-6 beta) was not detected in complexes utilizing extracts from the IL-1 nonproducing T cell line. These data are consistent with the requirement for C/EBP beta (NFIL-6) and C/EBP delta (NFIL-6 beta) in the activation of murine IL-1 beta gene expression by endotoxin.

An NFIL-6 sequence near the transcriptional initiation site is necessary for the lipopolysaccharide induction of murine interleukin-1 beta.

We have analyzed the interleukin-1 beta (IL-1 beta) promoter region near the cap site. Specific DNA sequences required for lipopolysaccharide (LPS) induction within this region were identified using transfection of reporter plasmids that contained portions of the proximal IL-1 beta 5'-flanking sequence. An LPS-responsive activation area was localized between nucleotides -50 to -100, and down-regulating sequences were present between nucleotides -100 and -2,111. A NFIL-6 site between -92 and -84 was identified in the functionally active region. Base substitutions within this single NFIL-6 site in the context of a 4.1-kb IL-1 beta promoter segment resulted in dramatic reduction of LPS-induced gene transcription. Introduction of multimers of this NFIL-6 sequence immediately 5' to minimal homologous or heterologous promoters conferred LPS inducibility in each case. Anti-C/EBP beta (NFIL-6) and anti-C/EBP delta (NFIL-6 beta) antibodies identified both of these proteins in complexes formed between the NFIL-6 site and mononuclear cell nuclear extracts. These data show that the proximal NFIL-6 site is required for the activation of murine IL-1 beta gene expression by endotoxin.

Identification of genes involved in utilization of acetate and acetoin in Bacillus subtilis.

The Bacillus subtilis ccpA gene has previously been shown to be involved in repression of amyE expression when cells are grown in excess glucose. The region of the B. subtilis chromosome downstream from ccpA was characterized to determine if additional genes involved in carbohydrate metabolism were present. Two open reading frames that exhibited sequence similarity to the Escherichia coli and B. subtilis motA and motB motility genes were found immediately downstream from ccpA; disruption of this region had no effect on growth, sporulation or motility. Two divergent transcriptional units containing the acsA and acuABC genes were also found in this region. The acsA gene encodes acetyl-CoA synthetase, and inactivation of this gene resulted in loss of the ability to utilize acetate as a carbon source for growth or sporulation. Disruption of the acuABC genes resulted in poor growth or sporulation on acetoin or butanediol. The acsA and acuABC promoter sequences were identified by primer extension, and are in close proximity. Two sequences resembling the amyO regulatory target site necessary for glucose repression of amyE were identified in the acsA-acuABC promoter regions.