Towards accurate exclusion of neonatal bacterial meningitis: a feasibility study of a novel 16S rDNA PCR assay.

BACKGROUND: PCRctic is an innovative assay based on 16S rDNA PCR technology that has been designed to detect a single intact bacterium in a specimen of cerebro-spinal fluid (CSF). The assay's potential for accurate, fast and inexpensive discrimination of bacteria-free CSF makes it an ideal adjunct for confident exclusion of bacterial meningitis in newborn babies where the negative predictive value of bacterial culture is poor. This study aimed to stress-test and optimize PCRctic in the "field conditions" to attain a clinically useful level of specificity. METHODS: The specificity of PCRctic was evaluated in CSF obtained from newborn babies investigated for meningitis on a tertiary neonatal unit. Following an interim analysis, the method of skin antisepsis was changed to increase bactericidal effect, and snap-top tubes (Eppendorf™) replaced standard universal containers for collection of CSF to reduce environmental contamination. RESULTS: The assay's specificity was 90.5% in CSF collected into the snap-top tubes - up from 60% in CSF in the universal containers. The method of skin antisepsis had no effect on the specificity. All CSF cultures were negative and no clinical cases of neonatal bacterial meningitis occurred during the study. CONCLUSIONS: A simple and inexpensive optimization of CSF collection resulted in a high specificity output. The low prevalence of neonatal bacterial meningitis means that a large multi-centre study will be required to validate the assay's sensitivity and its negative predictive value.

Tropomyosins: Potential Biomarkers for Urothelial Bladder Cancer.

Despite the incidence and prevalence of urothelial bladder cancer (UBC), few advances in treatment and diagnosis have been made in recent years. In this review, we discuss potential biomarker candidates: the tropomyosin family of genes, encoded by four loci in the human genome. The expression of these genes is tissue-specific. Tropomyosins are responsible for diverse cellular roles, most notably based upon their interplay with actin to maintain cellular processes, integrity and structure. Tropomyosins exhibit a large variety of splice forms, and altered isoform expression levels have been associated with cancer, including UBC. Notably, tropomyosin isoforms are detectable in urine, offering the potential for non-invasive diagnosis and risk-stratification. This review collates the basic knowledge on tropomyosin and its isoforms, and discusses their relationships with cancer-related phenomena, most specifically in UBC.

Functional Assessment of Clubfoot Associated HOXA9, TPM1, and TPM2 Variants Suggests a Potential Gene Regulation Mechanism.

BACKGROUND: Isolated nonsyndromic clubfoot is a common birth defect affecting 135,000 newborns worldwide each year. Although treatment has improved, substantial long-term morbidity persists. Genetic causes have been implicated in family-based studies but the genetic changes have eluded identification. Previously, using a candidate gene approach in our family-based dataset, we identified associations between clubfoot and four single nucleotide polymorphisms (SNPs) located in potential regulatory regions of genes involved in muscle development and patterning (HOXA9) and muscle function (TPM1 and TPM2) were identified. QUESTIONS/PURPOSES: Four SNPs, rs3801776/HOXA9, rs4075583/TPM1, rs2025126/TPM2, and rs2145925/TPM2, located in potential regulatory regions, were evaluated to determine whether they altered promoter activity. METHODS: Electrophoretic mobility shift assays were performed on these four SNPs to identify allele-specific DNA-protein interactions. SNPs showing differential banding patterns were assessed for effect on promoter activity by luciferase assay. Undifferentiated (for HOXA9) and differentiated (for TPM1 and TPM2) mouse cells were used in functional assays as a proxy for the in vivo developmental stage. RESULTS: Functional analyses showed that the ancestral alleles of rs3801776/HOXA9, rs4075583/TPM1, and rs2025126/TPM2 and the alternate allele of rs2145925/TPM2 created allele-specific nuclear protein interactions and caused higher promoter activity. Interestingly, while rs4075583/TPM1 showed an allele-specific nuclear protein interaction, an effect on promoter activity was observed only when rs4075583/TPM1 was expressed in the 1.7kb haplotype construct. CONCLUSION: Our results show that associated promoter variants in HOXA9, TPM1, and TPM2, alter promoter expression suggesting that they have a functional role. Moreover and importantly, we show that alterations in promoter activity may be observed only in the context of the genomic architecture. Therefore, future studies focusing on proteins binding to these regulatory SNPs may provide important key insights into gene regulation in clubfoot. CLINICAL RELEVANCE: Identifying the genetic risk signature for clubfoot is important to provide accurate genetic counseling for at-risk families, for development of prevention programs and new treatments.

Circulating microRNA as potential diagnostic and prognostic biomarkers of well-differentiated thyroid cancer: A review article.

Half of all people aged 50 and over develop a thyroid nodule in their lifetime, exclusion of cancer is required in each case. Nodule tissue sampling is performed by way of fine needle aspiration biopsy (FNAB), however a definite diagnosis is possible only in 30% of cases. The discovery of a diagnostic biomarker to discriminate between thyroid cancer and benign nodules would therefore greatly improve current clinical practice. Using the databases of Medline, Embase and Pubmed we identified 21 original research papers examining various microRNA as potential biomarkers. Currently, the most evidence supporting diagnostic utility exists for miRNA-222. It has been shown repeatedly to have potential in diagnosis of PTC & MTC as well as being linked with the most prognostic factors of all microRNA. To a lesser extent, evidence seems to support the diagnostic and prognostic utility of miR-146b, Let-7 family, miR-221 for PTC and miR-21 for PTC & FTC. MicroRNA appear to show promise as potential diagnostic and prognostic biomarkers, however there is still not enough data to produce a consensus. Continued research should be undertaken with streamlined protocols.

Functional structure of the promoter regions for the predominant low molecular weight isoforms of tropomyosin in human kidney cells.

High and low molecular weight (LMW) tropomyosin isoforms, by regulation of actin filaments, have a major role in the regulation of cell behaviour. They affect malignant transformation, motility, differentiation, metastasis and cell membrane protein presentation. Expression of LMW isoforms from the TPM1 and TPM3 genes have an important role in these effects but the regulation of their expression is unknown. Luciferase assays on a progressively truncated 1.7 kb fragment upstream of the exon 1b translation start site in the TPM1 and TPM3 genes in HEK-293 cells showed upstream activation sequences in TPM1 between -152 and -139 bp and in TPM3 between -154 and -102 bp. The effect of mutating candidate transcription factor binding sites identified an AML1-like transcription factor binding site in TPM1 and a cAMP response element in TPM3. Downstream from the primary activation sequence in TPM1 was a repressor region corresponding to two Sp/KLF family binding GC boxes. Band shift assays confirmed that deletion of these sites altered transcription factor binding and ChIP assays confirmed the presence of AML1 and CREB at the TPM1 and TPM3 activation sequences in the respective promoters. Expression of LMW isoforms from TPM1 and TPM3 genes is regulated very differently. This facilitates regulation of the many cell processes involving these proteins. In situations where these proteins have a critical role, such as cancer metastasis, it also facilitates specific intervention.

Prospective cohort study of induction of labor: Indications, outcome and postpartum hemorrhage.

INTRODUCTION: This study was undertaken because of the increasing rate of induction of labor (IOL) in our hospital and its associated higher caesarean section (CS) rates. The objective was to ascertain the incidence, indications, methods, outcome, and complications of IOL, in particular postpartum hemorrhage. METHODS: This was a prospective observational cohort study of women who underwent IOL in a medium-sized district general hospital. Blood loss was measured by the gravimetric method and correlated to postpartum hemoglobin level within 48 hours of birth. RESULTS: A total of 445 women needed IOL (incidence 33%). Common indications were: small for gestational age (SGA) or fetal growth restriction (FGR) (18%), spontaneous rupture of membrane (17%), reduced fetal movement (16%), prolonged pregnancy (15%), and diabetes (13%). In all, 67% women achieved spontaneous vaginal delivery and 18% underwent caesarean section. With regard to blood loss, 62 women (14%) had postpartum hemorrhage (PPH) of >1000 mL and 22 women (4.9%) had a blood loss >1500 mL. The caesarean section rate was higher than the overall emergency caesarean section rate in that year. Incidence of PPH in this cohort was higher than the normal incidence. CONCLUSIONS: Increasing trend of induction of labor (IOL) is due to the changing clinical policy on management of small for gestational age babies, spontaneous rupture of membrane, reduced fetal movement and other complications of pregnancy. There is conflicting evidence on the effect of IOL on caesarean section rates. IOL is a risk factor for PPH. Accurate measurement of blood loss is essential in detecting a fall in hemoglobin which in turn helps in the appropriate management of PPH.

Inflammatory Adipokines Decrease Expression of Two High Molecular Weight Isoforms of Tropomyosin Similar to the Change in Type 2 Diabetic Patients.

Cardiovascular disease and cancer are increased in Type 2 diabetes. TPM1 and TPM4 genes encode proteins associated with cardiovascular and neoplastic disease. High (HMW) and low (LMW) molecular weight isoforms from TPM1 and TPM4 are altered in several cancer cells and the 3'UTR of TPM1 mRNA is tumour suppressive. Leukocytes influence cardiovascular and neoplastic disease by immunosurveillance for cancer and by chronic inflammation in Type 2 diabetes and cardiovascular disease. The aim was to determine changes in expression of isoforms from TPM1 and TPM4 genes in leukocytes from Type 2 diabetic patients and to use the leukocyte cell line THP1 to identify possible mediators of changes in the patients. Gene expression was determined by RT-qPCR. In diabetes, expression of HMW isoforms from TPM1 were markedly decreased (0.55 v 1.00; p = 0.019) but HMW isoforms from TPM4 were not significantly different (0.76 v 1.00; p = 0.205). Within individual variance in expression of HMW isoforms was very high. The change in expression in HMW isoforms from TPM1 and TPM4 was replicated in THP1 cells treated with 1 ng/ml TNFα (0.10 and 0.12 v 1.00 respectively) or 10 ng/ml IL-1α (0.17 and 0.14 v 1.00 respectively). Increased insulin or glucose concentrations had no substantial effects on TPM1 or TPM4 expression. Decreased TPM1 mRNA resulted in decreases in HMW protein levels. Expression of HMW isoforms from TPM1 is decreased in Type 2 diabetes. This is probably due to increased levels of inflammatory cytokines TNFα and IL-1α in Type 2 diabetes. Lower levels of TPM1 mRNA reduce tumour suppression and could contribute to increased cancer risk in Type 2 diabetes. Decreased HMW tropomyosin isoforms are associated with cancer. Decreased HMW isoforms give rise to cells that are more plastic, motile, invasive and prone to dedifferentiation resulting in leukocytes that are more invasive but less functionally effective.

Polymorphisms in the tropomyosin TPM1 short isoform promoter alter gene expression and are associated with increased risk of metabolic syndrome.

BACKGROUND: Inflammation contributes to the development of atherosclerotic lesions in the metabolic syndrome. Tropomyosin isoform expression is altered in this disease and has a role in inflammatory cell plasticity, motility, and insulin sensitivity. We determined the frequency of haplotype carriage of three single-nucleotide polymorphisms (SNPs) in the short isoform promoter of the TPM1 gene in 300 normal controls and 500 metabolic syndrome patients. The effect of each haplotype on tropomyosin gene expression was assessed. METHODS: PCR-restriction fragment length polymorphism assays were developed for each polymorphism. Promoter activity was measured using luciferase assays in the insulin-sensitive human embryonic kidney (HEK) 293 and the monocyte THP-1 lines. RESULTS: The SNPs -111(T/C), -426(T/C), and -491(A/G), relative to the TPM1 short isoform transcription start site, occurred in haplotypes ATT, GCT, GTT, and GTC, and were in strong linkage disequilibrium. ATT had a frequency of 66%. The presence of -491G, which conforms to a predicted binding site for transcription factor AML-1, caused a decrease in gene expression of 24% in the HEK 293 cells. In the THP-1 cells, haplotypes GTC and GTT gave 24% lower expression, whereas haplotype GCT gave expression at wild-type levels. The carriage of a -491G allele gave an odds ratio of 1.4 (95% CI 1.02-1.8) for the metabolic syndrome (P < 0.03). CONCLUSIONS: A polymorphism in the TPM1 short isoform promoter region is predicted to alter transcription factor binding, alters gene expression and is associated with the metabolic syndrome. This could affect inflammatory cells and cytoskeleton-mediated insulin signaling.