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1.  Decreased myometrial p160 ROCK-1 expression in obese women at term pregnancy  
Background
Obesity is becoming an increasing problem in obstetric practice; it has led to an increase in the risk of caesarean delivery, prolonged pregnancy and dysfunctional labour. It has been postulated that many of these problems are as a result of abnormal myometrial contractility. The RhoA/Rho kinase pathway is involved in calcium sensitisation in the myometrium during labour and contributes to the phosphorylation of myosin phosphatase and thus continued myosin light chain activity, during uterine contractility. The aim of this study therefore, was to investigate the effect of obesity on the expression of various components of the RhoA/ROCK pathway in human myometrium at term pregnancy.
Methods
Protein was isolated from myometrial biopsies obtained at elective caesarean section, at term pregnancy from obese women and from those with a normal body mass index. Western blotting was performed using specific primary antibodies to RhoA/ Rho kinase associated proteins.
Results
The protein expression of p160 ROCK-1 was significantly decreased (P < 0.001) in the myometrium from women in the obese cohort (n = 22) at term pregnancy, compared to women of those of normal body mass index (n = 15). No alteration in expression of the other proteins investigated was noted.
Conclusions
The significant decrease in p160 ROCK-1 protein expression observed in the myometrium of obese women at late gestation may contribute to an inhibitory effect on contractility at labour, due to its contribution to calcium sensitisation and possibly other signalling pathways. These findings are relevant to the concept of compromised myometrial function in obese parturients.
doi:10.1186/1477-7827-11-79
PMCID: PMC3765888  PMID: 23948067
p160 ROCK 1 protein; Myometrium; Obesity; Term pregnancy
2.  Novel Approaches for Treating Musculoskeletal Diseases: Molecular Orthopedics and Systems Medicine 
Molecular medicine uses knowledge about cell structure and function for disease, diagnostics, stage characterisation and treatment. The advent of genomic technologies is considerably leading to developments in the field of molecular medicine. The accumulation of detailed information about gene expression, epigenetic variability, protein transcription and functional modulation is contributing to a new era in medicine. Rapid and early diagnostic procedures, molecular characterisation of degenerative and proliferative diseases and personalized therapies are predicted to lead to advancements in health prevention and treatment of disease. Diagnostic tools and therapies based on local and /or general modulation of cellular processes for traumatic or degenerative musculoskeletal conditions are becoming available. A logical consequence of the information derived from extensive data gathering, systems biology and systemic medicine has lead to significant improvements in understanding biological structure and function in a simultaneous bottom top and integrative, holistic manner. The description of disease mechanism at an intimate, subcellular level has a dual benefit. A thorough understanding of the crosstalk involved in molecular pathways both in the normal and the diseased state are expanding scientific knowledge and simultaneously are enabling design cell-targeted and individualized therapies. This paper presents a brief overview of current molecular based treatments available to the orthopedic surgeon and introduces the concept of systemic medicine from the perspective of musculoskeletal pathology.
doi:10.2174/1874325001307010144
PMCID: PMC3664448  PMID: 23798982
Systems biology; systems medicine; molecular biomarkers; gene therapy.
3.  Investigating a cluster of vulvar cancer in young women: a cross-sectional study of genital human papillomavirus prevalence 
BMC Infectious Diseases  2012;12:243.
Background
Vulvar cancer is a relatively rare malignancy, which occurs most often in postmenopausal women. We have previously identified a geographic cluster of vulvar cancer in young Indigenous women living in remote communities in the Arnhem Land region of Australia. In this population, we investigated the prevalence of oncogenic human papillomavirus (HPV) infection in anogenital samples (vulvar/vaginal/perianal area and cervix) and compared the overall, type-specific and multiple infection prevalence between sites.
Methods
A cross-sectional survey of 551 Indigenous women aged 18–60 years was undertaken in 9 Arnhem Land communities. Women were consented for HPV detection and genotyping collected by a combined vulvar/vaginal/perianal (VVP) sweep swab and a separate PreservCyt endocervical sample collected during Pap cytology screening. HPV DNA testing was undertaken using PCR with broad spectrum L1 consensus PGMY09/11 primers with genotyping of positive samples by Roche Linear Array. The primary outcomes were the prevalence of cervical and VVP high-risk (HR) HPV.
Results
The prevalence of VVP HR-HPV was 39%, which was significantly higher than the cervical HR-HPV prevalence (26%, p<0.0001). HPV-16 was the most common genotype detected in both sites (VVP 11%, cervical 6%). HPV-16 infection peaked in women aged <20 years; however, there was a marked decline in cervical HPV-16 prevalence with age (p=0.007), whereas following an initial decline, the prevalence of VVP HPV-16 remained constant in subsequent age-groups (p=0.835).
Conclusions
In this population experiencing a cluster of vulvar cancer, the prevalence of cervical oncogenic HPV infection was similar to that reported by studies of other Australian women; however there was a significantly higher prevalence of vulvar/vaginal/perianal infection to cervical. The large discrepancy in HPV prevalence between anogenital sites in this population may represent more persistent infection at the vulva. This needs further investigation, including the presence of possible environmental and/or genetic factors that may impair host immunity.
doi:10.1186/1471-2334-12-243
PMCID: PMC3507832  PMID: 23040203
Human papillomavirus; Population prevalence; Vulvar neoplasms; Young women; Indigenous women
5.  Uterine contractility of plants used to facilitate childbirth in Nigerian ethnomedicine 
Journal of Ethnopharmacology  2012;143(1):377-382.
Ethnopharmacological relevance
Pregnant women in Nigeria use plant preparations to facilitate childbirth and to reduce associated pain. The rationale for this is not known and requires pharmacological validation.
Aim of study
Obtain primary information regarding the traditional use of plants and analyze their uterine contractility at cellular level.
Materials and methods
Semi-structured, open interviews using questionnaires of traditional healthcare professionals and other informants triggered the collection and identification of medicinal plant species. The relative traditional importance of each medicinal plant was determined by its use-mention index. Extracts of these plants were analyzed for their uterotonic properties on an in vitro human uterine cell collagen model.
Result
The plants Calotropis procera, Commelina africana, Duranta repens, Hyptis suaveolens, Ocimum gratissimum, Saba comorensis, Sclerocarya birrea, Sida corymbosa and Vernonia amygdalina were documented and characterized. Aqueous extracts from these nine plants induced significant sustained increases in human myometrial smooth muscle cell contractility, with varying efficiencies, depending upon time and dose of exposure.
Conclusion
The folkloric use of several plant species during childbirth in Nigeria has been validated. Seven plants were for the first time characterized to have contractile properties on uterine myometrial cells. The results serve as ideal starting points in the search for safe, longer lasting, effective and tolerable uterotonic drug leads.
Graphical abstract
Pregnant woman in Nigeria rely on traditional herbal medicine to induce or ease labor, and to treat childbirth-related complications. Nine plant species have been documented and characterized for their uterotonic properties.
doi:10.1016/j.jep.2012.06.042
PMCID: PMC3430860  PMID: 22766472
UM, use-mentions; hTERT-HM, human uterine myometrial smooth muscle cells; Maternal healthcare; Uterus contractility; Labor; Postpartum care
6.  Uterotonic Plants and their Bioactive Constituents 
Planta medica  2010;77(3):207-220.
Abnormalities in the process of uterine muscle contractility during pregnancy and birth can have major clinical implications, including preterm labour, which is the single largest cause of maternal and prenatal mortality in the Western world and a major contributor to childhood developmental problems. In contrast, induction of labour may be necessary in certain conditions. Currently used interventional therapies to suppress (tocolytic agents) or to induce (uterotonic agents) uterine contractions lack potency and/or selectivity and can have harmful side effects for mother and baby. Nature’s diversity has always been, and still is, one of the biggest resources of therapeutic lead compounds. Many natural products exhibit biological activity against unrelated targets, thus providing researchers with starting points for drug development. In this review we will provide an overview of uterine muscle physiology, describe currently available biological screening procedures for testing of uterotonic plant compounds and will summarise traditionally-used uterotonic plants, their active components and their mechanisms, primarily focusing on uterotonic active circular plant peptides called cyclotides. Finally we will comment on the discovery of novel cyclotide-producing plant species and the possibility for the development of novel plant-derived uterotonic and tocolytic drugs.
doi:10.1055/s-0030-1250317
PMCID: PMC3407953  PMID: 20845261
women’s health; gynaecology; labour; myometrial smooth muscle; oxytocin; cyclotides; plants
8.  Ghrelin in the human myometrium 
Background
Ghrelin is a 28-amino acid octanolyated peptide, synthesised primarily in the stomach. It stimulates growth hormone release, food intake and exhibits many other diverse effects. Our group have previously determined that ghrelin inhibited human contractility in vitro. The aim of this study therefore, was to investigate the expression of ghrelin, its receptor, the growth hormone secretagogue receptor type 1 (GHS-R1), ghrelin O-acyltransferase (GOAT) which catalyses ghrelin octanoylation, prohormone convertase 1/3 (PC1/3) responsible for pro-ghrelin processing, in human myometrium, during pregnancy prior to labour, during labour and in the non-pregnant state. Modulation of ghrelin and ghrelin receptor expression in cultured myometrial cells was also investigated.
Methods
mRNA and protein were isolated from human myometrium and the myometrial smooth muscle cell line hTERT-HM; and real-time fluorescence RT-PCR, western blotting and fluorescence microscopy performed. The effects of β-Estradiol and bacterial lipopolysaccharide (LPS) on hTERT-HM gene expression were evaluated by western blotting.
Results
We have reported for the first time the expression and processing of ghrelin, GHS-R1, GOAT and PC1/3 expression in human myometrium, and also the down-regulation of ghrelin mRNA and protein expression during labour. Furthermore, GHS-R1 protein expression significantly decreased at labour. Myometrial GOAT expression significantly increased during term non-labouring pregnancy in comparison to both non-pregnant and labouring myometrium. Mature PC1/3 protein expression was significantly decreased at term pregnancy and labour in comparison to non-pregnant myometrium. Ghrelin, GHS-R1, GOAT and PC1/3 mRNA and protein expression was also detected in the hTERT-HM cells. Ghrelin protein expression decreased upon LPS treatment in these cells while β-Estradiol treatment increased GHS-R1 expression.
Conclusions
Ghrelin processing occurred in the human myometrium at term pregnancy and in the non-pregnant state. GOAT expression which increased during term non-labouring pregnancy demonstrating a similar expression pattern to prepro-ghrelin and GHS-R1, decreased at labour, signifying possible myometrial ghrelin acylation. Moreover, the presence of PC1/3 may contribute to pro-ghrelin processing. These results along with the previous in vitro data suggest that myometrially-produced and processed ghrelin plays a significant autocrine or paracrine role in the maintenance of relaxation in this tissue during pregnancy. Furthermore, the significant uterine modulators LPS and β-Estradiol are involved in the regulation of ghrelin and ghrelin receptor expression respectively, in the human myometrium.
doi:10.1186/1477-7827-8-55
PMCID: PMC2887880  PMID: 20509935
9.  Modulation of human uterine smooth muscle cell collagen contractility by thrombin, Y-27632, TNF alpha and indomethacin 
Background
Preterm labour occurs in approximately 10% of pregnancies and is a major cause of infant morbidity and mortality. However, the pathways involved in regulating contractility in normal and preterm labour are not fully elucidated. Our aim was to utilise a human myometrial contractility model to investigate the effect of a number of uterine specific contractility agents in this system. Therefore, we investigated the contractile response of human primary uterine smooth muscle cells or immortalised myometrial smooth muscle cells cultured within collagen lattices, to known mediators of uterine contractility, which included thrombin, the ROCK-1 inhibitor Y-27632, tumour necrosis factor alpha (TNF alpha) and the non-steroidal anti-inflammatory indomethacin.
Methods
Cell contractility was calculated over time, with the collagen gel contraction assay, utilising human primary uterine smooth muscle cells (hUtSMCs) and immortalised myometrial smooth muscle cells (hTERT-HM): a decrease in collagen gel area equated to an increase in contractility. RNA was isolated from collagen embedded cells and gene expression changes were analysed by real time fluorescence reverse transcription polymerase chain reaction. Scanning electron and fluorescence microscopy were employed to observe cell morphology and cell collagen gel interactions. Statistical analysis was performed using ANOVA followed by Tukey's post hoc tests.
Results
TNF alpha increased collagen contractility in comparison to the un-stimulated collagen embedded hUtSMC cells, which was inhibited by indomethacin, while indomethacin alone significantly inhibited contraction. Thrombin augmented the contractility of uterine smooth muscle cell and hTERT-HM collagen gels, this effect was inhibited by the thrombin specific inhibitor, hirudin. Y-27632 decreased both basal and thrombin-induced collagen contractility in the hTERT-HM embedded gels. mRNA expression of the thrombin receptor, F2R was up-regulated in hUtSMCs isolated from collagen gel lattices, following thrombin-stimulated contractility.
Conclusion
TNF alpha and thrombin increased uterine smooth muscle cell collagen contractility while indomethacin had the opposite effect. Thrombin-induced collagen contractility resulted in F2R activation which may in part be mediated by the ROCK-1 pathway. This study established the in vitro human myometrial model as a viable method to assess the effects of a range of uterotonic or uterorelaxant agents on contractility, and also permits investigation of the complex regulatory pathways involved in mediating myometrial contractility at labour.
doi:10.1186/1477-7827-7-2
PMCID: PMC2645409  PMID: 19133144
10.  Expression of RHOGTPase regulators in human myometrium 
Background
RHOGTPases play a significant role in modulating myometrial contractility in uterine smooth muscle. They are regulated by at least three families of proteins, RHO guanine nucleotide exchange factors (RHOGEFs), RHOGTPase-activating proteins (RHOGAPs) and RHO guanine nucleotide inhibitors (RHOGDIs). RHOGEFs activate RHOGTPases from the inactive GDP-bound to the active GTP-bound form. RHOGAPs deactivate RHOGTPases by accelerating the intrinsic GTPase activity of the RHOGTPases, converting them from the active to the inactive form. RHOGDIs bind to GDP-bound RHOGTPases and sequester them in the cytosol, thereby inhibiting their activity. Ezrin-Radixin-Moesin (ERM) proteins regulate the cortical actin cytoskeleton, and an ERM protein, moesin (MSN), is activated by and can also activate RHOGTPases.
Methods
We therefore investigated the expression of various RHOGEFs, RHOGAPs, a RHOGDI and MSN in human myometrium, by semi-quantitative reverse transcription PCR, real-time fluorescence RT-PCR, western blotting and immunofluorescence microscopy. Expression of these molecules was also examined in myometrial smooth muscle cells.
Results
ARHGEF1, ARHGEF11, ARHGEF12, ARHGAP5, ARHGAP24, ARHGDIA and MSN mRNA and protein expression was confirmed in human myometrium at term pregnancy, at labour and in the non-pregnant state. Furthermore, their expression was detected in myometrial smooth muscle cells. It was determined that ARHGAP24 mRNA expression significantly increased at labour in comparison to the non-labour state.
Conclusion
This study demonstrated for the first time the expression of the RHOGTPase regulators ARHGEF1, ARHGEF11, ARHGEF12, ARHGAP5, ARHGAP24, ARHGDIA and MSN in human myometrium, at term pregnancy, at labour, in the non-pregnant state and also in myometrial smooth muscle cells. ARHGAP24 mRNA expression significantly increased at labour in comparison to the non-labouring state. Further investigation of these molecules may enable us to further our knowledge of RHOGTPase regulation in human myometrium during pregnancy and labour.
doi:10.1186/1477-7827-6-1
PMCID: PMC2254629  PMID: 18190708
11.  Cloning and Characterization of a cDNA Encoding a Novel Extracellular Peroxidase from Trametes versicolor 
The white rot basidiomycete Trametes versicolor secretes a large number of peroxidases which are believed to be involved in the degradation of polymeric lignin. These peroxidases have been classified previously as lignin peroxidases or manganese peroxidases (MnP). We have isolated a novel extracellular peroxidase-encoding cDNA sequence from T. versicolor CU1, the transcript levels of which are repressed by low concentrations of Mn2+ and induced by nitrogen and carbon but not induced in response to a range of stresses which have been reported to induce MnP expression.
PMCID: PMC91187  PMID: 10049906
12.  Detection of Enteropathogenic Escherichia coli in Fecal Cultures by Use of a Modified Fluorescent-Antibody Technique 
Journal of Bacteriology  1965;89(3):570-573.
Martin, Alice J. (Nassau County Department of Health, Hempstead, N.Y.), and Margaret O'Brien. Detection of enteropathogenic Escherichia coli in fecal cultures by use of a modified fluorescent-antibody technique. J. Bacteriol. 89:570–573. 1965.—The application of the fluorescent-antibody (FA) technique to cultures of feces on blood-agar plates for the detection of enteropathogenic Escherichia coli serotypes is described. The results of a study of 364 fecal specimens, examined by both this modified technique and conventional cultural methods, are reported; the findings by the two methods are compared. The modified FA method offers several advantages over other techniques. The results of the examination can be reported sooner; more serotypes are discovered; it is simpler, easier, and quicker to prepare and examine growth from the blood plate than to examine fecal material; and the results are more definite. The results obtained by use of this modified FA procedure have shown that infection with multiple serotypes occurs much more frequently than cultural examinations alone indicate. Intensive cultural studies suggest that the inability to confirm serotypes which have been found by the FA method is probably due to inefficiencies in the cultural method.
PMCID: PMC277504  PMID: 14273630

Results 1-12 (12)