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High-grade serous ovarian cancers (HGSOCs) with homologous recombination deficiency (HRD) are initially responsive to poly (ADP-ribose) polymerase inhibitors (PARPi), but resistance ultimately emerges. HGSOC with CCNE1 amplification (CCNE1 amp) are associated with resistance to PARPi and platinum treatments. High replication stress in HRD and CCNE1 amp HGSOC leads to increased reliance on checkpoint kinase 1 (CHK1), a key regulator of cell cycle progression and the replication stress response. Here, we investigated the anti-tumor activity of the potent, highly selective, orally bioavailable CHK1 inhibitor (CHK1i), SRA737, in both acquired PARPi-resistant BRCA1/2 mutant and CCNE1 amp HGSOC models. We demonstrated that SRA737 increased replication stress and induced subsequent cell death in vitro. SRA737 monotherapy in vivo prolonged survival in CCNE1 amp models, suggesting a potential biomarker for CHK1i therapy. Combination SRA737 and PARPi therapy increased tumor regression in both PARPi-resistant and CCNE1 amp patient-derived xenograft models, warranting further study in these HGSOC subgroups.

Gestational diabetes mellitus (GDM) is a condition of pregnancy defined by new-onset hyperglycemia. GDM is associated with impaired maternal endothelial and vascular reactivity. Endothelin-1 (ET-1) is a potent vasoconstrictor that contributes to endothelial dysfunction, however, its abundance and actions in GDM are unclear. Maternal plasma was obtained from pregnancies complicated by GDM (n = 24) and gestation-matched controls (n = 42); circulating ET-1 levels were assessed by ELISA. Human omental arteries from healthy pregnancies and those complicated by GDM were dissected from omental fat biopsies and collected at cesarean section. mRNA expression of ET-1 and its receptors, ETA and ETB, in addition to vascular cell adhesion molecule-1 (VCAM1) and intercellular adhesion molecule-1 (ICAM1) were assessed by qPCR (n = 28). Using wire myography, we investigated vascular constriction to ET-1 (10-11-10-4 M) in omental arteries from pregnancies complicated by GDM, compared to gestation-matched controls (n = 7). GDM cases were stratified by clinical management, diet intervention (n = 5), or insulin treatment (n = 6). Additionally, arteries from healthy pregnancies were treated with insulin (1 mU/mL (n = 7) and 10 mU/mL (n = 5)) or vehicle control. Vasoactive response to ET-1 was measured via wire myography. Circulating ET-1 levels and mRNA expression of the ET-1 system in omental arteries were not found to be significantly different between pregnancies complicated by GDM compared to healthy controls. However, we found insulin treatment during pregnancy and in ex vivo models reduced ET-1 vasoconstriction of maternal vasculature in GDM. These data suggest insulin may improve vascular function in GDM, however, further investigation is needed to define the role of ET-1 in pregnancy.

Spontaneous preterm birth is the leading cause of perinatal morbidity and mortality. Tocolytics are drugs used in cases of imminent preterm birth to inhibit uterine contractions. Nifedipine is a calcium channel blocking agent used to delay threatened spontaneous preterm birth, however, has limited efficacy and lacks preclinical data regarding mechanisms of action. It is unknown if nifedipine affects the pro-inflammatory environment associated with preterm labour pathophysiology and we hypothesise nifedipine only targets myometrial contraction rather than also mitigating inflammation. We assessed anti-inflammatory and anti-contractile effects of nifedipine on human myometrium using in vitro and ex vivo techniques, and a mouse model of preterm birth. We show that nifedipine treatment inhibited contractions in myometrial in vitro contraction assays (P = 0.004 vs. vehicle control) and potently blocked spontaneous and oxytocin-induced contractions in ex vivo myometrial tissue in muscle myography studies (P = 0.01 vs. baseline). Nifedipine treatment did not reduce gene expression or protein secretion of pro-inflammatory cytokines in either cultured myometrial cells or ex vivo tissues. Although nifedipine could delay preterm birth in some mice, this was not consistent in all dams and was overall not statistically significant. Our data suggests nifedipine does not modulate preterm birth via inflammatory pathways in the myometrium, and this may account for its limited clinical efficacy.

BACKGROUND: High-grade serous tubo-ovarian cancer (HGSC) is the most common histological subtype of epithelial ovarian cancer, and highly lethal. Currently there is no effective screening test and prognosis is poor as the majority of cases are diagnosed at the advanced stage. Cell free RNAs including microRNAs (miRNAs) are dysregulated in ovarian cancer tissue and are detectable in the circulation. This study aimed to determine whether circulating cell free miRNAs may be potential biomarkers for the detection of HGSC. METHODS: Plasma was collected from women with HGSC (Grade 3, n = 24), and benign ovarian masses (n = 24). RNA was extracted from patient plasma and subjected to miRNA targeted next generation sequencing (NGS). A subsequent validation cohort was assessed using plasma collected from women with HGSC (n = 14) and controls (with a benign ovarian mass; n = 15). RNA was extracted and assessed using quantitative RT-PCR. RESULTS: Differential gene expression (DGE) of the NGS data revealed a significant increase in the miRNA, miR200c, in the circulation of women with HGSC (p less than 0.05) compared to controls. In the validation cohort miR200c expression by qPCR was found to also be increased in the circulation of women with HGSC compared to controls (p = 0.0023). CONCLUSIONS: Circulating miR200c may be a promising candidate biomarker for the detection of HGSC. Further larger cohort studies exploring earlier stages are needed to determine whether circulating miR200c may be a sensitive and specific biomarker of tubo-ovarian cancer.

Previously, we identified elevated transcripts for the gene Oleoyl-ACP Hydrolase (OLAH) in the maternal circulation of pregnancies complicated by preterm fetal growth restriction. As placental dysfunction is central to the pathogenesis of both fetal growth restriction and preeclampsia, we aimed to investigate OLAH levels and function in the human placenta. We assessed OLAH mRNA expression (qPCR) throughout pregnancy, finding placental expression increased as gestation progressed. OLAH mRNA and protein levels (Western blot) were elevated in placental tissue from cases of preterm preeclampsia, while OLAH protein levels in placenta from growth-restricted pregnancies were comparatively reduced in the preeclamptic cohort. OLAH expression was also elevated in placental explant tissue, but not isolated primary cytotrophoblast cultured under hypoxic conditions (as models of placental dysfunction). Further, we discovered that silencing cytotrophoblast OLAH reduced the expression of pro- and anti-apoptosis genes, BAX and BCL2, placental growth gene, IGF2, and oxidative stress gene, NOX4. Collectively, these findings suggest OLAH could play a role in placental dysfunction and may be a therapeutic target for mitigating diseases associated with this vital organ. Further research is required to establish the role of OLAH in the placenta, and whether these changes may be a maternal adaptation or consequence of disease.

New-onset maternal hypertension is a hallmark of preeclampsia, driven by widespread endothelial dysfunction and systemic vasoconstriction. Here, we set out to create a new ex vivo model using preeclamptic serum to cause injury to the endothelium, mimicking vascular dysfunction in preeclampsia and offering the potential to evaluate candidate therapeutic interventions. Human omental arteries were collected at caesarean section from normotensive pregnant patients at term (n = 9). Serum was collected from pregnancies complicated by preterm preeclampsia (birth < 34 weeks’ gestation, n = 16), term preeclampsia (birth > 37 weeks’ gestation, n = 5), and healthy gestation-matched controls (preterm n = 16, term n = 12). Using wire myography, we performed ex vivo whole vessel assessment where human omental arteries were treated with increasing doses of each serum treatment (2−20%) and vasoreactivity was assessed. All pregnant serum treatments successfully drove vasoconstriction; no significant difference was observed in the degree of vasoconstriction when exposed to preeclamptic or control serum. We further demonstrated the ability of esomeprazole (a candidate therapeutic for preeclampsia; 0.1−100 µM) to drive vasorelaxation of pre-constricted vessels (only with serum from preeclamptic patients). In summary, we describe a novel human physiological model of preeclamptic vascular constriction. We demonstrate its exciting potential to screen drugs for their therapeutic potential as treatment for vasoconstriction induced by preeclampsia.

Preeclampsia is a devastating, multisystem disorder of pregnancy. It has no cure except delivery, which if premature can impart significant neonatal morbidity. Efforts to repurpose pregnancy-safe therapeutics for the treatment of preeclampsia have led to the assessment of the proton pump inhibitor, esomeprazole. Preclinically, esomeprazole reduced placental secretion of anti-angiogenic sFlt-1, improved endothelial dysfunction, promoted vasorelaxation, and reduced maternal hypertension in a mouse model. Our understanding of the precise mechanisms through which esomeprazole works to reduce endothelial dysfunction and enhance vasoreactivity is limited. Evidence from earlier studies suggested esomeprazole might work via the nitric oxide pathway, upregulating endothelial nitric oxide synthase (eNOS). Here, we investigated the effect of esomeprazole in a mouse model of L-NAME-induced hypertension (decreased eNOS activity). We further antagonised the model by addition of diet-induced obesity, which is relevant to both preeclampsia and the nitric oxide pathway. Esomeprazole did not decrease blood pressure in this model, nor were there any alterations in vasoreactivity or changes in foetal outcomes in lean mice. We observed similar findings in the obese mouse cohort, except esomeprazole treatment enhanced ex vivo acetylcholine-induced vasorelaxation. As acetylcholine induces nitric oxide production, these findings hint at a function for esomeprazole in the nitric oxide pathway.

Previously, we demonstrated that the proton pump inhibitor, esomeprazole magnesium hydrate (MH), could have potential as a repurposed treatment against preeclampsia, a serious obstetric condition. In this study we investigate the difference in the preclinical effectiveness between 100 µM of esomeprazole MH and its hydration isomer, esomeprazole magnesium trihydrate (MTH). Here, we found that both treatments reduced secretion of sFLT-1 (anti-angiogenic factor) from primary cytotrophoblast, but only esomeprazole MH reduced sFLT-1 secretion from primary human umbilical vein endothelial cells (assessed via ELISA). Both drugs could mitigate expression of the endothelial dysfunction markers, vascular cell adhesion molecule-1 and endothelin-1 (via qPCR). Neither esomeprazole MH nor MTH quenched cytotrophoblast reactive oxygen species production in response to sodium azide (ROS assay). Finally, using wire myography, we demonstrated that both compounds were able to induce vasodilation of human omental arteries at 100 µM. Esomeprazole is safe to use in pregnancy and a candidate treatment for preeclampsia. Using primary human tissues and cells, we validated that esomeprazole is effective in enhancing vascular relaxation, and can reduce key factors associated with preeclampsia, including sFLT-1 and endothelial dysfunction. However, esomeprazole MH was more efficacious than esomeprazole MTH in our in vitro studies.

BACKGROUND: Dishevelled Associated Activator Of Morphogenesis 2 (DAAM2) levels are elevated in the maternal circulation and placenta in pregnancies complicated by fetal growth restriction. However, placental DAAM2 levels in cases of preeclampsia have not previously been explored. Here, we examined placental DAAM2 in pregnancies complicated by preterm preeclampsia, and whether candidate preeclampsia therapeutics altered its expression. METHODS: DAAM2 mRNA and protein levels were assessed in placental tissue from cases of preterm preeclampsia and gestation-matched controls (delivering ≤ 34 weeks; qPCR and western blot respectively). Short interfering RNAs were used to silence DAAM2 in isolated primary cytotrophoblast under normoxic (8 % O2) and hypoxic (1 % O2) conditions, and expression of anti-angiogenic sFLT-1, angiogenic PGF, antioxidant, fetal growth, and inflammatory genes assessed. DAAM2 expression was measured in placental explant tissue from pregnancies complicated by preeclampsia, treated with three proton pump inhibitors (100 µM esomeprazole, lansoprazole, and rabeprazole). RESULTS: DAAM2 expression was significantly reduced in preeclamptic placental tissue compared to controls, but protein production was unchanged. Silencing DAAM2 in hypoxic cytotrophoblast increased sFLT-i13 isoform expression, but did not alter sFLT-e15a or PGF expression, or sFLT-1 secretion. DAAM2 knockdown did not alter expression of antioxidant (NQO-1, TXN, GCLC), fetal growth (SPINT1), or inflammasome (NLRP3) genes. Esomeprazole and lansoprazole, but not rabeprazole, increased DAAM2 expression in placental explant tissue from cases of preeclampsia. CONCLUSION: Placental DAAM2 protein is not significantly altered in placental tissue in cases of preeclampsia, and its suppression does not alter sFLT-1 secretion. Hence, placental DAAM2 is unlikely to drive the pathogenesis associated with preeclampsia.

BACKGROUND: Congenital cytomegalovirus infection is the most common perinatal infection and a significant cause of sensorineural hearing loss, cerebral palsy, and neurodevelopmental disability. There is a paucity of human gene expression studies examining the pathophysiology of cytomegalovirus infection. OBJECTIVE: This study aimed to perform a whole transcriptomic assessment of amniotic fluid from pregnancies with live fetuses to identify differentially expressed genes and enriched Gene Ontology categories associated with congenital cytomegalovirus infection. STUDY DESIGN: Amniotic fluid supernatant was prospectively collected from pregnant women undergoing amniocentesis for suspected congenital cytomegalovirus infection because of first-trimester maternal primary infection or ultrasound features suggestive of fetal infection. Women who had received therapy to prevent fetal infection were excluded. Congenital cytomegalovirus infection was diagnosed via viral polymerase chain reaction of amniotic fluid; cytomegalovirus-infected fetuses were paired with noninfected controls, matched for gestational age and fetal sex. Paired-end RNA sequencing was performed on amniotic fluid cell-free RNA with the Novaseq 6000 at a depth of 30 million reads per sample. Following quality control and filtering, reads were mapped to the human genome and counts summarized across genes. Differentially expressed genes were identified using 2 approaches: voomWithQualityWeights in conjunction with limma and RUVSeq with edgeR. Genes with a false discovery rate <0.05 were considered statistically significant. Differential exon use was analyzed using DEXSeq. Functional analysis was performed using gene set enrichment analysis and Ingenuity Pathway Analysis. Manual curation of differentially regulated genes was also performed. RESULTS: Amniotic fluid samples were collected from 50 women; 16 (32%) had congenital cytomegalovirus infection confirmed by polymerase chain reaction. After excluding 3 sa

Preeclampsia is a multi-system disease that can have severe, even fatal implications for the mother and fetus. Abnormal placentation can lead to ischaemic tissue injury and placental inflammation. In turn, the placenta releases anti-angiogenic factors into the maternal circulation. These systemically act to neutralise angiogenic factors causing endothelial dysfunction causing preeclampsia. Hydroxychloroquine is an immune modulating drug that is considered safe in pregnancy. There is epidemiological evidence suggesting it may reduce the risk of preeclampsia. Here, we examined the effects hydroxychloroquine on the production and secretion of sFlt-1, soluble endoglin (sENG), placental growth factor (PlGF) and vascular endothelial growth factor (VEGF) in primary human placenta, cytotrophoblasts and umbilical vein endothelial cells (endothelial cell model). Hydroxychloroquine treatment decreased mRNA expression of two sFlt-1 isoforms and its protein secretion. sENG was not reduced. Hydroxychloroquine treatment increased secretion of pro-angiogenic factor PIGF from endothelial cells. It did not significantly reduce the expression of the endothelial cell inflammation marker, ET-1, and inflammation induced expression of the adhesion molecule, VCAM. Hydroxychloroquine could not overcome leukocyte adhesion to endothelial cells. Hydroxychloroquine mitigates features of preeclampsia, but it does not reduce key markers of endothelial dysfunction.

BACKGROUND: Metformin, widely used to treat diabetes, is now considered a candidate therapeutic for treatment of cardiovascular disease. This study aimed to assess whether metformin's non-glycaemic effects could mitigate cardiovascular disease indices in female mice consuming a high fat diet (HFD). METHODS: Four-week old female Arc:Arc(S) mice were placed on a standard (std) chow diet or Western-style HFD (22% fat, 0.15% cholesterol). At ∼8 months, the mice were administered 150 mg/kg metformin or vehicle (control) via intraperitoneal injection for 11 days. Blood pressure was measured (tail cuff plethysmography) at Day 9 and 11 of treatment. On Day 11, mice were weighed and culled. The mesenteric arcade and kidneys were collected for assessment of vascular reactivity (wire myography), and assessment of expression of cardiometabolic markers (qPCR), respectively. RESULTS: The HFD fed female mice were significantly heavier than those receiving the std diet at 1-12 weeks on diet, and at cull. Mice on a std diet with metformin treatment were significantly heavier at cull than the mice on a std diet administered the control treatment. Metformin treatment did not alter the weight of the mice receiving the HFD. Neither the HFD (compared to the std diet), nor metformin treatment (compared to control treatment) altered blood pressure, vascular reactivity, or expression of cardiometabolic markers in the kidney. CONCLUSION: Consumption of a Western-style HFD (without high salt/sugar levels) did not alter the cardiovascular markers measured. Further studies are required to establish the non-glycaemic, cardio-protective effects of metformin in high-risk cohorts.

Previously, we identified increased maternal circulating DAAM2 mRNA in pregnancies complicated by preterm fetal growth restriction (FGR). Here, we assessed whether circulating DAAM2 mRNA could detect FGR, and whether the DAAM2 gene, known to play roles in the Wnt signalling pathway is expressed in human placenta and associated with dysfunction and FGR. We performed linear regression analysis to calculate area under the ROC curve (AUC) for DAAM2 mRNA expression in the maternal circulation of pregnancies complicated by preterm FGR. DAAM2 mRNA expression was assessed across gestation by qPCR. DAAM2 protein and mRNA expression was assessed in preterm FGR placenta using western blot and qPCR. DAAM2 expression was assessed in term cytotrophoblasts and placental explant tissue cultured under hypoxic and normoxic conditions by qPCR. Small interfering RNAs were used to silence DAAM2 in term primary cytotrophoblasts. Expression of growth, apoptosis and oxidative stress genes were assessed by qPCR. Circulating DAAM2 mRNA was elevated in pregnancies complicated by preterm FGR [p < 0.0001, AUC = 0.83 (0.78–0.89)]. Placental DAAM2 mRNA was detectable across gestation, with highest expression at term. DAAM2 protein was increased in preterm FGR placentas but demonstrated no change in mRNA expression. DAAM2 mRNA expression was increased in cytotrophoblasts and placental explants under hypoxia. Silencing DAAM2 under hypoxia decreased expression of pro-survival gene, BCL2 and oxidative stress marker, NOX4, whilst increasing expression of antioxidant enzyme, HMOX-1. The increased DAAM2 associated with FGR and hypoxia implicates a potential role in placental dysfunction. Decreasing DAAM2 may have cytoprotective effects, but further research is required to elucidate its role in healthy and dysfunctional placentas.

Objectives: The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is upregulated in the maternal vasculature in preeclampsia, and contributes to oxidative stress and endothelial dysfunction. However, its function in the placenta is unclear. This paper investigated LOX-1 expression in models of placental dysfunction and preeclampsia, and whether candidate therapeutics for preeclampsia could alter its expression. Study design: Placentas were collected from preterm pregnancies and cases of preterm preeclampsia and fetal growth restriction. Blood was collected from participants whose pregnancies were complicated by preterm fetal growth restriction and/or preeclampsia. Primary cytotrophoblast and placental explant tissue were cultured under hypoxic (1% O2) or normoxic (8% O2) conditions. Cytotrophoblast were exposed to 10% preeclamptic or control serum. Cytotrophoblast and preeclamptic explant tissue were treated with 100 µM esomeprazole, lansoprazole or rabeprazole. Main outcome measures: LOX-1 expression was assessed in all samples via qPCR. Results: LOX-1 expression was reduced in placentas from cases of preterm preeclampsia, but not fetal growth restriction, compared to controls. LOX-1 expression was reduced in cytotrophoblast under hypoxia, but not in explant tissue. Treatment with preeclamptic serum in vitro did not alter cytotrophoblast LOX-1 expression. Circulating LOX-1 mRNA was unaltered in patients with fetal growth restriction, preeclampsia, and fetal hypoxia, compared to controls. Treatment with esomeprazole or lansoprazole in vitro increased placental LOX-1 expression. Conclusions: LOX-1 expression is reduced in preeclamptic placentas and hypoxic cytotrophoblast. Esomeprazole and lansoprazole increase placental LOX-1 expression. These findings demonstrate a role for LOX-1 in the placenta, and improve our understanding of maternal adaptations in pregnancy complications.

Background: Fetuses affected by placental insufficiency do not receive adequate nutrients and oxygenation, become growth restricted and acidemic, and can demise. Preterm fetal growth restriction is a severe form of placental insufficiency with a high risk of stillbirth. We set out to identify maternal circulating mRNA transcripts that are differentially expressed in preterm pregnancies complicated by very severe placental insufficiency, in utero fetal acidemia, and are at very high risk of stillbirth. Methods: We performed a cohort study across six hospitals in Australia and New Zealand, prospectively collecting blood from 128 pregnancies complicated by preterm fetal growth restriction (delivery < 34 weeks' gestation) and 42 controls. RNA-sequencing was done on all samples to discover circulating mRNAs associated with preterm fetal growth restriction and fetal acidemia in utero. We used RT-PCR to validate the associations between five lead candidate biomarkers of placental insufficiency in an independent cohort from Europe (46 with preterm fetal growth restriction) and in a third cohort of pregnancies ending in stillbirth. Results: In the Australia and New Zealand cohort, we identified five mRNAs that were highly differentially expressed among pregnancies with preterm fetal growth restriction: NR4A2, EMP1, PGM5, SKIL, and UGT2B1. Combining three yielded an area under the receiver operative curve (AUC) of 0.95. Circulating NR4A2 and RCBTB2 in the maternal blood were dysregulated in the presence of fetal acidemia in utero. We validated the association between preterm fetal growth restriction and circulating EMP1, NR4A2, and PGM5 mRNA in a cohort from Europe. Combining EMP1 and PGM5 identified fetal growth restriction with an AUC of 0.92. Several of these genes were differentially expressed in the presence of ultrasound parameters that reflect placental insufficiency. Circulating NR4A2, EMP1, and RCBTB2 mRNA were differentially regulated in another cohort destined for

Acidithiobacillus ferrooxidans is an acidophilic bacterium able to grow in environments with high concentrations of metals. It is a chemolithoautotroph able to form biofilms on the surface of solid minerals to obtain its energy. The response of both planktonic and sessile cells of A. ferrooxidans ATCC 23270 grown in elemental sulfur and adapted to high copper concentration was analyzed by quantitative proteomics. It was found that 137 proteins varied their abundance when comparing both lifestyles. Copper effllux proteins, some subunits of the ATP synthase complex, porins, and proteins involved in cell wall modification increased their abundance in copper-adapted sessile lifestyle cells. On the other hand, planktonic copper-adapted cells showed increased levels of proteins such as: cupreredoxins involved in copper cell sequestration, some proteins related to sulfur metabolism, those involved in biosynthesis and transport of lipopolysaccharides, and in assembly of type IV pili. During copper adaptation a decreased formation of biofilms was measured as determined by epifluorescence microscopy. This was apparently due not only to a diminished number of sessile cells but also to their exopolysaccharides production. This is the first study showing that copper, a prevalent metal in biomining environments causes dispersion of A. ferrooxidans biofilms.