Characterization of a chromatin-associated TCF7L1 complex in human embryonic stem cells.

Human embryonic stem cells (hESCs) resemble the pluripotent epiblast cells found in the early postimplantation human embryo and represent the "primed" state of pluripotency. One factor that helps primed pluripotent cells retain pluripotency and prepare genes for differentiation is the transcription factor TCF7L1, a member of a small family of proteins known as T cell factors/Lymphoid enhancer factors (TCF/LEF) that act as downstream components of the WNT signaling pathway. Transcriptional output of the WNT pathway is regulated, in part, by the activity of TCF/LEFs in conjunction with another component of the WNT pathway, ß-CATENIN. Because TCF7L1 plays an important role in regulating pluripotency, we began to characterize the protein complex associated with TCF7L1 when bound to chromatin in hESCs using rapid immunoprecipitation of endogenous proteins (RIME).  Data are available via ProteomeXchange with identifier PXD047582. These data identify known and new partners of TCF7L1 on chromatin and provide novel insights into how TCF7L1 and pluripotency itself might be regulated.

TCF7L1 suppresses primitive streak gene expression to support human embryonic stem cell pluripotency.

Human embryonic stem cells (hESCs) are exquisitely sensitive to WNT ligands, which rapidly cause differentiation. Therefore, hESC self-renewal requires robust mechanisms to keep the cells in a WNT inactive but responsive state. How they achieve this is largely unknown. We explored the role of transcriptional regulators of WNT signaling, the TCF/LEFs. As in mouse ESCs, TCF7L1 is the predominant family member expressed in hESCs. Genome-wide, it binds a gene cohort involved in primitive streak formation at gastrulation, including NODAL, BMP4 and WNT3 Comparing TCF7L1-bound sites with those bound by the WNT signaling effector ß-catenin indicates that TCF7L1 acts largely on the WNT signaling pathway. TCF7L1 overlaps less with the pluripotency regulators OCT4 and NANOG than in mouse ESCs. Gain- and loss-of-function studies indicate that TCF7L1 suppresses gene cohorts expressed in the primitive streak. Interestingly, we find that BMP4, another driver of hESC differentiation, downregulates TCF7L1, providing a mechanism of BMP and WNT pathway intersection. Together, our studies indicate that TCF7L1 plays a major role in maintaining hESC pluripotency, which has implications for human development during gastrulation.

Monitoring for valproate and phenytoin toxicity in hypoalbuminaemia: A retrospective cohort study.

AIMS: Equations to calculate albumin-adjusted total concentrations have been validated to correlate with measured free concentrations for both phenytoin and valproate, but there is a lack of data to assess correlation with clinical outcomes. We aimed to assess the association of hypoalbuminaemia and albumin-adjusted total concentrations with concentration-dependent toxicity for phenytoin and valproate and review the impact on management decisions following concentration monitoring in hypoalbuminaemia. METHODS: Patients undergoing concentration monitoring for phenytoin or valproate between January and December 2018 were included. Patients were identified using a centralised laboratory database with data extracted from medical records. RESULTS: Total phenytoin concentrations were measured for 144 patients, with hypoalbuminaemia (≤30 g L-1 ) recorded in 59 (41%) patients. Albumin-adjusted phenytoin concentration >20 mg L-1 was associated with increased neurological adverse effects (77% vs. 43%, P < .001). On logistic regression, higher albumin-adjusted phenytoin concentration was an independent risk factor for neurotoxicity (OR 1.06, 95% CI 1.01-1.12, P = .011). Total valproate concentrations were measured for 383 patients, with hypoalbuminaemia (≤30 g L-1 ) noted in 53 (14%) patients. For the valproate cohort, hypoalbuminaemia (42% vs. 28%, P = .039) and albumin-adjusted valproate concentration >100 mg L-1 (49% vs. 23%, P < .001) were both associated with increased neurotoxicity. On multiple logistic-regression, valproate daily dose (aOR = 1.01, 95% CI 1.00-1.02, P = .006) and albumin-adjusted valproate concentration (aOR 1.01, 95% CI 1.00-1.02, P = .033) were independent risk factors for neurotoxicity after accounting for confounders. CONCLUSION: While measuring free drug concentrations in hypoalbuminaemia would be ideal, the adjustment equations can help identify vulnerable patients needing further assessment of potential concentration-dependent toxicity.

Distribution and Function of Prostaglandin E2 Receptors in Mouse Uterus: Translational Value for Human Reproduction.

Prostaglandin (PG) E analogs are used clinically to ripen the cervix and induce labor. However, selective receptor agonists may have potential to improve induction response rates or manage unwanted uterine hypercontractility in conditions such as dysmenorrhea and preterm labor. To characterize their therapeutic value, PGE2 analogs were used to investigate the functional E-type prostanoid (EP) receptor population in isolated human uterus. Responsiveness in mouse tissues was also examined to validate its use as a preclinical model. Uterine samples were obtained from mice at dioestrus (n = 12), term gestation (n = 14), and labor (n = 12) and from the lower uterus of women undergoing hysterectomy (n = 12) or Caesarean section (n = 18). Vehicle and agonist effects were assessed using superfusion and immersion techniques. PGE2 evoked predominant excitatory responses in mouse and relaxation in human tissues. Selective EP4 agonists inhibited tissue activity in both nonpregnant species, while the EP2 mimetic CP533536 also attenuated uterine contractions throughout gestation. The uterotonic effects of the EP3/1 agonist sulprostone were more pronounced than the EP1 agonist ONO-D1-004, corresponding to abundant EP3 receptor expression in all samples. The contractile phenotype in mouse compared with human uteri may relate to regional differences as well as high expression of EP3 receptor transcripts. Similarities in nonpregnant and gestational tissues across species suggest that EP3 may represent a valuable translational drug target for preventing uterine hypercontractility by employing a selective antagonist. SIGNIFICANCE STATEMENT: This research validates the use of nonpregnant mice for preclinical drug discovery of uterine EP receptor targets. To determine the utility of novel drugs and delivery systems at term pregnancy and labor, pharmacological agents interacting with EP3 receptors have clear translational value.

Multidisciplinary quality improvement programme for older patients admitted to a vascular surgery ward.

BACKGROUND: Older vascular surgical patients are at high risk of hospital-associated complications and prolonged stays. AIMS: To implement a multidisciplinary co-management model for older vascular patients and evaluate impact on length of stay (LOS), delirium incidence, functional decline, medical complications and discharge destination. METHODS: Prospective pre-post evaluation of a quality improvement intervention, enrolling pre-intervention (August 2012-January 2013) and post-intervention cohort (September 2013-March 2014). Participants were consenting patients aged 65 years and over admitted to the vascular surgical ward of a metropolitan teaching hospital for at least 3 days. Intervention was physician-led co-management plus a multidisciplinary improvement programme targeting delirium and functional decline. Primary outcomes were LOS, delirium and functional decline. Secondary outcomes were medical complications and discharge destination. Process measures included documented consultation patterns. Administrative data were also compared for all patients aged 65 and older for 12 months pre- and post-intervention. RESULTS: We enrolled 112 participants pre-intervention and 123 participants post-intervention. LOS was reduced post-intervention (geometric mean 7.6 days vs 9.3 days; ratio of geometric means 0.82 (95% confidence interval CI0.68-1.00), P = 0.04). There was a trend to less delirium (18 (14.6%) vs 24 (21.4%), P = 0.17) and functional decline (18 (14.6%) vs 27 (24.3%), P = 0.06), with greatest reductions in the urgently admitted subgroup. Administrative data showed reduced median LOS (5.2 days vs 6 days, P = 0.03) and greater discharge home (72% vs 50%, P < 0.01). CONCLUSIONS: Physician-led co-management plus a multidisciplinary improvement programme may reduce LOS and improve functional outcomes in older vascular surgical patients.

Proteome Profile of Endogenous Retrotransposon-Associated Complexes in Human Embryonic Stem Cells.

Long Interspersed Element-1 (LINE-1 or L1) are transposable elements similar to retroviruses that have existed in the genome of primates for millions of years. They encode two Open Reading Frame (ORF) proteins (ORF1p and ORF2p) that bind L1 RNA to form a ribonucleoprotein (RNP) complex and are required for L1 integration into the host genome. Humans have evolved with L1 and found ways to limit L1 activity. To identify partners of the L1 RNP, previous studies used ectopic expression of L1 ORF1/2p or RNA in various cancer cells, which express low levels of the ORF proteins. Whether naturally occurring L1 RNP interacts with the same proteins in non-cancer cells is unknown. Here, the aim is to examine the natural assembly of endogenous L1 RNPs in normal human cells. L1 elements are expressed in human embryonic stem cells (hESCs), derived from pre-implantation embryos. Therefore, these cells are used to immunoprecipitate ORF1p followed by MS to identify proteins that associate with the naturally-occurring L1 ORF1p. Some of the same proteins as well as unique proteins are found interacting with the endogenous L1 ORF1p complexes. The analysis of ORF1p-associated proteins in hESCs can help address important questions in both retrotransposon biology and the biology of hESCs.

Variations in adjuvant chemotherapy and survival in women with epithelial ovarian cancer - a population-based study.

BACKGROUND: To investigate whether variations in primary chemotherapy were associated with survival in a nationally complete cohort of Australian women with epithelial ovarian cancer (EOC). MATERIAL AND METHODS: All 1192 women diagnosed with invasive EOC in Australia in 2005 were identified through state-based cancer registries. Medical record information including details of primary chemotherapy treatment was obtained and survival data updated in 2012. Those started on standard chemotherapy (carboplatin and paclitaxel given at three-weekly intervals) after primary cytoreductive surgery were included (n = 351) and the relative dose intensity (RDI) was calculated. Time interval between surgery and start of chemotherapy was analysed in weeks. Hazard ratios [HR, 95% confidence interval (CI)] were calculated using multivariable Cox proportional hazards models. RESULTS: Compared to women with RDI of 91-100%, those with RDI of ≤ 70% had significantly poor survival (HRadj = 1.62, 95% CI 1.05-2.49). This association was stronger among women with advanced (FIGO stage III/IV) disease at diagnosis (HRadj = 1.90, 95% CI 1.22-2.96). The interval between primary surgery and chemotherapy was not related to survival (HRadj = 0.98, 95% CI 0.93-1.03 for every week of delay), at least up to a period of five weeks. CONCLUSION: Our results suggest that RDI of 70% or less was associated with poorer survival, particularly in women with advanced stage EOC. In contrast, the interval duration between primary surgery and chemotherapy was not related to survival, irrespective of disease stage or residual disease. These results provide some reassurance that, at least up until five weeks post-surgery, timing of chemotherapy commencement has a negligible effect on survival.

Fez1/Lzts1 absence impairs Cdk1/Cdc25C interaction during mitosis and predisposes mice to cancer development.

The FEZ1/LZTS1 (LZTS1) protein is frequently downregulated in human cancers of different histotypes. LZTS1 is expressed in normal tissues, and its introduction in cancer cells inhibits cell growth and suppresses tumorigenicity, owing to an accumulation of cells in G2/M. Here, we define its role in cell cycle regulation and tumor progression by generating Lzts1 knockout mice. In Lzts1(-/-) mouse embryo fibroblasts (MEFs), Cdc25C degradation was increased during M phase, resulting in decreased Cdk1 activity. As a consequence, Lzts1(-/-) MEFs showed accelerated mitotic progression, resistance to taxol- and nocodazole-induced M phase arrest, and improper chromosome segregation. Accordingly, Lzts1 deficiency was associated with an increased incidence of both spontaneous and carcinogen-induced cancers in mice.

Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue.

We describe a label-free imaging method to monitor stem-cell metabolism that discriminates different states of stem cells as they differentiate in living tissues. In this method we use intrinsic fluorescence biomarkers and the phasor approach to fluorescence lifetime imaging microscopy in conjunction with image segmentation, which we use to introduce the concept of the cell phasor. In live tissues we are able to identify intrinsic fluorophores, such as collagen, retinol, retinoic acid, porphyrin, flavins, and free and bound NADH. We have exploited the cell phasor approach to detect a trend in metabolite concentrations along the main axis of the Caenorhabditis elegans germ line. This trend is consistent with known changes in metabolic states during differentiation. The cell phasor approach to lifetime imaging provides a label-free, fit-free, and sensitive method to identify different metabolic states of cells during differentiation, to sense small changes in the redox state of cells, and may identify symmetric and asymmetric divisions and predict cell fate. Our method is a promising noninvasive optical tool for monitoring metabolic pathways during differentiation or disease progression, and for cell sorting in unlabeled tissues.

Cdc25b phosphatase is required for resumption of meiosis during oocyte maturation.

In a wide variety of animal species, oocyte maturation is arrested temporarily at prophase of meiosis I (ref. 1). Resumption of meiosis requires activation of cyclin-dependent kinase-1 (CDK1, p34cdc2), one component of maturation-promoting factor (MPF). The dual specificity phosphatases Cdc25a, Cdc25b and Cdc25c are activators of cyclin-dependent kinases; consequently, they are postulated to regulate cell-cycle progression in meiosis and mitosis as well as the DNA-damage response. We generated Cdc25b-deficient (Cdc25b-/-) mice and found that they are viable. As compared with wildtype cells, fibroblasts from Cdc25b-/- mice grew vigorously in culture and arrested normally in response to DNA damage. Female Cdc25b-/- mice were sterile, and Cdc25b-/- oocytes remained arrested at prophase with low MPF activity. Microinjection of wildtype Cdc25b mRNA into Cdc25b-/- oocytes caused activation of MPF and resumption of meiosis. Thus, Cdc25b-/- female mice are sterile because of permanent meiotic arrest resulting from the inability to activate MPF. Cdc25b is therefore essential for meiotic resumption in female mice. Mice lacking Cdc25b provide the first genetic model for studying the mechanisms regulating prophase arrest in vertebrates.

The impact of transition to a digital hospital on medication errors (TIME study).

Digital transformation in healthcare improves the safety of health systems. Within our health service, a new digital hospital has been established and two wards from a neighbouring paper-based hospital transitioned into the new digital hospital. This created an opportunity to evaluate the impact of complete digital transformation on medication safety. Here we discuss the impact of transition from a paper-based to digital hospital on voluntarily reported medication incidents and prescribing errors. This study utilises an interrupted time-series design and takes place across two wards as they transition from a paper to a digital hospital. Two data sources are used to assess impacts on medication incidents and prescribing errors: (1) voluntarily reported medication incidents and 2) a chart audit of medications prescribed on the study wards. The chart audit collects data on procedural, dosing and therapeutic prescribing errors. There are 588 errors extracted from incident reporting software during the study period. The average monthly number of errors reduces from 12.5 pre- to 7.5 post-transition (p < 0.001). In the chart audit, 5072 medication orders are reviewed pre-transition and 3699 reviewed post-transition. The rates of orders with one or more error reduces significantly after transition (52.8% pre- vs. 15.7% post-, p < 0.001). There are significant reductions in procedural (32.1% pre- vs. 1.3% post-, p < 0.001), and dosing errors (32.3% pre- vs. 14% post-, p < 0.001), but not therapeutic errors (0.6% pre- vs. 0.7% post-, p = 0.478). Transition to a digital hospital is associated with reductions in voluntarily reported medication incidents and prescribing errors.

A novel role for an RNA polymerase III subunit POLR3G in regulating pluripotency in human embryonic stem cells.

The pluripotency of human embryonic stem cells (hESC) could have great potential for the development of cell replacement therapies. Previous studies have converged on the finding that OCT4, SOX2, and NANOG serve as key regulators in the maintenance of hESC. However, other signals that regulate hESC maintenance remain poorly studied. Here we describe a novel role of an RNA polymerase III (Pol III) subunit, POLR3G, in the maintenance of pluripotency in hESC. We demonstrate the presence of POLR3G in undifferentiated hESC, human induced pluripotent stem cells (hiPSC), and early mouse blastocysts. Downregulation of POLR3G is observed on differentiation of hESC and hiPSC, suggesting that POLR3G can be used as a molecular marker to readily identify undifferentiated pluripotent stem cells from their differentiated derivatives. Using an inducible shRNA lentiviral system, we found evidence that decreased levels of POLR3G result in loss of pluripotency and promote differentiation of hESC to all three germ layers but have no effect on cell apoptosis. On the other hand, overexpression of POLR3G has no effect on pluripotency and apoptosis in undifferentiated hESC. Interestingly, hESC expressing elevated levels of POLR3G are more resistant to differentiation. Furthermore, our experimental results show that POLR3G is a downstream target of OCT4 and NANOG, and our pharmacological study indicated that POLR3G expression can be readily regulated by the Erk1/2 signaling pathway. This study is the first to show an important role of POLR3G in the maintenance of hESC, suggesting a potential role of Pol III transcription in regulating hESC pluripotency.

RAD51C deficiency in mice results in early prophase I arrest in males and sister chromatid separation at metaphase II in females.

RAD51C is a member of the RecA/RAD51 protein family, which is known to play an important role in DNA repair by homologous recombination. In mice, it is essential for viability. Therefore, we have generated a hypomorphic allele of Rad51c in addition to a null allele. A subset of mice expressing the hypomorphic allele is infertile. This infertility is caused by sexually dimorphic defects in meiotic recombination, revealing its two distinct functions. Spermatocytes undergo a developmental arrest during the early stages of meiotic prophase I, providing evidence for the role of RAD51C in early stages of RAD51-mediated recombination. In contrast, oocytes can progress normally to metaphase I after superovulation but display precocious separation of sister chromatids, aneuploidy, and broken chromosomes at metaphase II. These defects suggest a possible late role of RAD51C in meiotic recombination. Based on the marked reduction in Holliday junction (HJ) resolution activity in Rad51c-null mouse embryonic fibroblasts, we propose that this late function may be associated with HJ resolution.

Rescue of radiation-induced cognitive impairment through cranial transplantation of human embryonic stem cells.

Cranial irradiation remains a frontline treatment for the control of tumor growth, and individuals surviving such treatments often manifest various degrees of cognitive dysfunction. Radiation-induced depletion of stem/precursor cell pools in the brain, particularly those residing in the neurogenic region of the hippocampus, is believed, in part, to be responsible for these often-unavoidable cognitive deficits. To explore the possibility of ameliorating radiation-induced cognitive impairment, athymic nude rats subjected to head only irradiation (10 Gy) were transplanted 2 days afterward with human embryonic stem cells (hESC) into the hippocampal formation and analyzed for stem cell survival, differentiation, and cognitive function. Animals receiving hESC transplantation exhibited superior performance on a hippocampal-dependent cognitive task 4 months postirradiation, compared to their irradiated surgical counterparts that did not receive hESCs. Significant stem cell survival was found at 1 and 4 months postirradiation, and transplanted cells showed robust migration to the subgranular zone throughout the dentate gyrus, exhibiting signs of neuron morphology within this neurogenic niche. These results demonstrate the capability to ameliorate radiation-induced normal tissue injury using hESCs, and suggest that such strategies may provide useful interventions for reducing the adverse effects of irradiation on cognition.

Specific loss of histone H3 lysine 9 trimethylation and HP1gamma/cohesin binding at D4Z4 repeats is associated with facioscapulohumeral dystrophy (FSHD).

Facioscapulohumeral dystrophy (FSHD) is an autosomal dominant muscular dystrophy in which no mutation of pathogenic gene(s) has been identified. Instead, the disease is, in most cases, genetically linked to a contraction in the number of 3.3 kb D4Z4 repeats on chromosome 4q. How contraction of the 4qter D4Z4 repeats causes muscular dystrophy is not understood. In addition, a smaller group of FSHD cases are not associated with D4Z4 repeat contraction (termed "phenotypic" FSHD), and their etiology remains undefined. We carried out chromatin immunoprecipitation analysis using D4Z4-specific PCR primers to examine the D4Z4 chromatin structure in normal and patient cells as well as in small interfering RNA (siRNA)-treated cells. We found that SUV39H1-mediated H3K9 trimethylation at D4Z4 seen in normal cells is lost in FSHD. Furthermore, the loss of this histone modification occurs not only at the contracted 4q D4Z4 allele, but also at the genetically intact D4Z4 alleles on both chromosomes 4q and 10q, providing the first evidence that the genetic change (contraction) of one 4qD4Z4 allele spreads its effect to other genomic regions. Importantly, this epigenetic change was also observed in the phenotypic FSHD cases with no D4Z4 contraction, but not in other types of muscular dystrophies tested. We found that HP1gamma and cohesin are co-recruited to D4Z4 in an H3K9me3-dependent and cell type-specific manner, which is disrupted in FSHD. The results indicate that cohesin plays an active role in HP1 recruitment and is involved in cell type-specific D4Z4 chromatin regulation. Taken together, we identified the loss of both histone H3K9 trimethylation and HP1gamma/cohesin binding at D4Z4 to be a faithful marker for the FSHD phenotype. Based on these results, we propose a new model in which the epigenetic change initiated at 4q D4Z4 spreads its effect to other genomic regions, which compromises muscle-specific gene regulation leading to FSHD pathogenesis.

Toilet Paper, Minced Meat and Diabetes Medicines: Australian Panic Buying Induced by COVID-19.

The COVID-19 pandemic has impacted the management of non-communicable diseases in health systems around the world. This study aimed to understand the impact of COVID-19 on diabetes medicines dispensed in Australia. Publicly available data from Australia's government subsidised medicines program (Pharmaceutical Benefits Scheme), detailing prescriptions by month dispensed to patients, drug item code and patient category, was obtained from January 2016 to November 2020. This study focused on medicines used in diabetes care (Anatomical Therapeutical Chemical code level 2 = A10). Number of prescriptions dispensed were plotted by month at a total level, by insulins and non-insulins, and by patient category (general, concessional). Total number of prescriptions dispensed between January and November of each year were compared. A peak in prescriptions dispensed in March 2020 was identified, an increase of 35% on March 2019, compared to average growth of 7.2% in previous years. Prescriptions dispensed subsequently fell in April and May 2020 to levels below the corresponding months in 2019. These trends were observed across insulins, non-insulins, general and concessional patient categories. The peak and subsequent dip in demand have resulted in a small unexpected overall increase for the period January to November 2020, compared to declining growth for the same months in prior years. The observed change in consumer behaviour prompted by COVID-19 and the resulting public health measures is important to understand in order to improve management of medicines supply during potential future waves of COVID-19 and other pandemics.

BRCA1 and BRCA2: breast/ovarian cancer susceptibility gene products and participants in DNA double-strand break repair.

BRCA1 and BRCA2 are tumor suppressor genes, familial mutations in which account for approximately 5% of breast cancer cases in the USA annually. Germ line mutations in BRCA1 that truncate or inactivate the protein lead to a cumulative risk of breast cancer, by age 70, of up to 80%, whereas the risk of ovarian cancer is 30-40%. For germ line BRCA2 mutations, the breast cancer cumulative risk approaches 50%, whereas for ovarian cancers, it is between 10 and 15%. Both BRCA1 and BRCA2 are involved in maintaining genome integrity at least in part by engaging in DNA repair, cell cycle checkpoint control and even the regulation of key mitotic or cell division steps. Unsurprisingly, the complete loss of function of either protein leads to a dramatic increase in genomic instability. How they function in maintaining genome integrity after the onset of DNA damage will be the focus of this review.

N-Hydroxybenzimidazole inhibitors of ExsA MAR transcription factor in Pseudomonas aeruginosa: In vitro anti-virulence activity and metabolic stability.

ExsA is a multiple adaptational response (MAR) transcription factor, regulating the expression of a virulence determinant, the type III secretion system (T3SS) in Pseudomonas aeruginosa. Non-cytotoxic, non-antibacterial N-hydroxybenzimidazoles were identified as effective inhibitors of ExsA-DNA binding, and their potential utility as anti-virulence agents for P. aeruginosa was demonstrated in a whole cell assay. Select N-hydroxybenzimidazole inhibitors were stable in an in vitro human liver microsomal assay.