Sleep Disturbances, Bowel Movement Kinetics, and Travel Interruption With Bowel Preparation: A Bowel CLEANsing National Initiative Substudy.

INTRODUCTION: We investigated sleep disturbances, bowel movement (BM) kinetics, and travel experience with different bowel preparation regimens in a substudy of patients enrolled in a randomized multicenter Canadian clinical trial. METHODS: Patients scheduled to have a colonoscopy between 7:30 am and 10:30 am (early morning) were randomized to (i) 4-L single-dose polyethylene glycol (PEG) given in the evening before, (ii) 2-L split-dose PEG (+bisacodyl 15 mg), or (iii) 4-L split-dose PEG. Patients scheduled to undergo a colonoscopy between 10:30 am and 4:30 pm (afternoon) were randomized to (iv) 2-L single-dose PEG (+bisacodyl 15 mg) in the morning, (v) 2-L split-dose PEG (+bisacodyl 15 mg), or (vi) 4-L split-dose PEG. Patients were asked to record information on BM kinetics, sleep, and travel to the endoscopy unit. Continuous and categorical variables were compared between groups using a Kruskal-Wallis test or χ 2 test, respectively. Intention-to-treat analyses were performed. RESULTS: Overall, 641 patients were included in this substudy. Patients undergoing early morning colonoscopies reported the most awakenings in the night when assigned to 4-L single-dose day-before PEG and the highest reduction in sleep hours when assigned to 4-L split-dose PEG. There were no significant between-group differences in urgent BMs, fecal incontinence episodes, or travel interruptions. Overall, 17% of those traveling for more than an hour had to stop for a BM during travel, with no significant difference between groups. DISCUSSION: Day-before and split-dose high-volume PEG regimens for colonoscopies scheduled before 10:30 am lead to the greatest sleep disturbance.

8-Oxo-guanine DNA damage induces transcription errors by escaping two distinct fidelity control checkpoints of RNA polymerase II.

RNA polymerase II (Pol II) has an intrinsic fidelity control mechanism to maintain faithful genetic information transfer during transcription. 8-Oxo-guanine (8OG), a commonly occurring damaged guanine base, promotes misincorporation of adenine into the RNA strand. Recent structural work has shown that adenine can pair with the syn conformation of 8OG directly upstream of the Pol II active site. However, it remains unknown how 8OG is accommodated in the active site as a template base for the incoming ATP. Here, we used molecular dynamics (MD) simulations to investigate two consecutive steps that may contribute to the adenine misincorporation by Pol II. First, the mismatch is located in the active site, contributing to initial incorporation of adenine. Second, the mismatch is in the adjacent upstream position, contributing to extension from the mismatched bp. These results are supported by an in vitro transcription assay, confirming that 8OG can induce adenine misincorporation. Our simulations further suggest that 8OG forms a stable bp with the mismatched adenine in both the active site and the adjacent upstream position. This stability predominantly originates from hydrogen bonding between the mismatched adenine and 8OG in a noncanonical syn conformation. Interestingly, we also found that an unstable bp present directly upstream of the active site, such as adenine paired with 8OG in the canonical anti conformation, largely disrupts the stability of the active site. Our findings have uncovered two main factors contributing to how 8OG induces transcriptional errors and escapes Pol II transcriptional fidelity control checkpoints.

Loss of mitochondrial pyruvate carrier 1 supports proline-dependent proliferation and collagen biosynthesis in ovarian cancer.

The pyruvate transporter MPC1 (mitochondrial pyruvate carrier 1) acts as a tumour-suppressor, loss of which correlates with a pro-tumorigenic phenotype and poor survival in several tumour types. In high-grade serous ovarian cancers (HGSOC), patients display copy number loss of MPC1 in around 78% of cases and reduced MPC1 mRNA expression. To explore the metabolic effect of reduced expression, we demonstrate that depleting MPC1 in HGSOC cell lines drives expression of key proline biosynthetic genes; PYCR1, PYCR2 and PYCR3, and biosynthesis of proline. We show that altered proline metabolism underpins cancer cell proliferation, reactive oxygen species (ROS) production, and type I and type VI collagen formation in ovarian cancer cells. Furthermore, exploring The Cancer Genome Atlas, we discovered the PYCR3 isozyme to be highly expressed in a third of HGSOC patients, which was associated with more aggressive disease and diagnosis at a younger age. Taken together, our study highlights that targeting proline metabolism is a potential therapeutic avenue for the treatment of HGSOC.

Expression of the Arabidopsis redox-related LEA protein, SAG21 is regulated by ERF, NAC and WRKY transcription factors.

SAG21/LEA5 is an unusual late embryogenesis abundant protein in Arabidopsis thaliana, that is primarily mitochondrially located and may be important in regulating translation in both chloroplasts and mitochondria. SAG21 expression is regulated by a plethora of abiotic and biotic stresses and plant growth regulators indicating a complex regulatory network. To identify key transcription factors regulating SAG21 expression, yeast-1-hybrid screens were used to identify transcription factors that bind the 1685 bp upstream of the SAG21 translational start site. Thirty-three transcription factors from nine different families bound to the SAG21 promoter, including members of the ERF, WRKY and NAC families. Key binding sites for both NAC and WRKY transcription factors were tested through site directed mutagenesis indicating the presence of cryptic binding sites for both these transcription factor families. Co-expression in protoplasts confirmed the activation of SAG21 by WRKY63/ABO3, and SAG21 upregulation elicited by oligogalacturonide elicitors was partially dependent on WRKY63, indicating its role in SAG21 pathogen responses. SAG21 upregulation by ethylene was abolished in the erf1 mutant, while wound-induced SAG21 expression was abolished in anac71 mutants, indicating SAG21 expression can be regulated by several distinct transcription factors depending on the stress condition.

Comparison of different definitions of metabolic syndrome and their associations with non-alcoholic fatty liver disease: a retrospective study.

Background: Metabolic syndrome (MetS) is considered an important risk factor for non-alcoholic fatty liver disease (NAFLD). The aim of this study was to measure the prevalence of MetS based on six different MetS definitions and compare the performance of various definitions for identifying diabetes, hypertension, and dyslipidemia among NAFLD patients. Methods: The definitions compared were those developed by the World Health Organization (WHO), National Cholesterol Education Program Adult Treatment Panel III (NCEP-ATP III), International Diabetes Federation (IDF), American Association of Clinical Endocrinologists (AACE), American Heart Association/National Heart, Lung and Blood Institute (AHA/NHLBI), and Interim Joint Statement "harmonized" criteria. Receiver operator characteristic (ROC) curves were plotted for the six MetS definitions with NAFLD diagnosis. The diagnosis for NAFLD was established based on liver imaging or biopsy compatible with fatty liver disease. Results: A total of 500 NAFLD patients were analyzed. The mean age was 61.2 (SD 13.2) years, and BMI was 32.7 (SD 8.0) kg/m2. The most prevalent MetS component was dyslipidemia (83%), followed by hypertension (60%), obesity (61%), and diabetes (57%). The prevalence of MetS according to the WHO, NCEP/ATP-III, IDF, AACE, AHA/NHLBI, and harmonized criteria was 69%, 59%, 54%, 64%, 78%, and 79%, respectively. The highest area under the ROC curve for diabetes and hypertension was with the WHO definition (0.7405) and (0.8120), respectively. Conclusions: The prevalence of MetS in NAFLD patients varies according to the definitions of MetS employed. The modified WHO definition appeared to be most useful for the screening of MetS in NAFLD patients.

Pharmacological EZH2 inhibition combined with retinoic acid treatment promotes differentiation and apoptosis in rhabdomyosarcoma cells.

BACKGROUND: Rhabdomyosarcomas (RMS) are predominantly paediatric sarcomas thought to originate from muscle precursor cells due to impaired myogenic differentiation. Despite intensive treatment, 5-year survival for patients with advanced disease remains low (< 30%), highlighting a need for novel therapies to improve outcomes. Differentiation therapeutics are agents that induce differentiation of cancer cells from malignant to benign. The histone methyltransferase, Enhancer of Zeste Homolog 2 (EZH2) suppresses normal skeletal muscle differentiation and is highly expressed in RMS tumours. RESULTS: We demonstrate combining inhibition of the epigenetic modulator EZH2 with the differentiating agent retinoic acid (RA) is more effective at reducing cell proliferation in RMS cell lines than single agents alone. In PAX3-FOXO1 positive RMS cells this is due to an RA-driven induction of the interferon pathway resulting in apoptosis. In fusion negative RMS, combination therapy led to an EZH2i-driven upregulation of myogenic signalling resulting in differentiation. In both subtypes, EZH2 is significantly associated with enrichment of trimethylated lysine 27 on histone 3 (H3K27me3) in genes that are downregulated in untreated RMS cells and upregulated with EZH2 inhibitor treatment. These results provide insight into the mechanism that drives the anti-cancer effect of the EZH2/RA single agent and combination treatment and indicate that the reduction of EZH2 activity combined with the induction of RA signalling represents a potential novel therapeutic strategy to treat both subtypes of RMS. CONCLUSIONS: The results of this study demonstrate the potential utility of combining EZH2 inhibitors with differentiation agents for the treatment of paediatric rhabdomyosarcomas. As EZH2 inhibitors are currently undergoing clinical trials for adult and paediatric solid tumours and retinoic acid differentiation agents are already in clinical use this presents a readily translatable potential therapeutic strategy. Moreover, as inhibition of EZH2 in the poor prognosis FPRMS subtype results in an inflammatory response, it is conceivable that this strategy may also synergise with immunotherapies for a more effective treatment in these patients.

Trichosporon mucoides prosthetic valve endocarditis managed with antifungal suppression therapy.

A 63-year-old male with mechanical aortic valve replacement presents with Trichosporon mucoides endocarditis. Eosinophilia was noted, which has recently been described in invasive trichosporonosis. He was treated successfully with combination voriconazole and terbinafine therapy. He was deemed not to be a cardiac surgery candidate, due to excessive estimated procedural mortality.

Differential Infectivity of Original and Delta Variants of SARS-CoV-2 in Children Compared to Adults.

Although children of all ages are susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, they have not been implicated as major drivers of transmission thus far. However, it is still unknown if this finding holds true with new variants of concern (VOC), such as Delta (B.1.617.2). This study aimed to examine differences in both viral RNA (as measured by cycle threshold [CT]) and viable-virus levels from children infected with Delta and those infected with original variants (OV). Furthermore, we aimed to compare the pediatric population infection trends to those in adults. We obtained 690 SARS-CoV-2 RT-PCR positive nasopharyngeal swabs from across Manitoba, Canada, which were further screened for mutations characteristic of VOC. Aliquots of sample were then provided for TCID50 (50% tissue culture infective dose) assays to determine infectious titers. Using a variety of statistical analyses we compared CT and infectivity of VOC in different age demographics. Comparing 122 Delta- to 175 OV-positive nasopharyngeal swab samples from children, we found that those infected with Delta are 2.7 times more likely to produce viable SARS-CoV-2 with higher titers (in TCID50 per milliliter), regardless of viral RNA levels. Moreover, comparing the pediatric samples to 130 OV- and 263 Delta-positive samples from adults, we found only that the Delta pediatric culture-positive samples had titers (TCID50 per milliliter) similar to those of culture-positive adult samples. IMPORTANCE These important findings show that children may play a larger role in viral transmission of Delta than for previously circulating SARS-CoV-2 variants. Additionally, they may suggest a mechanism for why Delta has evolved to be the predominant circulating variant.

Phytochemical Targeting of STAT3 Orchestrated Lipid Metabolism in Therapy-Resistant Cancers.

Lipids are critical for maintaining homeostasis and cellular metabolism. However, the dysregulation of lipid metabolism contributes to the pathogenesis of chronic inflammatory diseases and is a hallmark of several cancer types. Tumours exist in a microenvironment of poor vascularization-depleted oxygen and restricted nutrients. Under these conditions, tumours have been shown to increasingly depend on the metabolism of fatty acids for sustained proliferation and survival. Signal transducer and activator of transcription 3 (STAT3) plays a key role in cellular processes such as cell growth, apoptosis and lipid metabolism. Aberrant STAT3 activity, as seen in several cancer types, is associated with tumour progression and malignancy, in addition to propagating crosstalk between tumour cells and the microenvironment. Furthermore, STAT3-regulated lipid metabolism is critical for cancer stem cell self-renewal and therapy resistance. Plant-derived compounds known as phytochemicals are a potential source for novel cancer therapeutic drugs. Dietary phytochemicals are known to modulate key cellular signalling pathways involved in lipid homeostasis and metabolism, including the STAT3 signalling pathways. Targeting STAT3 orchestrated lipid metabolism has shown therapeutic promise in human cancer models. In this review, we summarize the antitumour activity of phytochemicals with an emphasis placed on their effect on STAT3-regulated lipid metabolism and their role in abrogating therapy resistance.

Intrinsic Cleavage of RNA Polymerase II Adopts a Nucleobase-independent Mechanism Assisted by Transcript Phosphate.

RNA polymerase II (Pol II) utilises the same active site for polymerization and intrinsic cleavage. Pol II proofreads the nascent transcript by its intrinsic nuclease activity to maintain high transcriptional fidelity critical for cell growth and viability. The detailed catalytic mechanism of intrinsic cleavage remains unknown. Here, we combined ab initio quantum mechanics/molecular mechanics studies and biochemical cleavage assays to show that Pol II utilises downstream phosphate oxygen to activate the attacking nucleophile in hydrolysis, while the newly formed 3'-end is protonated through active-site water without a defined general acid. Experimentally, alteration of downstream phosphate oxygen either by 2'-5' sugar linkage or stereo-specific thio-substitution of phosphate oxygen drastically reduced cleavage rate. We showed by N7-modification that guanine nucleobase does not directly involve as acid-base catalyst. Our proposed mechanism provides important insights into the understanding of intrinsic transcriptional cleavage reaction, an essential step of transcriptional fidelity control.

Molecular mechanisms of RNA polymerase II transcription elongation elucidated by kinetic network models.

Transcription elongation cycle (TEC) of RNA polymerase II (Pol II) is a process of adding a nucleoside triphosphate to the growing messenger RNA chain. Due to the long timescale events in Pol II TEC, an advanced computational technique, such as Markov State Model (MSM), is needed to provide atomistic mechanism and reaction rates. The combination of MSM and experimental results can be used to build a kinetic network model (KNM) of the whole TEC. This review provides a brief protocol to build MSM and KNM of the whole TEC, along with the latest findings of MSM and other computational studies of Pol II TEC. Lastly, we offer a perspective on potentially using a sequence dependent KNM to predict genome-wide transcription error.

The parasporal crystals of Bacillus pumilus strain 15.1: a potential virulence factor?

Bacillus pumilus strain 15.1 was previously found to cause larval mortality in the Med-fly Ceratitis capitata and was shown to produce crystals in association with the spore. As parasporal crystals are well-known as invertebrate-active toxins in entomopathogenic bacteria such as Bacillus thuringiensis (Cry and Cyt toxins) and Lysinibacillus sphaericus (Bin and Cry toxins), the B. pumilus crystals were characterized. The crystals were composed of a 45 kDa protein that was identified as an oxalate decarboxylase by peptide mass fingerprinting, N-terminal sequencing and by comparison with the genome sequence of strain 15.1. Synthesis of crystals by a plasmid-cured derivative of strain 15.1 (produced using a novel curing strategy), demonstrated that the oxalate decarboxylase was encoded chromosomally. Crystals spontaneously solubilized when kept at low temperatures, and the protein produced was resistant to trypsin treatment. The insoluble crystals produced by B. pumilus 15.1 did not show significant toxicity when bioassayed against C. capitata larvae, but once the OxdD protein was solubilized, an increase of toxicity was observed. We also demonstrate that the OxdD present in the crystals has oxalate decarboxylate activity as the formation of formate was detected, which suggests a possible mechanism for B. pumilus 15.1 activity. To our knowledge, the characterization of the B. pumilus crystals as oxalate decarboxylase is the first report of the natural production of parasporal inclusions of an enzyme.

Nutritional assessment of charitable meal programmes serving homeless people in Toronto.

OBJECTIVES: To assess the potential nutritional contribution of meals provided in a sample of community programmes for homeless individuals, to determine the effect of food donations on meal quality and to develop food-based guidance for meals that would meet adults' total nutrient needs. SETTING: Toronto, Canada. DESIGN: An analysis of weighed meal records from eighteen programmes. The energy and nutrient contents of meals were compared to requirement estimates to assess contribution to total needs, given that homeless people have limited access to nutritious foods. Mixed linear modelling was applied to determine the relationship between the use of food donations and meal quality. The composition of meals that would meet adults' nutrient requirements was determined by constructing simulated meals, drawing on the selection of foods available to programmes. SAMPLE: In all, seventy meals, sampled from eighteen programmes serving homeless individuals. RESULTS: On average, the meals contained 2.6 servings of grain products, 1.7 servings of meat and alternatives, 4.1 servings of vegetables and fruits and 0.4 servings of milk products. The energy and nutrient contents of most meals were below adults' average daily requirements. Most meals included both purchased and donated foods; the vitamin C content of meals was positively associated with the percentage of energy from donations. Increasing portion sizes improved the nutrient contribution of meals, but the provision of more milk products and fruits and vegetables was required to meet adults' nutrient requirements. CONCLUSIONS: The meals assessed were inadequate to meet adults' nutrient requirements. Improving the nutritional quality of meals requires additional resources.

Low-power laser stimulation of tissue engineered cartilage tissue formed on a porous calcium polyphosphate scaffold.

BACKGROUND AND OBJECTIVE: Forming cartilage tissue in vitro that resembles native tissue is one of the challenges of cartilage tissue engineering. The aim of this study was to determine whether low-power laser stimulation would improve the formation of cartilage tissue in vitro. STUDY DESIGN/MATERIALS AND METHODS: Bovine articular chondrocytes were seeded on the top surface of porous calcium polyphosphate substrates. After 2 days, laser stimulation was applied daily at a wavelength of 650 nm using a laser diode with energy densities of either 1.75 or 3 J/cm(2) for 4 weeks. Proteoglycan and collagen synthesis and matrix content were determined. Cartilage tissue morphology was evaluated histologically. RESULTS: Histologically, there was no difference in the appearance or cellularity of the tissues that formed in the presence or absence of laser stimulation at either dosage. There were no differences in DNA content between treated and untreated constructs and live-dead assay confirmed that this treatment was not toxic to the cells. Laser stimulation at 3 J/cm(2) enhanced matrix synthesis resulting in significantly more tissue formation than laser stimulation at 1.75 J/cm(2) or untreated cultures. CONCLUSION: Short exposures to low-power laser stimulation using a laser diode with 3 J/cm(2) dose improves cartilage tissue formation.