Cancer immunity by tissue-resident type 1 innate lymphoid cells and killer innate-like T cells.

Cancer progression can be restrained by tumor-infiltrating lymphocytes in a process termed cancer immunosurveillance. Based on how lymphocytes are activated and recruited to the tumor tissue, cancer immunity is either pre-wired, in which innate lymphocytes and innate-like T cells are directly recruited to and activated in tumors following their differentiation in primary lymphoid organs; or priming-dependent, in which conventional adaptive T cells are first primed by cognate antigens in secondary lymphoid organs before homing to and reactivated in tumors. While priming-dependent cancer immunity has been a focus of cancer immunology research for decades, in part due to historical preconception of cancer theory and tumor model choice as well as clinical success of conventional adaptive T cell-directed therapeutic programs, recent studies have revealed that pre-wired cancer immunity mediated by tissue-resident type 1 innate lymphoid cells (ILC1s) and killer innate-like T cells (ILTCKs) is an integral component of the cancer immunosurveillance process. Herein we review the distinct ontogenies and cancer-sensing mechanisms of ILC1s and ILTCKs in murine genetic cancer models as well as the conspicuously conserved responses in human malignancies. How ILC1s and ILTCKs may be targeted to broaden the scope of cancer immunotherapy beyond conventional adaptive T cells is also discussed.

Serine starvation silences estrogen receptor signaling through histone hypoacetylation.

Loss of estrogen receptor (ER) pathway activity promotes breast cancer progression, yet how this occurs remains poorly understood. Here, we show that serine starvation, a metabolic stress often found in breast cancer, represses estrogen receptor alpha (ERα) signaling by reprogramming glucose metabolism and epigenetics. Using isotope tracing and time-resolved metabolomic analyses, we demonstrate that serine is required to maintain glucose flux through glycolysis and the TCA cycle to support acetyl-CoA generation for histone acetylation. Consequently, limiting serine depletes histone H3 lysine 27 acetylation (H3K27ac), particularly at the promoter region of ER pathway genes including the gene encoding ERα, ESR1. Mechanistically, serine starvation impairs acetyl-CoA-dependent gene expression by inhibiting the entry of glycolytic carbon into the TCA cycle and down-regulating the mitochondrial citrate exporter SLC25A1, a critical enzyme in the production of nucleocytosolic acetyl-CoA from glucose. Consistent with this model, total H3K27ac and ERα expression are suppressed by SLC25A1 inhibition and restored by acetate, an alternate source of acetyl-CoA, in serine-free conditions. We thus uncover an unexpected role for serine in sustaining ER signaling through the regulation of acetyl-CoA metabolism.

Objective monitoring tools for improved management of childhood asthma.

Asthma is a common chronic disease amongst children. Epidemiological studies showed that the mortality rate of asthma in children is still high worldwide. Asthma control is therefore essential to minimize asthma exacerbations, which can be fatal if the condition is poorly controlled. Frequent monitoring could help to detect asthma progression and ensure treatment effectiveness. Although subjective asthma monitoring tools are available, the results vary as they rely on patients' self-perception. Emerging evidence suggests several objective tools could have the potential for monitoring purposes. However, there is no consensus to standardise the use of objective monitoring tools. In this review, we start with the prevalence and severity of childhood asthma worldwide. Then, we detail the latest available objective monitoring tools, focusing on their effectiveness in paediatric asthma management. Publications of spirometry, fractional exhaled nitric oxide (FeNO), hyperresponsiveness tests and electronic monitoring devices (EMDs) between 2016 and 2023 were included. The potential advantages and limitations of each tool were also discussed. Overall, this review provides a summary for researchers dedicated to further improving objective paediatric asthma monitoring and provides insights for clinicians to incorporate different objective monitoring tools in clinical practices.

Which Localizing Strategy is the Most Cost-Effective in Reoperative Primary Hyperparathyroidism?

INTRODUCTION: 2%-10% of patients with primary hyperparathyroidism (PHPT) who undergo parathyroidectomy develop persistent/recurrent disease. The aim of this study was to determine which preoperative localization method is most cost-effective in reoperative PHPT. METHODS: Clinical decision analytic models comparing cost-effectiveness of localizing studies in reoperative PHPT were constructed using TreeAge Pro. Cost and probability assumptions were varied via Probabilistic Sensitivity Analysis (PSA) to test the robustness of the base case models. RESULTS: Base case analysis of model 1 revealed ultrasound (US)-guided fine-needle aspiration with PTH assay as most cost-effective after localizing US. This was confirmed on PSA of model 1. Model 2 showed four-dimensional computed tomography (4D-CT) as most cost-effective after negative US. If not localized by US, on PSA, 4D-CT was the next most cost-effective test. CONCLUSIONS: US-guided FNA with PTH is the most cost-effective confirmatory test after US localization. 4D-CT should be considered as the next best test after negative US.

Cross-Binding of Four Adenosine/ATP Aptamers to Caffeine, Theophylline, and Other Methylxanthines.

The classical DNA aptamer for adenosine and ATP was selected twice using ATP as the target in 1995 and 2005, respectively. In 2022, this motif appeared four more times from selections using adenosine, ATP, theophylline, and caffeine as targets, suggesting that this aptamer can also bind methylxanthines. In this work, using thioflavin T fluorescence spectroscopy, this classical DNA aptamer showed Kd values for adenosine, theophylline, and caffeine of 9.5, 101, and 131 µM, respectively, and similar Kd values were obtained using isothermal titration calorimetry. Binding to the methylxanthines was also observed for the newly selected Ade1301 aptamer but not for the Ade1304 aptamer. The RNA aptamer for ATP also had no binding to the methylxanthines. Molecular dynamics simulations were performed using the classical DNA and RNA aptamers based on their NMR structures, and the simulation results were consistent with the experimental observations, explaining the selectivity profiles. This study suggests that a broader range of target analogues need to be tested for aptamers. For the detection of adenosine and ATP, the Ade1304 aptamer is a better choice due to its better selectivity.

Cross-Binding of Adenosine by Aptamers Selected Using Theophylline.

We recently reported that some adenosine binding aptamers can also bind caffeine and theophylline with around 20-fold lower affinities. This discovery led to the current work to examine the cross-binding of adenosine to theophylline aptamers. For the DNA aptamer for theophylline, cross-binding to adenosine was observed, and the affinity was 18 to 38-fold lower for adenosine based on assays using isothermal titration calorimetry and ThT fluorescence spectroscopy. The binding complexes were characterized using NMR spectroscopy, and both adenosine and theophylline showed an overall similar binding structure to the DNA theophylline aptamer, although small differences were also observed. In contrast, the RNA aptamer did not show binding to adenosine, although both aptamers have very similar relative selectivity for various methylxanthines including caffeine. After a negative selection, a few new aptamers with completely different primary sequences for theophylline were obtained and they did not show binding to adenosine. Thus, there are many ways for aptamers to bind theophylline and some can have cross-binding to adenosine. In biology, theophylline, caffeine, and adenosine can bind to the same protein receptors to regulate sleep, and their binding to the same DNA motifs may suggest an early role of nucleic acids in similar regulatory functions.

Subtyping at-risk adolescents for predicting response toward insomnia prevention program.

BACKGROUND: Previous study has shown that a brief cognitive-behavioral prevention insomnia program could reduce 71% risk of developing insomnia among at-risk adolescents. This study aimed to evaluate the differential response to insomnia prevention in subgroups of at-risk adolescents. METHODS: Adolescents with a family history of insomnia and subthreshold insomnia symptoms were randomly assigned to a 4-week insomnia prevention program or nonactive control group. Assessments were conducted at baseline, 1 week, and 6- and 12-month after the intervention. Baseline sleep, daytime, and mood profiles were used to determine different subgroups by using latent class analysis (LCA). Analyses were conducted based on the intention-to-treat approach. RESULTS: LCA identified three subgroups: (a) insomnia symptoms only, (b) insomnia symptoms with daytime sleepiness and mild anxiety, and (c) insomnia symptoms with daytime sleepiness, mild anxiety, and depression. The incidence rate of insomnia disorder over the 12-month follow-up was significantly reduced for adolescents receiving intervention in subgroup 3 compared with the controls (hazard ratio [HR] = 0.37; 95% confidence interval [CI]: 0.13-0.99; p = .049) and marginally for subgroup 2 (HR = 0.14; 95% CI: 0.02-1.08; p = .059). In addition, adolescents who received intervention in subgroups 2 and 3 had a reduced risk of excessive daytime sleepiness (subgroup 2: adjusted OR [AdjOR] = 0.45, 95% CI: 0.23-0.87; subgroup 3: AdjOR = 0.32, 95% CI: 0.13-0.76) and possible anxiety (subgroup 2: AdjOR = 0.47, 95% CI: 0.27-0.82; subgroup 3: AdjOR = 0.33, 95% CI: 0.14-0.78) compared with the controls over the 12-month follow-up. CONCLUSIONS: Adolescents at risk for insomnia can be classified into different subgroups according to their psychological profiles, which were associated with differential responses to the insomnia prevention program. These findings indicate the need for further phenotyping and subgrouping at-risk adolescents to develop personalized insomnia prevention.

Assessing the clinical viability of micro 3 pronuclei zygotes.

PURPOSE: What is the rate of euploidy and clinical viability of embryos resulting from micro 3 pronuclei zygotes? METHODS: Retrospective cohort analysis in a single, academic in vitro fertilization (IVF) center from March 2018 to June 2021. Cohorts were separated by fertilization as either a 2 pronuclear zygote (2PN) or micro 3 pronuclear zygote (micro 3PN). PGT-A was performed to identify embryonic ploidy rates in embryos created from micro 3PN zygotes. The clinical outcomes of all transferred euploid micro 3PN zygotes were evaluated from frozen embryo transfer (FET) cycles. RESULTS: During the designated study period, 75,903 mature oocytes were retrieved and underwent ICSI. Of these, 60,161 were fertilized as 2PN zygotes (79.3%) and 183 fertilized as micro 3PN zygotes (0.24%). Of the micro 3PN-derived embryos that underwent biopsy, 27.5% (n=11/42) were deemed euploid by PGT-A, compared to 51.4% (n=12,301/23,923) of 2PN-derived embryos, p=0.06. Four micro 3PN-derived embryos were transferred in subsequent single euploid FET cycles, which includes one live birth and one ongoing pregnancy. CONCLUSION: Micro 3PN zygotes that develop to the blastocyst stage and meet the criteria for embryo biopsy have the potential to be euploid by preimplantation genetic testing for aneuploidy (PGT-A) and if selected for transfer can achieve a live birth. Although there are a significantly lower number of micro 3PN embryos that make it to blastocyst biopsy, the potential to continue to culture abnormally fertilized oocytes may give these patients a chance at pregnancy that they previously did not have.

Peer feedback and Chinese medical students' English academic writing development: a longitudinal intervention study.

BACKGROUND: Studies have documented that utilizing peer feedback can enhance students' English academic writing skills. Little is known, however, about the effects of incorporating peer feedback to enhance English as a second language (L2) medical students' academic writing performance. METHODS: This longitudinal interventional study examines Chinese medical students' English academic writing skills development via peer feedback in four parallel classes over an 18-week semester between the experimental and control groups (n = 124). RESULTS: Significant increases in the experimental group's performance in the post-test were found after 18-week instructions (pre- vs. post-test: overall score, p < .001; task response, p < .001; coherence and cohesion, p < .001; lexical resource, p < .001; grammatical range and accuracy, p < .001), and the effects were retained in the delayed post-test 6 weeks later (post- vs. delayed post-test: overall score, p = .561; task response, p = .585; coherence and cohesion, p = .533; lexical resource, p = .796; grammatical range and accuracy, p = .670). Little improvement was found in the control group in the post-test (pre- vs. post-test: overall score, p = .213; task response, p = .275; coherence and cohesion, p = .383; lexical resource, p = .367; grammatical range and accuracy, p = .180) or the delayed post-test (post- vs. delayed post-test: overall score, p = .835; task response, p = .742; coherence and cohesion, p = .901; lexical resource, p = .897; grammatical range and accuracy, p = .695). Between-group comparisons indicate that the experimental group outperformed the control group in the post- and the delayed post-tests, as shown in their overall score and scores on the four components. CONCLUSIONS: Incorporating peer feedback into process-oriented medical English writing classroom teaching can effectively enhance Chinese medical students' English academic writing skills over time, while the traditional product-oriented writing instructions had little help in improving Chinese medical students' academic writing skills. This longitudinal intervention study develops our understanding of the effectiveness of peer feedback in L2 academic writing pedagogy. It offers instructional implications for L2 writing teachers to teach English academic writing among medical students in China and beyond. Limitations and suggestions for future studies are discussed.

Advances in Animal Models and Cutting-Edge Research in Alternatives: Proceedings of the Third International Conference on 3Rs Research and Progress, Vishakhapatnam, 2022.

Animal experimentation has been integral to drug discovery and development and safety assessment for many years, since it provides insights into the mechanisms of drug efficacy and toxicity (e.g. pharmacology, pharmacokinetics and pharmacodynamics). However, due to species differences in physiology, metabolism and sensitivity to drugs, the animal models can often fail to replicate the effects of drugs and chemicals in human patients, workers and consumers. Researchers across the globe are increasingly applying the Three Rs principles by employing innovative methods in research and testing. The Three Rs concept focuses on: the replacement of animal models (e.g. with in vitro and in silico models or human studies), on the reduction of the number of animals required to achieve research objectives, and on the refinement of existing experimental practices (e.g. eliminating distress and enhancing animal wellbeing). For the last two years, Oncoseek Bio-Acasta Health, a 3-D cell culture-based cutting-edge translational biotechnology company, has organised an annual International Conference on 3Rs Research and Progress. This series of global conferences aims to bring together researchers with diverse expertise and interests, and provides a platform where they can share and discuss their research to promote practices according to the Three Rs principles. In November 2022, the 3rd international conference, Advances in Animal Models and Cutting-Edge Research in Alternatives, took place at the GITAM University in Vishakhapatnam (AP, India) in a hybrid format (i.e. online and in-person). These conference proceedings provide details of the presentations, which were categorised under five different topic sessions. It also describes a special interactive session on in silico strategies for preclinical research in oncology, which was held at the end of the first day.

Evaluation of Human Hepatocyte Drug Metabolism Carrying High-Risk or Protection-Associated Liver Disease Genetic Variants.

Metabolic-dysfunction-associated steatotic liver disease (MASLD), which affects 30 million people in the US and is anticipated to reach over 100 million by 2030, places a significant financial strain on the healthcare system. There is presently no FDA-approved treatment for MASLD despite its public health significance and financial burden. Understanding the connection between point mutations, liver enzymes, and MASLD is important for comprehending drug toxicity in healthy or diseased individuals. Multiple genetic variations have been linked to MASLD susceptibility through genome-wide association studies (GWAS), either increasing MASLD risk or protecting against it, such as PNPLA3 rs738409, MBOAT7 rs641738, GCKR rs780094, HSD17B13 rs72613567, and MTARC1 rs2642438. As the impact of genetic variants on the levels of drug-metabolizing cytochrome P450 (CYP) enzymes in human hepatocytes has not been thoroughly investigated, this study aims to describe the analysis of metabolic functions for selected phase I and phase II liver enzymes in human hepatocytes. For this purpose, fresh isolated primary hepatocytes were obtained from healthy liver donors (n = 126), and liquid chromatography-mass spectrometry (LC-MS) was performed. For the cohorts, participants were classified into minor homozygotes and nonminor homozygotes (major homozygotes + heterozygotes) for five gene polymorphisms. For phase I liver enzymes, we found a significant difference in the activity of CYP1A2 in human hepatocytes carrying MBOAT7 (p = 0.011) and of CYP2C8 in human hepatocytes carrying PNPLA3 (p = 0.004). It was also observed that the activity of CYP2C9 was significantly lower in human hepatocytes carrying HSD17B13 (p = 0.001) minor homozygous compared to nonminor homozygous. No significant difference in activity of CYP2E1, CYP2C8, CYP2D6, CYP2E1, CYP3A4, ECOD, FMO, MAO, AO, and CES2 and in any of the phase II liver enzymes between human hepatocytes carrying genetic variants for PNPLA3 rs738409, MBOAT7 rs641738, GCKR rs780094, HSD17B13 rs72613567, and MTARC1 rs2642438 were observed. These findings offer a preliminary assessment of the influence of genetic variations on drug-metabolizing cytochrome P450 (CYP) enzymes in healthy human hepatocytes, which may be useful for future drug discovery investigations.

Hyperbaric oxygen therapy for central retinal artery occlusion: Visual acuity and time to treatment.

Purpose: To evaluate the effectiveness of hyperbaric oxygen (HBO2) therapy as a treatment for central retinal artery occlusion (CRAO). Methods: A total of 38 patients who underwent HBO2 for non-arteritic CRAO were identified. Patients with arteritic CRAO, branch retinal artery occlusion, ophthalmic artery occlusion, and other diagnoses were excluded from the analysis. The main outcome measured was the change in visual acuity at the most recent follow-up exam compared to the visual acuity at presentation before the initiation of HBO2 therapy. Results: The overall visual acuity after HBO2 compared with the visual acuity at presentation showed a mean improvement of 0.5 logMAR from 2.2 to 1.7 logMAR (p=0.0003). Patients who presented with hand motion and light perception vision had a mean improvement of 0.4 logMAR (p=0.06) and 0.8 logMAR (p=0.004) after HBO2, respectively. An average visual acuity improvement of 0.5 logMAR (p=0.01) was observed when patients underwent HBO2 earlier than 24 hours of symptom onset. This mean improvement increased to 0.9 logMAR (p=0.009) if HBO2 was initiated within eight hours. Conclusions: HBO2 may be an effective treatment for non-arteritic CRAO, especially if patients are treated early and present with salvageable vision. The time to treatment and the presenting visual acuity may be predictive factors on the visual prognosis following HBO2. Further studies with a prospective design and more patients are necessary to determine the long-term outcomes and the optimal protocol for HBO2 in CRAO patients.

Airway inflammation in severe asthmatics with acid gastro-oesophageal reflux.

The relationship between childhood asthma and gastro-oesophageal reflux (GOR) is contentious. Recent studies in adult asthmatics suggest that GOR is associated with worse control and differences in sputum proteomics related to epithelial integrity, systemic inflammation and host defence. We assessed 127 children with severe asthma undergoing bronchoscopy and pH study. There were no differences in asthma control or measures of airway inflammation or remodelling when those with acid GOR were compared with those without. These results suggest that acid GOR is not an important comorbidity in paediatric severe asthma.

Permeabilized Cryopreserved Human Hepatocytes as an Exogenous Metabolic System in a Novel Metabolism-Dependent Cytotoxicity Assay for the Evaluation of Metabolic Activation and Detoxification of Drugs Associated with Drug-Induced Liver Injuries: Results with Acetaminophen, Amiodarone, Cyclophosphamide, Ketoconazole, Nefazodone, and Troglitazone.

We report here a novel in vitro experimental system, the metabolism-dependent cytotoxicity assay (MDCA), for the definition of the roles of hepatic drug metabolism in toxicity. MDCA employs permeabilized cofactor-supplemented cryopreserved human hepatocytes (MetMax Human Hepatocytes, MMHH), as an exogenous metabolic activating system, and human embryonic kidney 293 (HEK293) cells, a cell line devoid of drug-metabolizing enzyme activity, as target cells for the quantification of drug toxicity. The assay was performed in the presence and absence of cofactors for key drug metabolism pathways known to play key roles in drug toxicity: NADPH/NAD+ for phase 1 oxidation, uridine 5'-diphosphoglucuronic acid (UDPGA) for uridine 5'-diphospho-glucuronosyltransferase (UGT) mediated glucuronidation, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) for cytosolic sulfotransferase (SULT) mediated sulfation, and glutathione (GSH) for glutathione S-transferase (GST) mediated GSH conjugation. Six drugs with clinically significant hepatoxicity, resulting in liver failure or a need for liver transplantation: acetaminophen, amiodarone, cyclophosphamide, ketoconazole, nefazodone, and troglitazone were evaluated. All six drugs exhibited cytotoxicity enhancement by NADPH/NAD+, suggesting metabolic activation via phase 1 oxidation. Attenuation of cytotoxicity by UDPGA was observed for acetaminophen, ketoconazole, and troglitazone, by PAPS for acetaminophen, ketoconazole, and troglitazone, and by GSH for all six drugs. Our results suggest that MDCA can be applied toward the elucidation of metabolic activation and detoxification pathways, providing information that can be applied in drug development to guide structure optimization to reduce toxicity and to aid the assessment of metabolism-based risk factors for drug toxicity. GSH detoxification represents an endpoint for the identification of drugs forming cytotoxic reactive metabolites, a key property of drugs with idiosyncratic hepatotoxicity. SIGNIFICANCE STATEMENT: Application of the metabolism-dependent cytotoxicity assay (MDCA) for the elucidation of the roles of metabolic activation and detoxification pathways in drug toxicity may provide information to guide structure optimization in drug development to reduce hepatotoxic potential and to aid the assessment of metabolism-based risk factors. Glutathione (GSH) detoxification represents an endpoint for the identification of drugs forming cytotoxic reactive metabolites that may be applied toward the evaluation of idiosyncratic hepatotoxicity.

Effects of Overexpression of Fibroblast Growth Factor 15/19 on Hepatic Drug Metabolizing Enzymes.

Fibroblast growth factors 15 (FGF15) and 19 (FGF19) are endocrine growth factors that play an important role in maintaining bile acid homeostasis. FGF15/19-based therapies are currently being tested in clinical trials for the treatment of nonalcoholic steatohepatitis and cholestatic liver diseases. To determine the physiologic impact of long-term elevations of FGF15/19, a transgenic mouse model with overexpression of Fgf15 (Fgf15 Tg) was used in the current study. The RNA sequencing (RNA-seq) analysis revealed elevations of the expression of several genes encoding phase I drug metabolizing enzymes (DMEs), including Cyp2b10 and Cyp3a11, in Fgf15 Tg mice. We found that the induction of several Cyp2b isoforms resulted in increased function of CYP2B in microsomal metabolism and pharmacokinetics studies. Because the CYP2B family is known to be induced by constitutive androstane receptor (CAR), to determine the role of CAR in the observed inductions, we crossed Fgf15 Tg mice with CAR knockout mice and found that CAR played a minor role in the observed alterations in DME expression. Interestingly, we found that the overexpression of Fgf15 in male mice resulted in a phenotypical switch from the male hepatic expression pattern of DMEs to that of female mice. Differences in secretion of growth hormone (GH) between male and female mice are known to drive sexually dimorphic, STAT5b-dependent expression patterns of hepatic genes. We found that male Fgf15 Tg mice presented with many features similar to GH deficiency, including lowered body length and weight, Igf-1 and Igfals expression, and STAT5 signaling. SIGNIFICANCE STATEMENT: The overexpression of Fgf15 in mice causes an alteration in DMEs at the mRNA, protein, and functional levels, which is not entirely due to CAR activation but associated with lower GH signaling.

Iron status of full-term infants in early infancy is not associated with maternal ferritin levels nor infant feeding practice.

Iron deficiency (ID) in early life is associated with morbidities. Most fetal iron required for infant growth is acquired in the third trimester from maternal iron store. However, how prenatal iron level affects ferritin level in early infancy remains controversial. This study aimed to examine the associations between maternal ferritin levels and cord blood serum ferritin (CBSF) and to compare the ferritin levels between different feeding practices in early infancy. Healthy Chinese mothers with uncomplicated pregnancy and their infants were followed up at 3 months post-delivery for questionnaire completion and infant blood collection. Infants who were predominantly breastfed and those who were predominantly formula fed were included in this analysis. Serum ferritin levels were measured in maternal blood samples collected upon delivery, cord blood and infant blood samples at 3 months of age. Ninety-seven mother-baby dyads were included. Maternal ID is common (56 %) while the CBSF levels were significantly higher than maternal ferritin levels. Only three infants (3 %) had ID at 3 months of age. There were no significant correlations between maternal ferritin levels with CBSF (r 0·168, P = 0·108) nor with infant ferritin levels at 3 months of age (r 0·023, P = 0·828). Infant ferritin levels at 3 months were significantly and independently associated with CBSF (P = 0·007) and birth weight (P < 0·001) after adjusting for maternal age, parity, maternal education, infant sex and feeding practice. In conclusion, maternal ID was common upon delivery. However, maternal ferritin levels were not significantly associated with CBSF concentrations nor infant ferritin concentrations at 3 months of age.

Messenger RNA Expression of Albumin, Transferrin, Transthyretin, Asialoglycoprotein Receptor, Cytochrome P450 Isoform, Uptake Transporter, and Efflux Transporter Genes as a Function of Culture Duration in Prolonged Cultured Cryopreserved Human Hepatocytes as Collagen-Matrigel Sandwich Cultures: Evidence for Redifferentiation upon Prolonged Culturing.

Hepatic gene expression as a function of culture duration was evaluated in prolonged cultured human hepatocytes. Human hepatocytes from seven donors were maintained as near-confluent collagen-Matrigelsandwich cultures, with messenger RNA expression for genes responsible for key hepatic functions quantified by real-time polymerase chain reaction at culture durations of 0 (day of plating), 2, 7, 9, 16, 23, 26, 29, 36, and 43 days. Key hepatocyte genes were evaluated, including the differentiation markers albumin, transferrin, and transthyretin; the hepatocyte-specific asialoglycoprotein receptor 1 cytochrome P450 isoforms CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A7; uptake transporter isoforms SLC10A1, SLC22A1, SLC22A7, SLCO1B1, SLCO1B3, and SLCO2B1; efflux transporter isoforms ATP binding cassette (ABC)B1, ABCB11, ABCC2, ABCC3, ABCC4, and ABCG2; and the nonspecific housekeeping gene hypoxanthine ribosyl transferase 1 (HPRT1). The well established dedifferentiation phenomenon was observed on day 2, with substantial (>80%) decreases in gene expression in day 2 cultures observed for all genes evaluated except HPRT1 and efflux transporters ABCB1, ABCC2, ABCC3 (<50% decrease in expression), ABCC4 (>400% increase in expression), and ABCG2 (no decrease in expression). All genes with a >80% decrease in expression were found to have increased levels of expression on day 7, with peak expression observed on either day 7 or day 9, followed by a gradual decrease in expression up to the longest duration evaluated of 43 days. Our results provide evidence that cultured human hepatocytes undergo redifferentiation upon prolonged culturing. SIGNIFICANCE STATEMENT: This study reports that although human hepatocytes underwent dedifferentiation upon 2 days of culture, prolonged culturing resulted in redifferentiation based on gene expression of differentiation markers, uptake and efflux transporters, and cytochrome P450 isoforms. The observed redifferentiation suggests that prolonged (>7 days) culturing of human hepatocyte cultures may represent an experimental approach to overcome the initial dedifferentiation process, resulting in "stabilized" hepatocytes that can be applied toward the evaluation of drug properties requiring an extended period of treatment and evaluation.

Intermediate phenotypes of childhood obstructive sleep apnea.

An intermediate phenotype of a disease is a trait in the path of pathogenesis from genetic predisposition to disease manifestation. Identifying intermediate phenotypes with high heritability is helpful in delineating the genetics of a disorder. In this study, we aimed to examine various traits with regards to obesity, cardiovascular risk and upper airway structure to identify potential intermediate phenotypes of childhood obstructive sleep apnea (OSA). Children aged between 6 and 18 years and their parents and siblings were recruited. All subjects underwent anthropometric measurements, cardiovascular risk assessment, sonographic measurement of lateral parapharyngeal wall (LPW) thickness, X-ray cephalometry and overnight polysomnography. A total of 34 phenotypes were examined. One hundred and one families consisting of 127 children (46 overweight) and 198 adults (84 overweight) were recruited. Heritability of obstructive apnea-hypopnea index (OAHI) was significant in overweight (h2  = 0.54) but not normal-weight individuals (h2  = 0.12). LPW thickness (h2  = 0.68) and resting blood pressure (h2  = 0.36 and 0.43 for systolic blood pressure [SBP] and diastolic blood pressure [DBP], respectively) were significantly heritable and associated with OAHI. Moreover, these traits were found to have shared genetic variance with OAHI in the overweight subgroup. Hyoid bone position also had significant heritability (h2  = 0.55) and association with OAHI but genetic correlation with OSA severity was not demonstrated. These findings suggest that LPW thickness and resting blood pressure are possible intermediate phenotypes of OSA independent of body mass index, especially in overweight patients. Identifying genes relevant to these phenotypes may help to elucidate the genetic susceptibility of OSA.

Prenatal methylmercury exposure is associated with decrease heart rate variability in children.

BACKGROUND: Although several epidemiological studies have suggested mercury (Hg) might be associated with cardiotoxicity, the impact of Hg exposure on cardiac autonomic activity and blood pressure in children has not been investigated at Hg exposure levels equivalent to the Environmental Protection Agency (EPA) reference dose. OBJECTIVE: To investigate the association between low dose prenatal and recent methylmercury (MeHg) exposures and cardiac autonomic function and blood pressure with adjustment for factors such as fish consumption among children from a high fish consumption coastal city. METHODS: Children aged 7-8 years were recruited from the birth cohort of our previous study. Heart rate variability (HRV), resting heart rate (RHR) and blood pressure were measured as surrogate markers of cardiac autonomic function. Cord blood and current whole blood Hg concentration were used as biomarkers of prenatal and recent MeHg exposure, respectively. Recent fish consumption information was estimated with a food frequency questionnaire. RESULTS: Among 604 children, median cord blood and whole blood Hg concentrations were 45.9 nmol/L (IQR: 32.8-65.03 nmol/L) and 13.57 nmol/L (IQR: 9.29-19.72 nmol/L), respectively. Our results demonstrated that prenatal MeHg exposure was associated with decreased HRV (i.e. low CVRR, SDRR, and RMSSD), reflecting reduced parasympathetic activity (i.e. low CCVHF and HF), and a sympathovagal balance shift toward sympathetic predominance (i.e. high %LF and LF/HF ratio). Adjustment of recent fish consumption further increased the significance and magnitude of the adverse associations of MeHg. CONCLUSION: The results of this study suggest that prenatal MeHg exposure is associated with decreased parasympathetic modulation of cardiac autonomic function in children.

Cardiovascular risks of children with primary snoring: A 5-year follow-up study.

BACKGROUND AND OBJECTIVE: This study aimed to investigate if childhood primary snoring (PS) was associated with adverse cardiovascular outcomes at 5-year follow-up. METHODS: This was a prospective matched cohort study. Subjects were recruited from a hospital-based cohort established from years 2006 to 2012 and they were aged 6-18 years at baseline. Each subject with PS was gender, age and BMI z-score matched with a control who had normal sleep study (obstructive apnoea-hypopnoea index [OAHI] < 1/h) and without habitual snoring (<3 nights/week) at baseline. All subjects underwent measurements of flow-mediated dilation (FMD) and carotid intima-media thickness (cIMT) and sleep study at baseline and follow-up visits. Twenty-four hour ambulatory blood pressure (ABP) was also recorded at follow-up. RESULTS: Fifty-five case-control pairs were recruited and the length of follow-up was 5.1 ± 1.3 years. At follow-up visit, subjects with PS at baseline had significantly lower FMD (-0.34% [-0.59 to -0.10]), greater cIMT (+0.01 mm [+0.001 to +0.013]), higher wake systolic blood pressure (SBP) (+2.6 mm Hg [+0.02 to +5.1]), sleep SBP (+3.0 mm Hg [+0.3 to +5.6]), sleep diastolic blood pressure (+2.2 mm Hg [+0.04 to +4.4]) and sleep mean arterial pressure (+2.2 mm Hg [+0.1 to +4.2]) compared to matched controls in the fully adjusted model for variables including change in OAHI and parental history of cardiovascular diseases. CONCLUSION: Childhood PS is associated with poorer endothelial function, greater cIMT and higher ABP at 5-year follow-up irrespective of change in obstructive sleep apnoea severity.