Endothermic physiology of extinct megatooth sharks.

The evolution of the extinct megatooth shark, Otodus megalodon, and its close phylogenetic relatives remains enigmatic. A central question persists regarding the thermophysiological origins of these large predatory sharks through geologic time, including whether O. megalodon was ectothermic or endothermic (including regional endothermy), and whether its thermophysiology could help to explain the iconic shark's gigantism and eventual demise during the Pliocene. To address these uncertainties, we present unique geochemical evidence for thermoregulation in O. megalodon from both clumped isotope paleothermometry and phosphate oxygen isotopes. Our results show that O. megalodon had an overall warmer body temperature compared with its ambient environment and other coexisting shark species, providing quantitative and experimental support for recent biophysical modeling studies that suggest endothermy was one of the key drivers for gigantism in O. megalodon and other lamniform sharks. The gigantic body size with high metabolic costs of having high body temperatures may have contributed to the vulnerability of Otodus species to extinction when compared to other sympatric sharks that survived the Pliocene epoch.

Photosynthetic biohybrid coculture for tandem and tunable CO2 and N2 fixation.

Solar-driven bioelectrosynthesis represents a promising approach for converting abundant resources into value-added chemicals with renewable energy. Microorganisms powered by electrochemical reducing equivalents assimilate CO2, H2O, and N2 building blocks. However, products from autotrophic whole-cell biocatalysts are limited. Furthermore, biocatalysts tasked with N2 reduction are constrained by simultaneous energy-intensive autotrophy. To overcome these challenges, we designed a biohybrid coculture for tandem and tunable CO2 and N2 fixation to value-added products, allowing the different species to distribute bioconversion steps and reduce the individual metabolic burden. This consortium involves acetogen Sporomusa ovata, which reduces CO2 to acetate, and diazotrophic Rhodopseudomonas palustris, which uses the acetate both to fuel N2 fixation and for the generation of a biopolyester. We demonstrate that the coculture platform provides a robust ecosystem for continuous CO2 and N2 fixation, and its outputs are directed by substrate gas composition. Moreover, we show the ability to support the coculture on a high-surface area silicon nanowire cathodic platform. The biohybrid coculture achieved peak faradaic efficiencies of 100, 19.1, and 6.3% for acetate, nitrogen in biomass, and ammonia, respectively, while maintaining product tunability. Finally, we established full solar to chemical conversion driven by a photovoltaic device, resulting in solar to chemical efficiencies of 1.78, 0.51, and 0.08% for acetate, nitrogenous biomass, and ammonia, correspondingly. Ultimately, our work demonstrates the ability to employ and electrochemically manipulate bacterial communities on demand to expand the suite of CO2 and N2 bioelectrosynthesis products.

Fecal Microbiota Transplantation for Sleep Disturbance in Postacute COVID-19 Syndrome.

BACKGROUND & AIMS: Postacute COVID-19 syndrome (PACS) is associated with sleep disturbance, but treatment options are limited. The etiology of PACS may be secondary to alterations in the gut microbiome. Here, we report the efficacy of fecal microbiota transplantation (FMT) in alleviating post-COVID insomnia symptoms in a nonrandomized, open-label prospective interventional study. METHODS: Between September 22, 2022, and May 22, 2023, we recruited 60 PACS patients with insomnia defined as Insomnia Severity Index (ISI) ≥8 and assigned them to the FMT group (FMT at weeks 0, 2, 4, and 8; n = 30) or the control group (n = 30). The primary outcome was clinical remission defined by an ISI of <8 at 12 weeks. Secondary outcomes included changes in the Pittsburgh Sleep Quality Index, Generalized Anxiety Disorder-7 scale, Epworth Sleepiness Scale, Multidimensional Fatigue Inventory, blood cortisol and melatonin, and gut microbiome analysis on metagenomic sequencing. RESULTS: At week 12, more patients in the FMT than the control group had insomnia remission (37.9% vs 10.0%; P = .018). The FMT group showed a decrease in ISI score (P < .0001), Pittsburgh Sleep Quality Index (P < .0001), Generalized Anxiety Disorder-7 scale (P = .0019), Epworth Sleepiness Scale (P = .0057), and blood cortisol concentration (P = .035) from baseline to week 12, but there was no significant change in the control group. There was enrichment of bacteria such as Gemmiger formicilis and depletion of microbial pathways producing menaquinol derivatives after FMT. The gut microbiome profile resembled that of the donor in FMT responders but not in nonresponders at week 12. There was no serious adverse event. CONCLUSIONS: This pilot study showed that FMT could be effective and safe in alleviating post-COVID insomnia, and further clinical trials are warranted. CLINICALTRIALS: gov, Number: NCT05556733.

Associations between obesity, a composite risk score for probable long COVID, and sleep problems in SARS-CoV-2 vaccinated individuals.

BACKGROUND: Preliminary data suggests that obesity might hasten the decline in mRNA vaccine-induced immunity against SARS-CoV-2. However, whether this renders individuals with obesity more susceptible to long COVID symptoms post-vaccination remains uncertain. Given sleep's critical role in immunity, exploring the associations between obesity, probable long COVID symptoms, and sleep disturbances is essential. METHODS: We analyzed data from a survey of 5919 adults aged 18 to 89, all of whom received two SARS-CoV-2 mRNA vaccinations. Participants were categorized into normal weight, overweight, and obesity groups based on ethnicity-specific BMI cutoffs. The probability of long COVID was evaluated using the Post-Acute Sequelae of SARS-CoV-2 (PASC) score, as our survey did not permit confirmation of acute SARS-CoV-2 infection through methods such as antibody testing. Additionally, sleep patterns were assessed through questionnaires. RESULTS: Participants with obesity exhibited a significantly higher adjusted odds ratio (OR) of having a PASC score of 12 or higher, indicative of probable long COVID in our study, compared to those with normal weight (OR: 1.55, 95% CI: 1.05, 2.28). No significant difference was observed for overweight individuals (OR: 0.92 [95% CI: 0.63, 1.33]). Both obesity and probable long COVID were associated with increased odds of experiencing a heightened sleep burden, such as the presence of obstructive sleep apnea or insomnia (P < 0.001). However, no significant interaction between BMI and probable long COVID status was found. CONCLUSIONS: Even post-vaccination, individuals with obesity may encounter a heightened risk of experiencing prolonged COVID-19 symptoms. However, confirming our observations necessitates comprehensive studies incorporating rigorous COVID infection testing, such as antibody assays - unavailable in our anonymous survey. Additionally, it is noteworthy that the correlation between probable long COVID and sleep disturbances appears to be independent of BMI.

Endometrial mesenchymal stromal/stem cells improve regeneration of injured endometrium in mice.

BACKGROUND: The monthly regeneration of human endometrial tissue is maintained by the presence of human endometrial mesenchymal stromal/stem cells (eMSC), a cell population co-expressing the perivascular markers CD140b and CD146. Endometrial regeneration is impaired in the presence of intrauterine adhesions, leading to infertility, recurrent pregnancy loss and placental abnormalities. Several types of somatic stem cells have been used to repair the damaged endometrium in animal models, reporting successful pregnancy. However, the ability of endometrial stem cells to repair the damaged endometrium remains unknown. METHODS: Electrocoagulation was applied to the left uterine horn of NOD/SCID mice causing endometrial injury. Human eMSC or PBS was then injected into the left injured horn while the right normal horn served as controls. Mice were sacrificed at different timepoints (Day 3, 7 and 14) and the endometrial morphological changes as well as the degree of endometrial injury and repair were observed by histological staining. Gene expression of various inflammatory markers was assessed using qPCR. The functionality of the repaired endometrium was evaluated by fertility test. RESULTS: Human eMSC successfully incorporated into the injured uterine horn, which displayed significant morphological restoration. Also, endometrium in the eMSC group showed better cell proliferation and glands formation than the PBS group. Although the number of blood vessels were similar between the two groups, gene expression of VEGF-α significantly increased in the eMSC group. Moreover, eMSC had a positive impact on the regeneration of both stromal and epithelial components of the mouse endometrium, indicated by significantly higher vimentin and CK19 protein expression. Reduced endometrial fibrosis and down-regulation of fibrosis markers were also observed in the eMSC group. The eMSC group had a significantly higher gene expression of anti-inflammatory factor Il-10 and lower mRNA level of pro-inflammatory factors Ifng and Il-2, indicating the role of eMSC in regulation of inflammatory reactions. The eMSC group showed higher implantation sites than the PBS group, suggesting better endometrial receptivity with the presence of newly emerged endometrial lining. CONCLUSIONS: Our findings suggest eMSC improves regeneration of injured endometrium in mice.

De Novo Sequencing of Synthetic Bis-cysteine Peptide Macrocycles Enabled by "Chemical Linearization" of Compound Mixtures.

A "chemical linearization" approach was applied to synthetic peptide macrocycles to enable their de novo sequencing from mixtures using nanoliquid chromatography-tandem mass spectrometry (nLC-MS/MS). This approach─previously applied to individual macrocycles but not to mixtures─involves cleavage of the peptide backbone at a defined position to give a product capable of generating sequence-determining fragment ions. Here, we first established the compatibility of "chemical linearization" by Edman degradation with a prominent macrocycle scaffold based on bis-Cys peptides cross-linked with the m-xylene linker, which are of major significance in therapeutics discovery. Then, using macrocycle libraries of known sequence composition, the ability to recover accurate de novo assignments to linearized products was critically tested using performance metrics unique to mixtures. Significantly, we show that linearized macrocycles can be sequenced with lower recall compared to linear peptides but with similar accuracy, which establishes the potential of using "chemical linearization" with synthetic libraries and selection procedures that yield compound mixtures. Sodiated precursor ions were identified as a significant source of high-scoring but inaccurate assignments, with potential implications for improving automated de novo sequencing more generally.

Chemical Exchange Saturation Transfer MRI for Differentiating Radiation Necrosis From Tumor Progression in Brain Metastasis-Application in a Clinical Setting.

BACKGROUND: High radiation doses of stereotactic radiosurgery (SRS) for brain metastases (BM) can increase the likelihood of radiation necrosis (RN). Advanced MRI sequences can improve the differentiation between RN and tumor progression (TP). PURPOSE: To use saturation transfer MRI methods including chemical exchange saturation transfer (CEST) and magnetization transfer (MT) to distinguish RN from TP. STUDY TYPE: Prospective cohort study. SUBJECTS: Seventy patients (median age 60; 73% females) with BM (75 lesions) post-SRS. FIELD STRENGTH/SEQUENCE: 3-T, CEST imaging using low/high-power (saturation B1  = 0.52 and 2.0 µT), quantitative MT imaging using B1  = 1.5, 3.0, and 5.0 µT, WAter Saturation Shift Referencing (WASSR), WAter Shift And B1 (WASABI), T1 , and T2 mapping. All used gradient echoes except T2 mapping (gradient and spin echo). ASSESSMENT: Voxel-wise metrics included: magnetization transfer ratio (MTR); apparent exchange-dependent relaxation (AREX); MTR asymmetry; normalized MT exchange rate and pool size product; direct water saturation peak width; and the observed T1 and T2 . Regions of interests (ROIs) were manually contoured on the post-Gd T1 w. The mean (of median ROI values) was compared between groups. Clinical outcomes were determined by clinical and radiologic follow-up or histopathology. STATISTICAL TESTS: t-Test, univariable and multivariable logistic regression, receiver operating characteristic, and area under the curve (AUC) with sensitivity/specificity values with the optimal cut point using the Youden index, Akaike information criterion (AIC), Cohen's d. P < 0.05 with Bonferroni correction was considered significant. RESULTS: Seven metrics showed significant differences between RN and TP. The high-power MTR showed the highest AUC of 0.88, followed by low-power MTR (AUC = 0.87). The combination of low-power CEST scans improved the separation compared to individual parameters (with an AIC of 70.3 for low-power MTR/AREX). Cohen's d effect size showed that the MTR provided the largest effect sizes among all metrics. DATA CONCLUSION: Significant differences between RN and TP were observed based on saturation transfer MRI. EVIDENCE LEVEL: 3 Technical Efficacy: Stage 2.

Relationship between apparent diffusion coefficient and survival as a function of distance from gross tumor volume on radiation planning MRI in newly diagnosed glioblastoma.

PURPOSE: To investigate the changes in apparent diffusion coefficient (ADC) within incrementally-increased margins beyond the gross tumor volume (GTV) on post-operative radiation planning MRI and their prognostic utility in glioblastoma. METHODS: Radiation planning MRIs of adult patients with newly diagnosed glioblastoma from 2017 to 2020 were assessed. The ADC values were normalized to contralateral normal white matter (nADC). Using 1 mm isotropic incremental margin increases from the GTV, the nADC values were calculated at each increment. Age, ECOG performance status, extent of resection and MGMT promoter methylation status were obtained from medical records. Using univariate and multivariable Cox regression analysis, association of nADC to progression-free and overall survival (PFS, OS) was assessed at each increment. RESULTS: Seventy consecutive patients with mean age of 53.6 ± 10.3 years, were evaluated. The MGMT promoter was methylated in 31 (44.3%), unmethylated in 36 (51.6%) and unknown in 3 (4.3%) patients. 11 (16%) underwent biopsy, 41 (44%) subtotal resection and 18 (26%) gross total resection. For each 1 mm increase in distance from GTV, the nADC decreased by 0.16% (p < 0.0001). At 1-5 mm increment, the nADC was associated with OS (p < 0.01). From 6 to 11 mm increment the nADC was associated with OS with the p-value gradually increasing from 0.018 to 0.046. nADC was not associated with PFS. CONCLUSION: The nADC values at 1-11 mm increments from the GTV margin were associated with OS. Future prospective multicenter studies are needed to validate the findings and to pave the way for the utilization of ADC for margin reduction in radiation planning.

RNA Polymerase II CTD phosphatase Rtr1 fine-tunes transcription termination.

RNA Polymerase II (RNAPII) transcription termination is regulated by the phosphorylation status of the C-terminal domain (CTD). The phosphatase Rtr1 has been shown to regulate serine 5 phosphorylation on the CTD; however, its role in the regulation of RNAPII termination has not been explored. As a consequence of RTR1 deletion, interactions within the termination machinery and between the termination machinery and RNAPII were altered as quantified by Disruption-Compensation (DisCo) network analysis. Of note, interactions between RNAPII and the cleavage factor IA (CF1A) subunit Pcf11 were reduced in rtr1Δ, whereas interactions with the CTD and RNA-binding termination factor Nrd1 were increased. Globally, rtr1Δ leads to decreases in numerous noncoding RNAs that are linked to the Nrd1, Nab3 and Sen1 (NNS) -dependent RNAPII termination pathway. Genome-wide analysis of RNAPII and Nrd1 occupancy suggests that loss of RTR1 leads to increased termination at noncoding genes. Additionally, premature RNAPII termination increases globally at protein-coding genes with a decrease in RNAPII occupancy occurring just after the peak of Nrd1 recruitment during early elongation. The effects of rtr1Δ on RNA expression levels were lost following deletion of the exosome subunit Rrp6, which works with the NNS complex to rapidly degrade a number of noncoding RNAs following termination. Overall, these data suggest that Rtr1 restricts the NNS-dependent termination pathway in WT cells to prevent premature termination of mRNAs and ncRNAs. Rtr1 facilitates low-level elongation of noncoding transcripts that impact RNAPII interference thereby shaping the transcriptome.

Effects of Tai Chi and cognitive behavioral therapy for insomnia on improving sleep in older adults: Study protocol for a non-inferiority trial.

Background: Insomnia is a prevailing health problem among older adults. Tai Chi, a popular mind-body exercise practiced by older people in various oriental communities, has been shown to improve sleep. However, Tai Chi has not been directly compared to cognitive behavioral therapy for insomnia (CBT-I), which is the first-line non-pharmacological treatment for insomnia in older adults. This study aims to examine whether Tai Chi is non-inferior to CBT-I as a treatment for insomnia in older adults. Methods: This is a single-center, assessor-blinded, non-inferiority randomized controlled trial comparing Tai Chi and CBT-I in 180 older adults aged ≥50 years with chronic insomnia according to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition. Participants will be randomly assigned to either the Tai Chi or CBT-I group. Interventions will last for 3 months with a 12-month follow-up. The primary outcome is self-perceived insomnia severity measured by Insomnia Severity Index (ISI) at 3 months and at 15 months. The secondary outcomes include the remission rate of chronic insomnia, insomnia treatment response, subjective sleep quantity and quality, 7-day actigraphy, 7-day sleep diary, sleep medication, health-related quality of life, mental health, body balance and lower extremity function, adverse events, habitual physical activity, and dietary intake. Measurements will be conducted at baseline, 3 months, and 15 months by outcome assessors who are blinded to the group allocation. Discussion: This will be the first non-inferiority randomized controlled trial to compare the efficacy and long-term outcomes of Tai Chi versus CBT-I for treating insomnia in older adults. This study will be of clinical importance as it supports the use of Tai Chi as an alternative non-pharmacological approach for insomnia treatment and sustainable management.

Three-dimensional culture models of human endometrium for studying trophoblast-endometrium interaction during implantation.

During implantation, a symphony of interaction between the trophoblast originated from the trophectoderm of the implanting blastocyst and the endometrium leads to a successful pregnancy. Defective interaction between the trophoblast and endometrium often results in implantation failure, pregnancy loss, and a number of pregnancy complications. Owing to ethical concerns of using in vivo approaches to study human embryo implantation, various in vitro culture models of endometrium were established in the past decade ranging from two-dimensional cell-based to three-dimensional extracellular matrix (ECM)/tissue-based culture systems. Advanced organoid systems have also been established for recapitulation of different cellular components of the maternal-fetal interface, including the endometrial glandular organoids, trophoblast organoids and blastoids. However, there is no single ideal model to study the whole implantation process leaving more research to be done pursuing the establishment of a comprehensive in vitro model that can recapitulate the biology of trophoblast-endometrium interaction during early pregnancy. This would allow us to have better understanding of the physiological and pathological process of trophoblast-endometrium interaction during implantation.

Segmentation and genome annotation algorithms for identifying chromatin state and other genomic patterns.

Segmentation and genome annotation (SAGA) algorithms are widely used to understand genome activity and gene regulation. These algorithms take as input epigenomic datasets, such as chromatin immunoprecipitation-sequencing (ChIP-seq) measurements of histone modifications or transcription factor binding. They partition the genome and assign a label to each segment such that positions with the same label exhibit similar patterns of input data. SAGA algorithms discover categories of activity such as promoters, enhancers, or parts of genes without prior knowledge of known genomic elements. In this sense, they generally act in an unsupervised fashion like clustering algorithms, but with the additional simultaneous function of segmenting the genome. Here, we review the common methodological framework that underlies these methods, review variants of and improvements upon this basic framework, and discuss the outlook for future work. This review is intended for those interested in applying SAGA methods and for computational researchers interested in improving upon them.

Evaluating the effectiveness of ensemble voting in improving the accuracy of consensus signals produced by various DTWA algorithms from step-current signals generated during nanopore sequencing.

Nanopore sequencing device analysis systems simultaneously generate multiple picoamperage current signals representing the passage of DNA or RNA nucleotides ratcheted through a biomolecule nanopore array by motor proteins. Squiggles are a noisy and time-distorted representation of an underlying nucleotide sequence, "gold standard model", due to experimental and algorithmic artefacts. Other research fields use dynamic time warped-space averaging (DTWA) algorithms to produce a consensus signal from multiple time-warped sources while preserving key features distorted by standard, linear-averaging approaches. We compared the ability of DTW Barycentre averaging (DBA), minimize mean (MM) and stochastic sub-gradient descent (SSG) DTWA algorithms to generate a consensus signal from squiggle-space ensembles of RNA molecules Enolase, Sequin R1-71-1 and Sequin R2-55-3 without knowledge of their associated gold standard model. We propose techniques to identify the leader and distorted squiggle features prior to DTWA consensus generation. New visualization and warping-path metrics are introduced to compare consensus signals and the best estimate of the "true" consensus, the study's gold standard model. The DBA consensus was the best match to the gold standard for both Sequin studies but was outperformed in the Enolase study. Given an underlying common characteristic across a squiggle ensemble, we objectively evaluate a novel "voting scheme" that improves the local similarity between the consensus signal and a given fraction of the squiggle ensemble. While the gold standard is not used during voting, the increase in the match of the final voted-on consensus to the underlying Enolase and Sequin gold standard sequences provides an indirect success measure for the proposed voting procedure in two ways: First is the decreased least squares warped distance between the final consensus and the gold model, and second, the voting generates a final consensus length closer to the known underlying RNA biomolecule length. The results suggest considerable potential in marrying squiggle analysis and voted-on DTWA consensus signals to provide low-noise, low-distortion signals. This will lead to improved accuracy in detecting nucleotides and their deviation model due to chemical modifications (a.k.a. epigenetic information). The proposed combination of ensemble voting and DTWA has application in other research fields involving time-distorted, high entropy signals.

Association between frailty and clinical outcomes in surgical patients admitted to intensive care units: a systematic review and meta-analysis.

BACKGROUND: Preoperative frailty may be a strong predictor of adverse postoperative outcomes. We investigated the association between frailty and clinical outcomes in surgical patients admitted to the ICU. METHODS: PubMed, Embase, and Ovid MEDLINE were searched for relevant articles. We included full-text original English articles that used any frailty measure, reporting results of surgical adult patients (≥18 yr old) admitted to ICUs with mortality as the main outcome. Data on mortality, duration of mechanical ventilation, ICU and hospital length of stay, and discharge destination were extracted. The quality of included studies and risk of bias were assessed using the Newcastle Ottawa Scale. Data were synthesised according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines. RESULTS: Thirteen observational studies met inclusion criteria. In total, 58 757 patients were included; 22 793 (39.4%) were frail. Frailty was associated with an increased risk of short-term (risk ratio [RR]=2.66; 95% confidence interval [CI]: 1.99-3.56) and long-term mortality (RR=2.66; 95% CI: 1.32-5.37). Frail patients had longer ICU length of stay (mean difference [MD]=1.5 days; 95% CI: 0.8-2.2) and hospital length of stay (MD=3.9 days; 95% CI: 1.4-6.5). Duration of mechanical ventilation was longer in frail patients (MD=22 h; 95% CI: 1.7-42.3) and they were more likely to be discharged to a healthcare facility (RR=2.34; 95% CI: 1.36-4.01). CONCLUSION: Patients with frailty requiring postoperative ICU admission for elective and non-elective surgeries had increased risk of mortality, lengthier admissions, and increased likelihood of non-home discharge. Preoperative frailty assessments and risk stratification are essential in patient and clinician planning, and critical care resource utilisation. CLINICAL TRIAL REGISTRATION: PROSPERO CRD42020210121.

Hypoxia Regulates the Self-Renewal of Endometrial Mesenchymal Stromal/Stem-like Cells via Notch Signaling.

Human endometrium is an incredibly dynamic tissue undergoing cyclic regeneration and shedding during a woman's reproductive life. Endometrial mesenchymal stromal/stem-like cells (eMSC) contribute to this process. A hypoxic niche with low oxygen levels has been reported in multiple somatic stem cell types. However, the knowledge of hypoxia on eMSC remains limited. In mice, stromal stem/progenitor cells can be identified by the label-retaining technique. We examined the relationship between the label-retaining stromal cells (LRSC) and hypoxia during tissue breakdown in a mouse model of simulated menses. Our results demonstrated that LRSC resided in a hypoxic microenvironment during endometrial breakdown and early repair. Immunofluorescence staining revealed that the hypoxic-located LRSC underwent proliferation and was highly colocalized with Notch1. In vitro studies illustrated that hypoxia activated Notch signaling in eMSC, leading to enhanced self-renewal, clonogenicity and proliferation of cells. More importantly, HIF-1α played an essential role in the hypoxia-mediated maintenance of eMSC through the activation of Notch signaling. In conclusion, our findings show that some endometrial stem/progenitor cells reside in a hypoxic niche during menstruation, and hypoxia can regulate the self-renewal activity of eMSC via Notch signaling.

Deuterium MRS of early treatment-induced changes in tumour lactate in vitro.

Elevated production of lactate is a key characteristic of aberrant tumour cell metabolism and can be non-invasively measured as an early marker of tumour response using deuterium (2 H) MRS. Following treatment, changes in the 2 H-labelled lactate signal could identify tumour cell death or impaired metabolic function, which precede morphological changes conventionally used to assess tumour response. In this work, the association between apoptotic cell death, extracellular lactate concentration, and early treatment-induced changes in the 2 H-labelled lactate signal was established in an in vitro tumour model. Experiments were conducted at 7 T on acute myeloid leukaemia (AML) cells, which had been treated with 10 µg/mL of the chemotherapeutic agent cisplatin. At 24 and 48 h after cisplatin treatment the cells were supplied with 20 mM of [6,6'-2 H2 ]glucose and scanned over 2 h using a two-dimensional 2 H MR spectroscopic imaging sequence. The resulting signals from 2 H-labelled glucose, lactate, and water were quantified using a spectral fitting algorithm implemented on the Oxford Spectroscopy Analysis MATLAB toolbox. After scanning, the cells were processed for histological stains (terminal deoxynucleotidyl transferase UTP nick end labelling and haematoxylin and eosin) to assess apoptotic area fraction and cell morphology respectively, while a colorimetric assay was used to measure extracellular lactate concentrations in the supernatant. Significantly lower levels of 2 H-labelled lactate were observed in the 48 h treated cells compared with the untreated and 24 h treated cells, and these changes were significantly correlated with an increase in apoptotic fraction and a decrease in extracellular lactate. By establishing the biological processes associated with treatment-induced changes in the 2 H-labelled lactate signal, these findings suggest that 2 H MRS of lactate may be valuable in evaluating early tumour response.

Delineation of molecular findings by whole-exome sequencing for suspected cases of paediatric-onset mitochondrial diseases in the Southern Chinese population.

BACKGROUND: Mitochondrial diseases (MDs) are a group of clinically and genetically heterogeneous disorders characterized by defects in oxidative phosphorylation. Since clinical phenotypes of MDs may be non-specific, genetic diagnosis is crucial for guiding disease management. In the current study, whole-exome sequencing (WES) was performed for our paediatric-onset MD cohort of a Southern Chinese origin, with the aim of identifying key disease-causing variants in the Chinese patients with MDs. METHODS: We recruited Chinese patients who had paediatric-onset MDs and a minimum mitochondrial disease criteria (MDC) score of 3. Patients with positive target gene or mitochondrial DNA sequencing results were excluded. WES was performed, variants with population frequency ≤ 1% were analysed for pathogenicity on the basis of the American College of Medical Genetics and Genomics guidelines. RESULTS: Sixty-six patients with pre-biopsy MDC scores of 3-8 were recruited. The overall diagnostic yield was 35% (23/66). Eleven patients (17%) were found to have mutations in MD-related genes, with COQ4 having the highest mutation rate owing to the Chinese-specific founder mutation (4/66, 6%). Twelve patients (12/66, 18%) had mutations in non-MD-related genes: ATP1A3 (n = 3, two were siblings), ALDH5A1, ARX, FA2H, KCNT1, LDHD, NEFL, NKX2-2, TBCK, and WAC. CONCLUSIONS: We confirmed that the COQ4:c.370G>A, p.(Gly124Ser) variant, was a founder mutation among the Southern Chinese population. Screening for this mutation should therefore be considered while diagnosing Chinese patients suspected to have MDs. Furthermore, WES has proven to be useful in detecting variants in patients suspected to have MDs because it helps to obtain an unbiased and precise genetic diagnosis for these diseases, which are genetically heterogeneous.

Quantitative CEST and MT at 1.5T for monitoring treatment response in glioblastoma: early and late tumor progression during chemoradiation.

PURPOSE: Quantitative MRI (qMRI) was performed using a 1.5T protocol that includes a novel chemical exchange saturation transfer/magnetization transfer (CEST/MT) approach. The purpose of this prospective study was to determine if qMRI metrics at baseline, at the 10th and 20th fraction during a 30 fraction/6 week standard chemoradiation (CRT) schedule, and at 1 month following treatment could be an early indicator of response for glioblastoma (GBM). METHODS: The study included 51 newly diagnosed GBM patients. Four regions-of-interest (ROI) were analyzed: (i) the radiation defined clinical target volume (CTV), (ii) radiation defined gross tumor volume (GTV), (iii) enhancing-tumor regions, and (iv) FLAIR-hyperintense regions. Quantitative CEST, MT, T1 and T2 parameters were compared between those patients progressing within 6.9 months (early), and those progressing after CRT (late), using mixed modelling. Exploratory predictive modelling was performed to identify significant predictors of early progression using a multivariable LASSO model. RESULTS: Results were dependent on the specific tumor ROI analyzed and the imaging time point. The baseline CEST asymmetry within the CTV was significantly higher in the early progression cohort. Other significant predictors included the T2 of the MT pools (for semi-solid at fraction 20 and water at 1 month after CRT), the exchange rate (at fraction 20) and the MGMT methylation status. CONCLUSIONS: We observe the potential for multiparametric qMRI, including a novel pulsed CEST/MT approach, to show potential in distinguishing early from late progression GBM cohorts. Ultimately, the goal is to personalize therapeutic decisions and treatment adaptation based on non-invasive imaging-based biomarkers.

Dynamic Remodeling of Membranes and Their Lipids during Acute Hormone-Induced Steroidogenesis in MA-10 Mouse Leydig Tumor Cells.

Lipids play essential roles in numerous cellular processes, including membrane remodeling, signal transduction, the modulation of hormone activity, and steroidogenesis. We chose steroidogenic MA-10 mouse tumor Leydig cells to investigate subcellular lipid localization during steroidogenesis. Electron microscopy showed that cAMP stimulation increased associations between the plasma membrane (PM) and the endoplasmic reticulum (ER) and between the ER and mitochondria. cAMP stimulation also increased the movement of cholesterol from the PM compared to untreated cells, which was partially inhibited when ATPase family AAA-domain containing protein 3 A (ATAD3A), which functions in ER and mitochondria interactions, was knocked down. Mitochondria, ER, cytoplasm, PM, PM-associated membranes (PAMs), and mitochondria-associated membranes (MAMs) were isolated from control and hormone-stimulated cells. Lipidomic analyses revealed that each isolated compartment had a unique lipid composition, and the induction of steroidogenesis caused the significant remodeling of its lipidome. cAMP-induced changes in lipid composition included an increase in phosphatidylserine and cardiolipin levels in PAM and PM compartments, respectively; an increase in phosphatidylinositol in the ER, mitochondria, and MAMs; and a reorganization of phosphatidic acid, cholesterol ester, ceramide, and phosphatidylethanolamine. Abundant lipids, such as phosphatidylcholine, were not affected by hormone treatment. Our data suggested that PM-ER-mitochondria tethering may be involved in lipid trafficking between organelles and indicated that hormone-induced acute steroid production involves extensive organelle remodeling.

Myometrial Cells Stimulate Self-Renewal of Endometrial Mesenchymal Stem-Like Cells Through WNT5A/ß-Catenin Signaling.

Human endometrium undergoes cycles of proliferation and differentiation throughout the reproductive years of women. The endometrial stem/progenitor cells contribute to this regenerative process. They lie in the basalis layer of the endometrium next to the myometrium. We hypothesized that human myometrial cells provide niche signals regulating the activities of endometrial mesenchymal stem-like cells (eMSCs). In vitro coculture of myometrial cells enhanced the colony-forming and self-renewal ability of eMSCs. The cocultured eMSCs retained their multipotent characteristic and exhibited a greater total cell output when compared with medium alone culture. The expression of active ß-catenin in eMSCs increased significantly after coculture with myometrial cells, suggesting activation of WNT/ß-catenin signaling. Secretory factors in spent medium from myometrial cell culture produced the same stimulatory effects on eMSCs. The involvement of WNT/ß-catenin signaling in self-renewal of eMSCs was confirmed with the use of WNT activator (Wnt3A conditioned medium) and WNT inhibitors (XAV939 and inhibitor of Wnt Production-2 [IWP-2]). The myometrial cells expressed more WNT5A than other WNT ligands. Recombinant WNT5A stimulated whereas anti-WNT5A antibody suppressed the colony formation, self-renewal, and T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcriptional activities of eMSCs. Moreover, eMSCs expressed FZD4 and LRP5. WNT5A is known to activate the canonical WNT signaling in the presence of these receptor components. WNT antagonist, DKK1, binds to LRP5/6. Consistently, DKK1 treatment nullified the stimulatory effect of myometrial cell coculture. In conclusion, our findings show that the myometrial cells are niche components of eMSCs, modulating the self-renewal activity of eMSCs by WNT5A-dependent activation of WNT/ß-catenin signaling. Stem Cells 2019;37:1455-1466.