Assembling a Hippo: the evolutionary emergence of an animal developmental signaling pathway.

Decades of work in developmental genetics has given us a deep mechanistic understanding of the fundamental signaling pathways underlying animal development. However, little is known about how these pathways emerged and changed over evolutionary time. Here, we review our current understanding of the evolutionary emergence of the Hippo pathway, a conserved signaling pathway that regulates tissue size in animals. This pathway has deep evolutionary roots, emerging piece by piece in the unicellular ancestors of animals, with a complete core pathway predating the origin of animals. Recent functional studies in close unicellular relatives of animals and early-branching animals suggest an ancestral function Hippo pathway of cytoskeletal regulation, which was subsequently co-opted to regulate proliferation and animal tissue size.

Transient Structural Dynamics of Glycogen Phosphorylase from Nonequilibrium Hydrogen/Deuterium-Exchange Mass Spectrometry.

It remains a major challenge to ascertain the specific structurally dynamic changes that underpin protein functional switching. There is a growing need in molecular biology and drug discovery to complement structural models with the ability to determine the dynamic structural changes that occur as these proteins are regulated and function. The archetypal allosteric enzyme glycogen phosphorylase is a clinical target of great interest to treat type II diabetes and metastatic cancers. Here, we developed a time-resolved nonequilibrium millisecond hydrogen/deuterium-exchange mass spectrometry (HDX-MS) approach capable of precisely locating dynamic structural changes during allosteric activation and inhibition of glycogen phosphorylase. We resolved obligate transient changes in the localized structure that are absent when directly comparing active/inactive states of the enzyme and show that they are common to allosteric activation by AMP and inhibition by caffeine, operating at different sites. This indicates that opposing allosteric regulation by inhibitor and activator ligands is mediated by pathways that intersect with a common structurally dynamic motif. This mass spectrometry approach uniquely stands to discover local transient structural dynamics and could be used broadly to identify features that influence the structural transitions of proteins.

CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis.

BACKGROUND: Transthyretin amyloidosis, also called ATTR amyloidosis, is a life-threatening disease characterized by progressive accumulation of misfolded transthyretin (TTR) protein in tissues, predominantly the nerves and heart. NTLA-2001 is an in vivo gene-editing therapeutic agent that is designed to treat ATTR amyloidosis by reducing the concentration of TTR in serum. It is based on the clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease (CRISPR-Cas9) system and comprises a lipid nanoparticle encapsulating messenger RNA for Cas9 protein and a single guide RNA targeting TTR. METHODS: After conducting preclinical in vitro and in vivo studies, we evaluated the safety and pharmacodynamic effects of single escalating doses of NTLA-2001 in six patients with hereditary ATTR amyloidosis with polyneuropathy, three in each of the two initial dose groups (0.1 mg per kilogram and 0.3 mg per kilogram), within an ongoing phase 1 clinical study. RESULTS: Preclinical studies showed durable knockout of TTR after a single dose. Serial assessments of safety during the first 28 days after infusion in patients revealed few adverse events, and those that did occur were mild in grade. Dose-dependent pharmacodynamic effects were observed. At day 28, the mean reduction from baseline in serum TTR protein concentration was 52% (range, 47 to 56) in the group that received a dose of 0.1 mg per kilogram and was 87% (range, 80 to 96) in the group that received a dose of 0.3 mg per kilogram. CONCLUSIONS: In a small group of patients with hereditary ATTR amyloidosis with polyneuropathy, administration of NTLA-2001 was associated with only mild adverse events and led to decreases in serum TTR protein concentrations through targeted knockout of TTR. (Funded by Intellia Therapeutics and Regeneron Pharmaceuticals; ClinicalTrials.gov number, NCT04601051.).

Randomised trial of genetic testing and targeted intervention to prevent the development and progression of Paget's disease of bone.

INTRODUCTION: Paget's disease of bone (PDB) frequently presents at an advanced stage with irreversible skeletal damage. Clinical outcomes might be improved by earlier diagnosis and prophylactic treatment. METHODS: We randomised 222 individuals at increased risk of PDB because of pathogenic SQSTM1 variants to receive 5 mg zoledronic acid (ZA) or placebo. The primary outcome was new bone lesions assessed by radionuclide bone scan. Secondary outcomes included change in existing lesions, biochemical markers of bone turnover and skeletal events related to PDB. RESULTS: The median duration of follow-up was 84 months (range 0-127) and 180 participants (81%) completed the study. At baseline, 9 (8.1%) of the ZA group had PDB lesions vs 12 (10.8%) of the placebo group. Two of the placebo group developed new lesions versus none in the ZA group (OR 0.41, 95% CI 0.00 to 3.43, p=0.25). Eight of the placebo group had a poor outcome (lesions which were new, unchanged or progressing) compared with none of the ZA group (OR 0.08, 95% CI 0.00 to 0.42, p=0.003). At the study end, 1 participant in the ZA group had lesions compared with 11 in the placebo group. Biochemical markers of bone turnover were significantly reduced in the ZA group. One participant allocated to placebo required rescue therapy with ZA because of symptomatic disease. The number and severity of adverse events did not differ between groups. CONCLUSIONS: Genetic testing for pathogenic SQSTM1 variants coupled with intervention with ZA is well tolerated and has favourable effects on the progression of early PDB. TRIAL REGISTRATION NUMBER: ISRCTN11616770.

Embedded Complexity of Evolutionary Sequences.

Multiple pathways and outcomes are common in evolutionary sequences for biological and other environmental systems due to nonlinear complexity, historical contingency, and disturbances. From any starting point, multiple evolutionary pathways are possible. From an endpoint or observed state, multiple possibilities exist for the sequence of events that created it. However, for any observed historical sequence-e.g., ecological or soil chronosequences, stratigraphic records, or lineages-only one historical sequence actually occurred. Here, a measure of the embedded complexity of historical sequences based on algebraic graph theory is introduced. Sequences are represented as system states S(t), such that S(t - 1) ≠ S(t) ≠ S(t + 1). Each sequence of N states contains nested subgraph sequences of length 2, 3, …, N - 1. The embedded complexity index (which can also be interpreted in terms of embedded information) compares the complexity (based on the spectral radius λ1) of the entire sequence to the cumulative complexity of the constituent subsequences. The spectral radius is closely linked to graph entropy, so the index also reflects information in the sequence. The analysis is also applied to ecological state-and-transition models (STM), which represent observed transitions, along with information on their causes or triggers. As historical sequences are lengthened (by the passage of time and additional transitions or by improved resolutions or new observations of historical changes), the overall complexity asymptotically approaches λ1 = 2, while the embedded complexity increases as N2.6. Four case studies are presented, representing coastal benthic community shifts determined from biostratigraphy, ecological succession on glacial forelands, vegetation community changes in longleaf pine woodlands, and habitat changes in a delta.

Allosteric Regulation of Glycogen Phosphorylase by Order/Disorder Transition of the 250' and 280s Loops.

Allostery is a fundamental mechanism of protein activation, yet the precise dynamic changes that underlie functional regulation of allosteric enzymes, such as glycogen phosphorylase (GlyP), remain poorly understood. Despite being the first allosteric enzyme described, its structural regulation is still a challenging problem: the key regulatory loops of the GlyP active site (250' and 280s) are weakly stable and often missing density or have large b-factors in structural models. This led to the longstanding hypothesis that GlyP regulation is achieved through gating of the active site by (dis)order transitions, as first proposed by Barford and Johnson. However, testing this requires a quantitative measurement of weakly stable local structure which, to date, has been technically challenging in such a large protein. Hydrogen-deuterium-exchange mass spectrometry (HDX-MS) is a powerful tool for studying protein dynamics, and millisecond HDX-MS has the ability to measure site-localized stability differences in weakly stable structures, making it particularly valuable for investigating allosteric regulation in GlyP. Here, we used millisecond HDX-MS to measure the local structural perturbations of glycogen phosphorylase b (GlyPb), the phosphorylated active form (GlyPa), and the inhibited glucose-6 phosphate complex (GlyPb:G6P) at near-amino acid resolution. Our results support the Barford and Johnson hypothesis for GlyP regulation by providing insight into the dynamic changes of the key regulatory loops.

Online Fully Automated System for Hydrogen/Deuterium-Exchange Mass Spectrometry with Millisecond Time Resolution.

Amide hydrogen/deuterium-exchange mass spectrometry (HDX-MS) is a powerful tool for analyzing the conformational dynamics of proteins in a solution. Current conventional methods have a measurement limit starting from several seconds and are solely reliant on the speed of manual pipetting or a liquid handling robot. Weakly protected regions of polypeptides, such as in short peptides, exposed loops and intrinsically disordered the protein exchange on the millisecond timescale. Typical HDX methods often cannot resolve the structural dynamics and stability in these cases. Numerous academic laboratories have demonstrated the considerable utility of acquiring HDX-MS data in the sub-second regimes. Here, we describe the development of a fully automated HDX-MS apparatus to resolve amide exchange on the millisecond timescale. Like conventional systems, this instrument boasts automated sample injection with software selection of labeling times, online flow mixing and quenching, while being fully integrated with a liquid chromatography-MS system for existing standard "bottom-up" workflows. HDX-MS's rapid exchange kinetics of several peptides demonstrate the repeatability, reproducibility, back-exchange, and mixing kinetics achieved with the system. Comparably, peptide coverage of 96.4% with 273 peptides was achieved, supporting the equivalence of the system to standard robotics. Additionally, time windows of 50 ms-300 s allowed full kinetic transitions to be observed for many amide groups; especially important are short time points (50-150 ms) for regions that are likely highly dynamic and solvent- exposed. We demonstrate that information on structural dynamics and stability can be measured for stretches of weakly stable polypeptides in small peptides and in local regions of a large enzyme, glycogen phosphorylase.

Periodic hypoxia, intermittent pain, and caffeine in male and female neonatal rats: corticosterone, insulin resistance, and hepatic gene expression.

Preterm infants experience multiple stressors including periodic neonatal hypoxia, maternal/caregiver separation, and acute pain from clinical procedures. Although neonatal hypoxia or interventional pain are associated with sexually dimorphic effects that may last into adulthood, the interaction of these common preterm stressors and caffeine pretreatment remains unknown. We hypothesize that an interaction of acute neonatal hypoxia, isolation, and pain modeling the experience of the preterm infant will augment the acute stress response and that caffeine routinely given to preterm infants will alter this response. Male and female rat pups were isolated and exposed to six cycles of periodic hypoxia (10% O2) or normoxia (room air control) and/or intermittent pain by administering needle pricks (or touch control) to the paw on postnatal (PD) days 1-4. An additional set of rat pups was pretreated with caffeine citrate (80 mg/kg ip) and studied on PD1. Plasma corticosterone, fasting glucose, and insulin were measured to calculate homeostatic model assessment for insulin resistance (HOMA-IR) (index of insulin resistance). Glucocorticoid-, insulin-, and caffeine-sensitive gene mRNAs were analyzed in the PD1 liver and hypothalamus to evaluate downstream markers of glucocorticoid action. Acute pain with periodic hypoxia led to a large increase in plasma corticosterone, which was attenuated by pretreatment with caffeine. Pain with periodic hypoxia led to a 10-fold increase in hepatic Per1 mRNA expression in males, which was attenuated with caffeine. The augmentation of corticosterone and HOMA-IR at PD1 after periodic hypoxia with pain suggests early intervention to attenuate the stress response may mitigate the programming effects of neonatal stress.

Improved stability second harmonic conversion of a diode-pumped Yb:YAG laser at the 0.5 kW level.

We report on efficient and stable, type-I phase-matched second harmonic conversion of a nanosecond high-energy, diode-pumped, Yb:YAG laser. With a frequency-doubling crystal in an enclosed, temperature controller with optical windows, 0.5% energy stability was achieved for approximately half an hour. This resulted in 48.9 J pulses at 10 Hz (489 W) and a conversion efficiency of 73.8%. These results are particularly important for stable and reliable operation of high-energy, frequency-doubled lasers.

Loss of SORCS2 is Associated with Neuronal DNA Double-Strand Breaks.

SORCS2 is one of five proteins that constitute the Vps10p-domain receptor family. Members of this family play important roles in cellular processes linked to neuronal survival, differentiation and function. Genetic and functional studies implicate SORCS2 in cognitive function, as well as in neurodegenerative and psychiatric disorders. DNA damage and DNA repair deficits are linked to ageing and neurodegeneration, and transient neuronal DNA double-strand breaks (DSBs) also occur as a result of neuronal activity. Here, we report a novel role for SORCS2 in DSB formation. We show that SorCS2 loss is associated with elevated DSB levels in the mouse dentate gyrus and that knocking out SORCS2 in a human neuronal cell line increased Topoisomerase IIß-dependent DSB formation and reduced neuronal viability. Neuronal stimulation had no impact on levels of DNA breaks in vitro, suggesting that the observed differences may not be the result of aberrant neuronal activity in these cells. Our findings are consistent with studies linking the VPS10 receptors and DNA damage to neurodegenerative conditions.

Feasibility of recruiting in prisons during a randomized controlled trial with people with serious mental illness.

BACKGROUND: Successful participant recruitment is vital to the feasibly of intervention research. In the behavioral and social sciences, intervention researchers face a myriad of recruitment barriers, many of which stem from working in real-world settings and among hard-to-access populations. Optimizing recruitment efforts requires being intentional about study planning and resource allocation, carefully documenting the outcomes of recruitment efforts, and developing and implementing procedures and strategies to overcome anticipated recruitment barriers. METHODS: The current article presents recruitment flowcharts to illustrate (a) the multistep recruitment process and (b) the points of potential participant attrition during recruitment from a two-phase group-based intervention study conducted among individuals with serious mental illness incarcerated in a state prison system in the U.S. In addition, qualitative methods are used to examine strategies employed during the study to support recruitment efforts. RESULTS: Despite challenges, this study was able to achieve recruitment goals. Analyses found the majority of potential participant attrition occurred prior to informed consent, highlighting the need for studies to track recruitment efforts in more detail than is currently recommended by commonly used guidelines. Strategies to optimize recruitment efforts included maximizing recruiter availability, developing a responsive communication approach, demonstrating respect for facility procedures and operations, and ensuring peak preparedness. CONCLUSION: Careful documentation of recruitment efforts and the early deployment of recruitment strategies is vital to the feasibility of intervention studies conducted in real-world settings with hard-to-access populations. The publication of recruitment procedures and outcomes can help future researchers anticipate recruitment challenges and inform recruitment goals, timelines, and strategies.

Posterior cervical spinal fusion in the pediatric population using modern adult instrumentation - clinical outcome and safety.

PURPOSE: Traditionally, less rigid fixation techniques have been applied to the pediatric cervical spine. There is a lack of long-term outcome data for rigid fixation techniques. The purpose of this study was to define the clinical outcome and safety of posterior instrumented fusion in the pediatric population using adult posterior instrumentation. METHODS: A multicenter, retrospective review of pediatric patients who underwent posterior cervical fusion using a 3.5 mm posterior cervical system for any indication was performed. Outcome parameters included complications, revision and fusion rates, operative time (OR), blood loss, and postoperative neurologic status. Outcomes were compared between patient groups (posterior only versus anterior/posterior approach, short versus intermediate versus long fusion, and between different etiologies) using Mann-Whitney and chi-square test. RESULTS: Seventy-nine patients with a mean age of 9.9 years and mean follow-up of 2.8 years were included. At baseline 44 (56%) had an abnormal neurologic exam. Congenital deformities and basilar invagination were the most common indications for surgery. Posterior-only surgery was performed in 71 (90%) cases; mean number of levels fused was 4 (range 1-15). Overall, 4 (5%) operative complications and 4 (5%) revisions were reported at an average postoperative time of 2.6 years. Neurologic status remained unchanged in 74%, improved in 23%, and worsened in 3%. When comparing outcome measures between the various groups, 2 significant differences were found: OR was longer in the anterior/posterior approach group and decline of neuro status was more frequent in the long fusion group. CONCLUSION: Posterior cervical fusion with an adult 3.5 mm posterior cervical system was safe in this cohort of 79 pediatric patients irrespective of surgical technique, fusion length, and etiology, resulting in a high fusion and low complication/revision rate.

Ultrasensitive Surface Plasmon Resonance Sensor with a Feature of Dynamically Tunable Sensitivity and High Figure of Merit for Cancer Detection.

Cancer is one of the leading causes of death worldwide, and it is well known that an early detection of cancer in a human body will provide an opportunity to cure the cancer. Early detection of cancer depends on the sensitivity of the measuring device and method, where the lowest detectable concentration of the cancerous cell in a test sample becomes a matter of high importance. Recently, Surface Plasmon Resonance (SPR) has proven to be a promising method to detect cancerous cells. The SPR method is based on the detection of changes in refractive indices of samples under testing and the sensitivity of such a SPR based sensor is related to the smallest detectable change in the refractive index of the sample. There exist many techniques where different combinations of metals, metal alloys and different configurations have been shown to lead to high sensitivities of the SPR sensors. Based on the difference in the refractive index between a normal healthy cell and a cancerous cell, recently, SPR method has been shown to be applicable to detect different types of cancers. In this work, we propose a new sensor surface configuration that comprises of gold-silver-graphene-black phosphorus to detect different cancerous cells based on the SPR method. Additionally, recently we proposed that the application of electric field across gold-graphene layers that form the SPR sensor surface can provide enhanced sensitivity than that is possible without the application of electrical bias. We utilized the same concept and numerically studied the impact of electrical bias across the gold-graphene layers combined with silver and black Phosphorus layers which forms the SPR sensor surface. Our numerical results have shown that electrical bias across the sensor surface in this new heterostructure can provide enhanced sensitivity compared to the original unbiased sensor surface. Not only that, our results have shown that as the electrical bias increases, the sensitivity increases up to a certain value and stabilizes at a still improved sensitivity value. Such dependence of sensitivity on the applied bias provides a dynamic tunability of the sensitivity and figure-of-merit (FOM) of the sensor to detect different types of cancer. In this work, we used the proposed heterostructure to detect six different types of cancers: Basal, Hela, Jurkat, PC12, MDA-MB-231, and MCF-7. Comparing our results to work published recently, we were able to achieve an enhanced sensitivity ranging from 97.2 to 1851.4 (deg/RIU) and FOM values ranging from 62.13 to 89.81 far above the values presented recently by other researchers.

Gap balanced adjusted mechanical alignment versus measured resection mechanical alignment: a randomised controlled trial.

INTRODUCTION: Alignment goals in total knee replacement (TKR) is a topical subject. This study compares the short-term functional outcomes and patient reported outcome measures (PROMs) of two philosophies for knee arthroplasty alignment: measured resection (MR) and an individualised alignment philosophy, with the tibia mechanically aligned and an instrumented gap balancer (GB) to align the femur in both flexion and extension. PATIENTS AND METHODS: 94 knees were enrolled in this randomised controlled trial. The surgical protocol used a MR technique for mechanical alignment or a GB technique for individualised alignment. Primary outcome was quadriceps strength. Secondary outcomes included validated functional tests and PROMs as well as patient satisfaction. Outcomes were assessed pre-operatively, at 6 weeks, 3, 6 and 12 months post-operatively. RESULTS: At 12-month follow-up, there was no significant difference in the change from baseline mean quadriceps peak torque between the two groups (p = 0.988). Significant improvement in the change in range of motion (ROM) in the GB group compared to the MR group at 3 months (13° vs 6° p = 0.028) but this improvement was not significant at 1 year (20° vs 17° p = 0.21). The functional test of balance showed statistically significant improvement at 6 weeks (p = 0.03) in the GB group but this difference was not maintained. PROMs favoured the GB group, with the KOOS pain scoring statistically better (p ≤ 0.05) at 6 weeks, 3, 6 and 12 months. CONCLUSIONS: Individualised alignment philosophy utilising a GB technique did not demonstrate an improvement in the primary outcome measure quadriceps peak torque. Improvement was seen in the GB group in PROM pain scores that was significant, both statistically and clinically, out to at least 1 year. Gains that were seen in functional assessment with GB, although significant at some time points, were no longer significant at 1 year and no difference was seen in quads strength. Compared to a MR technique, the individualised GB technique appears to confer some improvement in pain, ROM and some functional tests following TKR in the short-term.

HDfleX: Software for Flexible High Structural Resolution of Hydrogen/Deuterium-Exchange Mass Spectrometry Data.

Hydrogen/deuterium-exchange mass spectrometry (HDX-MS) experiments on protein structures can be performed at three levels: (1) by enzymatically digesting labeled proteins and analyzing the peptides (bottom-up), (2) by further fragmenting peptides following digestion (middle-down), and (3) by fragmenting the intact labeled protein (top-down) using soft gas-phase fragmentation methods, such as electron transfer dissociation (ETD). However, to the best of our knowledge, the software packages currently available for the analysis of HDX-MS data do not enable the peptide- and ETD-levels to be combined; they can only be analyzed separately. Thus, we developed HDfleX, a standalone application for the analysis of flexible high structural resolution of HDX-MS data, which allows data at any level of structural resolution (intact protein, peptide, fragment) to be merged. HDfleX features rapid experimental data fitting, robust statistical significance analyses, and optional methods for theoretical intrinsic calculations and a novel empirical correction for comparison between solution conditions.

Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry Approach to Correlate Local Structure and Aggregation in α-Synuclein.

In Parkinson's disease and other synucleinopathies, α-synuclein misfolds and aggregates. Its intrinsically disordered nature, however, causes it to adopt several meta-stable conformations stabilized by internal hydrogen bonding. Because they interconvert on short timescales, monomeric conformations of disordered proteins are difficult to characterize using common structural techniques. Few techniques can measure the conformations of monomeric α-synuclein, including millisecond hydrogen/deuterium-exchange mass spectrometry (HDX-MS). Here, we demonstrate a new approach correlating millisecond HDX-MS data with aggregation kinetics to determine the localized structural dynamics that underpin the self-assembly process in full-length wild-type monomeric α-synuclein. Our custom instrumentation and software enabled measurement of the amide hydrogen-exchange rates on the millisecond timescale for wild-type α-synuclein monomer up to residue resolution and under physiological conditions, mimicking those in the extracellular, intracellular, and lysosomal cellular compartments. We applied an empirical correction to normalize measured hydrogen-exchange rates and thus allow comparison between drastically different solution conditions. We characterized the aggregation kinetics and morphology of the resulting fibrils and correlate these with structural changes in the monomer. Applying a correlative approach to connect molecular conformation to aggregation in α-synuclein for the first time, we found that the central C-terminal residues of α-synuclein are driving its nucleation and thus its aggregation. We provide a new approach to link the local structural dynamics of intrinsically disordered proteins to functional attributes, which we evidence with new details on our current understanding of the relationship between the local chemical environment and conformational ensemble bias of monomeric α-synuclein.

Communicating Absolute Fracture Risk Reduction and the Acceptance of Treatment for Osteoporosis.

Healthcare professionals frequently communicate the benefits of treatments as a relative risk reduction (RRR) in the likelihood of an event occurring. Here we evaluated whether presenting the benefits of osteoporosis treatment as a RRR in fractures compared with an absolute risk reduction (ARR) changed the patient's attitudes towards accepting treatment. We surveyed 160 individuals attending a specialised osteoporosis clinic for face-to-face consultations between May 2018 and Jan 2021. They were presented with information on RRR for the treatment being considered followed by ARR and after each question were asked about how likely they would be to start treatment on a 5-point scale (1 = very likely, 5 = very unlikely). Participants were less likely to accept treatment when it was presented as ARR (mean score 2.02 vs. 2.67, p < 0.001, 95% CI for difference - 0.82 vs - 0.47) and thirty-eight participants (23.7%) declined treatment with knowledge of their ARR when they would have accepted the same treatment based on the RRR. Individuals who declined treatment had a lower 5-year risk of fracture than those who accepted treatment (9.0 vs. 12.5%, p < 0.001, 95% CI - 5.0 to - 1.6) and as fracture risk decreased, the participant was less likely to accept treatment (Spearman r - 0.32, 95% CI - 0.46 to - 0.17, p ≤ 0.001). Whilst presentation of data as ARR more accurately reflects individual benefit and helps facilitate shared decision-making, clinicians should be aware that this will lead to a proportion of patients with lower fracture risk declining treatment for osteoporosis.

Interaction of chronic pain, obesity and time of day on cortisol in female human adolescents.

Adolescent obesity augments and impedes the treatment of chronic pain. This is associated with increased systemic inflammation and is more prominent in females. In addition, pain and obesity each independently affect the hypothalamic-pituitary-adrenal (HPA) axis. However, the interaction of pain and obesity on the HPA axis and the potential for sexual dimorphism in this phenomenon is not established. We hypothesized that dysregulation of the HPA axis occurs in female human adolescents with chronic pain, obesity, or the combination of the two and is associated with gonadal steroids. We measured serum cortisol, estradiol, and testosterone in 13-17-year-old adolescent females (N = 79) from venous blood drawn during the daytime (0830-1730 h) and analyzed the data in toto and partitioned by morning vs. afternoon sampling time. Subjects were categorized as healthy weight/no pain (controls; BMI = 56th percentile [37-71]), healthy weight with chronic pain, obese without pain (BMI = 97th percentile [95-99]), or the combination of obesity and chronic pain. Serum cortisol was lower with chronic pain and/or obesity compared to healthy controls and was lower with chronic pain and obesity compared to chronic pain alone (healthy weight). The lower serum cortisol in the pain alone group was more prominent in the morning compared to the afternoon. There was no relationship between serum estradiol and testosterone and study group. The decrease in the anti-inflammatory and other pain-ameliorating effects of cortisol may contribute to chronic pain and its resistance to treatment with concurrent obesity in female adolescents.

Mode of action and human relevance assessment of male CD-1 mouse renal adenocarcinoma associated with lifetime exposure to empagliflozin.

Inhibition of sodium-glucose cotransporter-2 (SGLT2) has been shown to be a safe and efficacious approach to support managing Type 2 diabetes. In the 2-year carcinogenicity study with the SGLT2 inhibitor empagliflozin in CD-1 mice, an increased incidence of renal tubular adenomas and carcinomas was identified in the male high-dose group but was not observed in female mice. An integrated review of available nonclinical data was conducted to establish a mode-of-action hypothesis for male mouse-specific tumorigenesis. Five key events were identified through systematic analysis to form the proposed mode-of-action: (1) Background kidney pathology in CD-1 mice sensitizes the strain to (2) pharmacology-related diuretic effects associated with SGLT2 inhib ition. (3) In male mice, metabolic demand increases with the formation of a sex- and species-specific empagliflozin metabolite. These features converge to (4) deplete oxidative stress handling reserve, driving (5) constitutive cellular proliferation in male CD-1 mice. The proposed mode of action requires all five key events for empagliflozin to present a carcinogenicity risk in the CD-1 mouse. Considering that empagliflozin is not genotoxic in the standard battery of genotoxicity tests, and not all five key events are present in the context of female mice, rats, or humans, nor for other osmotic diuretics or other SGLT2 inhibitors, the observed male mouse renal tumors are not considered relevant to humans.

Use of a Wearable Biosensor to Study Heart Rate Variability in Chronic Obstructive Pulmonary Disease and Its Relationship to Disease Severity.

The purpose of this study was to explore the relationships between heart rate variability (HRV) and various phenotypic measures that relate to health and functional status in chronic obstructive pulmonary disease (COPD), and secondly, to demonstrate the feasibility of ascertaining HRV via a chest-worn wearable biosensor in COPD patients. HRV analysis was performed using SDNN (standard deviation of the mean of all normal R-R intervals), low frequency (LF), high frequency (HF), and LF/HF ratio. We evaluated the associations between HRV and COPD severity, class of bronchodilator therapy prescribed, and patient reported outcomes. Seventy-nine participants with COPD were enrolled. There were no differences in SDNN, HF, and LF/HF ratio according to COPD severity. The SDNN in participants treated with concurrent beta-agonists and muscarinic antagonists was lower than that in other participants after adjusting heart rate (beta coefficient -3.980, p = 0.019). The SDNN was positively correlated with Veterans Specific Activity Questionnaire (VSAQ) score (r = 0.308, p = 0.006) and handgrip strength (r = 0.285, p = 0.011), and negatively correlated with dyspnea by modified Medical Research Council (mMRC) questionnaire (r = -0.234, p = 0.039), health status by Saint George's Respiratory Questionnaire (SGRQ) (r = -0.298, p = 0.008), symptoms by COPD Assessment Test (CAT) (r = -0.280, p = 0.012), and BODE index (r = -0.269, p = 0.020). When measured by a chest-worn wearable device, reduced HRV was observed in COPD participants receiving inhaled beta-sympathomimetic agonist and muscarinic antagonists. HRV was also correlated with various health status and performance measures.