Obesity alters POMC and kisspeptin neuron crosstalk leading to reduced luteinizing hormone in male mice.

Obesity is associated with hypogonadism in males, characterized by low testosterone and sperm number. Previous studies determined that these stem from dysregulation of hypothalamic circuitry that regulates reproduction, by unknown mechanisms. Herein, we used mice fed chronic high-fat diet, that mimics human obesity, to determine mechanisms of impairment at the level of the hypothalamus, in particular gonadotropin-releasing hormone (GnRH) neurons that regulate luteinizing hormone (LH), which then regulates testosterone. Consistent with obese humans, we demonstrated lower LH, and lower pulse frequency of LH secretion, but unchanged pituitary responsiveness to GnRH. LH pulse frequency is regulated by pulsatile GnRH secretion, which is controlled by kisspeptin. Peripheral and central kisspeptin injections, and DREADD-mediated activation of kisspeptin neurons, demonstrated that kisspeptin neurons were suppressed in obese mice. Thus, we investigated regulators of kisspeptin secretion. We determined that the LH response to NMDA was lower in obese mice, corresponding to fewer glutamate receptors in kisspeptin neurons, which may be critical for kisspeptin synchronization. Given that kisspeptin neurons also interact with POMC neurons, which regulate satiety and are affected by obesity, we examined their crosstalk, and determined that the LH response to either DREADD-mediated activation of POMC neurons or central injection of αMSH, a product of POMC, is abolished in obese mice. This was accompanied by diminished levels of αMSH receptor, MC4R, in kisspeptin neurons. Together, our studies determined that obesity leads to the downregulation of receptors that regulate kisspeptin neurons, which is associated with lower LH pulse frequency, leading to lower LH and hypogonadism.Significance Statement Obesity presents a significant health concern, with multiple comorbidities, including impaired reproduction. However, mechanisms are not clear, and studies are confounded by the chronic nature of this condition that leads to synaptic changes and alterations in neuron responsiveness to stimuli. Here, we demonstrate that the interaction between feeding circuitry and reproductive circuitry is altered by chronic obesity. The reason may be that chronically higher activity of POMC neurons in response to higher leptin in obesity, downregulates αMSH receptors on target neurons, including kisspeptin. This may lead to the suppression of kisspeptin neurons, and their inability to regulate pulsatile secretion of GnRH, which then lowers LH pulse frequency, leading to lower LH in the circulation, lower testosterone, and lower sperm count.

A Surgical Desirability of Outcome Ranking (DOOR) Reveals Complex Relationships Between Race/Ethnicity, Insurance Type, and Neighborhood Deprivation.

OBJECTIVE: Develop an ordinal Desirability of Outcome Ranking (DOOR) for surgical outcomes to examine complex associations of Social Determinants of Health. BACKGROUND: Studies focused on single or binary composite outcomes may not detect health disparities. METHODS: Three health care system cohort study using NSQIP (2013-2019) linked with EHR and risk-adjusted for frailty, preoperative acute serious conditions (PASC), case status and operative stress assessing associations of multilevel Social Determinants of Health of race/ethnicity, insurance type (Private 13,957; Medicare 15,198; Medicaid 2835; Uninsured 2963) and Area Deprivation Index (ADI) on DOOR and the binary Textbook Outcomes (TO). RESULTS: Patients living in highly deprived neighborhoods (ADI>85) had higher odds of PASC [adjusted odds ratio (aOR)=1.13, CI=1.02-1.25, P <0.001] and urgent/emergent cases (aOR=1.23, CI=1.16-1.31, P <0.001). Increased odds of higher/less desirable DOOR scores were associated with patients identifying as Black versus White and on Medicare, Medicaid or Uninsured versus Private insurance. Patients with ADI>85 had lower odds of TO (aOR=0.91, CI=0.85-0.97, P =0.006) until adjusting for insurance. In contrast, patients with ADI>85 had increased odds of higher DOOR (aOR=1.07, CI=1.01-1.14, P <0.021) after adjusting for insurance but similar odds after adjusting for PASC and urgent/emergent cases. CONCLUSIONS: DOOR revealed complex interactions between race/ethnicity, insurance type and neighborhood deprivation. ADI>85 was associated with higher odds of worse DOOR outcomes while TO failed to capture the effect of ADI. Our results suggest that presentation acuity is a critical determinant of worse outcomes in patients in highly deprived neighborhoods and without insurance. Including risk adjustment for living in deprived neighborhoods and urgent/emergent surgeries could improve the accuracy of quality metrics.

Photo-expansion microscopy enables super-resolution imaging of cells embedded in 3D hydrogels.

Hydrogels are extensively used as tunable, biomimetic three-dimensional cell culture matrices, but optically deep, high-resolution images are often difficult to obtain, limiting nanoscale quantification of cell-matrix interactions and outside-in signalling. Here we present photopolymerized hydrogels for expansion microscopy that enable optical clearance and tunable ×4.6-6.7 homogeneous expansion of not only monolayer cell cultures and tissue sections, but cells embedded within hydrogels. The photopolymerized hydrogels for expansion microscopy formulation relies on a rapid photoinitiated thiol/acrylate mixed-mode polymerization that is not inhibited by oxygen and decouples monomer diffusion from polymerization, which is particularly beneficial when expanding cells embedded within hydrogels. Using this technology, we visualize human mesenchymal stem cells and their interactions with nascently deposited proteins at <120 nm resolution when cultured in proteolytically degradable synthetic polyethylene glycol hydrogels. Results support the notion that focal adhesion maturation requires cellular fibronectin deposition; nuclear deformation precedes cellular spreading; and human mesenchymal stem cells display cell-surface metalloproteinases for matrix remodelling.

Characterization of biotinylated human ACE2 and SARS-CoV-2 Omicron BA.4/5 spike protein reference materials.

Accurate diagnostic and serology assays are required for the continued management of the COVID-19 pandemic yet spike protein mutations and intellectual property concerns with antigens and antibodies used in various test kits render comparability assessments difficult. As the use of common, well-characterized reagents can help address this lack of standardization, the National Research Council Canada has produced two protein reference materials (RMs) for use in SARS-CoV-2 serology assays: biotinylated human angiotensin-converting enzyme 2 RM, ACE2-1, and SARS-CoV-2 Omicron BA.4/5 spike protein RM, OMIC-1. Reference values were assigned through a combination of amino acid analysis via isotope dilution liquid chromatography tandem mass spectrometry following acid hydrolysis, and ultraviolet-visible (UV-Vis) spectrophotometry at 280 nm. Vial-to-vial homogeneity was established using UV-Vis measurements, and protein oligomeric status, monitored by size exclusion liquid chromatography (LC-SEC), was used to evaluate transportation, storage, and freeze-thaw stabilities. The molar protein concentration in ACE2-1 was 25.3 ± 1.7 µmol L-1 (k = 2, 95% CI) and consisted almost exclusively (98%) of monomeric ACE2, while OMIC-1 contained 5.4 ± 0.5 µmol L-1 (k = 2) spike protein in a mostly (82%) trimeric form. Glycoprotein molar mass determination by LC-SEC with multi-angle light scattering detection facilitated calculation of corresponding mass concentrations. To confirm protein functionality, the binding of OMIC-1 to immobilized ACE2-1 was investigated with surface plasmon resonance and the resulting dissociation constant, KD ~ 4.4 nM, was consistent with literature values.

TDP-43 and RNA form amyloid-like myo-granules in regenerating muscle.

A dominant histopathological feature in neuromuscular diseases, including amyotrophic lateral sclerosis and inclusion body myopathy, is cytoplasmic aggregation of the RNA-binding protein TDP-43. Although rare mutations in TARDBP-the gene that encodes TDP-43-that lead to protein misfolding often cause protein aggregation, most patients do not have any mutations in TARDBP. Therefore, aggregates of wild-type TDP-43 arise in most patients by an unknown mechanism. Here we show that TDP-43 is an essential protein for normal skeletal muscle formation that unexpectedly forms cytoplasmic, amyloid-like oligomeric assemblies, which we call myo-granules, during regeneration of skeletal muscle in mice and humans. Myo-granules bind to mRNAs that encode sarcomeric proteins and are cleared as myofibres mature. Although myo-granules occur during normal skeletal-muscle regeneration, myo-granules can seed TDP-43 amyloid fibrils in vitro and are increased in a mouse model of inclusion body myopathy. Therefore, increased assembly or decreased clearance of functionally normal myo-granules could be the source of cytoplasmic TDP-43 aggregates that commonly occur in neuromuscular disease.

Engineered biosynthesis of plant heteroyohimbine and corynantheine alkaloids in Saccharomyces cerevisiae.

Monoterpene indole alkaloids (MIAs) are a class of natural products comprised of thousands of structurally unique bioactive compounds with significant therapeutic values. Due to difficulties associated with isolation from native plant species and organic synthesis of these structurally complex molecules, microbial production of MIAs using engineered hosts are highly desired. In this work, we report the engineering of fully integrated Saccharomyces cerevisiae strains that allow de novo access to strictosidine, the universal precursor to thousands of MIAs at 30-40 mg/L. The optimization efforts were based on a previously reported yeast strain that is engineered to produce high titers of the monoterpene precursor geraniol through compartmentalization of mevalonate pathway in the mitochondria. Our approaches here included the use of CRISPR-dCas9 interference to identify mitochondria diphosphate transporters that negatively impact the titer of the monoterpene, followed by genetic inactivation; the overexpression of transcriptional regulators that increase cellular respiration and mitochondria biogenesis. Strain construction included the strategic integration of genes encoding both MIA biosynthetic and accessory enzymes into the genome under a variety of constitutive and inducible promoters. Following successful de novo production of strictosidine, complex alkaloids belonging to heteroyohimbine and corynantheine families were reconstituted in the host with introduction of additional downstream enzymes. We demonstrate that the serpentine/alstonine pair can be produced at ∼5 mg/L titer, while corynantheidine, the precursor to mitragynine can be produced at ∼1 mg/L titer. Feeding of halogenated tryptamine led to the biosynthesis of analogs of alkaloids in both families. Collectively, our yeast strain represents an excellent starting point to further engineer biosynthetic bottlenecks in this pathway and to access additional MIAs and analogs through microbial fermentation. ONE SENTENCE SUMMARY: An Saccharomyces cerevisiae-based microbial platform was developed for the biosynthesis of monoterpene indole alkaloids, including the universal precursor strictosidine and further modified heteroyohimbine and corynantheidine alkaloids.

Contemporary Assessment of Adhesiolysis and Resection for Adhesive Small Bowel Obstruction in the State of New York.

BACKGROUND: Adhesive small bowel obstruction (aSBO) is a common surgical problem, with some advocating for a more aggressive operative approach to avoid recurrence. Contemporary outcomes in a real-world setting were examined. STUDY DESIGN: A retrospective cohort study was performed using the New York Statewide Planning and Research Cooperative database to identify adults admitted with aSBO, 2016-2020. Patients were stratified by the presence of inflammatory bowel disease (IBD) and cancer history. Diagnoses usually requiring resection were excluded. Patients were categorized into four groups: non-operative, adhesiolysis, resection, and 'other' procedures. In-hospital mortality, major complications, and odds of undergoing resection were compared. RESULTS: 58,976 patients were included. 50,000 (84.8%) underwent non-operative management. Adhesiolysis was the most common procedure performed (n = 4,990, 8.46%), followed by resection (n = 3,078, 5.22%). In-hospital mortality in the lysis and resection groups was 2.2% and 5.9% respectively. Non-IBD patients undergoing operation on the day of admission required intestinal resection 29.9% of the time. Adjusted odds of resection were highest for those with a prior aSBO episode (OR 1.29 95%CI 1.11-1.49), delay to operation ≥3 days (OR1.78 95%CI 1.58-1.99), and non-New York City (NYC) residents being treated at NYC hospitals (OR1.57 95%CI 1.19-2.07). CONCLUSION: Adhesiolysis is currently the most common surgery for aSBO, however nearly one-third of patients will undergo a more extensive procedure, with an increased risk of mortality. Innovative therapies are needed to reduce the risk of resection.

Integration of transcriptomics and spatial biology analyses reveals Galactomyces ferment filtrate promotes epidermal interconnectivity via induction of keratinocyte differentiation, proliferation and cellular bioenergetics.

OBJECTIVE: Human skin is the first line of defence from environmental factors such as solar radiation and is susceptible to premature ageing, including a disruption in epidermal differentiation and homeostasis. We evaluated the impact of a Galactomyces Ferment Filtrate (GFF) on epidermal differentiation and response to oxidative stress. METHODS: We used transcriptomics, both spatial and traditional, to assess the impact of GFF on epidermal biology and homeostasis in keratinocytes (primary or immortalized) and in ex vivo skin explant tissue. The effect of GFF on cell adhesion rates, cellular ATP levels and proliferation rates were quantitated. Oxidative phosphorylation and glycolytic rates were measured under normal and stress-induced conditions. RESULTS: Transcriptomics from keratinocytes and ex vivo skin explants from multiple donors show GFF induces keratinocyte differentiation, skin barrier development and cell adhesion while simultaneously repressing cellular stress and inflammatory related processes. Spatial transcriptomics profiling of ex vivo skin indicated basal keratinocytes at the epidermal-dermal junction and cornifying keratinocytes in the top layer of the epidermis as the primary cell types influenced by GFF treatment. Additionally, GFF significantly increases crosstalk between suprabasal and basal keratinocytes. To support these findings, we show that GFF can significantly increase cell adhesion and proliferation in keratinocytes. GFF also protected overall cellular bioenergetics under metabolic or oxidative stress conditions. CONCLUSION: Our findings provide novel insights into cellular differences and epidermal spatial localization in response to GFF, supporting previous findings that this filtrate has a significant impact on epidermal biology and homeostasis, particularly on spatially defined crosstalk. We propose that GFF can help maintain epidermal health by enhancing keratinocyte crosstalk and differentiation/proliferation balance as well as promoting an enhanced response to stress.

Kisspeptin neuron electrophysiology: Intrinsic properties, hormonal modulation, and regulation of homeostatic circuits.

The obligatory role of kisspeptin (KISS1) and its receptor (KISS1R) in regulating the hypothalamic-pituitary-gonadal axis, puberty and fertility was uncovered in 2003. In the few years that followed, an impressive body of work undertaken in many species established that neurons producing kisspeptin orchestrate gonadotropin-releasing hormone (GnRH) neuron activity and subsequent GnRH and gonadotropin hormone secretory patterns, through kisspeptin-KISS1R signaling, and mediate many aspects of gonadal steroid hormone feedback regulation of GnRH neurons. Here, we review knowledge accrued over the past decade, mainly in genetically modified mouse models, of the electrophysiological properties of kisspeptin neurons and their regulation by hormonal feedback. We also discuss recent progress in our understanding of the role of these cells within neuronal circuits that control GnRH neuron activity and GnRH secretion, energy balance and, potentially, other homeostatic and reproductive functions.

Association of Insurance Type With Inpatient Surgery 30-Day Complications and Costs.

INTRODUCTION: Safety-net hospitals (SNHs) have higher postoperative complications and costs versus low-burden hospitals. Do low socioeconomic status/vulnerable patients receive care at lower-quality hospitals or are there factors beyond providers' control? We studied the association of private, Medicare, and vulnerable insurance type with complications/costs in a high-burden SNH. METHODS: Retrospective inpatient cohort study using National Surgical Quality Improvement Program (NSQIP) data (2013-2019) with cost data risk-adjusted by frailty, preoperative serious acute conditions (PASC), case status, and expanded operative stress score (OSS) to evaluate 30-day unplanned reoperations, any complication, Clavien-Dindo IV (CDIV) complications, and hospitalization variable costs. RESULTS: Cases (Private 1517; Medicare 1224; Vulnerable 3648) with patient mean age 52.3 y [standard deviation = 14.7] and 47.3% male. Adjusting for frailty and OSS, vulnerable patients had higher odds of PASC (aOR = 1.71, CI = 1.39-2.10, P < 0.001) versus private. Adjusting for frailty, PASC and OSS, Medicare (aOR = 1.27, CI = 1.06-1.53, P = 0.009), and vulnerable (aOR = 2.44, CI = 2.13-2.79, P < 0.001) patients were more likely to undergo urgent/emergent surgeries. Vulnerable patients had increased odds of reoperation and any complications versus private. Variable cost percentage change was similar between private and vulnerable after adjusting for case status. Urgent/emergent case status increased percentage change costs by 32.31%. We simulated "switching" numbers of private (3648) versus vulnerable (1517) cases resulting in an estimated variable cost of $49.275 million, a 25.2% decrease from the original $65.859 million. CONCLUSIONS: Increased presentation acuity (PASC and urgent/emergent surgeries) in vulnerable patients drive increased odds of complications and costs versus private, suggesting factors beyond providers' control. The greatest impact on outcomes may be from decreasing the incidence of urgent/emergent surgeries by improving access to care.

Sex-Related Differences in Acuity and Postoperative Complications, Mortality and Failure to Rescue.

INTRODUCTION: Yentl syndrome describing sex-related disparities has been extensively studied in medical conditions but not after surgery. This retrospective cohort study assessed the association of sex, frailty, presenting with preoperative acute serious conditions (PASC), and the expanded Operative Stress Score (OSS) with postoperative complications, mortality, and failure-to-rescue. METHODS: The National Surgical Quality Improvement Program from 2015 to 2019 evaluating 30-d complications, mortality, and failure-to-rescue. RESULTS: Of 4,860,308 cases (43% were male; mean [standard deviation] age of 56 [17] y), 6.0 and 0.8% were frail and very frail, respectively. Frailty score distribution was higher in men versus women (P < 0.001). Most cases were low-stress OSS2 (44.9%) or moderate-stress OSS3 (44.5%) surgeries. While unadjusted 30-d mortality rates were higher (P < 0.001) in males (1.1%) versus females (0.8%), males had lower odds of mortality (adjusted odds ratio (aOR) = 0.92, 95% confidence interval [CI] = 0.90-0.94, P < 0.001) after adjusting for frailty, OSS, case status, PASC, and Clavien-Dindo IV (CDIV) complications. Males have higher odds of PASC (aOR = 1.33, CI = 1.31-1.35, P < 0.001) and CDIV complications (aOR = 1.13, CI = 1.12-1.15, P < 0.001). Male-PASC (aOR = 0.76, CI = 0.72-0.80, P < 0.001) and male-CDIV (aOR = 0.87, CI = 0.83-0.91, P < 0.001) interaction terms demonstrated that the increased odds of mortality associated with PASC or CDIV complications/failure-to-rescue were lower in males versus females. CONCLUSIONS: Our study provides a comprehensive analysis of sex-related surgical outcomes across a wide range of procedures and health care systems. Females presenting with PASC or experiencing CDIV complications had higher odds of mortality/failure to rescue suggesting sex-related care differences. Yentl syndrome may be present in surgical patients; possibly related to differences in presenting symptoms, patient care preferences, or less aggressive care in female patients and deserves further study.

Theory of chaotic orbital variations confirmed by Cretaceous geological evidence.

Variations in the Earth's orbit and spin vector are a primary control on insolation and climate; their recognition in the geological record has revolutionized our understanding of palaeoclimate dynamics, and has catalysed improvements in the accuracy and precision of the geological timescale. Yet the secular evolution of the planetary orbits beyond 50 million years ago remains highly uncertain, and the chaotic dynamical nature of the Solar System predicted by theoretical models has yet to be rigorously confirmed by well constrained (radioisotopically calibrated and anchored) geological data. Here we present geological evidence for a chaotic resonance transition associated with interactions between the orbits of Mars and the Earth, using an integrated radioisotopic and astronomical timescale from the Cretaceous Western Interior Basin of what is now North America. This analysis confirms the predicted chaotic dynamical behaviour of the Solar System, and provides a constraint for refining numerical solutions for insolation, which will enable a more precise and accurate geological timescale to be produced.

Early onset of senescence and imbalanced epidermal homeostasis across the decades in photoexposed human skin: Fingerprints of inflammaging.

Inflammaging is a theory of ageing which purports that low-level chronic inflammation leads to cellular dysfunction and premature ageing of surrounding tissue. Skin is susceptible to inflammaging because it is the first line of defence from the environment, particularly solar radiation. To better understand the impact of ageing and photoexposure on epidermal biology, we performed a system biology-based analysis of photoexposed face and arm, and photoprotected buttock sites, from women between the ages of 20s to 70s. Biopsies were analysed by histology, transcriptomics, and proteomics and skin surface biomarkers collected from tape strips. We identified morphological changes with age of epidermal thinning, rete ridge pathlength loss and stratum corneum thickening. The SASP biomarkers IL-8 and IL-1RA/IL1-α were consistently elevated in face across age and cis/trans-urocanic acid were elevated in arms and face with age. In older arms, the DNA damage response biomarker 53BP1 showed higher puncti numbers in basal layers and epigenetic ageing were accelerated. Genes associated with differentiation and senescence showed increasing expression in the 30s whereas genes associated with hypoxia and glycolysis increased in the 50's. Proteomics comparing 60's vs 20's confirmed elevated levels of differentiation and glycolytic-related proteins. Representative immunostaining for proteins of differentiation, senescence and oxygen sensing/hypoxia showed similar relationships. This system biology-based analysis provides a body of evidence that young photoexposed skin is undergoing inflammaging. We propose the presence of chronic inflammation in young skin contributes to an imbalance of epidermal homeostasis that leads to a prematurely aged appearance during later life.

Export of Organic Carbon from Reduced Fine-Grained Zones Governs Biogeochemical Reactivity in a Simulated Aquifer.

Sediment interfaces in alluvial aquifers have a disproportionately large influence on biogeochemical activity and, therefore, on groundwater quality. Previous work showed that exports from fine-grained, organic-rich zones sustain reducing conditions in downstream coarse-grained aquifers beyond the influence of reduced aqueous products alone. Here, we show that sustained anaerobic activity can be attributed to the export of organic carbon, including live microorganisms, from fine-grained zones. We used a dual-domain column system with ferrihydrite-coated sand and embedded reduced, fine-grained lenses from Slate River (Crested Butte, CO) and Wind River (Riverton, WY) floodplains. After 50 d of groundwater flow, 8.8 ± 0.7% and 14.8 ± 3.1% of the total organic carbon exported from the Slate and Wind River lenses, respectively, had accumulated in the sand downstream. Furthermore, higher concentrations of dissolved Fe(II) and lower concentrations of dissolved organic carbon in the sand compared to total aqueous transport from the lenses suggest that Fe(II) was produced in situ by microbial oxidation of organic carbon coupled to iron reduction. This was further supported by an elevated abundance of 16S rRNA and iron-reducing (gltA) gene copies. These findings suggest that organic carbon transport across interfaces contributes to downstream biogeochemical reactions in natural alluvial aquifers.

Characterization of a SARS-CoV-2 spike protein reference material.

Development of diagnostic testing capability has advanced with unprecedented pace in response to the COVID-19 pandemic. An undesirable effect of such speed is a lack of standardization, often leading to unreliable test results. To assist the research community surmount this challenge, the National Research Council Canada has prepared a SARS-CoV-2 spike protein reference material, SMT1-1, as a buffered solution. Value assignment was achieved by amino acid analysis (AAA) by double isotope dilution liquid chromatography-tandem mass spectrometry (LC-ID-MS/MS) following acid hydrolysis of the protein, in combination with ultraviolet-visible spectrophotometry (UV-Vis) based on tryptophan and tyrosine absorbance at 280 nm. Homogeneity of the material was established through spectrophotometric absorbance readings at 280 nm. Transportation and long-term storage stabilities were assessed by monitoring relative changes in oligomeric state by size-exclusion liquid chromatography (LC-SEC) with UV detection. The molar concentration of the spike protein in SMT1-1 was 5.68 ± 0.22 µmol L-1 (k = 2, 95% CI), with the native trimeric form accounting for ~ 94% of the relative abundance. Reference mass concentration and mass fraction values were calculated using the protein molecular weight and density of the SMT1-1 solution. The spike protein is highly glycosylated which leads to analyte ambiguity when reporting the more commonly used mass concentration. After glycoprotein molar mass determination by LC-SEC with multi-angle light scattering detection, we thus reported mass concentration values for both the protein-only portion and intact glycoprotein as 0.813 ± 0.030 and 1.050 ± 0.068 mg mL-1 (k = 2), respectively.

Intralesional therapies for vascular malformations of the head and neck.

Vascular malformations of the head and neck represent a spectrum of complex vascular anomalies, requiring a multidisciplinary approach toward diagnosis and treatment. Several intralesional therapeutic agents have been devised and pioneered over the years, some of which are now primary and standard of care for the management of these lesions. In this article, the authors discuss the currently available intralesional therapeutic agents for the management of vascular malformations in the head and neck region.

Sulforaphane Does Not Protect Right Ventricular Systolic and Diastolic Functions in Nrf2 Knockout Pulmonary Artery Hypertension Mice.

PURPOSE: Nrf2 is a nuclear transcription factor and plays an important role in the regulation of oxidative stress and inflammation. We recently demonstrated that sulforaphane (SFN) protected mice from developing pulmonary arterial hypertension (PAH) and right ventricular (RV) dysfunction by elevating cardiac Nrf2 expression and function. Here we further investigate Nrf2 dependence for SFN-mediated prevention of PAH and RV dysfunction in an Nrf2 knockout mouse model. METHODS: We used male global Nrf2-knockout mice and male C57/6 J wild type mice in the following groups: Control group received room air and vehicle control; SuHx group received SU5416 and 10% hypoxia for 4 weeks to induce PAH; SuHx+SFN group received both SuHx and sulforaphane, a Nrf2 activator, for 4 weeks. Transthoracic echocardiography was performed to quantify RV function and estimate pulmonary vascular resistance over 4 weeks. PAH was confirmed using invasive RV systolic pressure measurement at 4 weeks. RESULTS: All Nrf2 knockout mice survived the 4-week SuHx induction of PAH. SuHx caused progressive RV diastolic/systolic dysfunction and increased RV systolic pressure. The development of RV diastolic dysfunction occurred earlier in the Nrf2 knockout PAH mice when compared with the wide type PAH mice. SFN partially or completely reversed SuHx-induced RV diastolic/systolic dysfunction and increased RV systolic pressure in wild-type mice, but not in Nrf2 knockout mice. CONCLUSION: Our findings demonstrated the essential role of Nrf2 in SFN-mediated prevention of RV dysfunction and PAH, and increasing Nrf2 activity in patients with PAH may have therapeutic potential.

Activation of ENS Circuits in Mouse Colon: Coordination in the Mouse Colonic Motor Complex as a Robust, Distributed Control System.

The characteristic motor patterns of the colon are coordinated by the enteric nervous system (ENS) and involve enterochromaffin (EC) cells, enteric glia, smooth muscle fibers, and interstitial cells. While the fundamental control mechanisms of colonic motor patterns are understood, greater complexity in the circuitry underlying motor patterns has been revealed by recent advances in the field. We review these recent advances and new findings from our laboratories that provide insights into how the ENS coordinates motor patterns in the isolated mouse colon. We contextualize these observations by describing the neuromuscular system underling the colonic motor complex (CMC) as a robust, distributed control system. Framing the colonic motor complex as a control system reveals a new perspective on the coordinated motor patterns in the colon. We test the control system by applying electrical stimulation in the isolated mouse colon to disrupt the coordination and propagation of the colonic motor complex.

Protonation-Induced Chirality Drives Separation by Differential Ion Mobility Spectrometry.

Upon development of a workflow to analyze (±)-Verapamil and its metabolites using differential mobility spectrometry (DMS), we noticed that the ionogram of protonated Verapamil consisted of two peaks. This was inconsistent with its metabolites, as each exhibited only a single peak in the respective ionograms. The unique behaviour of Verapamil was attributed to protonation at its tertiary amino moiety, which generated a stereogenic quaternary amine. The introduction of additional chirality upon N-protonation of Verapamil renders four possible stereochemical configurations for the protonated ion: (R,R), (S,S), (R,S), or (S,R). The (R,R)/(S,S) and (R,S)/(S,R) enantiomeric pairs are diastereomeric and thus exhibit unique conformations that are resolvable by linear and differential ion mobility techniques. Protonation-induced chirality appears to be a general phenomenon, as N-protonation of 12 additional chiral amines generated diastereomers that were readily resolved by DMS.

Neoadjuvant durvalumab with or without stereotactic body radiotherapy in patients with early-stage non-small-cell lung cancer: a single-centre, randomised phase 2 trial.

BACKGROUND: Previous phase 2 trials of neoadjuvant anti-PD-1 or anti-PD-L1 monotherapy in patients with early-stage non-small-cell lung cancer have reported major pathological response rates in the range of 15-45%. Evidence suggests that stereotactic body radiotherapy might be a potent immunomodulator in advanced non-small-cell lung cancer (NSCLC). In this trial, we aimed to evaluate the use of stereotactic body radiotherapy in patients with early-stage NSCLC as an immunomodulator to enhance the anti-tumour immune response associated with the anti-PD-L1 antibody durvalumab. METHODS: We did a single-centre, open-label, randomised, controlled, phase 2 trial, comparing neoadjuvant durvalumab alone with neoadjuvant durvalumab plus stereotactic radiotherapy in patients with early-stage NSCLC, at NewYork-Presbyterian and Weill Cornell Medical Center (New York, NY, USA). We enrolled patients with potentially resectable early-stage NSCLC (clinical stages I-IIIA as per the 7th edition of the American Joint Committee on Cancer) who were aged 18 years or older with an Eastern Cooperative Oncology Group performance status of 0 or 1. Eligible patients were randomly assigned (1:1) to either neoadjuvant durvalumab monotherapy or neoadjuvant durvalumab plus stereotactic body radiotherapy (8 Gy × 3 fractions), using permuted blocks with varied sizes and no stratification for clinical or molecular variables. Patients, treating physicians, and all study personnel were unmasked to treatment assignment after all patients were randomly assigned. All patients received two cycles of durvalumab 3 weeks apart at a dose of 1·12 g by intravenous infusion over 60 min. Those in the durvalumab plus radiotherapy group also received three consecutive daily fractions of 8 Gy stereotactic body radiotherapy delivered to the primary tumour immediately before the first cycle of durvalumab. Patients without systemic disease progression proceeded to surgical resection. The primary endpoint was major pathological response in the primary tumour. All analyses were done on an intention-to-treat basis. This trial is registered with ClinicalTrial.gov, NCT02904954, and is ongoing but closed to accrual. FINDINGS: Between Jan 25, 2017, and Sept 15, 2020, 96 patients were screened and 60 were enrolled and randomly assigned to either the durvalumab monotherapy group (n=30) or the durvalumab plus radiotherapy group (n=30). 26 (87%) of 30 patients in each group had their tumours surgically resected. Major pathological response was observed in two (6·7% [95% CI 0·8-22·1]) of 30 patients in the durvalumab monotherapy group and 16 (53·3% [34·3-71·7]) of 30 patients in the durvalumab plus radiotherapy group. The difference in the major pathological response rates between both groups was significant (crude odds ratio 16·0 [95% CI 3·2-79·6]; p<0·0001). In the 16 patients in the dual therapy group with a major pathological response, eight (50%) had a complete pathological response. The second cycle of durvalumab was withheld in three (10%) of 30 patients in the dual therapy group due to immune-related adverse events (grade 3 hepatitis, grade 2 pancreatitis, and grade 3 fatigue and thrombocytopaenia). Grade 3-4 adverse events occurred in five (17%) of 30 patients in the durvalumab monotherapy group and six (20%) of 30 patients in the durvalumab plus radiotherapy group. The most frequent grade 3-4 events were hyponatraemia (three [10%] patients in the durvalumab monotherapy group) and hyperlipasaemia (three [10%] patients in the durvalumab plus radiotherapy group). Two patients in each group had serious adverse events (pulmonary embolism [n=1] and stroke [n=1] in the durvalumab monotherapy group, and pancreatitis [n=1] and fatigue [n=1] in the durvalumab plus radiotherapy group). No treatment-related deaths or deaths within 30 days of surgery were reported. INTERPRETATION: Neoadjuvant durvalumab combined with stereotactic body radiotherapy is well tolerated, safe, and associated with a high major pathological response rate. This neoadjuvant strategy should be validated in a larger trial. FUNDING: AstraZeneca.