The impact of geriatric-specific variables on long-term outcomes in patients with hepatopancreatobiliary and colorectal cancer selected for resection.

BACKGROUND: Preoperative geriatric-specific variables (GSV) influence short-term morbidity in surgical patients, but their impact on long-term survival in elderly patients with cancer remains undefined. STUDY DESIGN: This observational cohort study included patients ≥65 years who underwent hepatopancreatobiliary or colorectal operations for malignancy between 2014 and 2020. Individual patient data included merged ACS NSQIP data, Procedure Targeted, and Geriatric Surgery Research variables. Patients were stratified by age: 65-74, 75-84, and ≥85 and presence of these GSVs: mobility aid, preoperative falls, surrogate signed consent, and living alone. Bivariable and multivariable analyses were used to evaluate 1-year mortality and postoperative discharge to facility. RESULTS: 577 patients were included: 62.6 % were 65-74 years old, 31.7 % 75-84, and 5.7 % ≥ 85. 96 patients were discharged to a facility with frequency increasing with age group (11.4 % vs 22.4 % vs 42.4 %, respectively, p < 0.001). 73 patients (12.7 %) died during 1-year follow-up, 32.9 % from cancer recurrence. One-year mortality was associated with undergoing hepatopancreatobiliary operations (p = 0.017), discharge to a facility (p = 0.047), and a surrogate signing consent (p = 0.035). Increasing age (p < 0.001), hepatopancreatobiliary resection (p = 0.002), living home alone (p < 0.001), and mobility aid use (p < 0.001) were associated with discharge to a facility. CONCLUSION: Geriatric-specific variables, living alone and use of a mobility aid, were associated with discharge to a facility. A surrogate signing consent and discharge to a facility were associated with 1-year mortality. These findings underscore the importance of preoperative patient selection and optimization, efficacious discharge planning, and informed decision-making in the care of elderly cancer patients.

Sensor-Assisted Analysis of Autonomic and Cerebrovascular Dysregulation following Concussion in an Individual with a History of Ten Concussions: A Case Study.

INTRODUCTION: Concussion is known to cause transient autonomic and cerebrovascular dysregulation that generally recovers; however, few studies have focused on individuals with an extensive concussion history. METHOD: The case was a 26-year-old male with a history of 10 concussions, diagnosed for bipolar type II disorder, mild attention-deficit hyperactivity disorder, and a history of migraines/headaches. The case was medicated with Valproic Acid and Escitalopram. Sensor-based baseline data were collected within six months of his injury and on days 1-5, 10, and 14 post-injury. Symptom reporting, heart rate variability (HRV), neurovascular coupling (NVC), and dynamic cerebral autoregulation (dCA) assessments were completed using numerous biomedical devices (i.e., transcranial Doppler ultrasound, 3-lead electrocardiography, finger photoplethysmography). RESULTS: Total symptom and symptom severity scores were higher for the first-week post-injury, with physical and emotional symptoms being the most impacted. The NVC response showed lowered activation in the first three days post-injury, while autonomic (HRV) and autoregulation (dCA) were impaired across all testing visits occurring in the first 14 days following his concussion. CONCLUSIONS: Despite symptom resolution, the case demonstrated ongoing autonomic and autoregulatory dysfunction. Larger samples examining individuals with an extensive history of concussion are warranted to understand the chronic physiological changes that occur following cumulative concussions through biosensing devices.

Fluorometric Methods to Measure Bioavailable and Total Heme.

Heme b (iron protoporphyrin IX) is an essential but potentially cytotoxic cofactor, signaling molecule, and nutritional source of iron. Its importance in cell biology and metabolism is underscored by the fact that numerous diseases, including various cancers, neurodegenerative disorders, infectious diseases, anemias, and porphyrias, are associated with the dysregulation of heme synthesis, degradation, trafficking, and/or transport. Consequently, methods to measure, image, and quantify heme in cells are required to better understand the physiology and pathophysiology of heme. Herein, we describe fluorescence-based protocols to probe heme bioavailability and trafficking dynamics using genetically encoded fluorescent heme sensors in combination with various modalities, such as confocal microscopy, flow cytometry, and microplate readers. Additionally, we describe a protocol for measuring total heme and its precursor protoporphyrin IX using a fluorometric assay that exploits porphyrin fluorescence. Together, the methods described enable the monitoring of total and bioavailable heme to study heme homeostatic mechanisms in virtually any cell type and organism.

Socioeconomic vulnerability and access to community water fluoridation in Washington.

BACKGROUND: The aim of the authors was to determine whether socioeconomic vulnerability is associated with community water fluoridation (CWF). METHODS: The authors used US Census Bureau data to create 4 county-level vulnerability markers (percentages non-White, Hispanic or Latino, below the federal poverty threshold, education below high school), obtained county-level CWF data from the Washington State Department of Health, and evaluated associations using Spearman rank correlation coefficient and the Kruskal-Wallis rank sum test. The authors then interviewed 122 community members in Washington (December 2022-March 2023) and analyzed the interview data inductively. RESULTS: A higher percentage of non-White people at the county level was associated with a significantly higher level of CWF (Spearman rank correlation coefficient, 0.55; 95% CI, 0.29 to 0.82; P < .001), whereas county-level poverty was associated with significantly lower CWF (Spearman rank correlation coefficient, -0.36; 95% CI, -0.70 to -0.03; P = .02). High school completion was not associated with county-level CWF. Significantly larger proportions of Hispanics and Latinos lived in counties with higher CWF (P < .05). From the interviews, more participants thought tap water was healthy than unhealthy, but 41% had mixed feelings. Similarly, more participants thought CWF was acceptable than unacceptable, with 35% reporting mixed feelings. Negative views about tap water and CWF were more common among non-White participants. CONCLUSIONS: People in racially and ethnically diverse communities in Washington appear to have greater access to CWF, whereas those in lower-income communities have poorer access. PRACTICAL IMPLICATIONS: CWF is an important population-level strategy to prevent caries. Additional work is needed to improve access to CWF, especially for people from low-income communities.

Exposure to Total Suspended Solids (TSS) Impacts Gill Structure and Function in Adult Zebrafish.

Total suspended solids (TSS) are a major contributor of anthropogenic impacts to aquatic systems. TSS exposure have been shown to affect the function of gills, but the mode of action is unclear. Zebrafish (Danio rerio) is emerging as an excellent model for mechanistic toxicology, and as there are no baseline studies on TSS effects in zebrafish gills, we tested the hypothesis that environmental concentrations of TSS damages gill structure and function in this species. Adult zebrafish were exposed to either 0, 10, 100, 500, 1000, or 2000 mg/L TSS for 4 days to assess the gill morphology. The minimal concentration that affected the gill structure was further tested for the distribution of key ion transporters, including Na+/K+- ATPase (NKA) and vacuolar-type H+-ATPase (VHA), using confocal microscopy. Our results reveal that TSS concentration as low as 100 mg/L alters the morphology of gills, including greater filament thickness, lamellae thickness, and epithelial lifting. This was also associated with a reduction in NKA immunoreactive (IR) cell count and intensity in the 100 mg/L TSS group, while there was neither a change in the VHA-IR cell count or expression nor the transcript abundance of atp6v1a and atp1a1a4 in the gills. Markers of stress response in these animals, including levels of cortisol, glucose, lactate, and glycogen were not altered after 4 days of TSS exposure. Overall, environmentally relevant concentrations of TSS can damage the gill structure and function in zebrafish and has the potential to enhance the toxicity of contaminants acting via the gills.

Biodegradability of unheated and laboratory heated dissolved organic matter.

Following wildfires, partially combusted biomass remains on the forest floor and erosion from the landscape can release dissolved pyrogenic organic matter (dPyOM) to surface waters. Therefore, post-fire alterations to dissolved organic matter (DOM) in aquatic systems may play a vital role in DOM stability and biogeochemical cycles. Dissolved PyOM biodegradation remains poorly understood and is expected to vary with combustion temperature and fuel source. In this study laboratory heating and leaching of forest floor materials (soil and litter) were used to compare the biodegradability of unheated, low (250 °C), and moderate (450 °C) temperature leachates. Inoculation experiments were performed with river microbes. Dissolved organic carbon (DOC) and nitrogen (DON), inorganic nitrogen, and DOM optical properties were monitored for 38 days. Inoculation experiments showed significantly greater DOC biodegradation of low and moderate temperature samples (64% and 71%, respectively) compared to unheated samples (32%). The greater DOC biodegradation may be explained by lower molecular weight DOM composition of heated leachates which was supported by higher initial E2/E3 ratios (absorbance at 250 nm/365 nm). Further, the observed decrease in the E2/E3 ratio after incubation suggests biodegradation of smaller compounds. This trend was greater for heated samples than unheated DOM. Specific ultraviolet absorbance increased after incubation, suggesting biodegradation of aliphatic compounds. Inoculated moderate temperature samples showed the greatest DON degradation (74%), followed by low temperature (58%) and unheated (51%) samples. Overall, results suggest that low and moderate temperature dPyOM was more biodegradable than unheated DOM, which may have implications for aquatic biogeochemical cycling, ecosystem function, and water quality in fire-impacted watersheds.

Routine Postclinical Event Debriefings on Inpatient Pediatric Units.

BACKGROUND AND OBJECTIVES: Debriefings are an underutilized opportunity to enhance team performance and safety culture. Little is known about the impact of postclinical event debriefing programs in Pediatric Hospital Medicine (PHM). We sought to develop a standardized debriefing process with multidisciplinary involvement after all clinical events on PHM service lines. Our primary aim was to achieve 75% debriefing completion rate over 12 months with debriefing duration less than 10 minutes. METHODS: A standardized postclinical event debriefing process was created at a large tertiary children's hospital. We aimed to debrief after clinical events on PHM services. The debriefing process was developed with key stakeholders and used a key driver diagram and Plan-Do-Study-Act cycles to refine the process. The project team reviewed the data monthly. RESULTS: During our 20-month study period, debriefing completion rate sustained a median of 66% with a median debriefing time of 7 minutes. Most debriefings (61%) had all core team members present with attending physicians (pediatric hospitalists) being absent most often. Barriers to debriefing with all core members present included service type, time of day, and shift change. Process changes were implemented based on concerns addressed in the debriefings. CONCLUSIONS: Multidisciplinary, postclinical event debriefings were successfully implemented on inpatient pediatric wards. Future steps include process implementation on non-PHM units in our hospital based on expressed interest and to further assess how debriefings optimize team performance and improve clinical outcomes.

An intranasal nanoparticle STING agonist protects against respiratory viruses in animal models.

Respiratory viral infections cause morbidity and mortality worldwide. Despite the success of vaccines, vaccination efficacy is weakened by the rapid emergence of viral variants with immunoevasive properties. The development of an off-the-shelf, effective, and safe therapy against respiratory viral infections is thus desirable. Here, we develop NanoSTING, a nanoparticle formulation of the endogenous STING agonist, 2'-3' cGAMP, to function as an immune activator and demonstrate its safety in mice and rats. A single intranasal dose of NanoSTING protects against pathogenic strains of SARS-CoV-2 (alpha and delta VOC) in hamsters. In transmission experiments, NanoSTING reduces the transmission of SARS-CoV-2 Omicron VOC to naïve hamsters. NanoSTING also protects against oseltamivir-sensitive and oseltamivir-resistant strains of influenza in mice. Mechanistically, NanoSTING upregulates locoregional interferon-dependent and interferon-independent pathways in mice, hamsters, as well as non-human primates. Our results thus implicate NanoSTING as a broad-spectrum immune activator for controlling respiratory virus infection.

The effects of socioeconomic status on complex ventral hernia repair operative decision-making and outcomes.

BACKGROUND: Patients from low socioeconomic backgrounds have greater rates of morbidity and mortality across disease processes. The Distressed Communities Index identified several socioeconomic components that were used to create a Distressed Communities Index score for every ZIP code, then broken into quintiles from prosperous to distressed. We aimed to explore whether socioeconomic distress as defined by the Distressed Communities Index affects the outcome of complex ventral hernia repair in the elderly population. METHODS: Retrospective analysis was performed using the Abdominal Core Health Collaborative data. Included were adults aged 65+ years undergoing elective complex ventral hernia repair from 2013 to 2021. Primary outcomes were postoperative outcomes and composite hernia recurrence by Distressed Communities Index quintile. The Cox proportional hazards model was used for composite recurrence, and logistic regression was used for postoperative outcomes. RESULTS: A total of 4,172 patients were included. Patients in distressed communities were more likely to identify as female or racial minority and had greater body mass index and American Society of Anesthesiologists class. Lower Distressed Communities Index quintile was associated with larger hernia (P = .012), open repair (P = .019), and 30-day complication (P = .05). There was no association between time to recurrence and Distressed Communities Index quintile (P = .24). After adjusted analysis, there was no significant difference for readmission, reoperation, recurrence, and complications. CONCLUSION: Patients from more distressed communities presented in worse clinical status with larger hernias. This likely contributed to greater rates of open repair and complications. However, when adjusted for these variables, outcomes were similar across Distressed Communities Index quintile. This supports the efficacy of complex hernia repair across socioeconomic classes.

Puberty classifications in beef heifers are moderately to highly heritable and associated with candidate genes related to cyclicity and timing of puberty.

Introduction: Pubertal attainment is critical to reproductive longevity in heifers. Previously, four heifer pubertal classifications were identified according to attainment of blood plasma progesterone concentrations > 1 ng/ml: 1) Early; 2) Typical; 3) Start-Stop; and 4) Non-Cycling. Early and Typical heifers initiated and maintained cyclicity, Start-Stop started and then stopped cyclicity and Non-Cycling never initiated cyclicity. Start-Stop heifers segregated into Start-Stop-Discontinuous (SSD) or Start-Stop-Start (SSS), with SSD having similar phenotypes to Non-Cycling and SSS to Typical heifers. We hypothesized that these pubertal classifications are heritable, and loci associated with pubertal classifications could be identified by genome wide association studies (GWAS). Methods: Heifers (n = 532; 2017 - 2022) genotyped on the Illumina Bovine SNP50 v2 or GGP Bovine 100K SNP panels were used for variant component estimation and GWAS. Heritability was estimated using a univariate Bayesian animal model. Results: When considering pubertal classifications: Early, Typical, SSS, SSD, and Non-Cycling, pubertal class was moderately heritable (0.38 ± 0.08). However, when heifers who initiated and maintained cyclicity were compared to those that did not cycle (Early+Typical vs. SSD+Non-Cycling) heritability was greater (0.59 ± 0.19). A GWAS did not identify single nucleotide polymorphisms (SNPs) significantly associated with pubertal classifications, indicating puberty is a polygenic trait. A candidate gene approach was used, which fitted SNPs within or nearby a set of 71 candidate genes previously associated with puberty, PCOS, cyclicity, regulation of hormone secretion, signal transduction, and methylation. Eight genes/regions were associated with pubertal classifications, and twenty-two genes/regions were associated with whether puberty was attained during the trial. Additionally, whole genome sequencing (WGS) data on 33 heifers were aligned to the reference genome (ARS-UCD1.2) to identify variants in FSHR, a gene critical to pubertal attainment. Fisher's exact test determined if FSHR SNPs segregated by pubertal classification. Two FSHR SNPs that were not on the bovine SNP panel were selected for additional genotyping and analysis, and one was associated with pubertal classifications and whether they cycled during the trial. Discussion: In summary, these pubertal classifications are moderately to highly heritable and polygenic. Consequently, genomic tools to inform selection/management of replacement heifers would be useful if informed by SNPs associated with cyclicity and early pubertal attainment.

Metachrony drives effective mucociliary transport via a calcium-dependent mechanism.

The mucociliary transport apparatus is critical for maintaining lung health via the coordinated movement of cilia to clear mucus and particulates. A metachronal wave propagates across the epithelium when cilia on adjacent multiciliated cells beat slightly out of phase along the proximal-distal axis of the airways in alignment with anatomically directed mucociliary clearance. We hypothesized that metachrony optimizes mucociliary transport (MCT) and that disruptions of calcium signaling would abolish metachrony and decrease MCT. We imaged bronchi from human explants and ferret tracheae using micro-Optical Coherence Tomography (µOCT) to evaluate airway surface liquid depth (ASL), periciliary liquid depth (PCL), cilia beat frequency (CBF), MCT, and metachrony in situ. We developed statistical models that included covariates of MCT. Ferret tracheae were treated with BAPTA-AM (chelator of intracellular Ca2+), lanthanum chloride (nonpermeable Ca2+channel competitive antagonist), and repaglinide (inhibitor of calaxin) to test calcium-dependence of metachrony. We demonstrated metachrony contributes to mucociliary transport of human and ferret airways. MCT was augmented in regions of metachrony compared to non-metachronous regions by 48.1%, P=0.0009 or 47.5%, P<0.0020 in humans and ferrets, respectively. PCL and metachrony were independent contributors to MCT rate in humans; ASL, CBF, and metachrony contribute to ferret MCT rates. Metachrony can be disrupted by interference with calcium signaling including intracellular, mechanosensitive channels, and calaxin. Our results support that the presence of metachrony augments MCT in a calcium-dependent mechanism.

Spinal Pathology and Muscle Morphologies with Chronic Low Back Pain and Lower Limb Amputation.

INTRODUCTION: Low back pain (LBP) is highly prevalent after lower limb amputation (LLA) and contributes to substantial reductions in quality of life and function. Towards understanding pathophysiological mechanisms underlying LBP after LLA, this article compares lumbar spine pathologies and muscle morphologies between individuals with LBP, with and without LLA. MATERIALS AND METHODS: We queried electronic medical records of Service members with and without LLA who sought care for LBP at military treatment facilities between January 2002 and May 2020. Two groups with cLBP, one with (n = 15) and one without unilateral transtibial LLA (n = 15), were identified and randomly chosen from a larger sample. Groups were matched by age, mass, and sex. Lumbar muscle morphology, Pfirrmann grades, Modic changes, facet arthrosis, Meyerding grades, and lordosis angle were determined from radiographs and magnetic resonance images available in the medical record. Independent t-tests compared variables between cohorts while multiple regression models determined if intramuscular fat influenced Pfirrmann grades. Chi-square determined differences in presence of spondylolysis and facet arthrosis. RESULTS: Lordosis angle was larger with LLA (P = 0.01). Spondylolysis was more prevalent with LLA (P = 0.008; 40%) whereas facet arthrosis was similar between cohorts (P = 0.3). Muscle area was not different between cohorts, yet intramuscular fat was greater with LLA (P ≤ 0.05). Intramuscular fat did not influence Pfirrmann grades (P > 0.15). CONCLUSIONS: Despite similar lumbar muscle size, those with unilateral LLA may be predisposed to progress to symptomatic spondylolisthesis and intramuscular fat. Surgical and/or rehabilitation interventions may mitigate long-term effects of diminished spinal health, decrease LBP-related disability, and improve function for individuals with LLA.

Comparison of Polypentenamer and Polynorbornene Bottlebrushes in Dilute Solution.

Bottlebrush (BB) polymers were synthesized via grafting-from-atom transfer radical polymerization (ATRP) of styrene on polypentenamer and polynorbornene macroinitiators with matched grafting density (n g = 4) and backbone degrees of polymerization (122 ≥ N bb ≥ 61) to produce a comparative study on their respective dilute solution properties as a function of increasing side chain degree of polymerization (116 ≥ N sc ≥ 5). The grafting-from technique produced near quantitative grafting efficiency and narrow dispersity N sc as evidenced by spectroscopic analysis and ring closing metathesis depolymerization of the polypentenamer BBs. The versatility of this synthetic approach permitted a comprehensive survey of power law expressions that arise from monitoring intrinsic viscosity, hydrodynamic radius, and radius of gyration as a function of increasing the molar mass of the BBs by increasing N sc. These values were compared to a series of linear (nongrafted, N sc = 0) macroinitiators in addition to linear grafts. This unique study allowed elucidation of the onset of bottlebrush behavior for two different types of bottlebrush backbones with identical grafting density but inherently different flexibility. In addition, grafting-from ATRP of methyl acrylate on a polypentenamer macroinitiator allowed the observation of the effects of graft chemistry in comparison to polystyrene. Differences in the observed scaling relationships in dilute solution as a function of each of these synthetic variants are discussed.

Combined genetic-pharmacologic inactivation of tightly linked ADAMTS proteases in temporally specific windows uncovers distinct roles for versican proteolysis and glypican-6 in cardiac development.

Extracellular matrix remodeling mechanisms are understudied in cardiac development and congenital heart defects. We show that matrix-degrading metalloproteases ADAMTS1 and ADAMTS5, are extensively co-expressed during mouse cardiac development. The mouse mutants of each gene have mild cardiac anomalies, however, their combined genetic inactivation to elicit cooperative roles is precluded by tight gene linkage. Therefore, we coupled Adamts1 inactivation with pharmacologic ADAMTS5 blockade to uncover stage-specific cooperative roles and investigated their potential substrates in mouse cardiac development. ADAMTS5 blockade was achieved in Adamts1 null mouse embryos using an activity-blocking monoclonal antibody during distinct developmental windows spanning myocardial compaction or cardiac septation and outflow tract rotation. Synchrotron imaging, RNA in situ hybridization, immunofluorescence microscopy and electron microscopy were used to determine the impact on cardiac development and compared to Gpc6 and ADAMTS-cleavage resistant versican mutants. Mass spectrometry-based N-terminomics was used to seek relevant substrates. Combined inactivation of ADAMTS1 and ADAMTS5 prior to 12.5 days of gestation led to dramatic accumulation of versican-rich cardiac jelly and inhibited formation of compact and trabecular myocardium, which was also observed in mice with ADAMTS cleavage-resistant versican. Combined inactivation after 12.5 days impaired outflow tract development and ventricular septal closure, generating a tetralogy of Fallot-like defect. N-terminomics of combined ADAMTS knockout and control hearts identified a cleaved glypican-6 peptide only in the controls. ADAMTS1 and ADAMTS5 expression in cells was associated with specific glypican-6 cleavages. Paradoxically, combined ADAMTS1 and ADAMTS5 inactivation reduced cardiac glypican-6 and outflow tract Gpc6 transcription. Notably, Gpc6-/- hearts demonstrated similar rotational defects as combined ADAMTS inactivated hearts and both had reduced hedgehog signaling. Thus, versican proteolysis in cardiac jelly at the canonical Glu441-Ala442 site is cooperatively mediated by ADAMTS1 and ADAMTS5 and required for proper ventricular cardiomyogenesis, whereas, reduced glypican-6 after combined ADAMTS inactivation impairs hedgehog signaling, leading to outflow tract malrotation.

A Randomized, Double-Blind, Placebo-Controlled Pilot Trial of the Acute Antisuicidal and Antidepressant Effects of Intranasal (R,S)-Ketamine in Severe Unipolar and Bipolar Depression With and Without Comorbid Alcohol Use Disorder.

Objective: Although individuals with a family history of alcohol use disorder (AUD) have a superior antidepressant response to ketamine, outcomes in patients with current AUD remain unclear. This study sought to investigate whether intranasal (IN) racemic (R,S)-ketamine had antisuicidal and antidepressant effects in unipolar and bipolar depression and whether comorbid AUD conferred superior antisuicidal outcomes for patients.Methods: This was a double-blind, randomized, placebo-controlled trial (May 2018 to January 2022) of single administration, fixed-dose (50 mg) IN (R,S)-ketamine (or saline comparator) in unmedicated inpatients meeting Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision, criteria for a current major depressive episode (bipolar or unipolar), with current suicidal ideation (SI) and past attempt. Patients with and without comorbid AUD were enrolled. Change in Scale for Suicide Ideation score was the primary outcome measure, and change in Montgomery-Åsberg Depression Rating Scale score was the secondary outcome measure.Results: No significant group × time effect was noted for SI (F = 1.1, P = .36). A statistical trend toward superior improvement in suicidality was observed in participants with comorbid AUD. The group × time interaction was significant for improvements in depression (F = 3.06, P = .03) and largely unaffected by comorbid AUD or primary mood disorder type. Within the ketamine group, a significant correlation was observed between improvement in depressive symptoms and SI for patients without comorbid AUD (r =0.927, P = .023) that was absent in patients with AUD (r = 0.39, P = .44).Conclusion: IN ketamine induced rapid antidepressant effects compared to placebo but did not significantly alter SI scores. The treatment was well tolerated. Continued investigation with IN ketamine as a practical alternative to current formulations is warranted.Trial Registration: ClinicalTrials.gov identifier: NCT03539887.

Inhibition of ribonucleotide reductase subunit M2 enhances the radiosensitivity of metastatic pancreatic neuroendocrine tumor.

Ribonucleotide Reductase (RNR) is a rate-limiting enzyme in the production of deoxyribonucleoside triphosphates (dNTPs), which are essential substrates for DNA repair after radiation damage. We explored the radiosensitization property of RNR and investigated a selective RRM2 inhibitor, 3-AP, as a radiosensitizer in the treatment of metastatic pNETs. We investigated the role of RNR subunit, RRM2, in pancreatic neuroendocrine (pNET) cells and responses to radiation in vitro. We also evaluated the selective RRM2 subunit inhibitor, 3-AP, as a radiosensitizer to treat pNET metastases in vivo. Knockdown of RNR subunits demonstrated that RRM1 and RRM2 subunits, but not p53R3, play significant roles in cell proliferation. RRM2 inhibition activated DDR pathways through phosphorylation of ATM and DNA-PK protein kinases but not ATR. RRM2 inhibition also induced Chk1 and Chk2 phosphorylation, resulting in G1/S phase cell cycle arrest. RRM2 inhibition sensitized pNET cells to radiotherapy and induced apoptosis in vitro. In vivo, we utilized pNET subcutaneous and lung metastasis models to examine the rationale for RNR-targeted therapy and 3-AP as a radiosensitizer in treating pNETs. Combination treatment significantly increased apoptosis of BON (human pNET) xenografts and significantly reduced the burden of lung metastases. Together, our results demonstrate that selective RRM2 inhibition induced radiosensitivity of metastatic pNETs both in vitro and in vivo. Therefore, treatment with the selective RRM2 inhibitor, 3-AP, is a promising radiosensitizer in the therapeutic armamentarium for metastatic pNETs.

Inhibition of NOTCH4 sensitizes FLT3/ITD acute myeloid leukemia cells to FLT3 tyrosine kinase inhibition.

Internal tandem duplication mutations of FLT3 (FLT3/ITD) confer poor prognosis in AML. FLT3 tyrosine kinase inhibitors (TKIs) alone have limited and transient clinical efficacy thus calling for new targets for more effective combination therapy. In a loss-of-function RNAi screen, we identified NOTCH4 as one such potential target whose inhibition proved cytotoxic to AML cells, and also sensitized them to FLT3 inhibition. Further investigation found increased NOTCH4 expression in FLT3/ITD AML cell lines and primary patient samples. Inhibition of NOTCH4 by shRNA knockdown, CRISPR-Cas9-based knockout or γ-secretase inhibitors synergized with FLT3 TKIs to kill FLT3/ITD AML cells in vitro. NOTCH4 inhibition sensitized TKI-resistant FLT3/ITD cells to FLT3 TKI inhibition. The combination reduced phospho-ERK and phospho-AKT, indicating inhibition of MAPK and PI3K/AKT signaling pathways. It also led to changes in expression of genes involved in regulating cell cycling, DNA repair and transcription. A patient-derived xenograft model showed that the combination reduced both the level of leukemic involvement of primary human FLT3/ITD AML cells and their ability to engraft secondary recipients. In summary, these results demonstrate that NOTCH4 inhibition synergizes with FLT3 TKIs to eliminate FLT3/ITD AML cells, providing a new therapeutic target for AML with FLT3/ITD mutations.

Seasonality Affects Low-Molecular-Weight Organic Acids and Phenolic Compounds' Composition in Scots Pine Litterfall.

BACKGROUND AND AIMS: Secondary plant metabolites, including organic acids and phenolic compounds, have a significant impact on the properties of organic matter in soil, influencing its structure and function. How the production of these compounds in foliage that falls to the forest floor as litterfall varies across tree age and seasonality are of considerable interest for advancing our understanding of organic matter dynamics. METHODS: Monthly, we collected fallen needles of Scots pine (Pinus sylvestris L.) across stands of five different age classes (20, 40, 60, 80, and 100 years) for one year and measured the organic acids and phenolic compounds. RESULTS: Seven low-molecular-weight organic acids and thirteen phenolic compounds were detected in the litterfall. No differences were observed across stand age. Significant seasonal differences were detected. Most compounds peaked during litterfall in the growing season. Succinic acid was the most prevalent organic acid in the litterfall, comprising 78% of total organic acids (351.27 ± 34.27 µg g- 1), and was 1.5 to 11.0 times greater in the summer than all other seasons. Sinapic acid was the most prevalent phenolic compound in the litterfall (42.15 µg g- 1), representing 11% of the total phenolic compounds, and was 39.8 times greater in spring and summer compared to autumn and winter. Growing season peaks in needle concentrations were observed for all thirteen phenolic compounds and two organic acids (lactic, succinic). Citric acid exhibited a definitive peak in late winter into early spring. CONCLUSIONS: Our results highlight the seasonal dynamics of the composition of secondary plant metabolites in litterfall, which is most different at the onset of the growing season. Fresh inputs of litterfall at this time of emerging biological activity likely have seasonal impacts on soil's organic matter composition as well.

Exploring the Role of the Private Sector in Tuberculosis Detection and Management in Lima, Peru: A Mixed-Methods Patient Pathway Analysis.

In Latin America, little is known about the involvement of private health-care providers in tuberculosis (TB) detection and management. We sought to gain a better understanding of current and potential roles of the private sector in delivering TB services in Peru. We conducted a mixed-methods study in North Lima, Peru. The quantitative component comprised a patient pathway analysis assessing the alignment of TB services with patient care-seeking behavior. The qualitative component comprised in-depth interviews with 18 private health-care providers and 5 key informants. We estimated that 77% of patients sought care initially at a facility with TB diagnostic capacity and 59% at a facility with TB treatment capacity. Among private facilities, 43% offered smear microscopy, 13% offered radiography, and none provided TB treatment. Among public-sector facilities, 100% offered smear microscopy, 26% offered radiography, and 99% provided TB treatment. Private providers believed they offered shorter wait times and a faster diagnosis, but they struggled with a lack of referral systems and communication with the public sector. Nonrecognition of private-sector tests by the public sector led to duplicate testing of referred patients. Although expressing willingness to collaborate with public-sector programs for diagnosis and referral, private providers had limited interest in treating TB. This study highlights the role of private providers in Peru as an entry point for TB care. Public-private collaboration is necessary to harness the potential of the private sector as an ally for early diagnosis.