Novel Definitive Hypofractionated Accelerated Radiation Dose-painting (HARD) for Unresected Soft Tissue Sarcomas.

Purpose: Soft tissue sarcomas (STS) are historically radioresistant, with surgery being an integral component of their treatment. With their low α/ß, STS may be more responsive to hypofractionated radiation therapy (RT), which is often limited by long-term toxicity risk to surrounding normal tissue. An isotoxic approach using a hypofractionated accelerated radiation dose-painting (HARD) regimen allows for dosing based on clinical risk while sparing adjacent organs at risk. Methods and Materials: We retrospectively identified patients from 2019 to 2022 with unresected STS who received HARD with dose-painting to high, intermediate, and low-risk regions of 3.0 Gy, 2.5 Gy, and 2.0 to 2.3 Gy, respectively, in 20 to 22 fractions. Clinical endpoints included local control, locoregional control, progression free survival, overall survival, and toxicity outcomes. Results: Twenty-seven consecutive patients were identified and had a median age of 68 years and tumor size of 7.0 cm (range, 1.2-21.0 cm). Tumors were most often high-grade (70%), stage IV (70%), located in the extremities (59%), and locally recurrent (52%). With a median follow-up of 33.4 months, there was a 3-year locoregional control rate of 100%. The 3-year overall and progression-free survival were 44.9% and 23.3%, respectively. There were 5 (19%) acute and 2 (7%) late grade 3 toxicities, and there were no grade 4 or 5 toxicities at any point. Conclusions: The HARD regimen is a safe method of dose-escalating STS, with durable 3-year locoregional control. This approach is a promising alternative for unresected STS, though further follow-up is required to determine long-term control and toxicity.

Multi-Institutional Study of Referral Patterns for Gynecologic Oncology Consultation.

PURPOSE: Evaluation by a gynecologic oncologist (GO) is associated with improved clinical outcomes for patients with gynecologic cancers, yet little is known about health care factors that influence patients' referrals to GO. METHODS: Medical records of 50 consecutive new patients seen in GO clinics at each of six referral centers across the United States were reviewed. Patient and disease characteristics were collected along with referral indication, evaluation and referral dates, diagnostic procedures, provider specialties, and zone improvement plan (ZIP) code of up to three referring providers per patient. The primary outcome was interval between first evaluation and referral. Univariate associations were evaluated with Chi-square and Wilcoxon rank-sum tests and multivariable associations with negative binomial regression models. Secondary outcome was prolonged time to GO referral, defined as greater than the 75th percentile. Logistic regression was used for multivariable modeling. RESULTS: Three hundred patient records were analyzed. The median time from first health care encounter to referral was 15 days (IQR, 5-43). The mean distance from residence to GO was 39.8 miles (standard deviation, 53.8). Seventy-one percent of GO referrals were initiated by obstetrician-gynecologists, 9% by family physicians, and 6% internists. Presentation-to-referral interval was 76% shorter for patients evaluated by an emergency medicine clinician (exp(Beta), 0.24; 95% CI, 0.11 to 0.53; P < .001). Public insurance was associated with 1.47 times longer time to referral compared with private insurance (exp(Beta), 1.47; 95% CI, 1.05 to 2.04; P = .001). Residents of nonmetropolitan ZIP codes were less likely to have prolonged time to referral (odds ratio [OR], 0.288; P = .017). Distance from residence to GO (per 10 miles) increased the likelihood of prolonged time to referral (OR, 1.10; P = .010). CONCLUSION: Interventions are needed to improve recognition and referral of patients for gynecologic oncology evaluation. Community outreach and engagement with obstetrician-gynecologists should be prioritized to improve times to referral.

Mapping medically relevant RNA isoform diversity in the aged human frontal cortex with deep long-read RNA-seq.

Determining whether the RNA isoforms from medically relevant genes have distinct functions could facilitate direct targeting of RNA isoforms for disease treatment. Here, as a step toward this goal for neurological diseases, we sequenced 12 postmortem, aged human frontal cortices (6 Alzheimer disease cases and 6 controls; 50% female) using one Oxford Nanopore PromethION flow cell per sample. We identified 1,917 medically relevant genes expressing multiple isoforms in the frontal cortex where 1,018 had multiple isoforms with different protein-coding sequences. Of these 1,018 genes, 57 are implicated in brain-related diseases including major depression, schizophrenia, Parkinson's disease and Alzheimer disease. Our study also uncovered 53 new RNA isoforms in medically relevant genes, including several where the new isoform was one of the most highly expressed for that gene. We also reported on five mitochondrially encoded, spliced RNA isoforms. We found 99 differentially expressed RNA isoforms between cases with Alzheimer disease and controls.

Habitat escalated adaptive therapy (HEAT): a phase 2 trial utilizing radiomic habitat-directed and genomic-adjusted radiation dose (GARD) optimization for high-grade soft tissue sarcoma.

BACKGROUND: Soft tissue sarcomas (STS), have significant inter- and intra-tumoral heterogeneity, with poor response to standard neoadjuvant radiotherapy (RT). Achieving a favorable pathologic response (FPR ≥ 95%) from RT is associated with improved patient outcome. Genomic adjusted radiation dose (GARD), a radiation-specific metric that quantifies the expected RT treatment effect as a function of tumor dose and genomics, proposed that STS is significantly underdosed. STS have significant radiomic heterogeneity, where radiomic habitats can delineate regions of intra-tumoral hypoxia and radioresistance. We designed a novel clinical trial, Habitat Escalated Adaptive Therapy (HEAT), utilizing radiomic habitats to identify areas of radioresistance within the tumor and targeting them with GARD-optimized doses, to improve FPR in high-grade STS. METHODS: Phase 2 non-randomized single-arm clinical trial includes non-metastatic, resectable high-grade STS patients. Pre-treatment multiparametric MRIs (mpMRI) delineate three distinct intra-tumoral habitats based on apparent diffusion coefficient (ADC) and dynamic contrast enhanced (DCE) sequences. GARD estimates that simultaneous integrated boost (SIB) doses of 70 and 60 Gy in 25 fractions to the highest and intermediate radioresistant habitats, while the remaining volume receives standard 50 Gy, would lead to a > 3 fold FPR increase to 24%. Pre-treatment CT guided biopsies of each habitat along with clip placement will be performed for pathologic evaluation, future genomic studies, and response assessment. An mpMRI taken between weeks two and three of treatment will be used for biological plan adaptation to account for tumor response, in addition to an mpMRI after the completion of radiotherapy in addition to pathologic response, toxicity, radiomic response, disease control, and survival will be evaluated as secondary endpoints. Furthermore, liquid biopsy will be performed with mpMRI for future ancillary studies. DISCUSSION: This is the first clinical trial to test a novel genomic-based RT dose optimization (GARD) and to utilize radiomic habitats to identify and target radioresistance regions, as a strategy to improve the outcome of RT-treated STS patients. Its success could usher in a new phase in radiation oncology, integrating genomic and radiomic insights into clinical practice and trial designs, and may reveal new radiomic and genomic biomarkers, refining personalized treatment strategies for STS. TRIAL REGISTRATION: NCT05301283. TRIAL STATUS: The trial started recruitment on March 17, 2022.

Z-selective dimerization of terminal alkynes by a (PNNP)FeII complex.

A tetradentate bis(amido)bis(phosphine) FeII complex, (PNNP)Fe, is shown to activate the terminal C-H bond of aryl alkynes across its Fe-Namide bonds. (PNNP)Fe is also shown to catalytically dimerize terminal aryl alkynes to produce 1,3-enynes with Z : E ratios as high as 96 : 4 with yields up to 95% and loadings as low as 1 mol% at 30 °C in 2 h. A plausible metal-ligand cooperative mechanism invoking a vinylidene intermediate is proposed.

AlphaFold-assisted structure determination of a bacterial protein of unknown function using X-ray and electron crystallography.

Macromolecular crystallography generally requires the recovery of missing phase information from diffraction data to reconstruct an electron-density map of the crystallized molecule. Most recent structures have been solved using molecular replacement as a phasing method, requiring an a priori structure that is closely related to the target protein to serve as a search model; when no such search model exists, molecular replacement is not possible. New advances in computational machine-learning methods, however, have resulted in major advances in protein structure predictions from sequence information. Methods that generate predicted structural models of sufficient accuracy provide a powerful approach to molecular replacement. Taking advantage of these advances, AlphaFold predictions were applied to enable structure determination of a bacterial protein of unknown function (UniProtKB Q63NT7, NCBI locus BPSS0212) based on diffraction data that had evaded phasing attempts using MIR and anomalous scattering methods. Using both X-ray and micro-electron (microED) diffraction data, it was possible to solve the structure of the main fragment of the protein using a predicted model of that domain as a starting point. The use of predicted structural models importantly expands the promise of electron diffraction, where structure determination relies critically on molecular replacement.

Ki-67 Testing in Breast Cancer: Assessing Variability With Scoring Methods and Specimen Types and the Potential Subsequent Impact on Therapy Eligibility.

Abemaciclib was originally FDA approved for patients with ER-positive/HER2-negative breast cancer with Ki-67 expression ≥20%. However, there were no guidelines provided on which specimen to test or which scoring method to use. We performed a comprehensive study evaluating the variation in Ki-67 expression in breast specimens from 50 consecutive patients who could have been eligible for abemaciclib therapy. Three pathologists with breast expertise each performed a blinded review with 3 different manual scoring methods [estimated (EST), unweighted (UNW), and weighted (WT) (WT recommended by the International Ki-67 in Breast Cancer Working Group)]. Quantitative image analysis (QIA) using the HALO platform was also performed. Three different specimen types [core needle biopsy (CNB) (n=63), resection (RES) (n=52), and axillary lymph node metastasis (ALN) (n=50)] were evaluated for each patient. The average Ki-67 for all specimens was 14.68% for EST, 14.46% for UNW, 14.15% for WT, and 11.15% for QIA. For the manual methods, the range between the lowest and highest Ki-67 for each specimen between the 3 pathologists was 8.44 for EST, 5.94 for WT, and 5.93 for UNW. The WT method limited interobserver variability with ICC1=0.959 (EST ICC1=0.922 and UNW=0.949). Using the aforementioned cutoff of Ki-67 ≥20% versus <20% to determine treatment eligibility, the averaged EST method yields 20 of 50 patients (40%) who would have been treatment-eligible, versus 15 (30%) for the UNW, 17 (34%) for the WT, and 12 (24%) for the QIA. There was no statistically significant difference in Ki-67 among the 3 specimen types. The average Ki-67 difference was 4.36 for CNB vs RES, 6.95 for CNB versus ALN, and RES versus ALN (P=0.93, 0.99, and 0.94, respectively). Our study concludes that further refinement in Ki-67 scoring is advisable to reduce clinically significant variation.

Effect of aromatic substituents on thermoresponsive functional polycaprolactone micellar carriers for doxorubicin delivery.

Amphiphilic functional polycaprolactone (PCL) diblock copolymers are excellent candidates for micellar drug delivery. The functional groups on the backbone significantly affect the properties of PCL. A systematic investigation of the effect of aromatic substituents on the self-assembly of γ-functionalized PCLs and the delivery of doxorubicin (DOX) is presented in this work. Three thermoresponsive amphiphilic diblock copolymers with poly(γ-benzyloxy-ε-caprolactone) (PBnCL), poly(γ-phenyl- ε-caprolactone) (PPhCL), poly(γ-(4-ethoxyphenyl)-ε-caprolactone) (PEtOPhCL), respectively, as hydrophobic block and γ-tri(ethylene glycol) functionalized PCL (PME3CL) as hydrophilic block were prepared through ring-opening polymerization (ROP). The thermoresponsivity, thermodynamic stability, micelle size, morphology, DOX-loading, and release profile were determined. The LCST values of amphiphilic diblock copolymers PME3CL-b-PBnCL, PME3CL-b-PPhCL, and PME3CL-b-PEtOPhCL are 74.2°C, 43.3°C, and 37.3°C, respectively. All three copolymers formed spherical micelles in phosphate-buffered saline (PBS, 1×, pH = 7.4) at low concentrations ranging from 8.7 × 10-4 g/L to 8.9 × 10-4 g/L. PME3CL-b-PBnCL micelles showed the highest DOX loading capacity of 3.01 ± 0.18 (wt%) and the lowest drug release, while PME3CL-b-PEtOPhCL micelles exhibited the lowest DOX loading capacity of 1.95 ± 0.05 (wt%) and the highest drug release. Cytotoxicity and cellular uptake of all three micelles were assessed in vitro using MDA-MB-231 breast cancer cells. All three empty micelles did not show significant toxicity to the cells at concentrations high up to 0.5 mg/mL. All three DOX-loaded micelles were uptaken into the cells, and DOX was internalized into the nucleus of the cells.

Novel Postoperative Hypofractionated Accelerated Radiation Dose-Painting Approach for Soft Tissue Sarcoma.

Purpose: Hypofractionated radiation therapy (RT) offers benefits in the treatment of soft tissue sarcomas (STS), including exploitation of the lower α/ß, patient convenience, and cost. This study evaluates the acute toxicity of a hypofractionated accelerated RT dose-painting (HARD) approach for postoperative treatment of STS. Methods and Materials: This is a retrospective review of 53 consecutive patients with STS who underwent resection followed by postoperative RT. Standard postoperative RT dosing for R0/R1/gross disease with sequential boost (50 Gy + 14/16/20 Gy in 32-35 fractions) were replaced with dose-painting, which adapts dose based on risk of disease burden, to 50.4 and 63, 64.4, 70 Gy in 28 fractions, respectively. The first 10 patients were replanned with a sequential boost RT approach and dosimetric indices were compared. Time-to-event outcomes, including local control, regional control, distant control, and overall survival, were estimated with Kaplan-Meier analysis. Results: Median follow-up was 25.2 months. Most patients had high-grade (59%) STS of the extremity (63%) who underwent resection with either R1 (40%) or close (36%) margins. Four patients experienced grade 3 acute dermatitis which resolved by the 3-month follow-up visit. The 2-year local control, regional control, distant control, and overall survival were 100%, 92%, 68%, and 86%, respectively. Compared with the sequential boost plan, HARD had a significantly lower field size (total V50 Gy; P = .002), bone V50 (P = .031), and maximum skin dose (P = .008). Overall treatment time was decreased by 4 to 7 fractions, which translated to a decrease in estimated average treatment cost of $3056 (range, $2651-$4335; P < .001). Conclusions: In addition to benefits in cost, convenience, and improved biologic effect in STS, HARD regimen offers a safe treatment approach with dosimetric advantages compared with conventional sequential boost, which may translate to improved long-term toxicity.

Solvent-Dependent Emissions Properties of a Model Aurone Enable Use in Biological Applications.

Fluorophores are powerful visualization tools and the development of novel small organic fluorophores are in great demand. Small organic fluorophores have been derived from the aurone skeleton, 2-benzylidenebenzofuran-3(2H)-one. In this study, we have utilized a model aurone derivative with a methoxy group at the 3' position and a hydroxyl group at the 4' position, termed vanillin aurone, to develop a foundational understanding of structural factors impacting aurone fluorescence properties. The fluorescent behaviors of the model aurone were characterized in solvent environments differing in relative polarity and dielectric constant. These data suggested that hydrogen bonding or electrostatic interactions between excited state aurone and solvent directly impact emissions properties such as peak emission wavelength, emission intensity, and Stokes shift. Time-dependent Density Functional Theory (TD-DFT) model calculations suggest that quenched aurone emissions observed in water are a consequence of stabilization of a twisted excited state conformation that disrupts conjugation. In contrast, the calculations indicate that low polarity solvents such as toluene or acetone stabilize a brightly fluorescent planar state. Based on this, additional experiments were performed to demonstrate use as a turn-on probe in an aqueous environment in response to conditions leading to planar excited state stabilization. Vanillin aurone was observed to bind to a model ATP binding protein, YME1L, leading to enhanced emissions intensities with a dissociation equilibrium constant equal to ~ 30 µM. Separately, the aurone was observed to be cell permeable with significant toxicity at doses exceeding 6.25 µM. Taken together, these results suggest that aurones may be broadly useful as turn-on probes in aqueous environments that promote either a change in relative solvent polarity or through direct stabilization of a planar excited state through macromolecular binding.

Test-retest reliability and reliable change index of the Philips IntelliSpace Cognition digital test battery.

OBJECTIVE: This article provides the test-retest reliability and Reliable Change Indices (RCIs) of the Philips IntelliSpace Cognition (ISC) platform, which contains digitized versions of well-established neuropsychological tests. METHOD: 147 participants (ages 19 to 88) completed a digital cognitive test battery on the ISC platform or paper-pencil versions of the same test battery during two separate visits. Intraclass correlation coefficients (ICC) were calculated separately for the ISC and analog test versions to compare reliabilities between administration modalities. RCIs were calculated for the digital tests using the practice-adjusted RCI and standardized regression-based (SRB) method. RESULTS: Test-retest reliabilities for the ISC tests ranged from moderate to excellent and were comparable to the test-retest reliabilities for the paper-pencil tests. Baseline test performance, retest interval, age, and education predicted test performance at visit 2 with baseline test performance being the strongest predictor for all outcome measures. For most outcome measures, both methods for the calculation of RCIs show agreement on whether or not a reliable change was observed. CONCLUSIONS: RCIs for the digital tests enable clinicians to determine whether a measured change between assessments is due to real improvement or decline. Together, this contributes to the growing evidence for the clinical utility of the ISC platform.

Design of Ligand-Operable Protein-Cages That Open Upon Specific Protein Binding.

Protein nanocages have diverse applications in medicine and biotechnology, including molecular delivery. However, although numerous studies have demonstrated the ability of protein nanocages to encapsulate various molecular species, limited methods are available for subsequently opening a nanocage for cargo release under specific conditions. A modular platform with a specific protein-target-based mechanism of nanocage opening is notably lacking. To address this important technology gap, we present a new class of designed protein cages, the Ligand-Operable Cage (LOC). LOCs primarily comprise a protein nanocage core and a fused surface binding adaptor. The geometry of the LOC is designed so that binding of a target protein ligand (or multiple copies thereof) to the surface binder is sterically incompatible with retention of the assembled state of the cage. Therefore, the tight binding of a target ligand drives cage disassembly by mass action, subsequently exposing the encapsulated cargo. LOCs are modular; direct substitution of the surface binder sequence can reprogram the nanocage to open in response to any target protein ligand of interest. We demonstrate these design principles using both a natural and a designed protein cage as the core, with different proteins acting as the triggering ligand and with different reporter readouts─fluorescence unquenching and luminescence─for cage disassembly. These developments advance the critical problem of targeted molecular delivery and detection.

Interspecies Differences in 6PPD-Quinone Toxicity Across Seven Fish Species: Metabolite Identification and Semiquantification.

N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-Q) is a recently identified contaminant that originates from the oxidation of the tire antidegradant 6PPD. 6PPD-Q is acutely toxic to select salmonids at environmentally relevant concentrations, while other fish species display tolerance to concentrations that surpass those measured in the environment. The reasons for these marked differences in sensitivity are presently unknown. The objective of this research was to explore potential toxicokinetic drivers of species sensitivity by characterizing biliary metabolites of 6PPD-Q in sensitive and tolerant fishes. For the first time, we identified an O-glucuronide metabolite of 6PPD-Q using high-resolution mass spectrometry. The semiquantified levels of this metabolite in tolerant species or life stages, including white sturgeon (Acipenser transmontanus), chinook salmon (Oncorhynchus tshawytscha), westslope cutthroat trout (Oncorhynchus clarkii lewisi), and nonfry life stages of Atlantic salmon (Salmo salar), were greater than those in sensitive species, including coho salmon (Oncorhynchus kisutch), brook trout (Salvelinus fontinalis), and rainbow trout (Oncorhynchus mykiss), suggesting that tolerant species might detoxify 6PPD-Q more effectively. Thus, we hypothesize that differences in species sensitivity are a result of differences in basal expression of biotransformation enzyme across various fish species. Moreover, the semiquantification of 6PPD-Q metabolites in bile extracted from wild-caught fish might be a useful biomarker of exposure to 6PPD-Q, thereby being valuable to environmental monitoring and risk assessment.

Microglial Imaging in Alzheimer's Disease and Its Relationship to Brain Amyloid: A Human 18F-GE180 PET Study.

BACKGROUND: Emerging evidence suggests a potential causal role of neuroinflammation in Alzheimer's disease (AD). Using positron emission tomography (PET) to image overexpressed 18 kDA translocator protein (TSPO) by activated microglia has gained increasing interest. The uptake of 18F-GE180 TSPO PET was observed to co-localize with inflammatory markers and have a two-stage association with amyloid PET in mice. Very few studies evaluated the diagnostic power of 18F-GE180 PET in AD population and its interpretation in human remains controversial about whether it is a marker of microglial activation or merely reflects disrupted blood-brain barrier integrity in humans. OBJECTIVE: The goal of this study was to study human GE180 from the perspective of the previous animal observations. METHODS: With data from twenty-four participants having 18F-GE180 and 18F-AV45 PET scans, we evaluated the group differences of 18F-GE180 uptake between participants with and without cognitive impairment. An association analysis of 18F-GE180 and 18F-AV45 was then conducted to test if the relationship in humans is consistent with the two-stage association in AD mouse model. RESULTS: Elevated 18F-GE180 was observed in participants with cognitive impairment compared to those with normal cognition. No regions showed reduced 18F-GE180 uptake. Consistent with mouse model, a two-stage association between 18F-GE180 and 18F-AV45 was observed. CONCLUSIONS: 18F-GE180 PET imaging showed promising utility in detecting pathological alterations in a symptomatic AD population. Consistent two-stage association between 18F-GE180 and amyloid PET in human and mouse suggested that 18F-GE180 uptake in human might be considerably influenced by microglial activation.

Clinical and biological relevance of glial fibrillary acidic protein in Alzheimer's disease.

INTRODUCTION: There is a tremendous need for identifying reliable blood-based biomarkers for Alzheimer's disease (AD) that are tied to the biological ATN (amyloid, tau and neurodegeneration) framework as well as clinical assessment and progression. METHODS: One hundred forty-four elderly participants underwent 18F-AV45 positron emission tomography (PET) scan, structural magnetic resonance imaging (MRI) scan, and blood sample collection. The composite standardized uptake value ratio (SUVR) was derived from 18F-AV45 PET to assess brain amyloid burden, and the hippocampal volume was determined from structural MRI scans. Plasma glial fibrillary acidic protein (GFAP), phosphorylated tau-181 (ptau-181), and neurofilament light (NfL) measured by single molecular array (SIMOA) technology were assessed with respect to ATN framework, genetic risk factor, age, clinical assessment, and future functional decline among the participants. RESULTS: Among the three plasma markers, GFAP best discriminated participants stratified by clinical diagnosis and brain amyloid status. Age was strongly associated with NfL, followed by GFAP and ptau-181 at much weaker extent. Brain amyloid was strongly associated with plasma GFAP and ptau-181 and to a lesser extent with plasma NfL. Moderate association was observed between plasma markers. Hippocampal volume was weakly associated with all three markers. Elevated GFAP and ptau-181 were associated with worse cognition, and plasma GFAP was the most predictive of future functional decline. Combining GFAP and ptau-181 together was the best model to predict brain amyloid status across all participants (AUC = 0.86) or within cognitively impaired participants (AUC = 0.93); adding NfL as an additional predictor only had a marginal improvement. CONCLUSION: Our findings indicate that GFAP is of potential clinical utility in screening amyloid pathology and predicting future cognitive decline. GFAP, NfL, and ptau-181 were moderately associated with each other, with discrepant relevance to age, sex, and AD genetic risk, suggesting their relevant but differential roles for AD assessment. The combination of GFAP with ptau-181 provides an accurate model to predict brain amyloid status, with the superior performance of GFAP over ptau-181 when the prediction is limited to cognitively impaired participants.

Epidemiology of Trauma-Related Hemorrhage and Time to Definitive Care Across North America: Making the Case for Bleeding Control Education.

INTRODUCTION: Uncontrolled trauma-related hemorrhage remains the primary preventable cause of death among those with critical injury. STUDY OBJECTIVE: The purpose of this investigation was to evaluate the types of trauma associated with critical injury and trauma-related hemorrhage, and to determine the time to definitive care among patients treated at major trauma centers who were predicted to require massive transfusion. METHODS: A secondary analysis was performed of the Pragmatic, Randomized, Optimal Platelet and Plasma Ratios (PROPPR) trial data (N = 680). All patients included were predicted to require massive transfusion and admitted to one of 12 North American trauma centers. Descriptive statistics were used to characterize patients, including demographics, type and mechanism of injury, source of bleeding, and receipt of prehospital interventions. Patient time to definitive care was determined using the time from activation of emergency services to responder arrival on scene, and time from scene departure to emergency department (ED) arrival. Each interval was calculated and then summed for a total time to definitive care. RESULTS: Patients were primarily white (63.8%), male (80.3%), with a median age of 34 (IQR 24-51) years. Roughly one-half of patients experienced blunt (49.0%) versus penetrating (48.2%) injury. The most common types of blunt trauma were motor vehicle injuries (83.5%), followed by falls (9.3%), other (3.6%), assaults (1.8%), and incidents due to machinery (1.8%). The most common types of penetrating injuries were gunshot wounds (72.3%), stabbings (24.1%), other (2.1%), and impalements (1.5%). One-third of patients (34.5%) required some prehospital intervention, including intubation (77.4%), chest or needle decompression (18.8%), tourniquet (18.4%), and cardiopulmonary resuscitation (CPR; 5.6%). Sources of bleeding included the abdomen (44.3%), chest (20.4%), limb/extremity (18.2%), pelvis (11.4%), and other (5.7%). Patients waited for a median of six (IQR4-10) minutes for emergency responders to arrive at the scene of injury and traveled a median of 27 (IQR 19-42) minutes to an ED. Time to definitive care was a median of 57 (IQR 44-77) minutes, with a range of 12-232 minutes. Twenty-four-hour mortality was 15% (n = 100) with 81 patients dying due to exsanguination or hemorrhage. CONCLUSION: Patients who experience critical injury may experience lengthy times to receipt of definitive care and may benefit from bystander action for hemorrhage control to improve patient outcomes.

The Combined Effect of Copper Nanoparticles and Microplastics on Transcripts Involved in Oxidative Stress Pathway in Rainbow Trout (Oncorhynchus Mykiss) Hepatocytes.

Copper nanoparticles (CuNPs) and microplastics (MPs) are two emerging contaminants of freshwater systems. Despite their co-occurrence in many water bodies, the combined effects of CuNPs and MPs on aquatic organisms are not well-investigated. In this study, primary cultures of rainbow trout hepatocytes were exposed to dissolved Cu, CuNPs, MPs, or a combination of MPs and CuNPs for 48 h, and the transcript abundances of oxidative stress-related genes were investigated. Exposure to CuNPs or dissolved Cu resulted in a significant increase in the transcript abundances of two antioxidant enzymes, catalase (CAT) and superoxide dismutase (SOD). Exposure to CuNPs also led to an upregulation in the expression of Na+/K+ ATPase alpha 1 subunit (ATP1A1). Microplastics alone or in combination with CuNPs did not have a significant effect on abundances of the target gene transcripts. Overall, our findings suggested acute exposure to CuNPs or dissolved ions may induce oxidative stress in hepatocytes, and the Cu-induced effect on target gene transcripts was not associated with MPs.

Catalytic Site Constraints in the P450s Mediating Loganic Acid (7DLH) and Secologanic Acid Synthesis (SLAS) in Camptotheca.

Terpene indole alkaloids (TIAs) are plant-derived natural products synthesized in low levels in medicinal plants such as Catharanthus roseus and Camptotheca acuminata. TIA pathways species utilize several CYP72A subfamily members to form loganic acid from 7-deoxyloganic acid (a simple hydroxylation) as well as secologanin and secologanic acid from loganin and loganic acid (a C-C bond scission). Divergences in the specificities of these P450s have allowed Camptotheca secologanic acid synthases (SLASs) to become bifunctional enzymes capable of performing both reactions. In contrast, Catharanthus 7-deoxyloganic acid hydroxylase (7DLH) and secologanin synthase (SLS) have remained monofunctional enzymes capable either of monooxygenation or C-C bond scission. Our in vitro reconstitutions have now demonstrated that Camptotheca also contains a monofunctional 7DLH capable only of hydroxylating 7-deoxyloganic acid. Mutageneses aimed at evaluating residues important for the tight specificity of Camptotheca 7DLH (CYP72A729) and the broad specificity of SLAS (CYP72A564) have identified several residues where reciprocal switches substantially affect their activities: Lys128His in 7DLH increases hydroxylation of 7-deoxyloganic acid, and His132Lys in SLAS decreases this hydroxylation and C-C bond scissions of loganic acid and loganin; Gly321Ser in 7DLH does not affect hydroxylation of 7-deoxyloganic acid, whereas Ser324Gly in SLAS significantly increases C-C bond scission of loganic acid; Asp332Glu in the acid-alcohol pair of 7DLH increases hydroxylation of 7-deoxyloganic acid, whereas Glu335Asp in SLAS completely eliminates both of its activities. These mutations that enhance or eliminate these respective activities have significant potential to aid engineering efforts aimed at increasing TIA production in cell cultures, microbial systems, and/or other plants.

Using deep long-read RNAseq in Alzheimer's disease brain to assess medical relevance of RNA isoform diversity.

Due to alternative splicing, human protein-coding genes average over eight RNA isoforms, resulting in nearly four distinct protein coding sequences per gene. Long-read RNAseq (IsoSeq) enables more accurate quantification of isoforms, shedding light on their specific roles. To assess the medical relevance of measuring RNA isoform expression, we sequenced 12 aged human frontal cortices (6 Alzheimer's disease cases and 6 controls; 50% female) using one Oxford Nanopore PromethION flow cell per sample. Our study uncovered 53 new high-confidence RNA isoforms in medically relevant genes, including several where the new isoform was one of the most highly expressed for that gene. Specific examples include WDR4 (61%; microcephaly), MYL3 (44%; hypertrophic cardiomyopathy), and MTHFS (25%; major depression, schizophrenia, bipolar disorder). Other notable genes with new high-confidence isoforms include CPLX2 (10%; schizophrenia, epilepsy) and MAOB (9%; targeted for Parkinson's disease treatment). We identified 1,917 medically relevant genes expressing multiple isoforms in human frontal cortex, where 1,018 had multiple isoforms with different protein coding sequences, demonstrating the need to better understand how individual isoforms from a single gene body are involved in human health and disease, if at all. Exactly 98 of the 1,917 genes are implicated in brain-related diseases, including Alzheimer's disease genes such as APP (Aß precursor protein; five), MAPT (tau protein; four), and BIN1 (eight). As proof of concept, we also found 99 differentially expressed RNA isoforms between Alzheimer's cases and controls, despite the genes themselves not exhibiting differential expression. Our findings highlight the significant knowledge gaps in RNA isoform diversity and their medical relevance. Deep long-read RNA sequencing will be necessary going forward to fully comprehend the medical relevance of individual isoforms for a "single" gene.

Regioselective Direct C-H Bond Heteroarylation of Thiazoles Enabled by an Iminopyridine-Based α-Diimine Nickel(II) Complex Evaluated by DFT Studies.

Direct C-H bond arylation is a highly effective method for synthesizing arylated heteroaromatics. This method reduces the number of synthetic steps and minimizes the formation of impurities. We report an air- and moisture-stable iminopyridine-based α-diimine nickel(II) complex for direct C5-H bond arylation of thiazole derivatives. Under a low catalyst loading and performing the reactions at lower temperatures (80 °C) under aerobic conditions, we produced mono- and diarylated thiazole units. Competition experiments and density functional theory calculations revealed that the mechanism of C-H activation in 4-methylthiazole involves an electrophilic aromatic substitution.