Human SIRT5 variants with reduced stability and activity do not cause neuropathology in mice.

SIRT5 is a sirtuin deacylase that removes negatively charged lysine modifications, in the mitochondrial matrix and elsewhere in the cell. In benign cells and mouse models, under basal conditions, the phenotypes of SIRT5 deficiency are quite subtle. Here, we identify two homozygous SIRT5 variants in patients suspected to have mitochondrial disease. Both variants, P114T and L128V, are associated with reduced SIRT5 protein stability and impaired biochemical activity, with no evidence of neomorphic or dominant negative properties. The crystal structure of the P114T enzyme was solved and shows only subtle deviations from wild-type. Via CRISPR-Cas9, we generated a mouse model that recapitulates the human P114T mutation; homozygotes show reduced SIRT5 levels and activity, but no obvious metabolic abnormalities, neuropathology, or other gross phenotypes. We conclude that these human SIRT5 variants most likely represent severe hypomorphs, but are likely not by themselves the primary pathogenic cause of the neuropathology observed in the patients.

Beyond MABEL: An Integrative Approach to First in Human Dose Selection of Immunomodulators by the Health and Environmental Sciences Institute (HESI) Immuno-Safety Technical Committee (ITC).

Administration of a new drug candidate in a first-in-human (FIH) clinical trial is a particularly challenging phase in drug development and is especially true for immunomodulators, which are a diverse and complex class of drugs with a broad range of mechanisms of action and associated safety risks. Risk is generally greater for immunostimulators, in which safety concerns are associated with acute toxicity, compared to immunosuppressors, where the risks are related to chronic effects. Current methodologies for FIH dose selection for immunostimulators are focused primarily on identifying the minimum anticipated biological effect level (MABEL), which has often resulted in sub-therapeutic doses, leading to long and costly escalation phases. The Health and Environmental Sciences Institute (HESI) - Immuno-Safety Technical Committee (ITC) organized a project to address this issue through two complementary approaches: (i) an industry survey on FIH dose selection strategies and (ii) detailed case studies for immunomodulators in oncology and non-oncology indications. Key messages from the industry survey responses highlighted a preference toward more dynamic PK/PD approaches as in vitro assays are seemingly not representative of true physiological conditions for immunomodulators. These principles are highlighted in case studies. To address the above themes, we have proposed a revised decision tree, which expands on the guidance by the IQ MABEL Working Group (Leach et al. 2021). This approach facilitates a more refined recommendation of FIH dose selection for immunomodulators, allowing for a nuanced consideration of their mechanisms of action (MOAs) and the associated risk-to-benefit ratio, among other factors.

SIRT5 variants from patients with mitochondrial disease are associated with reduced SIRT5 stability and activity, but not with neuropathology.

SIRT5 is a sirtuin deacylase that represents the major activity responsible for removal of negatively-charged lysine modifications, in the mitochondrial matrix and elsewhere in the cell. In benign cells and mouse models, under basal non-stressed conditions, the phenotypes of SIRT5 deficiency are generally quite subtle. Here, we identify two homozygous SIRT5 variants in human patients suffering from severe mitochondrial disease. Both variants, P114T and L128V, are associated with reduced SIRT5 protein stability and impaired biochemical activity, with no evidence of neomorphic or dominant negative properties. The crystal structure of the P114T enzyme was solved and shows only subtle deviations from wild-type. Via CRISPR-Cas9, we generate a mouse model that recapitulates the human P114T mutation; homozygotes show reduced SIRT5 levels and activity, but no obvious metabolic abnormalities, neuropathology or other gross evidence of severe disease. We conclude that these human SIRT5 variants most likely represent severe hypomorphs, and are likely not the primary pathogenic cause of the neuropathology observed in the patients.

Disorders of Sodium.

Sodium imbalances are a common occurrence in the emergency department. Although recognition and diagnosis are relatively straightforward, discovering the cause and management should be approached systematically. The most important history items to ascertain is if the patient has symptoms and how long this imbalance has taken to develop. Treatment rapidity depends on severity of symptoms with the most rapid treatment occurring in only the severely symptomatic. Overcorrection has dire consequences and must be approached in a careful and systematic fashion in order to prevent these devastating consequences.

Triceps Rupture After Olecranon Fixation with Proximal Ulna Plate and Suture Augmentation: A Case Report.

CASE: Olecranon fractures treated with proximal ulna plate fixation and repairing the triceps with suture augmentation to the plate decrease the risk of "olecranon escape," but may lead to failure through triceps rupture. In this case report, a rare complication of triceps rupture occurred, and the patient underwent triceps repair. CONCLUSION: When fixing olecranon fractures, surgeons should minimize triceps dissection for hardware placement. If subjected to significant force, a surgical insult to the tendon footprint for a better plate contact on the bone and the presence of suture augmentation may change the construct failure mechanism and result in triceps rupture as opposed to fracture redisplacement.

Payload-Binding Fab Fragments Increase the Therapeutic Index of MMAE Antibody-Drug Conjugates.

Monomethyl auristatin E (MMAE) is a potent tubulin inhibitor that is used as the payload for four FDA-approved antibody-drug conjugates (ADC). Deconjugated MMAE readily diffuses into untargeted cells, resulting in off-target toxicity. Here, we report the development and evaluation of a humanized Fab fragment (ABC3315) that enhances the therapeutic selectivity of MMAE ADCs. ABC3315 increased the IC50 of MMAE against human cancer cell lines by > 500-fold with no impact on the cytotoxicity of MMAE ADCs, including polatuzumab vedotin (PV) and trastuzumab-vc-MMAE (TvcMMAE). Coadministration of ABC3315 did not reduce the efficacy of PV or TvcMMAE in xenograft tumor models. Coadministration of ABC3315 with 80 mg/kg TvcMMAE significantly (P < 0.0001) increased the cumulative amount of MMAE that was excreted in urine 0 to 4 days after administration from 789.4±19.0 nanograms (TvcMMAE alone) to 2625±206.8 nanograms (for mice receiving TvcMMAE with coadministration of ABC3315). Mice receiving 80 mg/kg TvcMMAE and PBS exhibited a significant drop in white blood cell counts (P = 0.025) and red blood cell counts (P = 0.0083) in comparison with control mice. No significant differences, relative to control mice, were found for white blood cell counts (P = 0.15) or for red blood cell counts (P = 0.23) for mice treated with 80 mg/kg TvcMMAE and ABC3315. Coadministration of ABC3315 with 120 mg/kg PV significantly (P = 0.045) decreased the percentage body weight loss at nadir for treated mice from 11.9%±7.0% to 4.1%±2.1%. Our results demonstrate that ABC3315, an anti-MMAE Fab fragment, decreases off-target toxicity while not decreasing antitumor efficacy, increasing the therapeutic window of MMAE ADCs.

Wildfires and the Changing Landscape of Air Pollution-related Health Burden in California.

Rationale: Wildfires are a growing source of pollution including particulate matter ⩽2.5 µm in aerodynamic diameter (PM2.5), but associated trends in health burden are not well characterized. Objectives: We investigated trends and disparities in PM2.5-related cardiorespiratory health burden (asthma, chronic obstructive pulmonary disease, and all-cause respiratory and cardiovascular emergency department [ED] visits and hospital admissions) for all days and wildfire smoke-affected days across California from 2008 to 2016. Methods: Using residential Zone Improvement Plan code and daily PM2.5 exposures, we estimated overall and subgroup-specific (age, gender, race and ethnicity) associations with cardiorespiratory outcomes. Health burden trends and disparities were evaluated on the basis of relative risk, attributable number, and attributable fraction by demographic and geographic factors and over time. Measurements and Main Results: PM2.5-attributed burden steadily decreased, whereas the fraction attributed to wildfire smoke varied by fire season intensity, constituting up to 15% of the annual PM2.5-burden. The highest relative risk and PM2.5-attributed burden (92 per 100,000 people) was observed for respiratory ED visits, accounting for 2.2% of the respiratory annual burden. Disparities in overall morbidity in the oldest age, Black, and "other" race groups were also reflected in PM2.5-attributed burden, whereas Asian populations had the highest risk rate in respiratory outcomes and thus the largest fraction of the total burden attributed to the exposure. In contrast, high wildfire PM2.5-attributed burden rates in rural, central, and northern California populations occurred because of differential exposure. Conclusions: In California, wildfires' impact on air quality offset the public health gains achieved through reductions in nonsmoke PM2.5. Disproportionate effects could be attributed to differences in subpopulation susceptibility, relative risk, and differential exposure.

Tunable Release of Calcium from Chitosan-Coated Bioglass.

Bioglass presents a standard biomaterial for regeneration of hard tissues in orthopedics and dentistry. The notable osteo-inductive properties of bioglass are largely due to the release of calcium ions from it. However, this release is not easily controllable and can often be excessive, especially during the initial interaction of the biomaterial with the surrounding tissues. Consequently, this excessive release can deplete the calcium content of the bioglass, ultimately reducing its overall bioactivity. In this study, we have tested if applying biopolymer chitosan coatings of different thicknesses would be able to mitigate and regulate the calcium ion release from monodisperse bioglass nanoparticles. Calcium release was assessed for four different chitosan coating thicknesses at different time points over the period of 28 days using a fluorescence quencher. Expectedly, chitosan-coated particles released less calcium as the concentration of chitosan in the coating solution increased, presumably due to the increased thickness of the chitosan coating around the bioglass particles. The mechanism of release remained constant for each coating thickness, corresponding to anomalous, non-Fickian diffusion, but the degree of anomalousness increased with the deposition of chitosan. Zeta potential testing showed an expected increase in the positive double layer charge following the deposition of the chitosan coating due to the surface exposure of the amine groups of chitosan. Less intuitively, the zeta potential became less positive as thickness of the chitosan coating increased, attesting to the lower density of the surface charges within thicker coatings than within the thinner ones. Overall, the findings of this study demonstrate that chitosan coating efficiently prevents the early release of calcium from bioglass. This coating procedure also allows for the tuning of the calcium release kinetics by controlling the chitosan concentration in the parent solution.

Integrated ECG Interpretation Course for the Improvement of Medical Student Electrocardiography Literacy.

Background: While undergraduate medical education curricula typically integrate electrocardiogram (ECG) interpretation during the preclinical phase, the skill can be challenging, and students may lack confidence to interpret ECGs in clinical practice. The purpose of this online curriculum was to provide a supplemental course which provides a practical, systematic approach to ECG reading and increases student confidence in recognizing basic patterns of pathology. Methods: We used a proprietary online learning content-management system to create a course for 4th-year medical students at a US-based MD program. The course consisted of 12 video modules which reviewed basic ECG interpretation and then provided practice ECG tracings for the students to interpret. Weekly online video calls with faculty were held to address any questions and to emphasize key concepts presented in that week's material. The students completed a pre- and post-course assessment on ECG pathology and reported their self-confidence in ECG interpretation skills. Results: The median score for the pre-test was 76.5/100 with a median self-confidence score of 3/10. After completion of the course, the students' post-test median score increased to 87.5 (p < 0.0001) with a median self-confidence score of 7.5 (p < 0.0001). As a result of this course, the students showed a significant improvement in grades and self-confidence. Discussion: An online course consisting of practice ECGs and limited direct faculty interaction helped the students improve ECG interpretation accuracy and self-confidence.

TAVR in the tricuspid domain: valve-in-valve transcatheter tricuspid valve replacement for bioprosthetic valve degeneration.

We report a novel use of transcatheter aortic valve replacement (TAVR) for valve-in-valve tricuspid valve replacement. A man in his 50s with prohibitive risks for surgical intervention underwent this procedure to improve severe, symptomatic tricuspid stenosis. Though current literature is limited to case reports, the Valve-in-Valve International Database (VIVID) reports similar mortality rates between surgical and transcutaneous replacement. As a novel, off-label procedure, there is limited operator experience. Nonetheless, in non-operative or high-risk patients, similar outcomes are noted in between transcatheter tricuspid valve replacement and surgical replacement. This registry sets the framework for further studies with the possibility of observing outcomes as operator experience increases, while highlighting the feasibility of the procedure.

Cell Penetrating Peptides Conjugated to Anti-Carcinoembryonic Antigen "Catch-and-Release" Monoclonal Antibodies Alter Plasma and Tissue Pharmacokinetics in Colorectal Cancer Xenograft Mice.

Cell penetrating peptides conjugated to delivery vehicles, such as nanoparticles or antibodies, can enhance the cytosolic delivery of macromolecules. The present study examines the effects of conjugation to cell penetrating and endosomal escape peptides (i.e., TAT, GALA, and H6CM18) on the pharmacokinetics and distribution of an anti-carcinoembryonic antigen "catch-and-release" monoclonal antibody, 10H6, in a murine model of colorectal cancer. GALA and TAT were conjugated to 10H6 using SoluLINK technology that allowed the evaluation of peptide-to-antibody ratio by ultraviolet spectroscopy. H6CM18 was conjugated to either NHS or maleimide-modified 10H6 using an azide-modified valine-citrulline linker and copper-free click chemistry. Unmodified and peptide-conjugated 10H6 preparations were administered intravenously at 6.67 nmol/kg to mice-bearing MC38CEA+ tumors. Unconjugated 10H6 demonstrated a clearance of 19.9 ± 1.36 mL/day/kg, with an apparent volume of distribution of 62.4 ± 7.78 mL/kg. All antibody-peptide conjugates exhibited significantly decreased plasma and tissue exposure, increased plasma clearance, and increased distribution volume. Examination of tissue-to-plasma exposure ratios showed an enhanced selectivity of 10H6-TAT for the GI tract (+25%), kidney (+24%), liver (+38%), muscle (+3%), and spleen (+33%). 10H6-GALA and 10H6-H6CM18 conjugates demonstrated decreased exposure in all tissues, relative to unmodified 10H6. All conjugates demonstrated decreased tumor exposure and selectivity; however, differences in tumor selectivity between 10H6 and 10H6-H6CM18 (maleimide) were not statistically significant. Relationships between the predicted peptide conjugate isoelectric point (pI) and pharmacokinetic parameters were bell-shaped, where pI values around 6.8-7 exhibit the slowest plasma clearance and smallest distribution volume. The data and analyses presented in this work may guide future efforts to develop immunoconjugates with cell penetrating and endosomal escape peptides.

Targeted Delivery of Endosomal Escape Peptides to Enhance Immunotoxin Potency and Anti-cancer Efficacy.

This work describes use of anti-carcinoembryonic antigen antibodies (10H6, T84.66) for targeted delivery of an endosomal escape peptide (H6CM18) and gelonin, a type I ribosome inactivating protein. The viability of colorectal cancer cells (LS174T, LoVo) was assessed following treatment with gelonin or gelonin immunotoxins, with or without co-treatment with T84.66-H6CM18. Fluorescent microscopy was used to visualize the escape of immunoconjugates from endosomes of treated cells, and efficacy and toxicity were assessed in vivo in xenograft tumor-bearing mice following single- and multiple-dose regimens. Application of 25 pM T84.66-H6CM18 combined with T84.66-gelonin increased gelonin potency by ~ 1,000-fold and by ~ 6,000-fold in LS174T and LoVo cells. Intravenous 10H6-gelonin at 1.0 mg/kg was well tolerated by LS174T tumor-bearing mice, while 10 and 25 mg/kg doses led to signs of toxicity. Single-dose administration of PBS, gelonin conjugated to T84.66 or 10H6, T84.66-H6CM18, or gelonin immunotoxins co-administered with T84.66-H6CM18 were evaluated. The combinations of T84.66-gelonin + 1.0 mg/kg T84.66-H6CM18 and 10H6-gelonin + 0.1 mg/kg T84.66-H6CM18 led to significant delays in LS174T growth. Use of a multiple-dose regimen allowed further anti-tumor effects, significantly extending median survival time by 33% and by 69%, for mice receiving 1 mg/kg 10H6-gelonin + 0.1 mg/kg T84.66-H6CM18 (p = 0.0072) and 1 mg/kg 10H6-gelonin + 1 mg/kg T84.66-H6CM18 (p = 0.0017). Combined administration of gelonin immunoconjugates with antibody-targeted endosomal escape peptides increased the delivery of gelonin to the cytoplasm of targeted cells, increased gelonin cell killing in vitro by 1,000-6,000 fold, and significantly increased in vivo efficacy.

Isolated hemangiopericytoma of the conjunctiva.

PURPOSE: To report a unique presentation of hemangiopericytoma and discuss the clinical course, pathological features, and management of this tumor. OBSERVATIONS: An otherwise healthy 54-year-old Caucasian female presented with a painless conjunctival mass. The lesion gradually enlarged over a three-week period and was unresponsive to corticosteroid treatment. The mass was surgically removed, and histopathologic findings were consistent with hemangiopericytoma. CONCLUSIONS AND IMPORTANCE: Conjunctival hemangiopericytoma should be considered in patients with conjunctival lesions unresponsive to medical management. Surgical excision is diagnostic and therapeutic and is the strongest predictor of clinical course. Incompletely excised lesions are at a greater risk of local recurrence and subsequent metastasis. Given the neoplasm's malignant potential, patients should be followed in the outpatient setting.

Dynamic Contrast-Enhanced Magnetic Resonance Imaging for the Prediction of Monoclonal Antibody Tumor Disposition.

The prediction of monoclonal antibody (mAb) disposition within solid tumors for individual patients is difficult due to inter-patient variability in tumor physiology. Improved a priori prediction of mAb pharmacokinetics in tumors may facilitate the development of patient-specific dosing protocols and facilitate improved selection of patients for treatment with anti-cancer mAb. Here, we report the use of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), with tumor penetration of the contrast agent gadobutrol used as a surrogate, to improve physiologically based pharmacokinetic model (PBPK) predictions of cetuximab pharmacokinetics in epidermal growth factor receptor (EGFR) positive xenografts. In the initial investigations, mice bearing Panc-1, NCI-N87, and LS174T xenografts underwent DCE-MRI imaging with the contrast agent gadobutrol, followed by intravenous dosing of an 125Iodine-labeled, non-binding mAb (8C2). Tumor concentrations of 8C2 were determined following the euthanasia of mice (3 h-6 days after 8C2 dosing). Potential predictor relationships between DCE-MRI kinetic parameters and 8C2 PBPK parameters were evaluated through covariate modeling. The addition of the DCE-MRI parameter Ktrans alone or Ktrans in combination with the DCE-MRI parameter Vp on the PBPK parameters for tumor blood flow (QTU) and tumor vasculature permeability (σTUV) led to the most significant improvement in the characterization of 8C2 pharmacokinetics in individual tumors. To test the utility of the DCE-MRI covariates on a priori prediction of the disposition of mAb with high-affinity tumor binding, a second group of tumor-bearing mice underwent DCE-MRI imaging with gadobutrol, followed by the administration of 125Iodine-labeled cetuximab (a high-affinity anti-EGFR mAb). The MRI-PBPK covariate relationships, which were established with the untargeted antibody 8C2, were implemented into the PBPK model with considerations for EGFR expression and cetuximab-EGFR interaction to predict the disposition of cetuximab in individual tumors (a priori). The incorporation of the Ktrans MRI parameter as a covariate on the PBPK parameters QTU and σTUV decreased the PBPK model prediction error for cetuximab tumor pharmacokinetics from 223.71 to 65.02%. DCE-MRI may be a useful clinical tool in improving the prediction of antibody pharmacokinetics in solid tumors. Further studies are warranted to evaluate the utility of the DCE-MRI approach to additional mAbs and additional drug modalities.

Evaluation of quantitative modeling methods in whole-body, dynamic [11C]-erlotinib PET.

BACKGROUND: [11C]-Erlotinib is a radiolabeled analogue of a tyrosine kinase inhibitor used to treat non-small cell lung cancer (NSCLC) which expresses specific kinase domain mutations of the epidermal growth factor receptor (EGFR). In this study, 10 subjects with NSCLC and assorted EGFR mutation status underwent a dynamic, multi-bed positron emission tomography (PET) scan using [11C]-erlotinib. Data were analyzed using a variety of quantitative techniques common in PET (graphical methods, kinetic models, and uptake value-based endpoints). Our primary goal was to determine the most reliable imaging endpoint given the need for maintaining minimal patient burden and recognizing the advantage of simple calculations in future trials. RESULTS: Standard uptake values (a semi-quantitative endpoint) were well correlated with both binding potential and volume of distribution (fully quantitative endpoints). Normalized tracer uptake was found to stabilize approximately 60 minutes post tracer injection. Conclusions: The kinetic properties of [11C]-erlotinib varied greatly across subjects. Our novel scanning protocol produced an important dataset which highlights the great heterogeneity of NSCLC and its apparent impact on [11C]-erlotinib kinetics. A lack of correlation between EGFR mutational status and quantitative endpoints appears to be due to disease heterogeneity and low tracer uptake. The most reliable fits of the dynamic data were based on the one-tissue compartmental model which were well correlated with mean SUV. Due to this correlation and good stability at late-time, SUV seems sufficiently well-suited to quantitative imaging of NSCLC lesions in the whole body with [11C]-erlotinib.

A prospective study to compare serial changes in pain scores for patients with and without a history of frequent ED utilization.

BACKGROUND: In the face of the opiate addiction epidemic, there is a paucity of research that evaluates limitations for our current pain rating methodologies for patient populations at risk for drug seeking behavior. OBJECTIVE: We hypothesized that VAS scores would be higher and show less serial improvement for patients with a history of frequent ED use. METHODS: This was a prospective, observational cohort study of a convenience sample of adult ED patients with chief complaint of pain. Initial VAS scores were recorded. Pain scores were subsequently updated 30-45 min after pain medication administration. ED frequenter defined as having >4 ED visits over a 1-year time period. Categorical data analyzed by chi-square; continuous data analyzed by t-tests. A multiple linear regression performed to control for confounding. RESULTS: 125 patients were enrolled; 51% ED frequenters. ED frequenters were similar to non-ED frequenters with respect to gender, mean age, Hispanic race, educational level, chief complaint type, and initial pain medication narcotic. ED frequenters more likely to have higher initial VAS score (9.17+/-1.25 vs. 8.51+/-1.68; p = 0.01) and higher second VAS scores (7.48+/-2.56 vs. 5.00+/-3.28; p <0.001) and significantly lower mean change in first to second VAS scores (1.69+/-2.17 vs. 3.51+/-3.25; p <0.001). Within our multiple linear regression model, only ED frequenter group (p < 0.001) and private insurance status (0.04) were associated with differences in mean reduction in pain scores. CONCLUSION: We found that ED frequenters had significantly less improvement between first and second VAS measurements.

Detection of Caenorhabditis elegans Germ Cell Apoptosis Following Exposure to Environmental Contaminant Mixtures: A Crude Oil-Dispersant Mixture Example.

Crude oil disasters, such as the Deepwater Horizon accident, have caused severe environmental contamination and damage, affecting the health of marine and terrestrial organisms. Some previous studies have demonstrated cleanup efforts using chemical dispersant induced more potent toxicities than oil alone due to an increase in bioavailability of crude oil components, such as PAHs. However, there still lacks a systematic procedure that provides methods to determine genotypic and phenotypic changes following exposure to environmental toxicants or toxicant mixture, such as dispersed crude oil. Here, we describe methods for identifying a mechanism of dispersed crude oil-induced reproductive toxicity in the model organisms, Caenorhabditis elegans (C. elegans). Due to the genetic malleability of C. elegans, two mutant strains outlined in this chapter were used to identify a pathway responsible for inducing apoptosis: MD701 bcIs39 [lim-7p::ced-1::GFP + lin-15(+)], a mutant strain that allows visualization of apoptotic bodies via a green fluorescent protein fused to CED-1; and TJ1 (cep-1(gk138) I.), a p53/CEP-1 defective strain that is unable to activate apoptosis via the p53/CEP-1 pathway. In addition, qRT-PCR was utilized to demonstrate the aberrant expression of apoptosis (ced-13, ced-3, ced-4, ced-9, cep-1, dpl-1, efl-1, efl-2, egl-1, egl-38, lin-35, pax-2, and sir-2.1) and cytochrome P450 (cyp14a3, cyp35a1, cyp35a2, cyp35a5, and cyp35c1) protein-coding genes following exposure to dispersed crude oil. The procedure outlined here can be applicable to determine whether environmental contaminants, most of time contaminant mixture, cause reproductive toxicity by activation of the proapoptotic, p53/CEP-1 pathway.

Control of sugar and amino acid feeding via pharyngeal taste neurons.

Insect gustatory systems comprise multiple taste organs for detecting chemicals that signal palatable or noxious quality. Although much is known about how taste neurons sense various chemicals, many questions remain about how individual taste neurons in each taste organ control feeding. Here, we use the Drosophila pharynx as a model to investigate how taste information is encoded at the cellular level to regulate consumption of sugars and amino acids. We first generate taste-blind animals and establish a critical role for pharyngeal input in food selection. We then investigate feeding behavior of both male and female flies in which only selected classes of pharyngeal neurons are restored via binary choice feeding preference assays as well as Fly Liquid-Food Interaction Counter (FLIC) assays. We find instances of integration as well as redundancy in how pharyngeal neurons control behavioral responses to sugars and amino acids. Additionally, we find that pharyngeal neurons drive sugar feeding preference based on sweet taste but not on nutritional value. Finally, we demonstrate functional specialization of pharyngeal and external neurons using optogenetic activation. Overall, our genetic taste neuron protection system in a taste-blind background provides a powerful approach to elucidate principles of pharyngeal taste coding and demonstrates functional overlap and subdivision among taste neurons.SIGNIFICANCE STATEMENTDietary intake of nutritious chemicals such as sugars and amino acids is essential for an animal's survival. In insects, distinct classes of taste neurons control acceptance or rejection of food sources. Here we develop a genetic system to investigate how individual taste neurons in the Drosophila pharynx encode specific tastants, focusing on sugars and amino acids. By examining flies in which only a single class of taste neurons is active, we find evidence for functional overlap as well as redundancy in responses to sugars and amino acids. We also uncover functional subdivision between pharyngeal and external neurons in driving feeding responses. Overall, we find that different pharyngeal neurons act together to control intake of the two categories of appetitive tastants.

The acylfulvene alkylating agent, LP-184, retains nanomolar potency in non-small cell lung cancer carrying otherwise therapy-refractory mutations.

More than 40% of non-small cell lung cancer (NSCLC) patients lack actionable targets and require non-targeted chemotherapeutics. Many become refractory to drugs due to underlying resistance-associated mutations. KEAP1 mutant NSCLCs further activate NRF2 and upregulate its client PTGR1. LP-184, a novel alkylating agent belonging to the acylfulvene class is a prodrug dependent upon PTGR1. We hypothesized that NSCLC with KEAP1 mutations would continue to remain sensitive to LP-184. LP-184 demonstrated highly potent anticancer activity both in primary NSCLC cell lines and in those originating from brain metastases of primary lung cancers. LP-184 activity correlated with PTGR1 transcript levels but was independent of mutations in key oncogenes (KRAS and KEAP1) and tumor suppressors (TP53 and STK11). LP-184 was orders of magnitude more potent in vitro than cisplatin and pemetrexed. Correlative analyses of sensitivity with cell line gene expression patterns indicated that alterations in NRF2, MET, EGFR and BRAF consistently modulated LP-184 sensitivity. These correlations were then extended to TCGA analysis of 517 lung adenocarcinoma patients, out of which 35% showed elevated PTGR1, and 40% of those further displayed statistically significant co-occurrence of KEAP1 mutations. The gene correlates of LP-184 sensitivity allow additional personalization of therapeutic options for future treatment of NSCLC.

A machine learning-based gene signature of response to the novel alkylating agent LP-184 distinguishes its potential tumor indications.

BACKGROUND: Non-targeted cytotoxics with anticancer activity are often developed through preclinical stages using response criteria observed in cell lines and xenografts. A panel of the NCI-60 cell lines is frequently the first line to define tumor types that are optimally responsive. Open data on the gene expression of the NCI-60 cell lines, provides a unique opportunity to add another dimension to the preclinical development of such drugs by interrogating correlations with gene expression patterns. Machine learning can be used to reduce the complexity of whole genome gene expression patterns to derive manageable signatures of response. Application of machine learning in early phases of preclinical development is likely to allow a better positioning and ultimate clinical success of molecules. LP-184 is a highly potent novel alkylating agent where the preclinical development is being guided by a dedicated machine learning-derived response signature. We show the feasibility and the accuracy of such a signature of response by accurately predicting the response to LP-184 validated using wet lab derived IC50s on a panel of cell lines. RESULTS: We applied our proprietary RADR® platform to an NCI-60 discovery dataset encompassing LP-184 IC50s and publicly available gene expression data. We used multiple feature selection layers followed by the XGBoost regression model and reduced the complexity of 20,000 gene expression values to generate a 16-gene signature leading to the identification of a set of predictive candidate biomarkers which form an LP-184 response gene signature. We further validated this signature and predicted response to an additional panel of cell lines. Considering fold change differences and correlation between actual and predicted LP-184 IC50 values as validation performance measures, we obtained 86% accuracy at four-fold cut-off, and a strong (r = 0.70) and significant (p value 1.36e-06) correlation between actual and predicted LP-184 sensitivity. In agreement with the perceived mechanism of action of LP-184, PTGR1 emerged as the top weighted gene. CONCLUSION: Integration of a machine learning-derived signature of response with in vitro assessment of LP-184 efficacy facilitated the derivation of manageable yet robust biomarkers which can be used to predict drug sensitivity with high accuracy and clinical value.