Reduced contextually induced muscle thermogenesis in rats with calorie restriction and lower aerobic fitness but not monogenic obesity.

We have previously identified predator odor as a potent stimulus activating thermogenesis in skeletal muscle in rats. As this may prove relevant for energy balance and weight loss, the current study investigated whether skeletal muscle thermogenesis was altered with negative energy balance, obesity propensity seen in association with low intrinsic aerobic fitness, and monogenic obesity. First, weight loss subsequent to 3 wk of 50% calorie restriction suppressed the muscle thermogenic response to predator odor. Next, we compared rats bred based on artificial selection for intrinsic aerobic fitness - high- and low-capacity runners (HCR, LCR) - that display robust leanness and obesity propensity, respectively. Aerobically fit HCR showed enhanced predator odor-induced muscle thermogenesis relative to the less-fit LCR. This contrasted with the profound monogenic obesity displayed by rats homozygous for a loss of function mutation in Melanocortin 4 receptor (Mc4rK3a,4X/K314X rats), which showed no discernable deficit in thermogenesis. Taken together, these data imply that body size or obesity per se are not associated with deficient muscle thermogenesis. Rather, the physiological phenotype associated with polygenic obesity propensity may encompass pleiotropic mechanisms in the thermogenic pathway. Adaptive thermogenesis associated with weight loss also likely alters muscle thermogenic mechanisms.

Loss-of-function variant in SPIN4 causes an X-linked overgrowth syndrome.

Overgrowth syndromes can be caused by pathogenic genetic variants in epigenetic writers, such as DNA and histone methyltransferases. However, no overgrowth disorder has previously been ascribed to variants in a gene that acts primarily as an epigenetic reader. Here, we studied a male individual with generalized overgrowth of prenatal onset. Exome sequencing identified a hemizygous frameshift variant in Spindlin 4 (SPIN4), with X-linked inheritance. We found evidence that SPIN4 binds specific histone modifications, promotes canonical WNT signaling, and inhibits cell proliferation in vitro and that the identified frameshift variant had lost all of these functions. Ablation of Spin4 in mice recapitulated the human phenotype with generalized overgrowth, including increased longitudinal bone growth. Growth plate analysis revealed increased cell proliferation in the proliferative zone and an increased number of progenitor chondrocytes in the resting zone. We also found evidence of decreased canonical Wnt signaling in growth plate chondrocytes, providing a potential explanation for the increased number of resting zone chondrocytes. Taken together, our findings provide strong evidence that SPIN4 is an epigenetic reader that negatively regulates mammalian body growth and that loss of SPIN4 causes an overgrowth syndrome in humans, expanding our knowledge of the epigenetic regulation of human growth.

Intravenous thrombolysis prior to mechanical thrombectomy does not affect clinical or procedural outcomes in patients with large vessel occlusion acute ischemic stroke.

Mechanical thrombectomy (MT) has revolutionized the care of large vessel occlusion acute ischemic strokes (LVOAIS). However, the benefit of intravenous thrombolysis prior to MT remains unproven. Two recent trials showed equivocal results regarding the benefits of pre-MT intravenous thrombolysis in predominantly Asian populations. We evaluated clinical outcomes and procedural metrics for patients with LVOAIS who were treated with MT alone compared to those who were treated with both intravenous tPA and MT. In a retrospective study, LVOAIS patients treated with MT, with or without preceding intravenous thrombolysis, between January of 2017 and December of 2019 were identified. Patients were treated according to contemporary guidelines. Baseline demographic and clinical characteristics, procedural metrics, and clinical outcomes were collected. Among LVOAIS patients, those treated with intravenous thrombolysis and MT did not differ from those with MT alone on clinical outcomes at three months. Further, the two groups did not differ on thrombectomy procedure times, recanalization rates, and symptomatic intracranial hemorrhage rates. In our patients with LVOAIS, intravenous thrombolysis combined with MT offered no advantage compared to MT alone in clinical outcomes or recanalization rates. Our results are consistent with earlier studies in other populations. In addition, our results suggest that IV tPA does not impact the ease of clot removal by MT. Further studies will evaluate how newly available thrombolytic agents may benefit patients eligible for MT.

Are Transmetatarsal Amputations a Durable Limb Salvage Option? A Single-Institution Descriptive Analysis.

In limb and life-threatening diabetic foot infections, transmetatarsal amputations are often indicated as a limb salvage procedure. The aim of this study is to analyze the long-term durability of initially successful transmetatarsal amputations in the diabetic population. We defined a successful transmetatarsal amputation as one which had clinical healing 1 year after surgery. A retrospective review of transmetatarsal amputations completed at our institution over an 11-year period was performed. We identified 83 amputations that met inclusion criteria. The mean follow-up was 4 years. The mean time to surgical healing was 109.8 days. After successfully healing the transmetatarsal amputation the long-term outcomes were analyzed. Re-ulcerations occurred in 44% of the transmetatarsal amputations a mean of 15 months after surgical healing. Patients who re-ulcerated were noted to be significantly younger (p value 0.02) with a significantly higher preprocedure hemoglobin A1c (p value < .001). Additional procedures after successful healing included 13 (15.66%) revision surgeries and 12 (14.46%) more proximal amputations. While transmetatarsal amputations remain a viable and durable limb preserving surgery, all patients who have undergone a transmetatarsal amputation should be monitored lifelong as they remain at risk for re-ulceration and more proximal amputation.

Enhanced weight and fat loss from long-term intermittent fasting in obesity-prone, low-fitness rats.

BACKGROUND: Intermittent fasting (IF) strategies have emerged as viable alternatives to traditional calorie-restricted diets. A key predictor of metabolic health and response to diet is cardiometabolic fitness, including intrinsic aerobic capacity. In a contrasting rat model of aerobic capacity-high- and low-capacity runners (HCR, LCR)-we found that the lean and physically active HCR were also more responsive to a standard calorie-restricted diet. Here, we assessed the ability of IF to induce weight loss on a background of high and low aerobic fitness accompanied by different levels of daily physical activity. METHODS: Female HCR and LCR (8 per line) were subjected to IF (alternate-day fasting) for 14 weeks. Outcomes included changes in body weight, fat and lean mass, daily physical activity, and food and water intake. After initial measurements, IF was continued, and measurements were repeated after one year of IF. RESULTS: All rats lost weight with IF, and LCR lost significantly more weight than HCR. This difference was primarily due to differential fat loss; loss of lean mass, on the other hand, was similar between HCR and LCR. Total food intake decreased with IF, and LCR showed lower intake than HCR only during the first 5 weeks of IF. Physical activity was suppressed by long-term IF. Physical activity increased on fed days compared to fasted days, and this pattern was more pronounced in HCR. The differential effects of IF in HCR and LCR persisted after one year of IF, with IF preventing the marked weight gain seen in ad libitum fed LCR during this time. CONCLUSION: Weight and fat loss from IF was more pronounced in obesity-prone, low-aerobic capacity LCR, despite the low activity levels seen in these rats. The possibility that aerobic capacity modulates response to IF in human participants remains unexplored.

Skeletal muscle thermogenesis induction by exposure to predator odor.

Non-shivering thermogenesis can promote negative energy balance and weight loss. In this study, we identified a contextual stimulus that induces rapid and robust thermogenesis in skeletal muscle. Rats exposed to the odor of a natural predator (ferret) showed elevated skeletal muscle temperatures detectable as quickly as 2 min after exposure, reaching maximum thermogenesis of >1.5°C at 10-15 min. Mice exhibited a similar thermogenic response to the same odor. Ferret odor induced a significantly larger and qualitatively different response from that of novel or aversive odors, fox odor or moderate restraint stress. Exposure to predator odor increased energy expenditure, and both the thermogenic and energetic effects persisted when physical activity levels were controlled. Predator odor-induced muscle thermogenesis is subject to associative learning as exposure to a conditioned stimulus provoked a rise in muscle temperature in the absence of the odor. The ability of predator odor to induce thermogenesis is predominantly controlled by sympathetic nervous system activation of ß-adrenergic receptors, as unilateral sympathetic lumbar denervation and a peripherally acting ß-adrenergic antagonist significantly inhibited predator odor-induced muscle thermogenesis. The potential survival value of predator odor-induced changes in muscle physiology is reflected in an enhanced resistance to running fatigue. Lastly, predator odor-induced muscle thermogenesis imparts a meaningful impact on energy expenditure as daily predator odor exposure significantly enhanced weight loss with mild calorie restriction. This evidence signifies contextually provoked, centrally mediated muscle thermogenesis that meaningfully impacts energy balance.

Compatible observable decompositions for coarse-grained representations of real molecular systems.

Coarse-grained (CG) observable expressions, such as pressure or potential energy, are generally different than their fine-grained (FG, e.g., atomistic) counterparts. Recently, we analyzed this so-called "representability problem" in Wagner et al. [J. Chem. Phys. 145, 044108 (2016)]. While the issue of representability was clearly and mathematically stated in that work, it was not made clear how to actually determine CG observable expressions from the underlying FG systems that can only be simulated numerically. In this work, we propose minimization targets for the CG observables of such systems. These CG observables are compatible with each other and with structural observables. Also, these CG observables are systematically improvable since they are variationally minimized. Our methods are local and data efficient because we decompose the observable contributions. Hence, our approaches are called the multiscale compatible observable decomposition (MS-CODE) and the relative entropy compatible observable decomposition (RE-CODE), which reflect two main approaches to the "bottom-up" coarse-graining of real FG systems. The parameterization of these CG observable expressions requires the introduction of new, symmetric basis sets and one-body terms. We apply MS-CODE and RE-CODE to 1-site and 2-site CG models of methanol for the case of pressure, as well as to 1-site methanol and acetonitrile models for potential energy.

Multiconfigurational Coarse-Grained Molecular Dynamics.

Standard low resolution coarse-grained modeling techniques have difficulty capturing multiple configurations of protein systems. Here, we present a method for creating accurate coarse-grained (CG) models with multiple configurations using a linear combination of functions or "states". Individual CG models are created to capture the individual states, and the approximate coupling between the two states is determined from an all-atom potential of mean force. We show that the resulting multiconfiguration coarse-graining (MCCG) method accurately captures the transition state as well as the free energy between the two states. We have tested this method on the folding of dodecaalanine, as well as the amphipathic helix of endophilin.

Quantitative Examination of Antibiotic Susceptibility of Neisseria gonorrhoeae Aggregates Using ATP-utilization Commercial Assays and Live/Dead Staining.

The emergence of antibiotic resistant Neisseria gonorrhoeae (GC) is a worldwide health threat and highlights the need to identify individuals who fail treatment. This Gram-negative bacterium causes gonorrhea exclusively in humans. During infection, it is able to form aggregates and/or biofilms. The minimum inhibitory concentration (MIC) test is used for to determine susceptibility to antibiotics and to define appropriate treatment. However, the mechanism of the eradication in vivo and its relationship to laboratory results are not known. A method that examines how GC aggregation affects antibiotic susceptibility and shows the relationship between aggregate size and antibiotic susceptibility was developed. When GC aggregate, they are more resistant to antibiotic killing, with bacteria in the center surviving ceftriaxone treatment better than those in the periphery. The data indicate that N. gonorrhoeae aggregation can reduce its susceptibility to ceftriaxone, which is not reflected using the standard agar plate-based MIC methods. The method used in this study will allow researchers to test bacterial susceptibility under clinically relevant conditions.

QRICH1 mutations cause a chondrodysplasia with developmental delay.

In many children with short stature, the etiology of the decreased linear growth remains unknown. We sought to identify the underlying genetic etiology in a patient with short stature, irregular growth plates of the proximal phalanges, developmental delay, and mildly dysmorphic facial features. Exome sequencing identified a de novo, heterozygous, nonsense mutation (c.1606C>T:p.R536X) in QRICH1. In vitro studies confirmed that the mutation impaired expression of the QRICH1 protein. SiRNA-mediated knockdown of Qrich1 in primary mouse epiphyseal chondrocytes caused downregulation of gene expression associated with hypertrophic differentiation. We then identified an unrelated individual with another heterozygous de novo nonsense mutation in QRICH1 who had a similar phenotype. A recently published study identified QRICH1 mutations in three patients with developmental delay, one of whom had short stature. Our findings indicate that QRICH1 mutations cause not only developmental delay but also a chondrodysplasia characterized by diminished linear growth and abnormal growth plate morphology due to impaired growth plate chondrocyte hypertrophic differentiation.

Rare but Recurrent ROS1 Fusions Resulting From Chromosome 6q22 Microdeletions are Targetable Oncogenes in Glioma.

PURPOSE: Gliomas, a genetically heterogeneous group of primary central nervous system tumors, continue to pose a significant clinical challenge. Discovery of chromosomal rearrangements involving kinase genes has enabled precision therapy, and improved outcomes in several malignancies. EXPERIMENTAL DESIGN: Positing that similar benefit could be accomplished for patients with brain cancer, we evaluated The Cancer Genome Atlas (TCGA) glioblastoma dataset. Functional validation of the oncogenic potential and inhibitory sensitivity of discovered ROS1 fusions was performed using three independent cell-based model systems, and an in vivo murine xenograft study. RESULTS: In silico analysis revealed previously unreported intrachromosomal 6q22 microdeletions that generate ROS1-fusions from TCGA glioblastoma dataset. ROS1 fusions in primary glioma and ependymoma were independently corroborated from MSK-IMPACT and Foundation Medicine clinical datasets. GOPC-ROS1 is a recurrent ROS1 fusion in primary central nervous system (CNS) tumors. CEP85L-ROS1 and GOPC-ROS1 are transforming oncogenes in cells of astrocytic lineage, and amenable to pharmacologic inhibition with several ROS1 inhibitors even when occurring concurrently with other cancer hotspot aberrations frequently associated with glioblastoma. Oral monotherapy with a brain-permeable ROS1 inhibitor, lorlatinib, significantly prolonged survival in an intracranially xenografted tumor model generated from a ROS1 fusion-positive glioblastoma cell line. CONCLUSIONS: Our findings highlight that CNS tumors should be specifically interrogated for these rare intrachromosomal 6q22 microdeletion events that generate actionable ROS1 fusions. ROS1 fusions in primary brain cancer may be amenable for clinical intervention with kinase inhibitors, and this holds the potential of novel treatment paradigms in these treatment-refractory cancer types, particularly in glioblastoma.

Quantum theory of multiscale coarse-graining.

Coarse-grained (CG) models serve as a powerful tool to simulate molecular systems at much longer temporal and spatial scales. Previously, CG models and methods have been built upon classical statistical mechanics. The present paper develops a theory and numerical methodology for coarse-graining in quantum statistical mechanics, by generalizing the multiscale coarse-graining (MS-CG) method to quantum Boltzmann statistics. A rigorous derivation of the sufficient thermodynamic consistency condition is first presented via imaginary time Feynman path integrals. It identifies the optimal choice of CG action functional and effective quantum CG (qCG) force field to generate a quantum MS-CG (qMS-CG) description of the equilibrium system that is consistent with the quantum fine-grained model projected onto the CG variables. A variational principle then provides a class of algorithms for optimally approximating the qMS-CG force fields. Specifically, a variational method based on force matching, which was also adopted in the classical MS-CG theory, is generalized to quantum Boltzmann statistics. The qMS-CG numerical algorithms and practical issues in implementing this variational minimization procedure are also discussed. Then, two numerical examples are presented to demonstrate the method. Finally, as an alternative strategy, a quasi-classical approximation for the thermal density matrix expressed in the CG variables is derived. This approach provides an interesting physical picture for coarse-graining in quantum Boltzmann statistical mechanics in which the consistency with the quantum particle delocalization is obviously manifest, and it opens up an avenue for using path integral centroid-based effective classical force fields in a coarse-graining methodology.

Peripheral blood detection of systemic graft-specific xeno-antibodies following transplantation of human neural progenitor cells into the porcine spinal cord.

Extensive pre-clinical and clinical studies have searched for therapeutic efficacy of cell-based therapeutics in diseases of the Central Nervous System (CNS) with no other viable options. Allogeneic cells represent the primary source of these therapies and immunosuppressive regimens have been empirically employed based on experience with solid organ transplantation, attempting to avoid immune mediated graft rejection. In this study, we aimed to 1) characterize the host immune response to stem cells transplanted into the CNS and 2) develop a non-invasive method for detecting immune response to transplanted cell grafts. Human neural progenitor cells were transplanted into the spinal cord of 10 Göttingen minipigs, of which 5 received no immunosuppression and 5 received Tacrolimus. Peripheral blood samples were collected longitudinally for flow cytometry cross match studies. Necropsy was performed at day 21 and spinal cord tissue analysis. We observed a transient increase in xeno-reactive antibodies was detected on post-operative day 7 and 14 in pigs that did not receive immunosuppression. This response was not detected in pigs that received Tacrolimus immunosuppression. No difference in graft survival was observed between the groups. Infiltration of numerous immune mediators including granulocytes, T lymphocytes, and activated microglia, and complement deposition were detected. In summary, a systemic immunologic response to stem cell grafts was detected for two weeks after transplantation using peripheral blood. This could be used as a non-invasive biomarker by investigators for detection of immunologic rejection. However, the absence of a detectable response in peripheral blood does not rule out a parenchymal immune response.

Extending the range and physical accuracy of coarse-grained models: Order parameter dependent interactions.

Order parameters (i.e., collective variables) are often used to describe the behavior of systems as they capture different features of the free energy surface. Yet, most coarse-grained (CG) models only employ two- or three-body non-bonded interactions between the CG particles. In situations where these interactions are insufficient for the CG model to reproduce the structural distributions of the underlying fine-grained (FG) model, additional interactions must be included. In this paper, we introduce an approach to expand the basis sets available in the multiscale coarse-graining (MS-CG) methodology by including order parameters. Then, we investigate the ability of an additive local order parameter (e.g., density) and an additive global order parameter (i.e., distance from a hard wall) to improve the description of CG models in interfacial systems. Specifically, we study methanol liquid-vapor coexistence, acetonitrile liquid-vapor coexistence, and acetonitrile liquid confined by hard-wall plates, all using single site CG models. We find that the use of order parameters dramatically improves the reproduction of structural properties of interfacial CG systems relative to the FG reference as compared with pairwise CG interactions alone.

Functional validation of the oncogenic cooperativity and targeting potential of tuberous sclerosis mutation in medulloblastoma using a MYC-amplified model cell line.

Medulloblastoma is the most common malignant brain tumor of childhood. To identify targetable vulnerabilities, we employed inhibitor screening that revealed mTOR inhibitor hypersensitivity in the MYC-overexpressing medulloblastoma cell line, D341. Concomitant exome sequencing unveiled an uncharacterized missense mutation, TSC2A415V , in these cells. We biochemically demonstrate that the TSC2A415V mutation is functionally deleterious, leading to shortened half-life and proteasome-mediated protein degradation. These data suggest that MYC cooperates with activated kinase pathways, enabling pharmacologic intervention in these treatment refractory tumors. We propose that identification of activated kinase pathways may allow for tailoring targeted therapy to improve survival and treatment-related morbidity in medulloblastoma.

On the representability problem and the physical meaning of coarse-grained models.

In coarse-grained (CG) models where certain fine-grained (FG, i.e., atomistic resolution) observables are not directly represented, one can nonetheless identify indirect the CG observables that capture the FG observable's dependence on CG coordinates. Often, in these cases it appears that a CG observable can be defined by analogy to an all-atom or FG observable, but the similarity is misleading and significantly undermines the interpretation of both bottom-up and top-down CG models. Such problems emerge especially clearly in the framework of the systematic bottom-up CG modeling, where a direct and transparent correspondence between FG and CG variables establishes precise conditions for consistency between CG observables and underlying FG models. Here we present and investigate these representability challenges and illustrate them via the bottom-up conceptual framework for several simple analytically tractable polymer models. The examples provide special focus on the observables of configurational internal energy, entropy, and pressure, which have been at the root of controversy in the CG literature, as well as discuss observables that would seem to be entirely missing in the CG representation but can nonetheless be correlated with CG behavior. Though we investigate these problems in the framework of systematic coarse-graining, the lessons apply to top-down CG modeling also, with crucial implications for simulation at constant pressure and surface tension and for the interpretations of structural and thermodynamic correlations for comparison to experiment.

Gene, Stem Cell, and Alternative Therapies for SCA 1.

Spinocerebellar ataxia 1 is an autosomal dominant disease characterized by neurodegeneration and motor dysfunction. In disease pathogenesis, polyglutamine expansion within Ataxin-1, a gene involved in transcriptional repression, causes protein nuclear inclusions to form. Most notably, neuronal dysfunction presents in Purkinje cells. However, the effect of mutant Ataxin-1 is not entirely understood. Two mouse models are employed to represent spinocerebellar ataxia 1, a B05 transgenic model that specifically expresses mutant Ataxin-1 in Purkinje cells, and a Sca1 154Q/2Q model that inserts the polyglutamine expansion into the mouse Ataxin-1 locus so that the mutant Ataxin-1 is expressed in all cells that express Ataxin-1. This review aims to summarize and evaluate the wide variety of therapies proposed for spinocerebellar ataxia 1, specifically gene and stem cell therapies.

High expression of orphan nuclear receptor NR4A1 in a subset of ovarian tumors with worse outcome.

OBJECTIVE: Nuclear receptors (NRs) play a vital role in the development and progression of several cancers including breast and prostate. Using TCGA data, we sought to identify critical nuclear receptors in high grade serous ovarian cancers (HGSOC) and to confirm these findings using in vitro approaches. METHODS: In silico analysis of TCGA data was performed to identify relevant NRs in HGSOC. Ovarian cancer cell lines were screened for NR expression and functional studies were performed to determine the significance of these NRs in ovarian cancers. NR expression was analyzed in ovarian cancer tissue samples using immunohistochemistry to identify correlations with histology and stage of disease. RESULTS: The NR4A family of NRs was identified as a potential driver of ovarian cancer pathogenesis. Overexpression of NR4A1 in particular correlated with worse progression free survival. Endogenous expression of NR4A1 in normal ovarian samples was relatively high compared to that of other tissue types, suggesting a unique role for this orphan receptor in the ovary. Expression of NR4A1 in HGSOC cell lines as well as in patient samples was variable. NR4A1 primarily localized to the nucleus in normal ovarian tissue while co-localization within the cytoplasm and nucleus was noted in ovarian cancer cell lines and patient tissues. CONCLUSIONS: NR4A1 is highly expressed in a subset of HGSOC samples from patients that have a worse progression free survival. Studies to target NR4A1 for therapeutic intervention should include HGSOC.

A Novel Crizotinib-Resistant Solvent-Front Mutation Responsive to Cabozantinib Therapy in a Patient with ROS1-Rearranged Lung Cancer.

PURPOSE: Rearranged ROS1 is a crizotinib-sensitive oncogenic driver in lung cancer. The development of acquired resistance, however, poses a serious clinical challenge. Consequently, experimental and clinical validation of resistance mechanisms and potential second-line therapies is essential. EXPERIMENTAL DESIGN: We report the discovery of a novel, solvent-front ROS1(D2033N) mutation in a patient with CD74-ROS1-rearranged lung adenocarcinoma and acquired resistance to crizotinib. Crizotinib resistance of CD74-ROS1(D2033N) was functionally evaluated using cell-based assays and structural modeling. RESULTS: In biochemical and cell-based assays, the CD74-ROS1(D2033N) mutant demonstrated significantly decreased sensitivity to crizotinib. Molecular dynamics simulation revealed compromised crizotinib binding due to drastic changes in the electrostatic interaction between the D2033 residue and crizotinib and reorientation of neighboring residues. In contrast, cabozantinib binding was unaffected by the D2033N substitution, and inhibitory potency against the mutant was retained. Notably, cabozantinib treatment resulted in a rapid clinical and near-complete radiographic response in this patient. CONCLUSIONS: These results provide the first example of successful therapeutic intervention with targeted therapy to overcome crizotinib resistance in a ROS1-rearranged cancer. Clin Cancer Res; 22(10); 2351-8. ©2015 AACR.

Predicting the Sensitivity of Multiscale Coarse-Grained Models to their Underlying Fine-Grained Model Parameters.

The sensitivity of a coarse-grained (CG) force field to changes in the underlying fine-grained (FG) model from which it was derived provides modeling insight for improving transferability across interaction parameters, transferability across temperature, and the calculation of thermodynamic derivatives. Methods in the literature, such as multi-trajectory finite differences and reweighted finite differences, are either too computationally demanding to calculate within acceptable noise tolerances or are too biased for practical accuracy. This work presents a new reweighting-free, single-simulation formula that allows for practical, high signal-to-noise calculations of CG model sensitivity with respect to FG model interaction parameters and thermodynamic state points. This formula, the self-consistent basis (SCB) single point formula, determines the many-body sensitivity in a single step by approximating the derivative of the many-body potential projected onto the same set of trial functions as the sensitivity. A related diagnostic formula also derived in this paper is the self-consistent iterative (SCI) single point formula, which is useful for identifying the importance of many-body sources of error and verifying CG representability of observables. The SCI formula determines the many-body sensitivity iteratively via a series of partially self-consistent, variational approximations to the complete many-body sensitivity. The new, computationally efficient SCB formula shows substantially less noise than previous methods when applied to single site methanol and solvent-free sodium chloride CG models, though bias can remain a problem. It represents a novel method for calculating alchemical transferability across interaction parameters at low computational cost and with high fidelity, and the results point to new understanding of the current limits of CG model transferability.