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Abstract Opioid overdose remains a problem in the United States despite pharmacotherapies, such as buprenorphine, in the treatment of opioid use disorder. This study characterized changes in buprenorphine use. Using the Drug Enforcement Administration's ARCOS, Medicaid, and Medicare claims databases, patterns in buprenorphine usage in the United States from 2018 to 2020 were analyzed by examining percentage changes in total grams distributed and changes in grams per 100 K people in year‐to‐year usage based on ZIP code and state levels. For ARCOS from 2018 to 2019 and 2019 to 2020, total buprenorphine distribution in grams increased by 16.2% and 12.6%, respectively. South Dakota showed the largest statewide percentage increase in both 2018–2019 (66.1%) and 2019–2020 (36.7%). From 2018 to 2019, the ZIP codes ND‐577 (156.4%) and VA‐222 (−82.1%) had the largest and smallest percentage changes, respectively. From 2019 to 2020, CA‐932 (250.2%) and IL‐603 (−36.8%) were the largest and smallest, respectively. In both 2018–2019 and 2019–2020, PA‐191 had the second highest increase in grams per 100K while OH‐452 was the only ZIP code to remain in the top three largest decreases in grams per 100K in both periods. Among Medicaid patients in 2018, there was a nearly 2000‐fold difference in prescriptions per 100k Medicaid enrollees between Kentucky (12 075) and Nebraska (6). Among Medicare enrollees in 2018, family medicine physicians and other primary care providers were the top buprenorphine prescribers. This study not only identified overall increases in buprenorphine availability but also pronounced state‐level differences. Such geographic analysis can be used to discern which public policies and regional factors impact buprenorphine access.

Objective: To evaluate the effectiveness and safety of an evidence-based urine culture stewardship program in reducing hospital catheter-associated urinary tract infections (CAUTIs) and the rate of CAUTIs across a 3-hospital system. Patients and Methods: This is a prospective, 2-year quality improvement program conducted from October 1, 2018, to September 30, 2020. An evidence-based urine culture stewardship program was designed, which consisted of the following: criteria for allowing or restricting urine cultures from catheterized patients, a best practice advisory integrated into the ordering system of an electronic medical record, and a systematic provider education and feedback program to ensure compliance. The system-wide rates of CAUTIs (total CAUTIs/catheter days×1000), changes in intercepts, trends, mortality, length of stay, rates of device utilization, and rates of hospital-onset sepsis were compared for 3 years before and 2 years after the launch of the program. Results: Catheter-associated urinary tract infections progressively decreased after the initiation of the program (B=−0.21, P=.001). When the trends before and after the initiation of the program were compared, there were no statistically significant increases in the ratio of actual to predicted hospital length of stay, intensive care unit length of stay, system-wide mortality, and intensive care unit mortality. Although the rates of hospital-acquired sepsis remained consistent after the implementation of the stewardship program through the first quarter of 2020, the rates showed an increase in the second and third quarters of 2020. However, hospital-onset sepsis events associated with the diagnosis of a urinary tract infection did not increase after the intervention. Conclusion: Urine culture stewardship is a safe and effective way to reduce CAUTIs among patients in a large multihospital health care system. Patient safety indicators appeared unchanged after the implementation of the program, and ongoing follow-up will improve confidence in the long-term sustainability of this strategy.

Abstract Aims To describe the natural history of SARS‐CoV‐2 infection in patients with hypertrophic cardiomyopathy (HCM) compared with a control group and to identify predictors of adverse events. Methods and results Three hundred and five patients [age 56.6 ± 16.9 years old, 191 (62.6%) male patients] with HCM and SARS‐Cov‐2 infection were enrolled. The control group consisted of 91 131 infected individuals. Endpoints were (i) SARS‐CoV‐2 related mortality and (ii) severe clinical course [death or intensive care unit (ICU) admission]. New onset of atrial fibrillation, ventricular arrhythmias, shock, stroke, and cardiac arrest were also recorded. Sixty‐nine (22.9%) HCM patients were hospitalized for non‐ICU level care, and 21 (7.0%) required ICU care. Seventeen (5.6%) died: eight (2.6%) of respiratory failure, four (1.3%) of heart failure, two (0.7%) suddenly, and three (1.0%) due to other SARS‐CoV‐2‐related complications. Covariates associated with mortality in the multivariable were age {odds ratio (OR) per 10 year increase 2.25 [95% confidence interval (CI): 1.12–4.51], P = 0.0229}, baseline New York Heart Association class [OR per one‐unit increase 4.01 (95%CI: 1.75–9.20), P = 0.0011], presence of left ventricular outflow tract obstruction [OR 5.59 (95%CI: 1.16–26.92), P = 0.0317], and left ventricular systolic impairment [OR 7.72 (95%CI: 1.20–49.79), P = 0.0316]. Controlling for age and sex and comparing HCM patients with a community‐based SARS‐CoV‐2 cohort, the presence of HCM was associated with a borderline significant increased risk of mortality OR 1.70 (95%CI: 0.98–2.91, P = 0.0600). Conclusions Over one‐fourth of HCM patients infected with SARS‐Cov‐2 required hospitalization, including 6% in an ICU setting. Age and cardiac features related to HCM, including baseline functional class, left ventricular outflow tract obstruction, and systolic impairment, conveyed increased risk of mortality.

We present the course of 4 pregnancies in 3 women with desmoplakin cardiomyopathy, with a focus on changes in left ventricular ejection fraction and N-terminal pro–B-type natriuretic peptide levels from the prepregnancy period through the postpartum period, as well as maternal cardiac, obstetric, and neonatal outcomes. (Level of Difficulty: Advanced.)

The Warburg effect, aerobic glycolysis, is a hallmark feature of cancer cells grown in culture. However, the relative roles of glycolysis and respiratory metabolism in supporting in vivo tumor growth and processes such as tumor dissemination and metastatic growth remain poorly understood, particularly on a systems level. Using a CRISPRi mini-library enriched for mitochondrial ribosomal protein and respiratory chain genes in multiple human lung cancer cell lines, we analyzed in vivo metabolic requirements in xenograft tumors grown in distinct anatomic contexts. While knockdown of mitochondrial ribosomal protein and respiratory chain genes (mito-respiratory genes) has little impact on growth in vitro, tumor cells depend heavily on these genes when grown in vivo as either flank or primary orthotopic lung tumor xenografts. In contrast, respiratory function is comparatively dispensable for metastatic tumor growth. RNA-Seq and metabolomics analysis of tumor cells expressing individual sgRNAs against mito-respiratory genes indicate overexpression of glycolytic genes and increased sensitivity of glycolytic inhibition compared to control when grown in vitro, but when grown in vivo as primary tumors these cells down-regulate glycolytic mechanisms. These studies demonstrate that discrete perturbations of mitochondrial respiratory chain function impact in vivo tumor growth in a context-specific manner with differential impacts on primary and metastatic tumors.

Cardiomyopathies are a heterogeneous collection of diseases that have in common primary functional and structural abnormalities of the heart muscle, often genetically determined. The most effective categorization of cardiomyopathies is based on the presenting phenotype, with hypertrophic, dilated, arrhythmogenic, and restrictive cardiomyopathy as the prototypes. Sex modulates the prevalence, morpho‐functional manifestations and clinical course of cardiomyopathies. Aspects as diverse as ion channel expression and left ventricular remodeling differ in male and female patients with myocardial disease, although the reasons for this are poorly understood. Moreover, clinical differences may also result from complex societal/environmental discrepancies between sexes that may disadvantage women. This review provides a state‐of‐the‐art appraisal of the influence of sex on cardiomyopathies, highlighting the many gaps in knowledge and open research questions.

Background We aimed to assess the association between number of pregnancies and long‐term progression of cardiac dysfunction, arrhythmias, and event‐free survival in women with pathogenic or likely pathogenic variants of gene encoding for Lamin A/C proteins ( LMNA+). Methods and Results We retrospectively included consecutive women with LMNA+ and recorded pregnancy data. We collected echocardiographic data, occurrence of atrial fibrillation, atrioventricular block, sustained ventricular arrhythmias, and implantation of cardiac electronic devices (implantable cardioverter defibrillator/cardiac resynchronization therapy defibrillator). We analyzed retrospectively complications during pregnancy and the peripartum period. We included 89 women with LMNA+ (28% probands, age 41±16 years), of which 60 had experienced pregnancy. Follow‐up time was 5 [interquartile range, 3–9] years. We analyzed 452 repeated echocardiographic examinations. Number of pregnancies was not associated with increased long‐term risk of atrial fibrillation, atrioventricular block, sustained ventricular arrhythmias, or implantable cardioverter defibrillator/cardiac resynchronization therapy defibrillator implantation. Women with previous pregnancy and nulliparous women had a similar annual deterioration of left ventricular ejection fraction (−0.5/year versus −0.3/year, P=0.37) and similar increase of left ventricular end‐diastolic diameter (0.1/year versus 0.2/year, P=0.09). Number of pregnancies did not decrease survival free from death, left ventricular assist device, or need for cardiac transplantation. Arrhythmias occurred during 9% of pregnancies. No increase in maternal and fetal complications was observed. Conclusions In our cohort of women with LMNA+, pregnancy did not seem associated with long‐term adverse disease progression or event‐free survival. Likewise, women with LMNA+ generally well‐tolerated pregnancy, with a small proportion of patients experiencing arrhythmias.

Energy metabolism and ATP levels are controlled by an interlocking network of pathways. Here, the authors apply a genome-wide CRISPR screen to define genes that increase or decrease ATP levels to define the “ATPome”, a map of pathways that contribute to cellular ATP regulation.

Sequestration of reactants in lipid vesicles is a strategy prevalent in biological systems to raise the rate and specificity of chemical reactions. Here, the authors show that micelle-assisted reactions facilitate native chemical ligation between a peptide-thioester and a Cys-peptide modified by a lipid-like moiety.

Sarcoidosis is a complex, multisystem inflammatory disease with a heterogeneous clinical spectrum. Approximately 25% of patients with systemic sarcoidosis will have cardiac involvement that portends a poorer outcome. The diagnosis, particularly of isolated cardiac sarcoidosis, can be challenging. A paucity of randomised data exist on who, when and how to treat myocardial inflammation in cardiac sarcoidosis. Despite this, corticosteroids continue to be the mainstay of therapy for the inflammatory phase, with an evolving role for steroid-sparing and biological agents. This review explores the immunopathogenesis of inflammation in sarcoidosis, current evidence-based treatment indications and commonly used immunosuppression agents. It explores a multidisciplinary treatment and monitoring approach to myocardial inflammation and outlines current gaps in our understanding of this condition, emerging research and future directions in this field.

The origin of phospholipids, the primary constituents of cell membranes, is uncertain. Here, the authors develop an in vitro system to synthesize phospholipid molecules from water-soluble single-chain amphiphilic precursors via a reaction catalysed by the mycobacterial ligase FadD10.

Cells communicate through chemical and mechanical signals but emulating these in non-living mimics has been challenging. Here the authors present a porous mimic with a DNA-hydrogel ‘nucleus’ that can communicate through diffusive protein signals.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-021-22508-2

Abstract Background Individuals with hypertrophic cardiomyopathy (HCM), even when asymptomatic, are at‐risk for sudden cardiac death and stroke from arrhythmias, making it imperative to identify individuals affected by this familial disorder. Consensus guidelines recommend that first‐degree relatives (FDRs) of a person with HCM undergo serial cardiovascular evaluations. Methods We determined the uptake of family screening in patients with HCM and developed an online video intervention to facilitate family communication and screening. Family screening and genetic testing data were collected through a prospective quality improvement initiative, a standardized clinical assessment and management plan (SCAMP), utilized in an established cardiovascular genetics clinic. Patients were prescribed an online video if screening of their FDRs was incomplete and a pilot study on video utilization and family communication was conducted. Results Two‐hundred and sixteen probands with HCM were enrolled in SCAMP Phase I and 190 were enrolled in SCAMP Phase II. In both phases, probands reported that 51% of FDRs had been screened (382/749 in Phase I, 258/504 in Phase II). Twenty patients participated in a pilot study on video utilization and family communication. Nine participants reported watching the video and six participants reported sharing the video with relatives; however only one participant reported sharing the video with relatives who were not yet aware of the diagnosis of HCM in the family. Conclusion Despite care in a specialized cardiovascular genetics clinic, approximately one half of FDRs of patients with HCM remained unscreened. Online interventions and videos may serve as supplemental tools for patients communicating genetic risk information to relatives.

Abstract Protein degradation is a fundamental process in all living cells and is essential to remove both damaged proteins and intact proteins that are no longer needed by the cell. We are interested in creating synthetic genetic circuits that function in a cell-free expression system. This will require not only an efficient protein expression platform but also a robust protein degradation system in cell extract. Therefore, we purified and tested the activity of E. coli ClpXP protease in cell-free transcription-translation (TX-TL) systems that used E. coli S30 cell extract. Surprisingly, our studies showed that purified ClpXP added to the TX-TL system has very low proteolytic activity. The low activity of ClpXP was correlated with the rapid consumption of adenosine triphosphate (ATP) in cell extract. We improved the activity of ClpXP in cell extract by adding exogenous ATP and an energy regeneration system. We then established conditions for both protein synthesis, and protein degradation by ClpXP to occur simultaneously in the TX-TL systems. The optimized conditions for ClpXP activity will be useful for creating tunable synthetic genetic circuits and in vitro synthetic biology.

Respiratory chain dysfunction can decrease ATP and increase reactive oxygen species (ROS) levels. Despite the importance of these metabolic parameters to a wide range of cellular functions and disease, we lack an integrated understanding of how they are differentially regulated. To address this question, we adapted a CRISPRi- and FACS-based platform to compare the effects of respiratory gene knockdown on ROS to their effects on ATP. Focusing on genes whose knockdown is known to decrease mitochondria-derived ATP, we showed that knockdown of genes in specific respiratory chain complexes (I, III, and CoQ10 biosynthesis) increased ROS, whereas knockdown of other low ATP hits either had no impact (mitochondrial ribosomal proteins) or actually decreased ROS (complex IV). Moreover, although shifting metabolic conditions profoundly altered mitochondria-derived ATP levels, it had little impact on mitochondrial or cytosolic ROS. In addition, knockdown of a subset of complex I subunits-including NDUFA8, NDUFB4, and NDUFS8-decreased complex I activity, mitochondria-derived ATP, and supercomplex level, but knockdown of these genes had differential effects on ROS. Conversely, we found an essential role for ether lipids in the dynamic regulation of mitochondrial ROS levels independent of ATP. Thus, our results identify specific metabolic regulators of cellular ATP and ROS balance that may help dissect the roles of these processes in disease and identify therapeutic strategies to independently target energy failure and oxidative stress.

Molecules that act as both autocatalysts and material precursors offer exciting prospects for self-synthesizing materials. Here, the authors design a triazole peptide that self-replicates and then self-assembles into nanostructures, coupling autocatalytic and assembly pathways to realize a reproducing supramolecular system.

Human pluripotent stem cell derived neurons have the potential for cell replacement therapy for brain injury and disease but problems on transplantation need to be overcome. Here, the authors use a microtopographic scaffold to graft neurons into both hippocampal organoids and the mouse brain striatum.

Insufficient or dysregulated energy metabolism may underlie diverse inherited and degenerative diseases, cancer, and even aging itself. ATP is the central energy carrier in cells, but critical pathways for regulating ATP levels are not systematically understood. We combined a pooled clustered regularly interspaced short palindromic repeats interference (CRISPRi) library enriched for mitochondrial genes, a fluorescent biosensor, and fluorescence-activated cell sorting (FACS) in a high-throughput genetic screen to assay ATP concentrations in live human cells. We identified genes not known to be involved in energy metabolism. Most mitochondrial ribosomal proteins are essential in maintaining ATP levels under respiratory conditions, and impaired respiration predicts poor growth. We also identified genes for which coenzyme Q10 (CoQ10) supplementation rescued ATP deficits caused by knockdown. These included CoQ10 biosynthetic genes associated with human disease and a subset of genes not linked to CoQ10 biosynthesis, indicating that increasing CoQ10 can preserve ATP in specific genetic contexts. This screening paradigm reveals mechanisms of metabolic control and genetic defects responsive to energy-based therapies.

BackgroundReperfusion in ischemia is believed to generate cytotoxic oxidative stress, which mediates reperfusion injury. These stress conditions can initiate lipid peroxidation and damage to proteins, as well as promote DNA strand breaks. As biliverdin and bilirubin produced by heme oxygenase isoform 1 (HO-1) have antioxidant properties, the production of both antioxidants by HO-1 may help increase the resistance of the ischemic brain to oxidative stress. In the present study, the survival effect of HO-1 was confirmed using hemin.MethodsTo confirm the roles of HO-1, carbon monoxide, and cyclic guanosine monophosphate further in the antioxidant effect of HO-1 and bilirubin, cells were treated with cycloheximide, desferoxamine, and zinc deuteroporphyrin IX 2,4 bis glycol, respectively.ResultsHO-1 itself acted as an antioxidant. Furthermore, iron, rather than carbon monoxide, was involved in the HO-1-mediated survival effect. HO-1 activity was also important in providing bilirubin as an antioxidant.ConclusionOur results suggested that HO-1 helped to increase the resistance of the ischemic brain to oxidative stress.

Bacterial tRNA guanine transglycosylases (TGTs) catalyze the exchange of guanine for the 7-deazaguanine queuine precursor, prequeuosine1 (preQ1). While the native nucleic acid substrate for bacterial TGTs is the anticodon loop of queuine-cognate tRNAs, the minimum recognition sequence for the enzyme is a structured hairpin containing the target G nucleobase in a "UGU" loop motif. Previous work has established an RNA modification system, RNA-TAG, in which Escherichia coli TGT exchanges the target G on an RNA of interest for chemically modified preQ1 substrates linked to a small-molecule reporter such as biotin or a fluorophore. While extending the substrate scope of RNA transglycosylases to include DNA would enable numerous applications, it has been previously reported that TGT is incapable of modifying native DNA. Here, we demonstrate that TGT can in fact recognize and label specific DNA substrates. Through iterative testing of rationally mutated DNA hairpin sequences, we determined the minimal sequence requirements for transglycosylation of unmodified DNA by E. coli TGT. Controlling steric constraint in the DNA hairpin dramatically affects labeling efficiency, and, when optimized, can lead to near-quantitative site-specific modification. We demonstrate the utility of our newly developed DNA-TAG system by rapidly synthesizing probes for fluorescent Northern blotting of spliceosomal U6 RNA and RNA FISH visualization of the long noncoding RNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). The ease and convenience of the DNA-TAG system will provide researchers with a tool for accessing a wide variety of versatile and affordable modified DNA substrates.

Cryphonectria parasitica is the causal agent of chestnut blight. Infection of this ascomycete with Cryphonectria hypovirus 1 (CHV1) results in reduction of virulence and sporulation of the fungus. The virus affects fungal gene expression and several of the CHV1 downregulated genes encode secreted proteins that contain consensus Kex2 processing signals. Additionally, CHV1 has been shown to colocalize in infected cells primarily with fungal trans-Golgi network vesicles containing the Kex2 protease. We report here the cloning, analysis, and possible role of the C. parasitica Kex2 gene (CpKex2). CpKex2 gene sequence analysis showed high similarity to other ascomycete kexin-like proteins. Southern blot analyses of CpKex2 showed a single copy of this gene in the fungal genome. In order to monitor the expression and evaluate the function of CpKex2, antibodies were raised against expressed protein and Kex2-silenced mutants were generated. Western blots indicate that the Kex2 protein was constitutively expressed. Growth rate of the fungus was not significantly affected in Kex2-silenced strains; however, these strains showed reduced virulence, reduced sexual and asexual sporulation, and reductions in mating and fertility. The reduced virulence was correlated with reduced Kex2 enzymatic activity and reduced relative mRNA transcript levels as measured by real time reverse-transcriptase polymerase chain reaction. These results suggest that secreted proteins processed by Kex2 are important in fungal development and virulence.

Interactions between angiogenesis and neurogenesis regulate embryonic brain development. However, a comprehensive understanding of the stages of vascular cell maturation is lacking, especially in the prenatal human brain. Using fluorescence-activated cell sorting, single-cell transcriptomics, and histological and ultrastructural analyses, we show that an ensemble of endothelial and mural cell subtypes tile the brain vasculature during the second trimester. These vascular cells follow distinct developmental trajectories and utilize diverse signaling mechanisms, including collagen, laminin, and midkine, to facilitate cell-cell communication and maturation. Interestingly, our results reveal that tip cells, a subtype of endothelial cells, are highly enriched near the ventricular zone, the site of active neurogenesis. Consistent with these observations, prenatal vascular cells transplanted into cortical organoids exhibit restricted lineage potential that favors tip cells, promotes neurogenesis, and reduces cellular stress. Together, our results uncover important mechanisms into vascular maturation during this critical period of human brain development.

Bioorthogonal cycloaddition reactions between tetrazines and strained dienophiles are widely used for protein, lipid and glycan labelling because of their extremely rapid kinetics. However, controlling this chemistry in the presence of living mammalian cells with a high degree of spatial and temporal precision remains a challenge. Here we demonstrate a versatile approach to light-activated formation of tetrazines from photocaged dihydrotetrazines. Photouncaging, followed by spontaneous transformation to reactive tetrazine, enables live-cell spatiotemporal control of rapid bioorthogonal cycloaddition with dienophiles such as trans-cyclooctenes. Photocaged dihydrotetrazines are stable in conditions that normally degrade tetrazines, enabling efficient early-stage incorporation of bioorthogonal handles into biomolecules such as peptides. Photocaged dihydrotetrazines allow the use of non-toxic light to trigger tetrazine ligations on living mammalian cells. By tagging reactive phospholipids with fluorophores, we demonstrate modification of HeLa cell membranes with single-cell spatial resolution. Finally, we show that photo-triggered therapy is possible by coupling tetrazine photoactivation with strategies that release prodrugs in response to tetrazine ligation.

Cardiovascular disease (CVD) is the leading cause of death worldwide. This article focuses on current guidelines for the primary prevention of CVD and addresses management of key risk factors. Dietary modification, weight loss, exercise, and tobacco use cessation are specific areas where focused efforts can successfully reduce CVD risk on both an individual and a societal level. Specific areas requiring management include dyslipidemia, hypertension, physical activity, diabetes, aspirin use, and alcohol intake. These preventive efforts have major public health implications. As the global population continues to grow, health care expenditures will also rise, with the potential to eventually overwhelm the health care system. Therefore it is imperative to apply our collective efforts on CVD prevention to improve the cardiovascular health of individuals, communities, and nations.

The main objective of this study was to detect fatigue-induced clinical symptoms of immune suppression in medical residents. Samples were collected from the subjects at rest, following the first night (low-stress), and the last night (high-stress) of night float. Computerized reaction tests, Epworth Sleepiness Scale, and Wellness Profile questionnaires were used to quantify fatigue level. DNA of human herpes viruses HSV-1, VZV, EBV, as well as cortisol and melatonin concentrations, were measured in saliva. Residents at the high-stress interval reported being sleepier compared to the rest interval. EBV DNA level increased significantly at both stress intervals, while VZV DNA level increased only at low-stress. DNA levels of HSV-1 decreased at low-stress but increased at high-stress. Combined assessment of the viral DNA showed significant effect of stress on herpes virus reactivation at both stress intervals. Cortisol concentrations at both stress intervals were significantly higher than those at rest.

Chemical cross-linking enables rapid identification of RNA-protein and RNA-nucleic acid inter- and intramolecular interactions. However, no method exists to site-specifically and covalently cross-link two user-defined sites within an RNA. Here, we develop RNA-CLAMP, which enables site-specific and enzymatic cross-linking (clamping) of two selected guanine residues within an RNA. Intramolecular clamping can disrupt normal RNA function, whereas subsequent photocleavage of the cross-linker restores activity. We used RNA-CLAMP to clamp two stem loops within the single-guide RNA (sgRNA) of the CRISPR-Cas9 gene editing system via a photocleavable cross-linker, completely inhibiting gene editing. Visible light irradiation cleaved the cross-linker and restored gene editing with high spatiotemporal resolution. Design of two photocleavable linkers responsive to different wavelengths of light allowed multiplexed photoactivation of gene editing in mammalian cells. This photoactivated CRISPR-Cas9 gene editing platform benefits from undetectable background activity, provides a choice of activation wavelengths, and has multiplexing capabilities.

The Warburg effect, aerobic glycolysis, is a hallmark feature of cancer cells grown in culture. However, the relative roles of glycolysis and respiratory metabolism in supporting in vivo tumor growth and processes such as tumor dissemination and metastatic growth remain poorly understood, particularly on a systems level. Using a CRISPRi mini-library enriched for mitochondrial ribosomal protein and respiratory chain genes in multiple human lung cancer cell lines, we analyzed in vivo metabolic requirements in xenograft tumors grown in distinct anatomic contexts. While knockdown of mitochondrial ribosomal protein and respiratory chain genes (mito-respiratory genes) has little impact on growth in vitro, tumor cells depend heavily on these genes when grown in vivo as either flank or primary orthotopic lung tumor xenografts. In contrast, respiratory function is comparatively dispensable for metastatic tumor growth. RNA-Seq and metabolomics analysis of tumor cells expressing individual sgRNAs against mito-respiratory genes indicate overexpression of glycolytic genes and increased sensitivity of glycolytic inhibition compared to control when grown in vitro, but when grown in vivo as primary tumors these cells down-regulate glycolytic mechanisms. These studies demonstrate that discrete perturbations of mitochondrial respiratory chain function impact in vivo tumor growth in a context-specific manner with differential impacts on primary and metastatic tumors.

Hypertrophic cardiomyopathy (HCM) is a disease of heart muscle, which affects ∼1 in 500 individuals and is characterized by increased left ventricular wall thickness. While HCM is caused by pathogenic variants in any one of eight sarcomere protein genes, clinical expression varies considerably, even among patients with the same pathogenic variant. To determine whether background genetic variation or environmental factors drive these differences, we studied disease progression in 11 pairs of monozygotic HCM twins. The twin pairs were followed for 5 to 14 y, and left ventricular wall thickness, left atrial diameter, and left ventricular ejection fraction were collected from echocardiograms at various time points. All nine twin pairs with sarcomere protein gene variants and two with unknown disease etiologies had discordant morphologic features of the heart, demonstrating the influence of nonhereditable factors on clinical expression of HCM. Whole genome sequencing analysis of the six monozygotic twins with discordant HCM phenotypes did not reveal notable somatic genetic variants that might explain their clinical differences. Discordant cardiac morphology of identical twins highlights a significant role for epigenetics and environment in HCM disease progression.

Glycerophospholipids (GPLs) perform numerous essential functions in biology, including forming key structural components of cellular membranes and acting as secondary messengers in signaling pathways. Developing biomimetic molecular devices that can detect specific GPLs would enable modulation of GPL-related processes. However, the compositional diversity of GPLs, combined with their hydrophobic nature, has made it challenging to develop synthetic scaffolds that can react with specific lipid species. By taking advantage of the host-guest chemistry of cyclodextrins, we have engineered a molecular device that can selectively hydrolyze GPLs under physiologically relevant conditions. A chemically modified α-cyclodextrin bearing amine functional groups was shown to hydrolyze lyso-GPLs, generating free fatty acids. Lyso-GPLs are preferentially hydrolyzed when part of a mixture of GPL lipid species, and reaction efficiency was dependent on lyso-GPL chemical structure. These findings lay the groundwork for the development of molecular devices capable of specifically manipulating lipid-related processes in living systems.