Disruption of Z-Disc Function Promotes Mechanical Dysfunction in Human Myocardium: Evidence for a Dual Myofilament Modulatory Role by Alpha-Actinin 2.

The ACTN2 gene encodes α-actinin 2, located in the Z-disc of the sarcomeres in striated muscle. In this study, we sought to investigate the effects of an ACTN2 missense variant of unknown significance (p.A868T) on cardiac muscle structure and function. Left ventricular free wall samples were obtained at the time of cardiac transplantation from a heart failure patient with the ACTN2 A868T heterozygous variant. This variant is in the EF 3-4 domain known to interact with titin and α-actinin. At the ultrastructural level, ACTN2 A868T cardiac samples presented small structural changes in cardiomyocytes when compared to healthy donor samples. However, contractile mechanics of permeabilized ACTN2 A868T variant cardiac tissue displayed higher myofilament Ca2+ sensitivity of isometric force, reduced sinusoidal stiffness, and faster rates of tension redevelopment at all Ca2+ levels. Small-angle X-ray diffraction indicated increased separation between thick and thin filaments, possibly contributing to changes in muscle kinetics. Molecular dynamics simulations indicated that while the mutation does not significantly impact the structure of α-actinin on its own, it likely alters the conformation associated with titin binding. Our results can be explained by two Z-disc mediated communication pathways: one pathway that involves α-actinin's interaction with actin, affecting thin filament regulation, and the other pathway that involves α-actinin's interaction with titin, affecting thick filament activation. This work establishes the role of α-actinin 2 in modulating cross-bridge kinetics and force development in the human myocardium as well as how it can be involved in the development of cardiac disease.

Method To Visualize the Intratumor Distribution and Impact of Gemcitabine in Pancreatic Ductal Adenocarcinoma by Multimodal Imaging.

Gemcitabine (dFdC) is a common treatment for pancreatic cancer; however, it is thought that treatment may fail because tumor stroma prevents drug distribution to tumor cells. Gemcitabine is a pro-drug with active metabolites generated intracellularly; therefore, visualizing the distribution of parent drug as well as its metabolites is important. A multimodal imaging approach was developed using spatially coregistered mass spectrometry imaging (MSI), imaging mass cytometry (IMC), multiplex immunofluorescence microscopy (mIF), and hematoxylin and eosin (H&E) staining to assess the local distribution and metabolism of gemcitabine in tumors from a genetically engineered mouse model of pancreatic cancer (KPC) allowing for comparisons between effects in the tumor tissue and its microenvironment. Mass spectrometry imaging (MSI) enabled the visualization of the distribution of gemcitabine (100 mg/kg), its phosphorylated metabolites dFdCMP, dFdCDP and dFdCTP, and the inactive metabolite dFdU. Distribution was compared to small-molecule ATR inhibitor AZD6738 (25 mg/kg), which was codosed. Gemcitabine metabolites showed heterogeneous distribution within the tumor, which was different from the parent compound. The highest abundance of dFdCMP, dFdCDP, and dFdCTP correlated with distribution of endogenous AMP, ADP, and ATP in viable tumor cell regions, showing that gemcitabine active metabolites are reaching the tumor cell compartment, while AZD6738 was located to nonviable tumor regions. The method revealed that the generation of active, phosphorylated dFdC metabolites as well as treatment-induced DNA damage primarily correlated with sites of high proliferation in KPC PDAC tumor tissue, rather than sites of high parent drug abundance.

Summer and Winter Marine Heatwaves Favor an Invasive Over Native Seaweeds.

Marine heatwaves (MHWs) are emerging as forceful agents of ecosystem change and are increasing in frequency, duration, and intensity with climate change. During MHWs, physiological thresholds of native species may be exceeded while the performance of invasive species with warm affinities may be enhanced. As a consequence, MHWs could significantly alter an ecosystem's invasive dynamics, but such interactions are poorly understood. Following a 10-d acclimation period, we investigated the physiological resistance and resilience of an intertidal rock pool assemblage invaded by the seaweed Sargassum muticum to realistic 14-d marine heatwave scenarios (+1.5°C, +2.0°C, +3.5°C) followed by a 14-d recovery period. We conducted mesocosm experiments in both summer and winter to investigate temporal variability of MHWs. MHW treatments had clear negative impacts on native seaweeds (Fucus serratus and Chondrus crispus) while enhancing the performance of S. muticum. This pattern was consistent across season indicating that acclimation to cooler ambient temperatures results in winter MHWs having significant impacts on native species. As climate warming advances, this may ultimately lead to changes in competitive interactions and potentially exclusion of native species, while invasive species may proliferate and become more conspicuous within temperate rocky shore environments.

The importance of feldspar for ice nucleation by mineral dust in mixed-phase clouds.

The amount of ice present in mixed-phase clouds, which contain both supercooled liquid water droplets and ice particles, affects cloud extent, lifetime, particle size and radiative properties. The freezing of cloud droplets can be catalysed by the presence of aerosol particles known as ice nuclei. One of the most important ice nuclei is thought to be mineral dust aerosol from arid regions. It is generally assumed that clay minerals, which contribute approximately two-thirds of the dust mass, dominate ice nucleation by mineral dust, and many experimental studies have therefore focused on these materials. Here we use an established droplet-freezing technique to show that feldspar minerals dominate ice nucleation by mineral dusts under mixed-phase cloud conditions, despite feldspar being a minor component of dust emitted from arid regions. We also find that clay minerals are relatively unimportant ice nuclei. Our results from a global aerosol model study suggest that feldspar ice nuclei are globally distributed and that feldspar particles may account for a large proportion of the ice nuclei in Earth's atmosphere that contribute to freezing at temperatures below about -15 °C.

Diminished reovirus capsid stability alters disease pathogenesis and littermate transmission.

Reovirus is a nonenveloped mammalian virus that provides a useful model system for studies of viral infections in the young. Following internalization into host cells, the outermost capsid of reovirus virions is removed by endosomal cathepsin proteases. Determinants of capsid disassembly kinetics reside in the viral σ3 protein. However, the contribution of capsid stability to reovirus-induced disease is unknown. In this study, we found that mice inoculated intramuscularly with a serotype 3 reovirus containing σ3-Y354H, a mutation that reduces viral capsid stability, succumbed at a higher rate than those infected with wild-type virus. At early times after inoculation, σ3-Y354H virus reached higher titers than wild-type virus at several sites within the host. Animals inoculated perorally with a serotype 1 reassortant reovirus containing σ3-Y354H developed exaggerated myocarditis accompanied by elaboration of pro-inflammatory cytokines. Surprisingly, unchallenged littermates of mice infected with σ3-Y354H virus displayed higher titers in the intestine, heart, and brain than littermates of mice inoculated with wild-type virus. Together, these findings suggest that diminished capsid stability enhances reovirus replication, dissemination, lethality, and host-to-host spread, establishing a new virulence determinant for nonenveloped viruses.

Mechanisms of Lipid Accumulation in the Bone Morphogenetic Protein Receptor Type 2 Mutant Right Ventricle.

RATIONALE: In heritable pulmonary arterial hypertension with germline mutation in the bone morphogenetic protein receptor type 2 (BMPR2) gene, right ventricle (RV) dysfunction is associated with RV lipotoxicity; however, the underlying mechanism for lipid accumulation is not known. OBJECTIVES: We hypothesized that lipid accumulation in cardiomyocytes with BMPR2 mutation occurs owing to alterations in lipid transport and impaired fatty acid oxidation (FAO), which is exacerbated by a high-lipid (Western) diet (WD). METHODS: We used a transgenic mouse model of pulmonary arterial hypertension with mutant BMPR2 and generated a cardiomyocyte cell line with BMPR2 mutation. Electron microscopy and metabolomic analysis were performed on mouse RVs. MEASUREMENTS AND MAIN RESULTS: By metabolomics analysis, we found an increase in long-chain fatty acids in BMPR2 mutant mouse RVs compared with controls, which correlated with cardiac index. BMPR2-mutant cardiomyocytes had increased lipid compared with controls. Direct measurement of FAO in the WD-fed BMPR2-mutant RV showed impaired palmitate-linked oxygen consumption, and metabolomics analysis showed reduced indices of FAO. Using both mutant BMPR2 mouse RVs and cardiomyocytes, we found an increase in the uptake of (14)C-palmitate and fatty acid transporter CD36 that was further exacerbated by WD. CONCLUSIONS: Taken together, our data suggest that impaired FAO and increased expression of the lipid transporter CD36 are key mechanisms underlying lipid deposition in the BMPR2-mutant RV, which are exacerbated in the presence of dietary lipids. These findings suggest important features leading to RV lipotoxicity in pulmonary arterial hypertension and may point to novel areas of therapeutic intervention.

Molecular Analysis of Sarcoidosis Granulomas Reveals Antimicrobial Targets.

Sarcoidosis is a granulomatous disease of unknown cause. Prior molecular and immunologic studies have confirmed the presence of mycobacterial virulence factors, such as catalase peroxidase and superoxide dismutase A, within sarcoidosis granulomas. Molecular analysis of granulomas can identify targets of known antibiotics classes. Currently, major antibiotics are directed against DNA synthesis, protein synthesis, and cell wall formation. We conducted molecular analysis of 40 sarcoidosis diagnostic specimens and compared them with 33 disease control specimens for the presence of mycobacterial genes that encode antibiotic targets. We assessed for genes involved in DNA synthesis (DNA gyrase A [gyrA] and DNA gyrase B), protein synthesis (RNA polymerase subunit ß), cell wall synthesis (embCAB operon and enoyl reductase), and catalase peroxidase. Immunohistochemical analysis was conducted to investigate the locale of mycobacterial genes such as gyrA within 12 sarcoidosis specimens and 12 disease controls. Mycobacterial DNA was detected in 33 of 39 sarcoidosis specimens by quantitative real-time polymerase chain reaction compared with 2 of 30 disease control specimens (P < 0.001, two-tailed Fisher's test). Twenty of 39 were positive for three or more mycobacterial genes, compared with 1 of 30 control specimens (P < 0.001, two-tailed Fisher's test). Immunohistochemistry analysis localized mycobacterial gyrA nucleic acids to sites of granuloma formation in 9 of 12 sarcoidosis specimens compared with 1 of 12 disease controls (P < 0.01). Microbial genes encoding enzymes that can be targeted by currently available antimycobacterial antibiotics are present in sarcoidosis specimens and localize to sites of granulomatous inflammation. Use of antimicrobials directed against target enzymes may be an innovative treatment alternative.

Prolonged hypoxia induces monocarboxylate transporter-4 expression in mesenchymal stem cells resulting in a secretome that is deleterious to cardiovascular repair.

MSCs encounter extended hypoxia in the wound microenvironment yet little is known about their adaptability to this prolonged hypoxic milieu. In this study, we evaluated the cellular and molecular response of MSCs in extended hypoxia (1% O2 ) versus normoxia (20% O2 ) culture. Prolonged hypoxia induced a switch toward anaerobic glycolysis transcriptome and a dramatic increase in the transcript and protein levels of monocarboxylate transporter-4 (MCT4) in MSCs. To clarify the impact of MCT4 upregulation on MSC biology, we generated MSCs which stably overexpressed MCT4 (MCT4-MSCs) at levels similar to wild-type MSCs following prolonged hypoxic culture. Consistent with its role to efflux lactate to maintain intracellular pH, MCT4-MSCs demonstrated reduced intracellular lactate. To explore the in vivo significance of MCT4 upregulation in MSC therapy, mice were injected intramuscularly following MI with control (GFP)-MSCs, MCT4-MSCs, or MSCs in which MCT4 expression was stably silenced (KDMCT4-MSCs). Overexpression of MCT4 worsened cardiac remodeling and cardiac function whereas silencing of MCT4 significantly improved cardiac function. MCT4-overexpressing MSC secretome induced reactive oxygen species-mediated cardiomyocyte but not fibroblast apoptosis in vitro and in vivo; lactate alone recapitulated the effects of the MCT4-MSC secretome. Our findings suggest that lactate extruded by MCT4-overexpressing MSCs preferentially induced cell death in cardiomyocytes but not in fibroblasts, leading ultimately to a decline in cardiac function and increased scar size. A better understanding of stem cells response to prolonged hypoxic stress and the resultant stem cell-myocyte/fibroblast cross-talk is necessary to optimize MSC-based therapy for cardiac regeneration.

Rate of Homogenous Nucleation of Ice in Supercooled Water.

The homogeneous freezing of water is of fundamental importance to a number of fields, including that of cloud formation. However, there is considerable scatter in homogeneous nucleation rate coefficients reported in the literature. Using a cold stage droplet system designed to minimize uncertainties in temperature measurements, we examined the freezing of over 1500 pure water droplets with diameters between 4 and 24 µm. Under the assumption that nucleation occurs within the bulk of the droplet, nucleation rate coefficients fall within the spread of literature data and are in good agreement with a subset of more recent measurements. To quantify the relative importance of surface and volume nucleation in our experiments, where droplets are supported by a hydrophobic surface and surrounded by oil, comparison of droplets with different surface area to volume ratios was performed. From our experiments it is shown that in droplets larger than 6 µm diameter (between 234.6 and 236.5 K), nucleation in the interior is more important than nucleation at the surface. At smaller sizes we cannot rule out a significant contribution of surface nucleation, and in order to further constrain surface nucleation, experiments with smaller droplets are necessary. Nevertheless, in our experiments, it is dominantly volume nucleation controlling the observed nucleation rate.

Identification of small proline-rich repeat protein 3 as a novel atheroprotective factor that promotes adaptive Akt signaling in vascular smooth muscle cells.

OBJECTIVE: Atherosclerosis is the primary driver of cardiovascular disease, the leading cause of death worldwide. Identification of naturally occurring atheroprotective genes has become a major goal for the development of interventions that will limit atheroma progression and associated adverse events. To this end, we have identified small proline-rich repeat protein (SPRR3) as selectively upregulated in vascular smooth muscle cells (VSMCs) of atheroma-bearing arterial tissue versus healthy arterial tissue. In this study, we sought to determine the role of SPRR3 in atheroma pathophysiology. APPROACH AND RESULTS: We found that atheroprone apolipoprotein E-null mice lacking SPRR3 developed significantly greater atheroma burden. To determine the cellular driver(s) of this increase, we evaluated SPRR3-dependent changes in bone marrow-derived cells, endothelial cells, and VSMCs. Bone marrow transplant of SPRR3-expressing cells into SPRR3(-/-)apolipoprotein E-deficient recipients failed to rescue atheroma burden. Similarly, endothelial cells did not exhibit a response to SPRR3 loss. However, atheromas from SPRR3-deficient mice exhibited increased TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)-positive VSMCs compared with control. Cell death in SPRR3-deficient VSMCs was significantly increased in vitro. Conversely, SPRR3-overexpressing VSMCs exhibited reduced apoptosis compared with control. We also observed a PI3K (phosphatidylinositol 3-kinase)/Akt-dependent positive association between SPRR3 expression and levels of active Akt in VSMCs. The survival advantage seen in SPRR3-overexpressing VSMCs was abrogated after the addition of a PI3K/Akt pathway inhibitor. CONCLUSIONS: These results indicate that SPRR3 protects the lesion from VSMC loss by promoting survival signaling in plaque VSMCs, thereby significantly decreasing atherosclerosis progression. As the first identified atheroma-specific VSMC prosurvival factor, SPRR3 represents a potential target for lesion-specific modulation of VSMC survival.

Three-leaf quantum interference clovers in a trigonal single-molecule magnet.

We report on a single-molecule magnet where the spatial arrangement of three manganese ions and their spin-orbit coupling tensor orientations result in threefold angular modulations of the magnetization tunneling rates and quantum interference patterns that mimic the form of a three-leaf clover. Although expected in all quantum tunneling of magnetization resonances for a trigonal molecular symmetry, the threefold modulation only appears at resonances for which a longitudinal magnetic field is applied (i.e., resonance numbers |k|>0). A sixfold transverse field modulation observed at resonance k = 0 manifests as a direct consequence of a threefold corrugation of the spin-orbit coupling energy landscape, creating an effective longitudinal field which varies the resonance condition in the presence of a transverse field. The observations allow for an association between the trigonal distortion of the local spin-orbit interactions and the spatial disposition of the constituent ions, a finding that can be extrapolated to other systems where spin-orbit coupling plays a significant role.

Whole-body retention of α-linolenic acid and its apparent conversion to other n-3 PUFA in growing pigs are reduced with the duration of feeding α-linolenic acid.

In the present study, fifteen growing pigs were used to determine the whole-body oxidation, retention efficiency (RE) and apparent conversion (AC) of α-linolenic acid (18 : 3n-3) to n-3 highly unsaturated fatty acids (HUFA), including EPA (20 : 5n-3) and DHA (22 : 6n-3). The pigs were fed a diet containing 10% flaxseed for 30 d. Whole-body fatty acid composition was determined at initial (27.7 (SE 1.9) kg), intermediate (day 15; 39.2 (SE 1.4) kg) and final (45.7 (SE 2.2) kg) body weight. On day 12, four pigs were fed 10 mg/kg of uniformly labelled (13)C-18 : 3n-3 (single-bolus dose) to determine the oxidation of 18 : 3n-3. Expired CO2 samples were collected for 24 h thereafter. The whole-body content of n-3 PUFA increased linearly (P< 0.0001) with time; however, the content of 22 : 6n-3 exhibited a quadratic response (P< 0.01) with a peak occurring at 15 h. As a proportion of intake, the RE of 18 : 3n-3 tended to reduce with time (P = 0.098). The AC of ingested 18 : 3n-3 to the sum of n-3 HUFA was reduced with time (P< 0.05; 12.2 v. 7.53 % for days 0-15 and days 15-30, respectively). The AC of 18 : 3n-3 to 20 : 5n-3 or 22 : 6n-3 was lower than that to 20 : 3n-3, both for days 0-15 (P < 0.05; 1.14 or 1.07 v. 7.06 %) and for days 15-30 (P< 0.05; 1.51 or 0.33 v. 4.29 %). The direct oxidation of 18 : 3n-3 was 7.91 (SE 0.98) % and was similar to the calculated disappearance of 18 : 3n-3 between days 0 and 30 (8.81 (SE 5.24) %). The oxidation of 18 : 3n-3 was much lower than that reported in other species. The AC of 18 : 3n-3 to n-3 HUFA was reduced over time and that to 20 : 3n-3 in the present study was much higher than that reported in other species and should be explored further.

LNK/SH2B3 loss of function increases susceptibility to murine and human atrial fibrillation.

AIMS: The lymphocyte adaptor protein (LNK) is a negative regulator of cytokine and growth factor signalling. The rs3184504 variant in SH2B3 reduces LNK function and is linked to cardiovascular, inflammatory, and haematologic disorders, including stroke. In mice, deletion of Lnk causes inflammation and oxidative stress. We hypothesized that Lnk-/- mice are susceptible to atrial fibrillation (AF) and that rs3184504 is associated with AF and AF-related stroke in humans. During inflammation, reactive lipid dicarbonyls are the major components of oxidative injury, and we further hypothesized that these mediators are critical drivers of the AF substrate in Lnk-/- mice. METHODS AND RESULTS: Lnk-/- or wild-type (WT) mice were treated with vehicle or 2-hydroxybenzylamine (2-HOBA), a dicarbonyl scavenger, for 3 months. Compared with WT, Lnk-/- mice displayed increased AF duration that was prevented by 2-HOBA. In the Lnk-/- atria, action potentials were prolonged with reduced transient outward K+ current, increased late Na+ current, and reduced peak Na+ current, pro-arrhythmic effects that were inhibited by 2-HOBA. Mitochondrial dysfunction, especially for Complex I, was evident in Lnk-/- atria, while scavenging lipid dicarbonyls prevented this abnormality. Tumour necrosis factor-α (TNF-α) and interleukin-1 beta (IL-1ß) were elevated in Lnk-/- plasma and atrial tissue, respectively, both of which caused electrical and bioenergetic remodelling in vitro. Inhibition of soluble TNF-α prevented electrical remodelling and AF susceptibility, while IL-1ß inhibition improved mitochondrial respiration but had no effect on AF susceptibility. In a large database of genotyped patients, rs3184504 was associated with AF, as well as AF-related stroke. CONCLUSION: These findings identify a novel role for LNK in the pathophysiology of AF in both experimental mice and humans. Moreover, reactive lipid dicarbonyls are critical to the inflammatory AF substrate in Lnk-/- mice and mediate the pro-arrhythmic effects of pro-inflammatory cytokines, primarily through electrical remodelling.

Epitope Lability of Phosphorylated Biomarkers of the DNA Damage Response Pathway Results in Increased Vulnerability to Effects of Delayed or Incomplete Formalin Fixation.

Phosphorylated biomarkers are crucial for our understanding of drug mechanism of action and dose selection during clinical trials, particularly for drugs that target protein kinases, such as DNA-damage-response (DDR) inhibitors. However, tissue fixation conditions needed to preserve DDR-specific phospho-biomarkers have not been previously investigated. Using xenograft tissues and tightly controlled formalin fixation conditions, we assessed how preanalytical factors affect phosphorylated DDR biomarkers pRAD50(Ser635), ɣH2AX(Ser139), pKAP1(Ser824), and non-phosphorylated biomarkers cMYC and ATM. Cold ischemia times ranged from 15 min to 6 hr, and the fixation duration ranged from 24 hr to 4 weeks. Epitopes pRAD50 and pKAP1 appeared the most labile assessed with staining loss after just 15 min of cold ischemia time, while ATM was more robust showing consistent expression up to 1 hr of cold ischemia. Notably, ɣH2AX expression was lost with formalin fixation over 48 hr. The use of core needle biopsies where possible and novel fixation methods such as the 2-step temperature-controlled formalin approach may improve phosphorylated biomarker preservation; however, practical challenges may affect wider clinical application. The most essential tissue-processing step when downstream analysis includes DDR phosphorylated biomarkers is immediate tissue submersion in formalin, without delay, upon excision from the patient, followed by room temperature fixation for 24 hr.

Idiopathic subglottic stenosis arises at the epithelial interface of host and pathogen.

Background: Idiopathic subglottic stenosis (iSGS) is a rare fibrotic disease of the proximal airway affecting adult Caucasian women nearly exclusively. Life-threatening ventilatory obstruction occurs secondary to pernicious subglottic mucosal scar. Disease rarity and wide geographic patient distribution has previously limited substantive mechanistic investigation into iSGS pathogenesis. Result: By harnessing pathogenic mucosa from an international iSGS patient cohort and single-cell RNA sequencing, we unbiasedly characterize the cell subsets in the proximal airway scar and detail their molecular phenotypes. Results show that the airway epithelium in iSGS patients is depleted of basal progenitor cells, and the residual epithelial cells acquire a mesenchymal phenotype. Observed displacement of bacteria beneath the lamina propria provides functional support for the molecular evidence of epithelial dysfunction. Matched tissue microbiomes support displacement of the native microbiome into the lamina propria of iSGS patients rather than disrupted bacterial community structure. However, animal models confirm that bacteria are necessary for pathologic proximal airway fibrosis and suggest an equally essential role for host adaptive immunity. Human samples from iSGS airway scar demonstrate adaptive immune activation in response to the proximal airway microbiome of both matched iSGS patients and healthy controls. Clinical outcome data from iSGS patients suggests surgical extirpation of airway scar and reconstitution with unaffected tracheal mucosa halts the progressive fibrosis. Conclusion: Our data support an iSGS disease model where epithelial alterations facilitate microbiome displacement, dysregulated immune activation, and localized fibrosis. These results refine our understanding of iSGS and implicate shared pathogenic mechanisms with distal airway fibrotic diseases.

Calmodulin kinase II inhibition protects against structural heart disease.

Beta-adrenergic receptor (betaAR) stimulation increases cytosolic Ca(2+) to physiologically augment cardiac contraction, whereas excessive betaAR activation causes adverse cardiac remodeling, including myocardial hypertrophy, dilation and dysfunction, in individuals with myocardial infarction. The Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) is a recently identified downstream element of the betaAR-initiated signaling cascade that is linked to pathological myocardial remodeling and to regulation of key proteins involved in cardiac excitation-contraction coupling. We developed a genetic mouse model of cardiac CaMKII inhibition to test the role of CaMKII in betaAR signaling in vivo. Here we show CaMKII inhibition substantially prevented maladaptive remodeling from excessive betaAR stimulation and myocardial infarction, and induced balanced changes in excitation-contraction coupling that preserved baseline and betaAR-stimulated physiological increases in cardiac function. These findings mark CaMKII as a determinant of clinically important heart disease phenotypes, and suggest CaMKII inhibition can be a highly selective approach for targeting adverse myocardial remodeling linked to betaAR signaling.

Fatal antimalarial-induced cardiomyopathy: report of 2 cases.

Chloroquine and hydroxychloroquine are used to chronically treat certain rheumatologic diseases and are generally considered safe. We describe 2 patients with skeletal myopathy and fatal cardiomyopathy-uncommon and underrecognized adverse effects of these agents. Both patients developed arrhythmias and heart failure, and 1 patient had documented diaphragmatic involvement. Muscle specimens showed typical vacuolar myopathy (indicative of impaired autophagy) with myeloid bodies in both patients and curvilinear bodies in 1 patient. Antimalarial-induced cardiomyopathy should be considered in patients receiving these medications with otherwise unexplained muscle weakness or cardiac symptoms. Whether autophagy enhancers can be used to manage such myopathies merits investigation.

AZD4625 is a Potent and Selective Inhibitor of KRASG12C.

AZD4625 is a potent, selective, and orally bioavailable inhibitor of oncogenic KRASG12C as demonstrated in cellular assays and in vivo in preclinical cell line-derived and patient-derived xenograft models. In vitro and cellular assays have shown selective binding and inhibition of the KRASG12C mutant isoform, which carries a glycine to cysteine mutation at residue 12, with no binding and inhibition of wild-type RAS or isoforms carrying non-KRASG12C mutations. The pharmacology of AZD4625 shows that it has the potential to provide therapeutic benefit to patients with KRASG12C mutant cancer as either a monotherapy treatment or in combination with other targeted drug agents.

The Wnt modulator sFRP2 enhances mesenchymal stem cell engraftment, granulation tissue formation and myocardial repair.

Cell-based therapies, using multipotent mesenchymal stem cells (MSCs) for organ regeneration, are being pursued for cardiac disease, orthopedic injuries and biomaterial fabrication. The molecular pathways that regulate MSC-mediated regeneration or enhance their therapeutic efficacy are, however, poorly understood. We compared MSCs isolated from MRL/MpJ mice, known to demonstrate enhanced regenerative capacity, to those from C57BL/6 (WT) mice. Compared with WT-MSCs, MRL-MSCs demonstrated increased proliferation, in vivo engraftment, experimental granulation tissue reconstitution, and tissue vascularity in a murine model of repair stimulation. The MRL-MSCs also reduced infarct size and improved function in a murine myocardial infarct model compared with WT-MSCs. Genomic and functional analysis indicated a downregulation of the canonical Wnt pathway in MRL-MSCs characterized by significant up-regulation of specific secreted frizzled-related proteins (sFRPs). Specific knockdown of sFRP2 by shRNA in MRL-MSCs decreased their proliferation and their engraftment in and the vascular density of MRL-MSC-generated experimental granulation tissue. These results led us to generate WT-MSCs overexpressing sFRP2 (sFRP2-MSCs) by retroviral transduction. sFRP2-MSCs maintained their ability for multilineage differentiation in vitro and, when implanted in vivo, recapitulated the MRL phenotype. Peri-infarct intramyocardial injection of sFRP2-MSCs resulted in enhanced engraftment, vascular density, reduced infarct size, and increased cardiac function after myocardial injury in mice. These findings implicate sFRP2 as a key molecule for the biogenesis of a superior regenerative phenotype in MSCs.

Holistic Characterization of a Salmonella Typhimurium Infection Model Using Integrated Molecular Imaging.

A more complete and holistic view on host-microbe interactions is needed to understand the physiological and cellular barriers that affect the efficacy of drug treatments and allow the discovery and development of new therapeutics. Here, we developed a multimodal imaging approach combining histopathology with mass spectrometry imaging (MSI) and same section imaging mass cytometry (IMC) to study the effects of Salmonella Typhimurium infection in the liver of a mouse model using the S. Typhimurium strains SL3261 and SL1344. This approach enables correlation of tissue morphology and specific cell phenotypes with molecular images of tissue metabolism. IMC revealed a marked increase in immune cell markers and localization in immune aggregates in infected tissues. A correlative computational method (network analysis) was deployed to find metabolic features associated with infection and revealed metabolic clusters of acetyl carnitines, as well as phosphatidylcholine and phosphatidylethanolamine plasmalogen species, which could be associated with pro-inflammatory immune cell types. By developing an IMC marker for the detection of Salmonella LPS, we were further able to identify and characterize those cell types which contained S. Typhimurium.