The RNA-binding protein QKI belongs to the hnRNP K-homology domain protein family, a well-known regulator of pre-mRNA alternative splicing and is associated with several neurodevelopmental disorders. Qki is found highly expressed in developing and adult hearts. By employing the human embryonic stem cell (hESC) to cardiomyocyte differentiation system and generating QKI-deficient hESCs (hESCs-QKIdel) using CRISPR/Cas9 gene editing technology, we analyze the physiological role of QKI in cardiomyocyte differentiation, maturation, and contractile function. hESCs-QKIdel largely maintain normal pluripotency and normal differentiation potential for the generation of early cardiogenic progenitors, but they fail to transition into functional cardiomyocytes. In this work, by using a series of transcriptomic, cell and biochemical analyses, and the Qki-deficient mouse model, we demonstrate that QKI is indispensable to cardiac sarcomerogenesis and cardiac function through its regulation of alternative splicing in genes involved in Z-disc formation and contractile physiology, suggesting that QKI is associated with the pathogenesis of certain forms of cardiomyopathies.

Histologically normal tissue adjacent to the tumor can provide insight of the microenvironmental alterations surrounding the cancerous lesion and affecting the progression of the disease. However, little is known about the molecular changes governing cancer initiation in cancer-free breast tissue. Here, we employed laser microdissection and whole-transcriptome profiling of the breast epithelium prior to and post tumor diagnosis to identify the earliest alterations in breast carcinogenesis. Furthermore, a comprehensive analysis of the three tissue compartments (microdissected epithelium, stroma, and adipose tissue) was performed on the breast donated by either healthy subjects or women prior to the clinical manifestation of cancer (labeled “susceptible normal tissue”). Although both susceptible and healthy breast tissues appeared histologically normal, the susceptible breast epithelium displayed a significant upregulation of genes involved in fatty acid uptake/transport (CD36 and AQP7), lipolysis (LIPE), and lipid peroxidation (AKR1C1). Upregulation of lipid metabolism- and fatty acid transport-related genes was observed also in the microdissected susceptible stromal and adipose tissue compartments, respectively, when compared with the matched healthy controls. Moreover, inter-compartmental co-expression analysis showed increased epithelium-adipose tissue crosstalk in the susceptible breasts as compared with healthy controls. Interestingly, reductions in natural killer (NK)-related gene signature and CD45+/CD20+ cell staining were also observed in the stromal compartment of susceptible breasts. Our study yields new insights into the cancer initiation process in the breast. The data suggest that in the early phase of cancer development, metabolic activation of the breast, together with increased epithelium-adipose tissue crosstalk may create a favorable environment for final cell transformation, proliferation, and survival.

Temperature sensitive (TS) missense mutants have been foundational for characterization of essentialgene function. However, an unbiased approach for analysis of biochemical and biophysical changes in TSmissense mutants within the context of their functional proteomes is lacking. We applied massspectrometry (MS) based thermal proteome profiling (TPP) to investigate the proteome-wide effects ofmissense mutations in an application that we refer to as mutant Thermal Proteome Profiling (mTPP).This study characterized global impacts of temperature sensitivity-inducing missense mutations in twodifferent subunits of the 26S proteasome. The majority of alterations identified by RNA-Seq and globalproteomics were similar between the mutants, which could suggest that a similar functional disruption isoccurring in both missense variants. Results from mTPP, however, provide unique insights into themechanisms that contribute to the TS phenotype in each mutant, revealing distinct changes that were notobtained using only steady-state transcriptome and proteome analyses. Computationally, multisite λ-dynamics simulations add clear support for mTPP experimental findings. This work shows that mTPP is aprecise approach to measure changes in missense mutant containing proteomes without the requirementfor large amounts of starting material, specific antibodies against proteins of interest, and/or geneticmanipulation of the biological system. Although experiments were performed under permissiveconditions, mTPP provided insights into the underlying protein stability changes that cause dramaticcellular phenotypes observed at non-permissive temperatures. Overall, mTPP provides uniquemechanistic insights into missense mutation dysfunction and connection of genotype to phenotype in arapid, non-biased fashion.

The secretion of prostaglandins (PGs) by bovine corpora lutea was investigated. Corpora lutea from the early, early-mid and late-mid stages of the luteal phase were dissociated by collagenase treatment and cultured in monolayer in Dulbecco's modified Eagle's medium containing 10% (v/v) fetal calf serum. Treatment with either LH (100 ng/ml) or dibutyryl cyclic AMP (dbcAMP; 1 mmol/l) had no effect on progesterone secretion by early luteal phase cells but stimulated progesterone secretion two-to fourfold by cells from the latter stages. The secretion rates, per \ g=m\ g cell protein, of 6-keto-PGF1\g=a\, PGE2 and PGF2\g=a\ were substantially greater in cells from the early luteal phase than in those from the latter stages, however, all changes in PG secretion in response to treatments were qualitatively similar between cells from the three stages of the luteal phase. The secretion rate of 6-keto\x=req-\ PGF1\g=a\ was greater than that of PGE2 or PGF2\g=a\ and was inhibited by treatment with indomethacin (28 \g=m\mol/l) but unaltered by treatment with LH, dbcAMP or butyrate (1 mmol/l). Secretion of PGE2 was inhibited by indomethacin but stimulated two\x=req-\ to threefold by treatment with either dbcAMP or butyrate. Secretion of PGF2\g=a\ was minimal and not inhibited further by treatment with indomethacin, but was stimulated 10-to 40-fold with dbcAMP. Indomethacin treatment inhibited the stimulatory effect of dbcAMP; butyrate had no effect on PGF2\g=a\ secretion. Treatment with LH had no effect on any of the PGs measured. In these experiments the secretion of progesterone appeared unrelated to any changes in the secretion of PGs. Thus it would appear that the production of progesterone by bovine luteal cells in culture is not related nor dependent upon the secretion of 6-keto-PGF1\g=a\, PGE2 or PGF2\g=a\, and that LH/cAMP does not regulate the secretion of PGs since LH had no effect on PG secretion and since the effects of dbcAMP appeared not to be through a cAMP-dependent pathway.

e Francisella philomiragia is a very uncommon pathogen of humans. Diseases caused by it are protean and have been reported largely in near-drowning victims and those with chronic granulomatous disease. We present a case of F. philomiragia pneumonia with peripheral edema and bacteremia in a renal transplant patient and review the diverse reports of F. philomiragia infections. CASE REPORTA 63-year-old female from Indiana presented to an Indianapolis hospital with worsening shortness of breath, nonproductive cough, and increasing bilateral peripheral edema. The patient was afebrile, normotensive, and normocardic but was tachypnic (40 bpm) and denied having fevers, chills, or other symptoms of an infectious process while at home. Significant medical history obtained at the time of presentation included a renal transplant secondary to polycystic kidney disease 14 years prior for which she receives chronic immunosuppressive therapy (tacrolimus and prednisone). The patient did not report recent travel outside Indiana, exposure to wild animals or recreational water sources, or exposure to sick individuals. Because of the possibility of acute transplant rejection, the patient was admitted to the intensive care unit for extensive evaluation. A chest X-ray performed at the time of admission revealed bilateral perihilar and upper-lobe infiltrates consistent with bilateral bronchopneumonia, prompting the collection of a set of blood cultures from the left arm and of another set from the right arm and initiation of empirical broad-spectrum antimicrobial therapy with vancomycin and piperacillin-tazobactam. Aside from blood cultures, no other microbiology testing was performed. Laboratory studies conducted at the time of admission revealed leukocytosis (11,600 cells/l) with 93% neutrophils, anemia (3.32 million cells/l), kidney failure (elevated levels of urea nitrogen [48 mg/dl] and creatinine [3.70 mg/dl]), and hyperglycemia (127 mg of glucose/dl). Elevated levels of procalcitonin (1.86 ng/ml), hematuria (25 cells/l), and proteinuria (500 mg/ dl) were also noted. Because of the possibility of acute-on-chronic kidney disease, a renal biopsy was subsequently performed, and it revealed acute allograft rejection.Following approximately 24 h of incubation in a continuousmonitoring blood culture instrument (BD Bactec 9240; BD Diagnostic Systems, Sparks, MD), the aerobic bottles from both sets of blood cultures signaled positively. Gram stains of broth from both bottles revealed pleomorphic, Gram-negative coccobacilli (Fig. 1A). Subcultures of the blood culture broth grew medium-sized (ϳ5-mm diameter), glossy, convex colonies resembling a member of the Enterobacteriaceae on sheep blood and chocolate agars after 48 h of incubation at 35°C in 5% CO 2 . A Gram stain of the colonies revealed organisms with morphologies identical to those seen in the blood culture broth smear. The isolate tested positive for cytochrome oxidase and catalase. Together, these observations ruled out possible select agents, including Francisella tularensis a...