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Objectives. To compare the clinicopathologic characteristics of urothelial carcinoma (UC), urothelial carcinoma with squamous differentiation (UCSD), and squamous cell carcinoma (SCC) of the bladder, which have been suggested to differ in terms of risk factors, immunophenotype, and prognosis. Methods. We evaluated the clinicopathologic features of radical cystectomy specimens between 1980 and 2015 with a diagnosis of SCC, UCSD, and UC. PD-L1 immunohistochemistry (clinically available clones 22C3, SP142, and SP263) was performed on SCC and UCSD. Multivariate Cox regression was used to identify prognostic factors. Kaplan–Meier curves were plotted to assess cancer-specific survival (CSS). Results. Of the 1478 cases, there were 1126 UC (76%), 217 UCSD (15%), and 135 SCC (9%). Bladder cancer was more common in men than women (80% vs 20%, P < .0001). However, a higher proportion of SCC and UCSD occurred in women (SCC-36%, UCSD-22%, UC-18%). Women were significantly more likely to be never smokers in all 3 cohorts (UC: 45% vs 16%, UCSD: 44% vs 12%, SCC: 40% vs 18%, P < .0001). Patients with SCC and UCSD were at a higher pathologic stage (>pT2) at the time of cystectomy (UCSD-74%, SCC 71%, UC-44%, P < .0001) and had worse CSS compared to patients with UC ( P = 0.006). SCC had higher PD-L1 scores (all clones) than UCSD ( P < .0001). PD-L1 22C3 ( P = .02, HR: 0.36) and SP142 scores ( P = .046, HR: 0.27) predicted CSS on Kaplan–Meier analysis for SCC cases. Conclusions. UC, UCSD, and SCC are associated with different risk factors, gender distributions, and clinical outcomes. PD-L1 is expressed in SCC and UCSD, suggesting some patients may benefit from targeted therapy.

To assess the role of telomerase activity and telomere length in pancreatic CSCs we used different CSC enrichment methods (CD133, ALDH, sphere formation) in primary patient-derived pancreatic cancer cells. We show that CSCs have higher telomerase activity and longer telomeres than bulk tumor cells. Inhibition of telomerase activity, using genetic knockdown or pharmacological inhibitor (BIBR1532), resulted in CSC marker depletion, abrogation of sphere formation in vitro and reduced tumorigenicity in vivo. Furthermore, we identify a positive feedback loop between stemness factors (NANOG, OCT3/4, SOX2, KLF4) and telomerase, which is essential for the self-renewal of CSCs. Disruption of the balance between telomerase activity and stemness factors eliminates CSCs via induction of DNA damage and apoptosis in primary patient-derived pancreatic cancer samples, opening future perspectives to avoid CSC-driven tumor relapse. In the present study, we demonstrate that telomerase regulation is critical for the “stemness” maintenance in pancreatic CSCs and examine the effects of telomerase inhibition as a potential treatment option of pancreatic cancer. This may significantly promote our understanding of PDAC tumor biology and may result in improved treatment for pancreatic cancer patients.

The participation of nonmuscle myosin in force maintenance is controversial. To participate in force maintenance, nonmuscle myosin activity must be regulated in smooth muscle. To investigate this question, phosphorylation of the regulatory light chains of smooth muscle myosin (SMLC) and nonmuscle myosin (NMLC) were examined by two dimensional SDS‐PAGE. Intact mouse aortic smooth muscle strips were stimulated with either KCl depolarization or angiotensin II, and 4 distinct LC spots were resolved by two dimensional SDS‐PAGE. Using mass spectroscopy, these spots were identified as four distinct proteins; phosphorylated NMLC, non‐phosphorylated NMLC, phosphorylated SMLC and non‐phosphorylated SMLC. During KCl depolarization, inhibition of Rho kinase or PKC did not affect the increases in SMLC and NMLC phosphorylation. During angiotensin II stimulation, inhibition of PKC or Rho kinase reduced SMLC phosphorylation, while only inhibition of Rho kinase reduced NMLC phosphorylation. These data suggest that in smooth muscle, nonmuscle myosin is regulated by Ca 2+ ‐calmodulin activated MLCK during depolarization, and a Rho kinase mediated pathway during agonist stimulation.

Overexpression of 11b‐hydroxysteroid dehydrogenase type 1 (11b‐HSD1) in adipose tissue leads to obesity in mice. We hypothesized that endogenous 11b‐HSD1 played a significant functional role in adipogenesis. Stable transfection of 3T3‐L1 cells with small hairpin RNA (shRNA), compared to a nucleotide‐substituted control, silenced 11b‐HSD1 expression and substantially attenuated the accumulation of lipid droplets and the expression of adipogenesis marker genes induced by a mixture containing either corticosterone or dexamethasone. 11b‐HSD1 silencing significantly elevated the concentration of 11‐dehydrocorticosterone in the culture supernatant, but did not affect the levels of corticosterone or dexamethasone, measured by LC‐MS/MS. 11b‐HSD1 silencing significantly attenuated the translocation of glucocorticoid receptors to the nucleus and reduced the number of cells entering the S phase in response to glucocorticoids (13 ± 1% vs. 30 ± 1% in control, n=5, P<0.05), a critical event in the initiation of adipogenesis. 11b‐HSD1 shRNA delivered by lentiviral vectors after the induction of differentiation reduced 11b‐HSD1 expression, but did not affect the further progression of adipogenesis. Our results established a functional and stage‐dependent role of 11b‐HSD1 in the initiation of 3T3‐L1 adipogenesis, and provided new insights into the development of obesity and related diseases.

Abstract Background Colon cancer (CC) incidence in young adults (age 20-49 years), termed early-onset CC (EO-CC), is increasing. Methods Individual patient data on 35 713 subjects with stage III colon cancer from 25 randomized studies in the Adjuvant Colon Cancer ENdpoint database were pooled. The distributions of demographics, clinicopathological features, biomarker status, and outcome data were summarized by age group. Overall survival, disease-free survival, time to recurrence, and survival after recurrence were assessed by Kaplan-Meier curves and Cox models stratified by treatment arms within studies, adjusting for sex, race, body mass index, performance status, disease stage, grade, risk group, number of lymph nodes examined, disease sidedness, and molecular markers. All statistical tests were 2-sided. Results Using a 5% difference between age groups as the clinically meaningful cutoff, patients with stage III EO-CC had similar sex, race, performance status, risk group, tumor sidedness, and T stage compared with patients with late-onset CC (age 50 years and older). EO-CC patients were less likely to be overweight (30.2% vs 36.2%) and more commonly had 12 or more lymph nodes resected (69.5% vs 58.7%). EO-CC tumors were more frequently mismatch repair deficient (16.4% vs 11.5%) and less likely to have BRAFV600E (5.6% vs 14.0%), suggesting a higher rate of Lynch syndrome in EO-CC. Patients with EO-CC had statistically significantly better overall survival (hazard ratio [HR] = 0.81, 95% confidence interval [CI] = 0.74 to 0.89; P &lt; .001), disease-free survival (HR = 0.91, 95% CI = 0.84 to 0.98; P = .01), and survival after recurrence (HR = 0.88, 95% CI = 0.80 to 0.97; P = .008) in the analysis without molecular markers; however, age at onset of CC lost its prognostic value when outcome was adjusted for molecular markers. Conclusion Tumor biology was found to be a more important prognostic factor than age of onset among stage III colon cancer patients in the Adjuvant Colon Cancer ENdpoint database.

We have recently demonstrated that vasodilation to the endothelium‐dependent agonist acetylcholine is amplified in contracting compared with quiescent skeletal muscle, highlighting the importance of the vascular endothelium in integrating vasomotor signals to regulate vascular tone in humans. In the present study, we tested the hypothesis that sensitivity to the endothelium‐dependent vasodilator ATP is augmented during exercise and systemic hypoxia, whereas sensitivity to the nitric oxide donor and endothelium‐independent dilator sodium nitroprusside (SNP) is not affected by these physiological conditions. In 12 young adults (7 M, 5 F), we measured forearm blood flow (FBF; Doppler ultrasound) and calculated changes in vascular conductance (FVC: quotient of FBF and mean arterial pressure) in response to local, intra‐arterial infusion of ATP or SNP during 1) control resting conditions (CON), 2) dynamic handgrip exercise (EX, 20 contractions per minute at 10% of maximal voluntary contraction), or 3) systemic hypoxia (HYP, 80% O 2 saturation). Three doses of each vasodilator were infused via a brachial artery catheter during each condition (ATP: 1.25, 2.50, and 5.00 μg·dl forearm volume −1 ·min −1 ; SNP: 0.25, 0.50, and 1.00 μg·dl forearm volume −1 ·min −1 ). The order of drugs and conditions was randomized. Within each trial, infusions began after 3 min of rest, steady‐state exercise, or hypoxia, and each dose was infused for 2 min while the exercise or hypoxic stimulus was sustained. Compared to control conditions, vasodilatory sensitivity to the high dose of ATP was enhanced by 53 ± 18% during mild handgrip exercise (Δ FVC, CON: 120 ± 16 vs. EX: 176 ± 28 ml·min −1 ·100 mmHg −1 ; P < 0.05), whereas vasodilatory sensitivity was not affected at lower doses of ATP ( P = NS; Dose × Condition interaction P < 0.05). In contrast, exercise had no effect on vascular sensitivity to any dose of SNP ( P = NS). Similarly, during systemic hypoxia, vasodilation to ATP was enhanced by 18 ± 10% at the medium dose (Δ FVC, CON: 100 ± 15 vs. HYP: 125 ± 24 ml·min −1 ·100 mmHg −1 ; P < 0.05) and 28 ± 11% at the high dose (Δ FVC, CON: 120 ± 16 vs. HYP: 158 ± 27 ml·min −1 ·100 mmHg −1 ; P < 0.05; Dose × Condition interaction P < 0.05), whereas vasodilation to SNP was not different from control conditions at any dose ( P = NS). These findings provide evidence that muscle contractions and hypoxia augment ATP‐mediated, endothelium‐dependent vasodilation in human skeletal muscle. Moreover, given that intravascular ATP increases under these physiological conditions, the results indicate that small changes in plasma ATP may result in augmented vasodilation and contribute to blood flow and oxygen delivery in humans. Support or Funding Information HL119337 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Angiotensin II produces oxidative stress and endothelial dysfunction. ACE2 converts angiotensin II to angiotensin 1–7 and may protect against effects of angiotensin II. We tested the hypothesis that ACE2 deficiency increases oxidative stress and vasomotor dysfunction in cerebral arteries, and examined the role of ACE2 in vascular aging. ACE2 was present at relatively low levels in cerebral arteries. Systolic arterial pressure was similar in adult and old ACE2 knockout (KO) and wild‐type (WT) mice. Maximal dilatation to acetylcholine (ACh) was impaired in the basilar artery from adult ACE2 KO mice compared to adult WT. In old mice, maximal dilatation to ACh was impaired in WT mice but severely impaired in ACE2 KO mice (P<0.05). The antioxidant tempol (1 mM) improved responses to ACh in adult and old ACE2 KO and WT mice. Nitrotyrosine staining in the basilar artery was increased in adult ACE2 KO mice and in old ACE2 KO and WT mice. Expression of NAD(P)H oxidase subunits Nox2 and p47phox, and of pro‐inflammatory molecules Rcan1 and TNFα, was increased in cerebral arteries from old ACE2 KO and WT animals. In summary, ACE2 deficiency impaired vasomotor function in cerebral arteries from adult mice and augments endothelial dysfunction during aging. Oxidative stress plays a critical role in cerebrovascular dysfunction induced by ACE2 deficiency and aging. NIH grants NS‐24621, HL‐38091, HL‐62984; AHA 0815525G, 10PRE3780044.

There are multiple conditions that can make children prone to having a sudden cardiac arrest (SCA) or sudden cardiac death (SCD). Efforts have been made by multiple organizations to screen children for cardiac conditions, but the emphasis has been on screening before athletic competition. This article is an update of the previous American Academy of Pediatrics policy statement of 2012 that addresses prevention of SCA and SCD. This update includes a comprehensive review of conditions that should prompt more attention and cardiology evaluation. The role of the primary care provider is of paramount importance in the evaluation of children, particularly as they enter middle school or junior high. There is discussion about whether screening should find any cardiac condition or just those that are associated with SCA and SCD. This update reviews the 4 main screening questions that are recommended, not just for athletes, but for all children. There is also discussion about how to handle post-SCA and SCD situations as well as discussion about genetic testing. It is the goal of this policy statement update to provide the primary care provider more assistance in how to screen for life-threatening conditions, regardless of athletic status.

During nitric oxide (NO) signaling, cGMP activates type Iα protein kinase G (PKGIα), which subsequently activates myosin light chain phosphatase (MLCP). The increase in MLCP activity decreases myosin phosphorylation which leads to smooth muscle relaxation. MLCP is a trimeric enzyme consisting of a myosin targeting subunit (MYPT1), a catalytic subunit and a subunit of unknown function. MYPT1 has two isoforms; alterative mRNA splicing of a 3’ exon results in the presence or absence of a C‐terminal leucine zipper (LZ) domain. Further, the MYPT1 LZ+ is required for PKGIα mediated activation of MLCP. Moreover, recent studies have demonstrated PKGIα phosphorylates MYPT1. This study examined the affect of MYPT1 structure for PKGIα phosphorylation. Two MYPT1 fragments differing only by the LZ domain (aa 1010–1039) were used: MYPT1FL (aa 500–1039, LZ+) and MYPT1TR2 (aa 500–1010, LZ−). Both the rate (0.0216±0.005 min −1 vs 0.0596±0.005 min −1 ) and extent of PKGIα mediated phosphorylation of MYPT1TR2 were lower when compared to MYPT1FL. In addition, two‐dimensional SDS‐PAGE demonstrated that following PKGIα phosphorylation, MYPT1TR2 existed primarily as the non‐phosphorylated protein whereas MYPT1FL was diphosphorylated. These results demonstrate that MYPT1 structure is important for determining the sensitivity of smooth muscle to NO mediated vasodilatation.