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The increased detection and treatment of early stage breast cancer as well as ductal carcinoma in situ (DCIS) has not led to significant survival benefits. Therefore, the current standard treatment of DCIS is questionable. An informed evidence-based treatment strategy, and likely de-escalation from the current standards requires new prognostic models built from more comprehensive characterization with objective criteria. Parallel profiling of the molecular landscape and micro-environment in pure DCIS remains challenging due to histological heterogeneity and the inevitable reliance on small archived specimens. Leveraging recent methodological advances, we characterized the mutational, transcriptional, histological and microenvironmental landscape across multiple micro-dissected regions from 39 cases to generate a multi-modal breast precancer atlas. The histological architecture was associated with grade, adiposity, and intrinsic expression subtypes. Similar to previous findings, high-grade lesions had higher mutational burden, including TP53 mutations, while low-grade lesions had more frequent 16q losses and GATA3 mutations. Multi-region analysis revealed most somatic alterations, including whole genome duplication events, were clonal, but genetic divergence increased with distance between regions. In 7/12 evaluable cases, somatic mutations in putative driver genes affected a subset of regions only. This genetic heterogeneity often accompanied phenotypic heterogeneity and regions with low risk features (Normal-like, Luminal A) occurred earlier than those with high-risk features (Her2-like, Basal or necrosis) according to the phylogenetic analysis. The immune-environment was evaluated using multiplex immuno-histochemistry to measure relative stromal and epithelial densities of B lymphocyte (B-cell), T lymphocyte (T-cell) and regulatory T cells (T-reg) and identify 3 immune-states: Active, Suppressed and Excluded (lower epithelial density). All states included both DCIS and adjacent benign regions, and none associated with intrinsic subtypes. The Excluded state was enriched in high-grade DCIS and, compared to benign areas, more likely acquired in DCIS, showing transcriptional evidence of stronger immune-suppression and possible evasion. The breast pre-cancer atlas therefore reveals correlated levels of phenotypic and genotypic heterogeneity, including at sub-histological resolution. These uniquely integrated observations will help scope future studies, prioritize candidate markers for progression risk modelling and identify functional similarities in precursor lesions from other types of adenocarcinomas.

The effects of acute ingestion of nitrate on short-duration repeated sprint performance (RSP) are unclear. This study investigated the effect of acute ingestion of beetroot juice on a test of RSP in team sport athletes. Sixteen male team sport athletes undertook four trials using a 40 m maximum shuttle run test (MST), which incorporates 10 × 40 m shuttle sprints with 30 s between the start of each sprint. Two familiarisation trials, followed by nitrate-rich beetroot juice (BR; ~6 mmol nitrate) and nitrate-depleted beetroot juice (PLA; ~0.0034 mmol nitrate) trials were completed in a randomised, double-blind manner. Ingestion of beetroot juice 3 h prior to exercise elevated plasma nitrate concentrations ~6-fold in BR (BR, 413 ± 56 μM; PLA, 69 ± 30 μM; P 0.001). RSP, assessed by sprint performance decrement (S

This study considered contextual factors (i.e., times, places, peers) associated with cannabis use. A total of 153 participants answered an anonymous online survey, completed the Cannabis Use Disorders Identification Test – Revised (CUDIT-R), and indicated their numbers of regular smoking partners, and times and places cannabis was normally purchased. Recent cannabis smokers had higher CUDIT-R scores and purchased cannabis from more places more often. Multiple regression considered subscales of the CUDIT-R. Greater cannabis consumption was associated with more smoking partners and purchases of cannabis at more times and places. Cannabis dependence was associated with cannabis purchases from more places and times and reports that there were more people prepared to do them favors. Harmful use was associated with more purchases at more locations. Patterns of cannabis foraging were compared with foraging behaviors previously observed for caffeine, nicotine, and alcohol. The data could inform the development and use of social media and location-aware services seeking to target risky substance use.

We live and to do so we must breathe and eat, so are we a combination of what we eat and breathe? Here, we will consider this question, and the role in this respect of the AMP-activated protein kinase (AMPK). Emerging evidence suggests that AMPK facilitates central and peripheral reflexes that coordinate breathing and oxygen supply, and contributes to the central regulation of feeding and food choice. We propose, therefore, that oxygen supply to the body is aligned with not only the quantity we eat, but also nutrient-based diet selection, and that the cell-specific expression pattern of AMPK subunit isoforms is critical to appropriate system alignment in this respect. Currently available information on how oxygen supply may be aligned with feeding and food choice, or vice versa, through our motivation to breathe and select particular nutrients is sparse, fragmented and lacks any integrated understanding. By addressing this, we aim to provide the foundations for a clinical perspective that reveals untapped potential, by highlighting how aberrant cell-specific changes in the expression of AMPK subunit isoforms could give rise, in part, to known associations between metabolic disease, such as obesity and type 2 diabetes, sleep-disordered breathing, pulmonary hypertension and acute respiratory distress syndrome.

The effects of acute ingestion of caffeine on short-duration high-intensity performance are equivocal, while studies of novel modes of delivery and the efficacy of low doses of caffeine are warranted. The aims of the present study were to investigate the effect of acute ingestion of caffeinated chewing gum on repeated sprint performance (RSP) in team sport athletes, and whether habitual caffeine consumption alters the ergogenic effect, if any, on RSP. A total of 18 male team sport athletes undertook four RSP trials using a 40-m maximum shuttle run test, which incorporates 10 × 40-m sprints with 30 s between the start of each sprint. Each participant completed two familiarization sessions, followed by caffeine (CAF; caffeinated chewing gum; 200 mg caffeine) and placebo (PLA; noncaffeinated chewing gum) trials in a randomized, double-blind manner. RSP, assessed by sprint performance decrement (%), did not differ (p = .209; effect size = 0.16; N = 18) between CAF (5.00 ± 2.84%) and PLA (5.43 ± 2.68%). Secondary analysis revealed that low habitual caffeine consumers (n = 10) experienced an attenuation of sprint performance decrement during CAF relative to PLA (5.53 ± 3.12% vs. 6.53 ± 2.91%, respectively; p = .049; effect size =0.33); an effect not observed in moderate/high habitual caffeine consumers (>130 mg/day, n = 6; 3.98 ± 2.57% vs. 3.80 ± 1.79%, respectively; p = .684; effect size = 0.08). The data suggest that a low dose of caffeine in the form of caffeinated chewing gum attenuates the sprint performance decrement during RSP by team sport athletes with low, but not moderate-to-high, habitual consumption of caffeine.

Ductal carcinoma in situ (DCIS) comprises ~20% of all breast cancer diagnoses in the United States. Nearly all DCIS are treated to prevent progression to potentially lethal invasive cancer, yet it is estimated about half of DCIS progress within 10 years making the systematic treatment controversial. This highlights an urgent need for reliable molecular markers to stratify patients by risk. Small, degraded biopsies have made molecular profiling historically challenging; furthermore, a single biopsy can have several regions varying in nuclear grade, hormone receptor status, and histological architecture requiring laser-capture microdissection. Recent advances in whole-exome sequencing now enable mutational profiling from limited quantities ( Whole-exome sequencing was performed on 59 regions across 31 pure DCIS cases varying in nuclear grade (including 19% low-grade), and histological architecture (52% cribriform, 39% solid). Driver analysis revealed breast cancer-like driver alterations in 30/31 cases, and evidence for multiple putative drivers in 26/31, even in low-grade DCIS. The most frequently mutated genes across patients were common breast cancer driver genes: PIK3CA (37%), TP53 (22%), and GATA3 (11%). Phylogenetic trees constructed on multi-region biopsies revealed that regions of the same biopsy shared 12-336 alterations, and all pathogenic driver point mutations. Nevertheless, 1-22 region-specific alterations were found in each biopsy, some of which were likely driver CNA. We identified several histological associations with genetic alterations including high grade (p=2x10-4) and solid DCIS (p=3x10-4) associated with high copy number burden, and a non-significant increase in GATA3 (13% vs 0%) and PIK3CA (50% vs 28%) pathogenic mutations in cribriform vs solid architecture. Lastly, the most genetically diverse DCIS were HER2+/ER- (p=0.07) and those displaying necrosis (p=0.006). Overall, using novel methodology we performed detailed multi-region mutational profiling on difficult to sequence precancer lesions, revealing varying genetic diversity within a biopsy and novel associations between histology and underlying genetic landscape. We illustrate multiple driver genetic alterations and diversity present even in low-grade, pure DCIS lesions, providing key insight into early breast carcinogenesis and representing a critical step towards the development of prognostic markers of progression. Citation Format: Daniela Nachmanson, Mark F. Evans, Joseph Steward, Adam Officer, Huazhen Yao, Thomas J. O'Keefe, Farnaz Hasteh, Gary S. Stein, Kristen Jepsen, Donald L. Weaver, Gillian L. Hirst, Brian L. Sprague, Laura J. Esserman, Alexander Borowsky, Janet L. Stein, Olivier Harismendy. Mutational profiling of premalignant breast microbiopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2176.

Optimising training and performance through nutrition strategies is central to supporting elite sportspeople, much of which has focused on manipulating the relative intake of carbohydrate and fat and their contributions as fuels for energy provision. The ketone bodies, namely acetoacetate, acetone and β-hydroxybutyrate (βHB), are produced in the liver during conditions of reduced carbohydrate availability and serve as an alternative fuel source for peripheral tissues including brain, heart and skeletal muscle. Ketone bodies are oxidised as a fuel source during exercise, are markedly elevated during the post-exercise recovery period, and the ability to utilise ketone bodies is higher in exercise-trained skeletal muscle. The metabolic actions of ketone bodies can alter fuel selection through attenuating glucose utilisation in peripheral tissues, anti-lipolytic effects on adipose tissue, and attenuation of proteolysis in skeletal muscle. Moreover, ketone bodies can act as signalling metabolites, with βHB acting as an inhibitor of histone deacetylases, an important regulator of the adaptive response to exercise in skeletal muscle. Recent development of ketone esters facilitates acute ingestion of βHB that results in nutritional ketosis without necessitating restrictive dietary practices. Initial reports suggest this strategy alters the metabolic response to exercise and improves exercise performance, while other lines of evidence suggest roles in recovery from exercise. The present review focuses on the physiology of ketone bodies during and after exercise and in response to training, with specific interest in exploring the physiological basis for exogenous ketone supplementation and potential benefits for performance and recovery in athletes.

BACKGROUND: Breast tumor stratification by recurrence-risk is a critical for deciding patient treatment. Here an approach combining cancer pathways microarray data complemented by RNA in situ hybridization (ISH) was investigated as a means for recurrence marker discovery and visualization in pathology specimens. METHODS: LncRNA and mRNA expressions in breast carcinomas with low (n=8) versus intermediate/high (n=10) recurrence-scores as estimated by 21-gene assay and pathology review were compared by microarray assay. Tissue microarrays were prepared from breast carcinomas (n=20) and ductal carcinoma in situ (DCIS) specimens (n=84 patients) with known outcomes. Thirteen RNA ISH assays were performed: lncRNAs (BBC3–1, FER3, RAD21-AS1, ZEB1–2) and mRNAs (GLO1, GLTSCR2, TGFB1, TLR2) [implicated by the microarray data]; MKI67; a pooled panel of recurrence-associated proliferation markers (BIRC5, Cyclin B1, MKI67, MYBL2, STK15); a pooled panel of non-proliferation recurrence-associated markers (CEACAM5, HTF9C, NDRG1, TP53, SLC7A5); and lncRNAs H19 and HOTAIR. RESULTS: Seven lncRNAs and 10 mRNAs showed significantly (P

Background and objectives Maladaptive decision-making strategies could contribute to cannabis-related problems, as some individuals may neither select safe patterns of cannabis use, nor seek treatment. Methods To explore decision-making styles and their relationship to cannabis-related harm, 153 respondents completed the Cannabis Use Disorders Identification Test-Revised (CUDIT-R), the Melbourne Decision Making Questionnaire (MDMQ), and answered questions about their willingness to seek online: (1) further information or (2) treatment for cannabis-related issues. Results Multiple regression considered relationships between problematic cannabis use, decision-making style, and cannabis use within the past month. Subscales of the CUDIT-R revealed that: (1) hazardous use was associated with higher hypervigilance and higher decisional self-esteem; (2) dependence symptoms were associated with lower vigilance and higher procrastination; and (3) harmful use was associated with higher procrastination. People with symptoms of CUD were less likely to seek further help or support online if prone to procrastination or buckpasssing. Discussion and conclusions Decisional style influenced cannabis use and symptoms. Those people with cannabis-related problems that did not seek online assistance were defensively avoidant. Scientific significance Procrastination is a feature of problematic cannabis use. Online offers of assistance may be ignored by defensively avoidant CUD clients. (Am J Addict 2019;00:1-7).

Introduction: Ductal carcinoma in situ (DCIS) of the breast is a premalignant lesion representing a spectrum of biology and risk. While many patients with DCIS undergo surgical resection with no survival benefit given the indolent nature of their disease, others do possess biologically aggressive DCIS that has the potential to evolve into invasive cancer if left untreated. Yet even among those patients with biologically aggressive DCIS, their risk of dying from metastatic breast cancer is only 3.3% compared to 30-40% among patients with biologically aggressive invasive cancer.1 The DEFENSE study, supported with funding from the NCI Molecular Characterization of Screen-Detected Lesions (MCL) consortium, was designed to identify molecular, immunological, and stromal-related factors that allow DCIS to develop high-risk features without ever becoming invasive breast cancer. Methods: The overall objective of the DEFENSE study is to compare 100 patients with invasive high-risk breast cancer enrolled on the I-SPY2 trial matched based upon age and tumor molecular profile (Mammaprint) to 100 patients with high-risk DCIS, defined as having at least two of the following characteristics: large (>5cm), high-grade, hormone receptor-negative status and/or HER2-positive status. Tumors obtained from each of these patients are divided into 22 sequential sections with regions of pathologic interest identified prior to undergoing whole exome DNA sequencing, SMART-3SEQ RNA sequencing, multiplex immunofluoresence (mIF) for immune cell infiltrates, and stromal profiling by IHC. Each profiling modality is performed by a different institution included in the MCL consortium (UCSF, UCD, UCSD, Stanford, and UVM). In order to evaluate the logistic and financial feasibility of our multi-institution protocol prior to expanding to our proposed full-scale study, we are conducting a pilot study of 11 high-risk DCIS specimens. Results: The 11 pilot specimens have been successfully sent and received by each institution. H&E images from the sectioned specimens have been uploaded to the NASA Jet Propulsion Lab (JPL) cloud-based platform and shared for pathologic annotation among institutions. mIF of the pilot cohort has been completed, but interpretation of associations between tumor subtype and immune cell infiltrate is limited by the small sample size. Whole exome DNA sequencing, SMART-3SEQ RNA sequencing, and stromal profiling of the pilot cohort are each ongoing. We anticipate being able to provide full results from each profiling modality performed from the pilot cohort, as well as results from an expanded cohort of 40 additional specimens, by December 2020. Conclusions: We have successfully shown that a multi-institution collaboration can effectively share pathologic data and conduct data analyses using a variety of tumor profiling modalities. We anticipate that data from our expanded cohort will allow us to differentiate the underlying biology of high-risk DCIS from invasive breast cancer, identifying mechanistic opportunities for future intervention. References:1 Narod SA et al (2015). Breast Cancer Mortality After a Diagnosis of Ductal Carcinoma in Situ. JAMAOncol. 1(7): 888-96. Citation Format: Alexa Glencer, Olivier Harismendy, Alexander Borowsky, Hidetoshi Mori, Michael Campbell, Gillian Hirst, Janet Stein, Mark Evans, Prachi Ghule, Robert West, Laura J. Esserman. Elucidating the biology of high-risk ductal carcinoma in situ (DCIS) through genomics and immunohistochemical profiling of the tumor microenvironment: The DEFENSE study [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-14.

Background: With the implementation of widespread breast cancer screening, the diagnosis of Ductal Carcinoma In Situ (DCIS) has increased 5 fold. Most cases are treated with combinations of surgery, radiation and endocrine therapy, reducing the risk of second events, including ipsilateral invasive cancer. Without standard markers to confidently identify the most indolent lesions, a subset of cases are likely over-treated. The mutational landscapes of DCIS and invasive ductal carcinoma (IDC) are similar and not sufficient to identify higher risk lesions with recent studies suggesting that clonal selection plays a limited role in progression. Histological analysis highlighted the role of the extracellular matrix and immune-surveillance to maintain duct integrity and limit progression. Due to the small size, limited availability and quality of research specimens, few biomarker studies investigated pure DCIS with adequate follow-up or genome-wide methods, let alone integrated more than one type of biomarker. Unlike breast cancer, there is no comprehensive, systematic, multi-modal atlas of DCIS, limiting the ability to test broad and novel hypotheses and characterize processes maintaining breast tissue homeostasis. Methods: Through sequential sectioning of pure DCIS archived specimens, a total of 70 histological regions from 40 cases were annotated and profiled using up to 3 platforms: multiplex immuno-histochemistry (mIHC), RNA-seq, and whole-exome sequencing. Stromal and epithelial spatial distribution of immune cells and states were quantified using mIHC. The epithelial compartments were microdissected and profiled using genome-wide gene expression, DNA mutations and copy number alterations. Results: Epithelial regions were classified according to expression subtypes consistent with histological markers, highlighting associations with the lesion architecture and grade. Compared to solid pattern, cribriform pattern DCIS has induced EMT processes and repressed proliferation processes, a trend reminiscent of low-recurrence-risk expression signatures measured in IDC. The DNA copy number burden increased with grade and the mutational burden was the highest in solid DCIS. Both were higher in Her2+ cases. The clustering of mutations at chromosome 17p - attributed to the APOBEC-driven Kataegis phenomenon - was observed in a subset of regions, suggesting this event can occur in pre-invasive lesions. All DCIS had somatic alterations of at least one known driver gene with some associated with grade (TP53) or expression subtype (ERBB2). Multi-region profiling available in a subset of samples revealed genetic heterogeneity of likely-driver events between proximal regions of similar histological characteristics. The density and proliferative states of selected immune cells - including T-cells, B-cells and Macrophages - highlights the diversity of the tumor immune environment with the highest densities observed in Her2+ ducts and stroma, minimal ductal infiltration in other lesions, fewer dividing B- and T-cells around the more proliferative areas and a small number of regions depleted from any adaptive immune cells. Conclusion: This first multi-modal profiling of pure DCIS reveals an unsuspected diversity of molecular and microenvironmental states and presents their association with progression risk factors. The observations support the need for stronger integration of molecular and clinicopathology features, especially at sub-histological levels, to ensure the findings can be interpreted in the correct clinical and phenotypic context. The compatibility of the approach with archived specimens supports the expansion to larger retrospective DCIS collections with outcomes. Citation Format: Olivier Harismendy, Daniela Nachmanson, Mark F. Evans, Hidetoshi Mori, Adam Officer, Christina Yau, Joseph Steward, Huazhen Yao, Thomas O'Keefe, Farnaz Hasteh, Gary S. Stein, Kristen Jepsen, Michael Campbell, Donald L. Weaver, Gillian L. Hirst, Brian L. Sprague, Laura J. Esserman, Jonathan A. Gordon, Alexander Borowsky, Janet L. Stein. The breast pre-cancer atlas illustrates the molecular and micro-environmental diversity of ductal carcinoma in situ [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr GS2-01.

Coxsackieviruses B (CV B) are known as the most common viral cause of human heart infections. Cardiac inflammations contribute to sudden unexpected death (SUD) significantly. The diagnosis remains difficult with the traditional diagnostic tests and must be substantially improved. This has prompted health professionals to seek new diagnostic procedures which may provide important clues regarding underlying etiology. The present study is based on patients with infectious heart diseases and SUD victims with no relevant pathologies. They were investigated for possible CV-B infection. Patients with coronary artery diseases and unnatural road and domestic accident victims served as controls. The samples were studied for CV-B applying PCR. Histopathology for inflammatory markers, immunohistochemistry (IHC) for immune inflammatory cells and the enteroviral VP1-capsid protein were performed. Overall, 102 patients and 87 SUD victims were studied. As controls, 100 patients and 54 SUD unnatural accident victims were enrolled. CV-B were detected in 28 patients and 15 SUD victims. The control group samples were completely virus negative. Compared to controls, IHC revealed a significant presence of T and B lymphocytes within the myocardium. Furthermore, enteroviral VP1-capsid protein were detected from samples by IHC. Applying a comprehensive combination of methods, our results demonstrate the involvement of CV-B in cases of heart infection suggesting they play a significant role in SUD. Our results emphasize the importance of opting for a combination of methods.

Sudden infant death (SID) is an unresolved problem of high relevance and previous studies have indicated a role of viral heart infections. The diagnosis remains difficult in clinical practice using routine diagnostic tests and must be substantially improved. A prospective study based on post-mortem samples from SID victims whose heart disease was not clinically recognized was conducted for 4 years in a Tunisian University Hospital. Pediatric cases of unnatural death served as controls. Both SID victims and controls were investigated for possible coxsackievirus-B (CV-B) infection in heart tissue. During the study period, 39 cases with a male predominance (77%) were reported. There was no positive family history of coronary artery disease among the victims. In 35 cases (90%), low birth weight and/or critical development period were reported. All SID victims had complained of mild fever and insomnia for a few days preceding death, which required infectious laboratory investigations marked with an elevated white blood cell count (WBC) and C-reactive protein (CRP). The cardiac biomarkers were also elevated. The histopathological investigations of the heart tissue samples revealed signs of myocardial and pericardial inflammation. Enterovirus was detected by immunohistochemistry (IHC) and PCR from myocardial samples from 6 cases (15.3%) having myocarditis and 3 cases (7.7%) having perimyocarditis. The current study is of great interest and is aimed at urging health professionals to adopt systematically long intensive heart care in infants with underlying vulnerability as well as new diagnostic approaches including histopathology complemented with IHC and molecular pathology.

Helicobacter pylori status influences the prognosis and management of gastric extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), so accurate determination of H pylori status is of clinical importance. The low rate of histologic H pylori positivity among gastric MALT lymphoma cases at our institution prompted investigation for possible causes. A case series of 24 patients as having gastric MALT lymphoma (with no diffuse large B-cell component) in a tertiary care setting between 1997 and 2010 was identified, and clinical records were reviewed. Immunohistochemical staining for H pylori and BCL10 was performed. This study received institutional review board approval (protocol number M13-033). Thirty-nine percent of cases (9/23) were H pylori positive by histology, and 4 additional patients had positive serologic results; overall, 57% of cases (13/23) were positive for H pylori. Treatment with antisecretory medications was associated with a lower likelihood of histologic positivity (13% among treated patients vs 75% among untreated; P = .04). Nuclear localization of BCL10 was seen in 2 cases and was not associated with H pylori status. Antisecretory medications decrease the likelihood of histologic detection of H pylori in gastric MALT lymphoma cases. Incorporation of results of serologic or other testing is needed to ensure correct classification with respect to H pylori status.

Introduction: Ductal carcinoma in situ (DCIS) of the breast is a premalignant lesion representing a spectrum of biology and risk. While many patients with DCIS undergo surgical resection with no survival benefit given the indolent nature of their disease, others do possess biologically aggressive DCIS that has the potential to evolve into invasive cancer if left untreated. Yet even among those patients with biologically aggressive DCIS, their risk of dying from metastatic breast cancer is only 3.3% compared to 30-40% among patients with biologically aggressive invasive cancer.1 We are interested in identifying molecular and tumor immune microenvironment factors that allow DCIS to develop high-risk features without ever becoming invasive breast cancer. Methods: We are conducting a retrospective pilot study (DEFENSE) of 10 patients with invasive high-risk breast cancer enrolled on the I-SPY2 trial matched based upon age and tumor molecular profile to 10 patients with high-risk DCIS, defined as having at least two of the following characteristics: large (>5cm), high-grade, hormone receptor-negative status and/or HER2-positive status. Tumors obtained from each of these patients will be divided into 22 sequential sections with regions of pathologic interest identified prior to undergoing whole exome DNA sequencing, SMART-3SEQ RNA sequencing, multiplex immunohistochemistry (mIHC) using three immune panels, and stromal profiling. Each profiling modality will be performed by a different institution included in the NIH Molecular Characterization of Screen-Detected Lesions (MCL) consortium. This pilot study will inform our decision to expand our study to a full-scale review of 200 patients (100 DCIS, 100 invasive cancer) among our institutions. Results: We have thus far demonstrated the feasibility of our pilot study with 10 blocks in total sent to and received by each institution. Whole exome DNA sequencing, SMART-3SEQ, mIHC, and stromal profiling work has begun and will be available for presentation, and we plan to make pathology, clinical and genomic data available through the MCL's collaborative partnership with the NASA Jet Propulsion Lab (JPL) cloud-based platform. Conclusions: We have successfully shown that a multi-institution collaborative can effectively share pathologic data and conduct data analyses using a variety of tumor profiling modalities. We anticipate that our data will allow us to differentiate the underlying biology of high-risk DCIS from invasive breast cancer, identifying mechanistic opportunities for future intervention. References: 1 Narod SA et al (2015). Breast Cancer Mortality After a Diagnosis of Ductal Carcinoma in Situ. JAMA Oncol. 1(7): 888-96. Citation Format: Alexa Glencer, Olivier Harismendy, Alexander Borowsky, Gillian L. Hirst, Janet Stein, Mark Evans, Donald Weaver, Robert West, Case Brabham, Hidetoshi Mori, Laura J. Esserman. Understanding the biology of high-risk ductal carcinoma in situ (DCIS) through genomics and the tumor immune microenvironment: The DEFENSE study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5440.

Two-pore segment channel 2 (TPC2) is a ubiquitously expressed, lysosomally targeted ion channel that aids in terminating autophagy and is inhibited upon its association with mechanistic target of rapamycin (mTOR). It is controversial whether TPC2 mediates lysosomal Ca2+ release or selectively conducts Na+ and whether the binding of nicotinic acid adenine dinucleotide phosphate (NAADP) or phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] is required for the activity of this ion channel. We show that TPC2 is required for intracellular Ca2+ signaling in response to NAADP or to mTOR inhibition by rapamycin. In pulmonary arterial myocytes, rapamycin and NAADP evoked global Ca2+ transients that were blocked by depletion of lysosomal Ca2+ stores. Preincubation of cells with high concentrations of rapamycin resulted in desensitization and blocked NAADP-evoked Ca2+ signals. Moreover, rapamycin and NAADP did not evoke discernable Ca2+ transients in myocytes derived from Tpcn2 knockout mice, which showed normal responses to other Ca2+-mobilizing signals. In HEK293 cells stably overexpressing human TPC2, shRNA-mediated knockdown of mTOR blocked rapamycin- and NAADP-evoked Ca2+ signals. Confocal imaging of a genetically encoded Ca2+ indicator fused to TPC2 demonstrated that rapamycin-evoked Ca2+ signals localized to lysosomes and were in close proximity to TPC2. Therefore, inactivation of mTOR may activate TPC2 and consequently lysosomal Ca2+ release.

Aim Long non-coding RNAs (lncRNAs) are potential biomarkers for breast cancer risk stratification. LncRNA expression has been investigated primarily by RNA sequencing, quantitative reverse transcription PCR or microarray techniques. In this study, six breast cancer-implicated lncRNAs were investigated by chromogenic in situ hybridisation (CISH). Methods Invasive breast carcinoma (IBC), ductal carcinoma in situ (DCIS) and normal adjacent (NA) breast tissues from 52 patients were screened by CISH. Staining was graded by modified Allred scoring. Results HOTAIR , H19 and KCNQ1OT1 had significantly higher expression levels in IBC and DCIS than NA (p HOTAIR and H19 were expressed more strongly in IBC than in DCIS tissues (p HOTAIR and KCNQ101T were expressed in tumour cells; H19 and MEG3 were expressed in stromal microenvironment cells; MALAT1 was expressed in all cells strongly and ZFAS1 was negative or weakly expressed in all specimens. Conclusion These data corroborate the involvement of three lncRNAs ( HOTAIR , H19 and KCNQ1OT1 ) in breast tumourigenesis and support lncRNA CISH as a potential clinical assay. Importantly, CISH allows identification of the tissue compartment expressing lncRNA.

Background: The endolysosomal TPC2 ion channel interacts with mTOR to regulate cellular energy utilization. Results: Mice lacking TPC2 display muscle atrophy phenotype with reduced muscle endurance, altered autophagy, and lysosomal enzymatic activities. Conclusion: TPC2 regulates autophagic signaling in skeletal muscle. Significance: TPC2 impacts protein turnover via regulating autophagy signaling in the process of tissue homeostasis and aging., Postnatal skeletal muscle mass is regulated by the balance between anabolic protein synthesis and catabolic protein degradation, and muscle atrophy occurs when protein homeostasis is disrupted. Autophagy has emerged as critical in clearing dysfunctional organelles and thus in regulating protein turnover. Here we show that endolysosomal two-pore channel subtype 2 (TPC2) contributes to autophagy signaling and protein homeostasis in skeletal muscle. Muscles derived from Tpcn2−/− mice exhibit an atrophic phenotype with exacerbated autophagy under starvation. Compared with wild types, animals lacking TPC2 demonstrated an enhanced autophagy flux characterized by increased accumulation of autophagosomes upon combined stress induction by starvation and colchicine treatment. In addition, deletion of TPC2 in muscle caused aberrant lysosomal pH homeostasis and reduced lysosomal protease activity. Association between mammalian target of rapamycin and TPC2 was detected in skeletal muscle, allowing for appropriate adjustments to cellular metabolic states and subsequent execution of autophagy. TPC2 therefore impacts mammalian target of rapamycin reactivation during the process of autophagy and contributes to maintenance of muscle homeostasis.

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link Cultivation of parasites is not a routine identification technique for human parasitic diseases but provides invaluable help in patient care, research and epidemiology, particularly in the diagnosis, management, control and prevention of these diseases. Moreover, culture facilitates students’ learning and understanding of the complex morphology, physiology, life cycle and host-parasite relationships of parasites. However, cultivation of parasites can be fastidious and requires specific techniques, resources and skills that may not be available in many biomedical laboratories. Thus, among other factors, cultivation of specific forms of some parasites or species requires in vitro culture of cells to be successful. For example, the emerging human protozoan pathogen Enterocytozoon bieneusi, the most frequently diagnosed microsporidial species in humans, has been successfully cultured only in short term cultures (6 months) and requires animal cells. An innovative teaching group of academics from De Montfort University (DMU), University of San Pablo CEU (USP-CEU, Spain) and University Miguel Hernández (Spain), in conjunction with clinicians and practising Biomedical Scientists from the UK National Health Service are developing on-line resources for teaching and learning the different steps and phases for cultivating mammalian cells (including human cells) and parasites in a biomedical laboratory. These resources or units will be a key part of the “virtual laboratory” section of the novel package DMU e-Parasitology, which will be publicly accessible through the DMU website later in 2018 (http://parasitology.dmu.ac.uk). Cell culture related units are being developed in close collaboration with academics that have built a real cell and parasite culture laboratory. Specific units that describe the basic equipment and resources to cultivate cells and these organisms in a standard biosafety level 2 (BSL-2) medical laboratory (e.g. biological safety cabinet class II, incubators, sterilization, cryogenic storage and inverted microscope) and its workflow are being developed. These units will be highly interactive and engaging and will present short videos of a technician/scientist working in real conditions with this equipment to enhance students’ understanding and learning. Short formative assessments will be introduced to facilitate the self-evaluation of users’ learning. Finally, photographs and short videos of different human parasites in different media and cultures are being produced and introduced in the “virtual microscope” section of the DMU e-Parasitology. Users will be able to zoom in and out and move around of each sample simulating real parasite cultures. Moreover, students will be able to gain a complete understanding of the different structures and characteristics of major human parasites for clinical diagnostic purposes. We consider that the novel teaching and learning resource DMU e-Parasitology will help students and academics around the world in the teaching and study of human parasitology, making this relevant subject more interesting. Academics will be able to enrich their strategies for teaching and make their sessions more appetising and stimulating. Finally, DMU e-Parasitology could help educators in course development and could be used for training purposes by future technicians that will work in a cell or parasite culture laboratory.

Canine obesity is now the number one health concern in dogs worldwide. Regular physical activity can improve health, and owners are advised to exercise their dogs on a regular basis. However, limited information exists about associations between overweight status of dogs and walking activity. An online survey was conducted between June and August in 2014, coinciding with the broadcast of a national UK television programme, exploring dog behaviour. Information gathered included signalment, overweight status, and owner-reported information on duration and frequency of dog walking. The University of Liverpool Ethics Committee approved the project, and owners consented to data use. Simple and multiple logistic regression analyses were used to determine associations between overweight status and dog walking activity. Data were available from 11 154 adult dogs, and 1801 (16·1 %) of these were reported as overweight by their owners. Dogs reported to be overweight dogs were more likely to be neutered (P P P P

Regular physical activity is an important means of promoting health, both in people and their pets. Walking is the most common method used for dogs, but there is a lack of clarity on how much daily activity different breeds of dog require. Data from an online survey of UK dog owners were collected between June and August in 2014. The University of Liverpool Ethics Committee approved the project, and owners consented to data use. The initial dataset (17 028 dogs) was first cleaned to remove erroneous data, and then edited to remove mixed breed dogs, leaving a total of 12 314 dogs from known pedigree breeds. Other information collected included sex, age, neuter status, breed, and amount and frequency of exercise. Exercise frequency and duration were estimated across different breeds, and compared with Kennel Club recommendations, usingχ2tests and binary logistic regression. The online survey data indicated differences amongst breeds in the amount of walking reported (P P P

In this chapter we examine the importance of cytoplasmic nanojunctions—nanometer scale appositions between organellar membranes including the molecular transporters therein—to the cell signaling machinery, with specific reference to Ca2+ transport and signaling in vascular smooth muscle and endothelial cells. More specifically, we will consider the extent to which quantitative modeling may aid in the development of our understanding of these processes. Testament to the requirement for such approaches lies in the fact that recent studies have provided evermore convincing evidence in support of the view that cytoplasmic nanospaces may be as significant to the process of Ca2+ signaling as the Ca2+ transporters, release channels, and Ca2+-storing organelles themselves. Moreover, the disruption and/or dysfunction of cytoplasmic nanospaces may be central to the origin of certain diseases. By way of introduction, we provide a historical perspective on the identification of smooth muscle cell plasma membrane (PM)-sarcoplasmic reticulum (SR) nanospaces and the early evidence in support of their role in the generation of asynchronous Ca2+ waves. We then summarize how stochastic modeling approaches can aid and guide the development of our understanding of two basic functional steps leading to healthy smooth muscle cell contraction. We furthermore outline how more sophisticated and realistic quantitative stochastic modeling may be employed not only to test working hypotheses, but also to lead in their development in a manner that informs further experimental investigation. Finally, we consider more recently defined nanospaces such as the lysosome-SR junction, by way of demonstrating the importance of quantitative stochastic modeling to our understanding of signaling mechanisms.

Human papillomavirus (HPV) infection, most commonly genotype 16 of the alpha-9 family, is implicated in the etiology of a subset of oropharyngeal squamous cell carcinomas (OPSC) worldwide. Data are scarce regarding OPSC in South Africans, and three prior studies suggest no significant etiologic role for HPV. We aimed to investigate for evidence of HPV etiology in OPSCs from black South Africans by polymerase chain reaction (PCR) methodologies with determination of HPV subtype by sequencing, in situ hybridization (ISH), and p16INK4a immunohistochemistry (IHC), as a surrogate marker for an HPV-driven tumor. It was hypothesized that HPV-driven tumors would be positive by PCR plus IHC and/or ISH whereas OPSCs with HPV background infections (HPV-passenger) would be positive by PCR alone. Formalin-fixed, paraffin embedded tissues from 51 OPSCs collected between 2005 and 2010 from 41 patients were analyzed for HPV by GP5?6? PCR (targeting the HPV L1 region), pU-1M/pU- 2R PCR (targeting the HPV E6/E7 region) and HPV-31 specific PCR (targeting the E5 region), chromogenic ISH, and p16INK4a IHC. All cases positive by PCR were subject to sequencing to determine HPV genotype. The patient mean age was 58.0 years and 88 % were male. Of the 51 evaluable tumors, 48 (94.1 %) were positive for HPV DNA by PCR: 25 (49.1 %) met criteria for an HPV-driven tumor, 23 (45.1 %) for HPV-passenger, and 3 (5.9 %) were HPV unrelated. Sequencing of the PCR-positive cases revealed the following genotypes: combined HPV-16 and 31 (41.7 %), HPV-31 (25.0 %), HPV-16 (22.9 %), combined HPV-16 and 18 (6.3 %), and a single case each of HPV 18 and HPV 33. Studies via ISH were negative in all cases. In accordance with worldwide trends but contrary to prior South African data, HPV likely plays an etiologic role in a significant subset (at least 49.1 %) of OPSC in black South Africans. We found that the alpha-9 HPV family, particularly HPV-16 and 31 either in combination or separately, to predominate in our sample tumors. The use of multiple PCR primers increased sensitivity of viral detection, and a HPV-31 specific primer confirmed the presence of this genotype in many samples. Further studies including HPV E6/E7 mRNA assays are needed to better elucidate the pathogenic role of HPV in black South African OPSCs.

This review focuses on how smooth muscle sarcoplasmic reticulum (SR), the major releasable Ca2+ store in these cells, performs its many functions by communicating with the plasma membrane (PM) and other organelles across cytoplasmic nanospaces, defined by membrane–membrane junctions less than 50 nm across. In spite of accumulating evidence in favour of the view that cytoplasmic nanospaces are a prerequisite for effective control of diverse cellular functions, our current understanding of how smooth muscle cells accomplish site- and function-specific Ca2+ signalling remains in its infancy. We first present evidence in support of the view that effective Ca2+ signalling depends on the restricted diffusion of Ca2+ within cytoplasmic nanospaces. We then develop an evidence-based model of the smooth muscle SR – the ‘pan-junctional SR’ model – that incorporates a network of tubules and quilts that are capable of auto-regulating their Ca2+ content and determining junctional [Ca2+]i through loading and unloading at membrane–membrane nanojunctions. Thereby, we provide a novel working hypothesis in order to inform future investigation into the control of a variety of cellular functions by local Ca2+ signals at junctional nanospaces, from contraction and energy metabolism to nuclear transcription. Based on the current literature, we discuss the molecular mechanisms whereby the SR mediates these multiple functions through the interaction of ion channels and pumps embedded in apposing membranes within inter-organellar junctions. We finally highlight the fact that although most current hypotheses are qualitatively supported by experimental data, solid quantitative simulations are seriously lacking. Considering that at physiological concentrations the number of calcium ions in a typical junctional nanospace between the PM and SR is of the order of 1, ion concentration variability plays a major role as the currency of information transfer and stochastic quantitative modelling will be required to both test and develop working hypotheses.

In this review, we describe the background and implications of our recent discovery that two-pore channels (TPCs) comprise a novel class of calcium release channels gated by the intracellular messenger nicotinic acid adenine dinucleotide phosphate (NAADP). Their localisation to the endolysosomal system highlights a new function for these organelles as targets for NAADP-mediated Ca2+ mobilisation. In addition, we describe how TPCs may also trigger further Ca2+ release by coupling to the endoplasmic reticular stores through activation of IP3 receptors and ryanodine receptors.

Previous studies of arterial smooth muscle have shown that inositol 1,4,5-trisphosphate (IP 3 ) and cyclic ADP-ribose mobilize Ca 2+ from the sarcoplasmic reticulum. In contrast, little is known about Ca 2+ mobilization by nicotinic acid adenine dinucleotide phosphate, a pyridine nucleotide derived from β-NADP + . We show here that intracellular dialysis of nicotinic acid adenine dinucleotide phosphate (NAADP) induces spatially restricted “bursts” of Ca 2+ release that initiate a global Ca 2+ wave and contraction in pulmonary artery smooth muscle cells. Depletion of sarcoplasmic reticulum Ca 2+ stores with thapsigargin and inhibition of ryanodine receptors with ryanodine, respectively, block the global Ca 2+ waves by NAADP. Under these conditions, however, localized Ca 2+ bursts are still observed. In contrast, xestospongin C, an IP 3 receptor antagonist, had no effect on Ca 2+ signals by NAADP. We propose that NAADP mobilizes Ca 2+ via a 2-pool mechanism, and that initial Ca 2+ bursts are amplified by subsequent sarcoplasmic reticulum Ca 2+ release via ryanodine receptors but not via IP 3 receptors.

Ca2+ mobilization from intracellular stores represents an important cell signalling process that is regulated, in mammalian cells, by inositol-1,4,5-trisphosphate (InsP3), cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate (NAADP). InsP3 and cyclic ADP ribose cause the release of Ca2+ from sarcoplasmic/endoplasmic reticulum stores by the activation of InsP3 and ryanodine receptors (InsP3Rs and RyRs). In contrast, the nature of the intracellular stores targeted by NAADP and the molecular identity of the NAADP receptors remain controversial, although evidence indicates that NAADP mobilizes Ca2+ from lysosome-related acidic compartments. Here we show that two-pore channels (TPCs) comprise a family of NAADP receptors, with human TPC1 (also known as TPCN1) and chicken TPC3 (TPCN3) being expressed on endosomal membranes, and human TPC2 (TPCN2) on lysosomal membranes when expressed in HEK293 cells. Membranes enriched with TPC2 show high affinity NAADP binding, and TPC2 underpins NAADP-induced Ca2+ release from lysosome-related stores that is subsequently amplified by Ca2+-induced Ca2+ release by InsP3Rs. Responses to NAADP were abolished by disrupting the lysosomal proton gradient and by ablating TPC2 expression, but were only attenuated by depleting endoplasmic reticulum Ca2+ stores or by blocking InsP3Rs. Thus, TPCs form NAADP receptors that release Ca2+ from acidic organelles, which can trigger further Ca2+ signals via sarcoplasmic/endoplasmic reticulum. TPCs therefore provide new insights into the regulation and organization of Ca2+ signals in animal cells, and will advance our understanding of the physiological role of NAADP.

BackgroundSarcomatoid renal cell carcinomas (SRCC) are composed of two cell populations, a sarcomatous component (SC) and a carcinomatous component (CC). SRCC are particularly aggressive and often present at an advanced stage at diagnosis. Epithelial–mesenchymal transition (EMT) has been proposed as a mechanism for the development of SC from CC.Aims and methodsE- to N-cadherin switching, localisation of β-catenin, and expression of Snail and secreted protein acidic and rich in cysteine (SPARC) (markers of EMT) were studied to determine whether SRCC is an example of EMT. Expression of these markers was analysed by immunohistochemistry on 21 cases of SRCC that had both SC and CC and scored according to intensity and extent.ResultsE-cadherin expression was decreased in SC (Wilcoxon signed-rank test, p=0.0004) while N-cadherin expression was high in both components (p=0.46). Membranous β-catenin expression was decreased in SC (pConclusionsE- to N-cadherin switching, dissociation of β-catenin from the membrane, and increased expression of Snail and SPARC in SC indicate that SRCC is an example of EMT. High expression of N-cadherin and Snail in CC suggest early involvement in initiating EMT. Once EMT is established, loss of E-cadherin, release of β-catenin into the cytoplasm, and expression of SPARC correspond with mesenchymal phenotypic expression.

Human papillomavirus (HPV) positive tonsillar squamous cell carcinoma (TSCC) is associated with a favorable clinical outcome. However, the HPV detected in a given tumor may be causal (driver HPV) or an incidental bystander (passenger HPV). There is a need to discriminate these forms of HPV in TSCCs to understand their impact on HPV as a biomarker for use in TSCC patient management. This study has compared the polymerase chain reaction (PCR), chromogenic in situ hybridization (CISH), and p16(INK4a) immunohistochemistry in the assessment of HPV status in TSCC. Archival specimens of TSCC from thirty patients were investigated. HPV was detected by PCR in 25/30 (83.3%) tumors; HPV16 (70.0%) and HPV52 (6.7%) were the most common types. HPV was corroborated by CISH in 22/25 (88.0%) specimens; integrated HPV was implicated by the presence of punctate signals in each of these cases. p16(INK4a) staining was found in 20/22 (90.9%) HPV PCR positive samples; two PCR/CISH HPV positive cases were p16(INK4a) negative and two HPV negative samples were p16(INK4a) positive. These data suggest that a minority of HPV positive TSCCs are positive for passenger HPV and that two or more assays may be required for diagnosing driver HPV status. Further studies are required to exam whether oropharyngeal tumors positive for passenger HPV have a less favorable prognosis than tumors that are driver HPV positive. The clinical significance of TSCCs that test HPV negative/p16(INK4a) positive, PCR and CISH HPV positive/p16 (INK4a) negative, or PCR HPV positive/p16 (INK4a) and CISH negative, also requires further investigation.

Virus-induced myocarditis is a common disease even in infants and young adults, but the diagnosis can be difficult according to the Dallas-criteria, which have been criticized as being too unreliable. The diagnosis has been substantially improved due to immunohistochemistry (IHC) for the detection of the VP1-capsid-protein of enterovirus as well as reversetranscriptase-polymerase chain reaction assays (RT-PCR) for viral genome detection. We report an unusual case of myocarditis in a young adult athlete whose heart disease was not clinically recognized and, thus, caused his sudden unexpected death (SUD). Histopathological investigations of heart tissue samples revealed signs of myocarditis. IHC was used to detect the VP1-capsid-protein of enterovirus. RT-PCR assays were used to detect enterovirus RNA. Enterovirus myocarditis was determined as a cause of death.

Hypoxic chemotransduction in the carotid body requires release of excitatory transmitters from type I cells that activate afferent sensory neurones. Transmitter release is dependent on voltage-gated Ca 2+ entry which is evoked by membrane depolarization. This excitatory response to hypoxia is initiated by inhibition of specific O 2 sensitive K + channels, of which several types have been reported. Here, we discuss mechanisms which have been put forward to account for hypoxic inhibition of type I cell K + channels. Whilst evidence indicates that one O 2 sensitive K + channel, BK Ca , may be regulated by gasotransmitters (CO and H 2 S) in an O 2 -dependent manner, other studies now indicate that activation of AMP-activated protein kinase (AMPK) accounts for inhibition of both BK Ca and ‘leak’ O 2 sensitive K + channels, and perhaps also other O 2 sensitive K + channels reported in different species. We propose that type I cell AMPK activation occurs as a result of inhibition of mitochondrial oxidative phosphorylation, and does not require increased production of reactive oxygen species. Thus, AMPK activation provides the basis for unifying the ‘membrane’ and ‘mitochondrial’ hypotheses, previously regarded as disparate, to account for hypoxic chemotransduction.

Aim: This study investigated the utility of visual inspection with acetic acid (VIA) as a method for cervical cancer screening in Thailand and examined the relationship of VIA to high-risk human papillomavirus (HR-HPV) status. Methods: Cervical cells were collected from 160 patients receiving a Pap smear. VIA was performed on the cervix of the patients by application of 5% acetic acid. HPV screening of DNA extracted from cytology samples was performed by PCR using the GP5+/6+ primer system followed by reverse line blot hybridization genotyping. Results: The majority (96.9%) of the patients were diagnosed with normal or inflammatory cytologic changes. 32.8% of normal cytology and 42.0% of inflammation cases showed positive acetowhite staining. 3.1%, 38.1% and 42.5% of subjects were positive for an abnormal Pap test, VIA test, and HPV DNA, respectively. VIA demonstrated 50% sensitivity and 66.7% specificity for abnormal histology with PPV and NPV values of 50% and 66.7%, respectively, whereas HPV DNA test showed 100% sensitivity. HPV16 was the most common (54.4%) and HR-HPV was detected in 36.3% of all cases. 48.5% of HR-HPV positive and 36.8% of HR-HPV negative cervices stained with acetowhite following the VIA test. Conclusion: The VIA test is a simple method for cervical cancer screening; however, a significant proportion of patients with normal or inflammatory cytology were positive by this test. Further, HR-HPV in women without acetowhite staining was demonstrated. Therefore, some form of HR-HPV detection test may be required for combination with cervical cell screening even in low-resource nations.

This study investigated the role of human papillomavirus (HPV) in Schneiderian papillomas (SPs) to determine whether HPV is associated with the pathogenesis of particular histologic subtypes and whether p16(INK4a) can be used as a surrogate marker for HPV detection. Twenty-seven papilloma specimens (19 inverted [IPs], 6 exophytic [EPs], 1 oncocytic [OP] and 1 mixed) were collected from 23 patients. Purified SP DNA extracts were tested for HPV by PCR using GP5 +/GP6 + primers; HPV genotyping was performed by dot blot hybridization. PCR positive specimens were screened for HPV by biotinyl-tyramide-based chromogenic in situ hybridization (CISH). Immunohistochemsistry (IHC) for the HPV L1 capsid protein and for p16(INK4a) was performed on all specimens. HPV was detected by PCR in 16/27 (59.3%) SPs; 9/19 (47.4%) IPs; 6/6 (100%) EPs [p = 0.051], and 1/1 (100%) mixed SP. HPV was not detected in the single OP. High risk genotypes were detected in 4/9 IPs (44.4%) and 0/6 EPs (0%) [p = 0.10]. Seven of 16 PCR positive SPs were also CISH positive for HPV: 5/6 EPs (83.3%) and 1/9 IP (11.1%) [p = 0.01]. IHC for the L1 capsid protein was positive in 2 SPs (1 EP and 1 mixed). p16(INK4a) staining was seen in 14/16 (87.5%) PCR positive SPs and in 10/11 (90.9%) PCR negative SPs (p = 1.00). In summary, this study demonstrates a strong association between HPV and EPs, however, its role in IPs remains less well-defined. Further, p16(INK4a) is not a useful surrogate marker for HPV detection across the various SPs.

Two-pore channels (TPCs or TPCNs) are novel members of the large superfamily of voltage-gated cation channels with slightly higher sequence homology to the pore-forming subunits of voltage-gated Ca2+ and Na+ channels than most other members. Recent studies demonstrate that TPCs locate to endosomes and lysosomes and form Ca2+ release channels that respond to activation by the Ca2+ mobilizing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). With multiple endolysosomal targeted NAADP receptors now identified, important new insights into the regulation of endolysosomal function in health and disease will therefore be unveiled. (C) 2010 Federation of European Biochemical Societies.

Vital homeostatic mechanisms monitor O2 supply and adjust respiratory and circulatory function to meet demand. The pulmonary arteries and carotid bodies are key systems in this respect. Hypoxic pulmonary vasoconstriction (HPV) aids ventilation-perfusion matching in the lung by diverting blood flow from areas with an O2 deficit to those rich in O2, while a fall in arterial pO2 increases sensory afferent discharge from the carotid body to elicit corrective changes in breathing patterns. We discuss here the new concept that hypoxia, by inhibiting oxidative phosphorylation, activates AMP-activated protein kinase (AMPK) leading to consequent phosphorylation of target proteins, such as ion channels, which initiate pulmonary artery constriction and carotid body activation. Consistent with this view, AMPK knockout mice exhibit an impaired ventilatory response to hypoxia. Thus, AMPK may be sufficient and necessary for hypoxia-response coupling and may regulate O2 and thereby energy (ATP) supply at the whole body as well as the cellular level.

Diagnosis of basaloid squamous carcinoma (BSCC) currently relies mainly on histological criteria, with variable immunohistochemical results reported in small series. We explored the use of a battery of immunohistochemical stains to elucidate this diagnosis on 45 cases of BSCC. To further elucidate the immunohistochemical profile of BSCC, to explore potential genetic pathways of malignant transformation using proliferation markers, and to investigate a possible link with Human Papillomavirus (HPV). Forty-five cases of BSCC and 34 site-matched cases of squamous cell carcinoma (SCC) were obtained from the archives of the pathology department at our institution. Extensive literature review was undertaken utilizing Medline. Ber-EP4 is a useful diagnostic marker for BSCC, positive in 82% (37/45) of the cases and in 68% (23/34) of SCC. An alternative is the combination of cytokeratins CK14 and CK7, known to be negative, and CK1, known to be positive, which achieves an accuracy of 73% (33/45) in BSCC and 88% (30/34) in SCC. The two diagnostic approaches were in agreement in 66% of the cases; both methods were equally accurate in the divergent cases. Increased expression of the proliferation markers supports the concept that BSCC is a rapidly growing tumor. Results of p16 stains support an etiological link between BSCC and HPV; interestingly, HPV was present significantly more in BSCC (71% (32/45)), than in SCC (59% (20/34)) in this study (P = 0.02).

BAY 50-4798, a novel, engineered form of interleukin (IL)-2, is a selective agonist for the high-affinity IL-2 receptor and induces the proliferation of activated human T cells with potency similar to recombinant IL-2 (rIL-2), but has reduced proliferative activity on natural killer cells and is associated with a diminished secondary cytokine cascade. In the current study, the transcriptional profiles of human peripheral blood mononuclear cells (PBMCs) stimulated in vitro with BAY 50-4798 and rIL-2 were compared using Affymetrix microarray technology in combination with Ingenuity Pathway Analysis (IPA) to determine whether there are quantitative or qualitative differences in the molecular networks activated by these IL-2 analogs. A total of 299 genes were differentially expressed in response to the two IL-2 analogs, with an increase in the number of differences over time. Consistent with the fact that BAY 50-4798 interacts with fewer forms of the IL-2 receptor than rIL-2 to activate fewer cell types, 169 genes were expressed at lower levels in PBMCs cultured with BAY 50-4798 compared with IL-2. These genes were mainly categorized as cytokines and chemokines, and were used to build multiple molecular interaction networks, the most significant of which centered around a subunit of NF-kappaB, which is known to play a pivotal role in inflammation, and was associated with cell death. Of the genes induced in response to BAY 50-4798, only 25% were expressed at lower levels than those induced by rIL-2. Moreover, despite its more selective receptor targeting compared with rIL-2, BAY 50-4798 caused higher levels of expression of 130 genes, which predominantly fell into categories associated with metabolism and transcription. We interpret these results as consistent with the expected transcriptional profile of a mutein engineered and demonstrated to have diminished inflammatory effects yet fully retain selected features of IL-2 activity. In addition to demonstrating that the responses to BAY 50-4798 are characterized by differential expression of genes known to be induced by IL-2, we report for the first time the induction of a significant number of genes not previously reported in the context of IL-2 biology.

Calcium signals determine, for example, smooth muscle contraction and changes in gene expression. How calcium signals select for these processes is enigmatic. We build on the "panjunctional sarcoplasmic reticulum" hypothesis, describing our view that different calcium pumps and release channels, with different kinetics and affinities for calcium, are strategically positioned within nanojunctions of the SR and help demarcate their respective cytoplasmic nanodomains. SERCA2b and RyR1 are preferentially targeted to the sarcoplasmic reticulum (SR) proximal to the plasma membrane (PM), i.e., to the superficial buffer barrier formed by PM-SR nanojunctions, and support vasodilation. In marked contrast, SERCA2a may be entirely restricted to the deep, perinuclear SR and may supply calcium to this sub-compartment in support of vasoconstriction. RyR3 is also preferentially targeted to the perinuclear SR, where its clusters associate with lysosome-SR nanojunctions. The distribution of RyR2 is more widespread and extends from this region to the wider cell. Therefore, perinuclear RyR3s most likely support the initiation of global calcium waves at L-SR junctions, which subsequently propagate by calcium-induced calcium release via RyR2 in order to elicit contraction. Data also suggest that unique SERCA and RyR are preferentially targeted to invaginations of the nuclear membrane. Site- and function-specific calcium signals may thus arise to modulate stimulus-response coupling and transcriptional cascades.

Perhaps the defining characteristic of pulmonary arteries is the process of hypoxic pulmonary vasoconstriction (HPV) which, under physiological conditions, supports ventilation-perfusion matching in the lung by diverting blood flow away from oxygen deprived areas of the lung to oxygen rich regions. However, when alveolar hypoxia is more widespread, either at altitude or with disease (e.g., cystic fibrosis), HPV may lead to hypoxic pulmonary hypertension. HPV is driven by the intrinsic response to hypoxia of pulmonary arterial smooth muscle and endothelial cells, which are acutely sensitive to relatively small changes in pO2 and have evolved to monitor oxygen supply and thus address ventilation-perfusion mismatch. There is now a consensus that the inhibition by hypoxia of mitochondrial oxidative phosphorylation represents a key step towards the induction of HPV, but the precise nature of the signalling pathway(s) engaged thereafter remains open to debate. We will consider the role of the AMP-activated protein kinase (AMPK) and liver kinase B1 (LKB1), an upstream kinase through which AMPK is intimately coupled to changes in oxygen supply via mitochondrial metabolism. A growing body of evidence, from our laboratory and others, suggests that modulation of the LKB1-AMPK signalling pathway underpins both hypoxic pulmonary vasoconstriction and the development of pulmonary hypertension.

Background and Aims: The investigation of human papillomavirus (HPV) physical status in pre-invasive cervical lesions has been restricted by the small amounts of tissue available for study. Multiple displacement amplification (MDA), a phi29 DNA polymerase based whole genome amplification technique, has the potential to help resolve this problem by yielding large amounts of high molecular weight DNA from tiny starting quantities. Methods: Firstly, a comparison was made of restriction endonuclease fragment patterns of DNA from seven different HPV types and corresponding MDA products. Secondly, E6/E7 and LCR sequencing data from HPV16 recombinant plasmid and MDA copy DNA were correlated. Thirdly, DNA and MDA products from cervical cell lines (CaSki, HeLa, and SiHa that contain integrated HPV) and an invasive cervical carcinoma were analysed by Southern blot hybridisation. Fourthly, MDA product from CaSki cell DNA mixed with HPV18-plasmid DNA was tested for the demonstration of both episomal and integrated HPV. Finally, MDA products from HPV16 positive abnormal cervical cytological samples were assayed for integration by Southern blot hybridisation. Results: DNA templates and MDA products yielded analogous data. Episomal and integrated HPV DNA were successfully detected by Southern blot assay of the cell line/HPV-plasmid model, and in MDA products of clinical samples. Conclusions: These data show that MDA has considerable potential to assist in the investigation of HPV physical status; abundant (>40 μg) DNA can be generated with high fidelity from minuscule (50 ng) starting quantities, and both episomal and integrated HPV DNA are distinguishable in MDA products from solid tumours and cytological materials.

All cells respond to metabolic stress. However, a variety of specialized cells, commonly referred to as O2-sensing cells, are acutely sensitive to relatively small changes in P. Within a variety of organisms such O2-sensing cells have evolved as vital homeostatic mechanisms that monitor O2 supply and alter respiratory and circulatory function, as well as the capacity of the blood to transport O2. Thereby, arterial P may be maintained within physiological limits. In mammals, for example, two key tissues that contribute to this process are the pulmonary arteries and the carotid bodies. Constriction of pulmonary arteries by hypoxia optimizes ventilation–perfusion matching in the lung, whilst carotid body excitation by hypoxia initiates corrective changes in breathing patterns via increased sensory afferent discharge to the brain stem. Despite extensive investigation, the precise mechanism(s) by which hypoxia mediates these responses has remained elusive. It is clear, however, that hypoxia inhibits mitochondrial function in O2-sensing cells over a range of P that has no such effect on other cell types. This raised the possibility that AMP-activated protein kinase might function to couple mitochondrial oxidative phosphorylation to Ca2+ signalling mechanisms in O2-sensing cells and thereby underpin pulmonary artery constriction and carotid body excitation by hypoxia. Our recent investigations have provided significant evidence in support of this view.

Specialized O2-sensing cells exhibit a particularly low threshold to regulation by O2 supply and function to maintain arterial pO2 within physiological limits. For example, hypoxic pulmonary vasoconstriction optimizes ventilation-perfusion matching in the lung, whereas carotid body excitation elicits corrective cardio-respiratory reflexes. It is generally accepted that relatively mild hypoxia inhibits mitochondrial oxidative phosphorylation in O2-sensing cells, thereby mediating, in part, cell activation. However, the mechanism by which this process couples to Ca2+ signaling mechanisms remains elusive, and investigation of previous hypotheses has generated contrary data and failed to unite the field. We propose that a rise in the cellular AMP/ATP ratio activates AMP-activated protein kinase and thereby evokes Ca2+ signals in O2-sensing cells. Co-immunoprecipitation identified three possible AMP-activated protein kinase subunit isoform combinations in pulmonary arterial myocytes, with alpha1 beta2 gamma1 predominant. Furthermore, their tissue-specific distribution suggested that the AMP-activated protein kinase-alpha1 catalytic isoform may contribute, via amplification of the metabolic signal, to the pulmonary selectivity required for hypoxic pulmonary vasoconstriction. Immunocytochemistry showed AMP-activated protein kinase-alpha1 to be located throughout the cytoplasm of pulmonary arterial myocytes. In contrast, it was targeted to the plasma membrane in carotid body glomus cells. Consistent with these observations and the effects of hypoxia, stimulation of AMP-activated protein kinase by phenformin or 5-aminoimidazole-4-carboxamide-riboside elicited discrete Ca2+ signaling mechanisms in each cell type, namely cyclic ADP-ribose-dependent Ca2+ mobilization from the sarcoplasmic reticulum via ryanodine receptors in pulmonary arterial myocytes and transmembrane Ca2+ influx into carotid body glomus cells. Thus, metabolic sensing by AMP-activated protein kinase may mediate chemotransduction by hypoxia.

Field and microstructural analyses of faults with millimeters to hundreds of meters of displacement within welded tuffs at Yucca Mountain, Nevada, has led us to identify four different fault zone architectures. We designate these fault zones as Classes A–D. We conclude that Classes A, C, and D are genetically related and that observed differences in their morphology and deformation mechanisms are related to differences in displacement magnitude and changes in the rheology of faulted rocks. We show that Class A, C, and D fault zones were formed by progressive cataclasis, leading to the development of a foliated gouge core and wide damage zone at the highest displacement magnitudes. By inference, displacement variations across individual high displacement faults are expected to produce significant lateral changes in fault zone architecture. The broad range of fault rock textures and architectures found on the Class A, C, and D continuum may produce important corresponding variability in hydrologic properties in ignimbrites. By contrast, Class B fault zones have unique mineralogies and microtextures relative to other Yucca Mountain fault zones. Most notable is the occurrence of distinctive jigsaw puzzle texture within fault core breccias, which consists of angular wall rock fragments floating in a coarsely crystalline secondary calcite cement matrix. The calcite cement constitutes more than 65% of the Class B fault zones. Comparisons with other occurrences of secondary minerals within open lithophysal cavities, which appear to have formed in unsaturated conditions, indicate that the secondary mineralization history at Yucca Mountain was complex and likely polygenetic. The age and origin of the secondary calcite minerals in the Class B fault zones remain undetermined.

Previous studies on pulmonary arterial smooth muscle cells have shown that nicotinic acid adenine dinucleotide phosphate (NAADP) evokes highly localized intracellular Ca(2+) signals by mobilizing thapsigargin-insensitive stores. Such localized Ca(2+) signals may initiate global Ca(2+) waves and contraction of the myocytes through the recruitment of ryanodine receptors on the sarcoplasmic reticulum via Ca(2+)-induced Ca(2+) release. Here we show that NAADP evokes localized Ca(2+) signals by mobilizing a bafilomycin A1-sensitive, lysosome-related Ca(2+) store. These lysosomal stores facilitate this process by co-localizing with a portion of the sarcoplasmic reticulum expressing ryanodine receptors to comprise a highly specialized trigger zone for NAADP-dependent Ca(2+) signaling by the vasoconstrictor hormone, endothelin-1. These findings further advance our understanding of how the spatial organization of discrete, organellar Ca(2+) stores may underpin the generation of differential Ca(2+) signaling patterns by different Ca(2+)-mobilizing messengers.

The clinical course of inflammatory bowel disease (IBD) in dogs is characterized by spontaneous exacerbations and remissions, which makes assessment of disease burden difficult. The objectives of this study were to develop a scoring system for evaluation of canine IBD activity and to validate this scoring method by correlating it to objective laboratory and histologic indices of intestinal inflammation. Fifty-eight dogs with IBD were evaluated prospectively and compared to 9 disease-free control dogs. Clinical disease activity was quantified by a simple scoring system, the canine IBD activity index (CIBDAI), and compared to serum concentrations of C-reactive protein (CRP), haptoglobin (HAP), alpha-acid glycoprotein (AGP), and serum amyloid A (SAA), as well as histology scores derived from endoscopic biopsy specimens. Forty-six dogs were available for a reevaluation of the CIBDAI, CRP HAP, and AGP, and 34 dogs had repeat analysis of SAA performed after medical therapy. Serum concentrations of CRP were significantly (P.02) increased in dogs with CIBDAI scoresor = 5 (mild disease activity or greater) compared to controls. Among IBD dogs, the CIBDAI showed good correlation (r = 0.82, P.0001) to both histology and HAP scores, but CRP also was a strong co-correlate of disease activity. The IBD dogs showed significantly (P.0001) decreased CIBDAI and CRP values but significantly (P.0001) increased HAP concentrations after medical therapy compared to pretreatment values. We conclude that the CIBDAI is a reliable measure of inflammatory activity in canine IBD and that CRP is suitable for laboratory evaluation of the effect of therapy in these patients.

The European Standards Committee on Oxidative DNA Damage (ESCODD) was set up to resolve the problems associated with the measurement of background levels of oxidative DNA damage (in particular 8-oxo-7,8-dihydroguanine, or 8-oxoGua) in human cells. A tendency for DNA oxidation to occur during sample preparation prior to chromatography has been recognized as the source of a very substantial artefact. To assess the success of attempts to eliminate the artefact, ESCODD has distributed to its members standard samples of pig liver and HeLa cells for analysis. Estimates of 8-oxoGua in pig liver, using chromatographic techniques, ranged from 2.23 to 441 per 10(6) guanines, with a median of 10.47 per 10(6) guanines. Chromatographic analysis of HeLa cell DNA gave a range of 1.84 to 214 per 10(6) guanines with a median of 5.23 per 10(6) guanines. HeLa cell DNA was also analysed by an enzymic approach, in which whole cell DNA was treated with formamidopyrimidine DNA glycosylase, which nicks DNA at sites of 8-oxoGua, and the breaks measured with the comet assay, alkaline unwinding or alkaline elution. Values with these methods ranged from 0.06 to 4.988-oxoGua per 10(6) guanines, with a median of 0.79 per 10(6) guanines. Although there are clearly still serious discrepancies between methods and laboratories, the lowest estimates by chromatography (arguably those in which the artefact was best controlled) are only 2.5 times higher than the median value obtained with the enzymic approach.

Hypoxic pulmonary vasoconstriction (HPV) is unique to pulmonary arteries, and supports ventilation/perfusion matching. However, in diseases such as emphysema, HPV can promote hypoxic pulmonary hypertension (HPH), which ultimately leads to right heart failure. Since it was first described, the mechanisms underpinning HPV have remained obscure, and current therapies for HPH are poor. Previous investigations have suggested that HPV may be mediated by processes intrinsic to the pulmonary artery smooth muscle, and by the release of a vasoconstrictor(s) from the endothelium. It was thought that oxygen-sensitive ion channels in the smooth muscle cell membrane triggered HPV, and it has been argued that the endothelium-derived vasoconstrictor is endothelin-1. However, these proposals remain controversial. This review discusses the regulation by hypoxia of cyclic adenosine diphosphate-ribose production and Ca 2+ release from the sarcoplasmic reticulum in pulmonary artery smooth muscle. The role of these processes in triggering maintained HPV is then related to its subsequent progression due to vasoconstrictor(s) release from the endothelium.

We have shown that hypoxia reduces plasma insulin, which correlates with corticotropin-releasing hormone (CRH) receptor 1 (CRHR1) in rats, but the mechanism remains unclear. Here, we report that hypobaric hypoxia at an altitude of 5,000 m for 8 h enhances rat plasma CRH, corticosterone, and glucose levels, whereas the plasma insulin and pancreatic ATP/ADP ratio is reduced. In islets cultured under normoxia, CRH stimulated insulin release in a glucose- and CRH-level–dependent manner by activating CRHR1 and thus the cAMP-dependent protein kinase pathway and calcium influx through L-type channels. In islets cultured under hypoxia, however, the insulinotropic effect of CRH was inactivated due to reduced ATP and cAMP and coincident loss of intracellular calcium oscillations. Serum and glucocorticoid-inducible kinase 1 (SGK1) also played an inhibitory role. In human volunteers rapidly ascended to 3,860 m, plasma CRH and glucose levels increased without a detectable change in plasma insulin. By contrast, volunteers with acute mountain sickness (AMS) exhibited a marked decrease in HOMA insulin sensitivity (HOMA-IS) and enhanced plasma CRH. In conclusion, hypoxia may attenuate the CRH-insulinotropic effect by reducing cellular ATP/ADP ratio, cAMP and calcium influx, and upregulated SGK1. Hypoxia may not affect HOMA-IS in healthy volunteers but reduces it in AMS volunteers.

Further to advancements in instrumentation and fluorescent dye technologies, there has been a resurgence of interest in the flow cytometric assay of formalin-fixed, paraffin-embedded specimens. Here we present a novel, simple and effective alternative to whole block sectioning that allows selective multisampling of tissues within a specimen block and the investigation of intratumoral heterogeneity. Formalin-fixed, paraffin-embedded breast carcinoma specimens were core-punched using 1.0 mm diameter needles and assayed by flow cytometry using a modified Hedley method. Intratumoral heterogeneity for DNA index and per cent S-phase fraction was detected in 10 of 23 (44%) and 11 of 23 (47%) specimens respectively. Macro-level genomic heterogeneity is common in breast cancer even within a single surgical specimen block. Studies investigating the relationship of DNA content heterogeneity to other markers of genomic instability such as mutations, deletions, insertions and translocations are warranted.