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Background: Musculoskeletal deficits of the craniocervical region play an important role in causing various cervical disorders. Cervical disorders and neck pain are almost as prevalent as low back pain and like low back pain; in most of the cases it is difficult to determine the actual cause of neck pain hence it is regarded as “non-specific neck pain”. This study was conducted to determine the correlation between deep neck flexor endurance, the intensity of pain, and functional disability in individuals with chronic non-specific neck pain. Materials and methods: A cross-sectional observational study was conducted on twenty individuals with chronic non-specific neck pain. Numerical Pain Rating Scale (NPRS), Neck Disability Index (NDI), and Craniocervical Flexion Test (CCFT) were used to measure neck pain intensity, neck disability, and deep neck flexor endurance respectively. The correlation was found using the Pearson Correlation Coefficient test. Result: The results showed that there was a positive correlation found between NPRS and NDI, and CCFT has negative correlations with NPRS and NDI respectively. Conclusion: The findings suggest that the lesser deep neck flexor endurance corresponded to greater neck pain intensity and disability. Keywords: cervical spine, neck disability index, neck pain, craniocervical flexion test, deep neck flexors endurance, correlation.

Altered RNA expression of repetitive sequences and retrotransposition are frequently seen in colorectal cancer, implicating a functional importance of repeat activity in cancer progression. We show the nucleoside reverse transcriptase inhibitor 3TC targets activities of these repeat elements in colorectal cancer preclinical models with a preferential effect in p53-mutant cell lines linked with direct binding of p53 to repeat elements. We translate these findings to a human phase II trial of single-agent 3TC treatment in metastatic colorectal cancer with demonstration of clinical benefit in 9 of 32 patients. Analysis of 3TC effects on colorectal cancer tumorspheres demonstrates accumulation of immunogenic RNA:DNA hybrids linked with induction of interferon response genes and DNA damage response. Epigenetic and DNA-damaging agents induce repeat RNAs and have enhanced cytotoxicity with 3TC. These findings identify a vulnerability in colorectal cancer by targeting the viral mimicry of repeat elements.Significance:Colorectal cancers express abundant repeat elements that have a viral-like life cycle that can be therapeutically targeted with nucleoside reverse transcriptase inhibitors (NRTI) commonly used for viral diseases. NRTIs induce DNA damage and interferon response that provide a new anticancer therapeutic strategy.This article is highlighted in the In This Issue feature, p. 1397

Supplementary Figure from Reverse Transcriptase Inhibition Disrupts Repeat Element Life Cycle in Colorectal Cancer

Supplementary Data from Reverse Transcriptase Inhibition Disrupts Repeat Element Life Cycle in Colorectal Cancer

Altered RNA expression of repetitive sequences and retrotransposition are frequently seen in colorectal cancer, implicating a functional importance of repeat activity in cancer progression. We show the nucleoside reverse transcriptase inhibitor 3TC targets activities of these repeat elements in colorectal cancer preclinical models with a preferential effect in p53-mutant cell lines linked with direct binding of p53 to repeat elements. We translate these findings to a human phase II trial of single-agent 3TC treatment in metastatic colorectal cancer with demonstration of clinical benefit in 9 of 32 patients. Analysis of 3TC effects on colorectal cancer tumorspheres demonstrates accumulation of immunogenic RNA:DNA hybrids linked with induction of interferon response genes and DNA damage response. Epigenetic and DNA-damaging agents induce repeat RNAs and have enhanced cytotoxicity with 3TC. These findings identify a vulnerability in colorectal cancer by targeting the viral mimicry of repeat elements.Significance:Colorectal cancers express abundant repeat elements that have a viral-like life cycle that can be therapeutically targeted with nucleoside reverse transcriptase inhibitors (NRTI) commonly used for viral diseases. NRTIs induce DNA damage and interferon response that provide a new anticancer therapeutic strategy.This article is highlighted in the In This Issue feature, p. 1397

Of the over 5000 exoplanets that have been detected, only about a dozen have ever been directly imaged. Earth-like exoplanets are on the order of 10 billion times fainter than their host star in visible and near-infrared, requiring a coronagraph instrument to block primary starlight and allow for the imaging of nearby orbiting planets. In the pursuit of direct imaging of exoplanets, scalar vortex coronagraphs (SVCs) are an attractive alternative to vector vortex coronagraphs (VVCs). VVCs have demonstrated 2e-9 raw contrast in broadband light but have several limitations due to their polarization properties. SVCs imprint the same phase ramp as VVCs on the incoming light and do not require polarization splitting, but they are inherently chromatic. Discretized phase ramp patterns such as a wrapped staircase help reduce SVC chromaticity and simulations show it outperforms a chromatic classical vortex in broadband light. We designed, fabricated, and tested a wrapped staircase SVC, and here we present the broadband characterization on the high contrast spectroscopy testbed. We also performed wavefront correction on the in-air coronagraph testbed at NASA's Jet Propulsion Laboratory and achieved an average raw contrasts of 3.2e-8 in monochromatic light and 2.2e-7 across a 10% bandwidth., Comment: arXiv admin note: text overlap with arXiv:2212.02633

The detection and characterization of Earth-like exoplanets around Sun-like stars for future flagship missions requires coronagraphs to achieve contrasts on the order of 1e-10 at close angular separations and over large spectral bandwidths (>=20%). We present our progress thus far on exploring the potential for scalar vortex coronagraphs (SVCs) in direct exoplanet imaging. SVCs are an attractive alternative to vector vortex coronagraphs (VVCs), which have recently demonstrated 6e-9 raw contrast in 20% broadband light but are polarization dependent. SVCs imprint the same phase ramp on the incoming light and do not require polarization splitting, but are inherently limited by their chromatic behavior. Several SVC designs have been proposed in recent years to solve this issue by modulating or wrapping the azimuthal phase function according to specific patterns. For one such design, the staircase SVC, we present our best experimental SVC results demonstrating raw contrast of 2e-7 in 10% broadband light. Since SVC broadband performance and aberration sensitivities are highly dependent on topology, we conducted a comparative study of several SVC designs to optimize for high contrast across a range of bandwidths. Furthermore, we present a new coronagraph optimization tool to predict performance in order to find an achromatic solution.

The successful application of antibody-based therapeutics in either primary or metastatic cancer depends upon the selection of rare cell surface epitopes that distinguish cancer cells from surrounding normal epithelial cells. By contrast, as circulating tumor cells (CTCs) transit through the bloodstream, they are surrounded by hematopoietic cells with dramatically distinct cell surface proteins, greatly expanding the number of targetable epitopes. Here, we show that an antibody (23C6) against cadherin proteins effectively suppresses blood-borne metastasis in mouse isogenic and xenograft models of triple negative breast and pancreatic cancers. The 23C6 antibody is remarkable in that it recognizes both the epithelial E-cadherin (CDH1) and mesenchymal OB-cadherin (CDH11), thus overcoming considerable heterogeneity across tumor cells. Despite its efficacy against single cells in circulation, the antibody does not suppress primary tumor formation, nor does it elicit detectable toxicity in normal epithelial organs, where cadherins may be engaged within intercellular junctions and hence inaccessible for antibody binding. Antibody-mediated suppression of metastasis is comparable in matched immunocompetent and immunodeficient mouse models. Together, these studies raise the possibility of antibody targeting CTCs within the vasculature, thereby suppressing blood-borne metastasis.

Little is known about the effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the coronavirus disease 2019 (COVID-19) on pregnant mothers and their infants. Moreover, there is no definitive evidence that SARS CoV- 2 can be vertically transmitted from an infected mother to the unborn fetus.

Coronavirus disease-19 (COVID-19) is caused by a newly discovered coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although SARS-CoV-2 is visualized on electron microscopy, there is an increasing demand for widely applicable techniques to visualize viral components within tissue specimens. Viral protein and RNA can be detected on formalin-fixed paraffin-embedded (FFPE) tissue using immunohistochemistry (IHC) and in situ hybridization (ISH), respectively. Herein, we evaluate the staining performance of ISH for SARS-CoV-2 and an IHC directed at the SARS-CoV nucleocapsid protein and compare these results to a gold standard, tissue quantitative real-time polymerase chain reaction (qRT-PCR). We evaluated FFPE sections from 8 COVID-19 autopsies, including 19 pulmonary and 39 extrapulmonary samples including the heart, liver, kidney, small intestine, skin, adipose tissue, and bone marrow. We performed RNA-ISH for SARS-CoV-2 on all cases with IHC for SARS-CoV and SARS-CoV-2 qRT-PCR performed on selected cases. Lungs from 37 autopsies performed before the COVID-19 pandemic served as negative controls. The ISH and IHC slides were reviewed by 4 observers to record a consensus opinion. Selected ISH and IHC slides were also reviewed by 4 independent observers. Evidence of SARS-CoV-2 was identified on both the IHC and ISH platforms. Within the postmortem lung, detected viral protein and RNA were often extracellular, predominantly within hyaline membranes in patients with diffuse alveolar damage. Among individual cases, there was regional variation in the amount of detectable virus in lung samples. Intracellular viral RNA and protein was localized to pneumocytes and immune cells. Viral RNA was detected on RNA-ISH in 13 of 19 (68%) pulmonary FFPE blocks from patients with COVID-19. Viral protein was detected on IHC in 8 of 9 (88%) pulmonary FFPE blocks from patients with COVID-19, although in 5 cases the stain was interpreted as equivocal. From the control cohort, FFPE blocks from all 37 patients were negative for SARS-CoV-2 RNA-ISH, whereas 5 of 13 cases were positive on IHC. Collectively, when compared with qRT-PCR on individual tissue blocks, the sensitivity and specificity for ISH was 86.7% and 100%, respectively, while those for IHC were 85.7% and 53.3%, respectively. The interobserver variability for ISH ranged from moderate to almost perfect, whereas that for IHC ranged from slight to moderate. All extrapulmonary samples from COVID-19-positive cases were negative for SARS-CoV-2 by ISH, IHC, and qRT-PCR. SARS-CoV-2 is detectable on both RNA-ISH and nucleocapsid IHC. In the lung, viral RNA and nucleocapsid protein is predominantly extracellular and within hyaline membranes in some cases, while intracellular locations are more prominent in others. The intracellular virus is detected within pneumocytes, bronchial epithelial cells, and possibly immune cells. The ISH platform is more specific, easier to analyze and the interpretation is associated with the improved interobserver agreement. ISH, IHC, and qRT-PCR failed to detect the virus in the heart, liver, and kidney.

Pancreatic ductal adenocarcinoma (PDAC) lethality is due to metastatic dissemination. Characterization of rare, heterogeneous circulating tumor cells (CTCs) can provide insight into metastasis and guide development of novel therapies. Using the CTC-iChip to purify CTCs from PDAC patients for RNA-seq characterization, we identify three major correlated gene sets, with stemness genes LIN28B/KLF4, WNT5A, and LGALS3 enriched in each correlated gene set; only LIN28B CTC expression was prognostic. CRISPR knockout of LIN28B—an oncofetal RNA-binding protein exerting diverse effects via negative regulation of let-7 miRNAs and other RNA targets—in cell and animal models confers a less aggressive/metastatic phenotype. This correlates with de-repression of let-7 miRNAs and is mimicked by silencing of downstream let-7 target HMGA2 or chemical inhibition of LIN28B/let-7 binding. Molecular characterization of CTCs provides a unique opportunity to correlated gene set metastatic profiles, identify drivers of dissemination, and develop therapies targeting the “seeds” of metastasis., Metastatic dissemination contributes to the lethality in pancreatic ductal adenocarcinoma (PDAC). Here, the authors perform RNA-sequencing on patient derived circulating tumor cells (CTCs) and identify three major CTC subgroups, and show the therapeutic potential of targeting LIN28B/let-7 pathway to halt cancer metastasis.

For direct imaging of exoplanets, Scalar Vortex Coronagraphs (SVCs) are an attractive alternative to the popularly used Vector Vortex Coronagraphs (VVCs). This is primarily because they are able to induce the same phase ramp regardless of the incoming light's polarization state. We tested a set of stepped SVC staircase masks in the Exoplanet Technology Laboratory (ET Lab) at Caltech on the High-Contrast Spectroscopy Testbed (HCST). Here we present some preliminary findings of their starlight suppression ability, achieving raw contrasts on the order of 1e-5 for 7 to 9 lambda/D. We also characterized their chromatic performance and performed wavefront control to achieve preliminary contrasts on the order of 1e-7 with EFC. These initial experimental results with SVCs have shown scalar vortex technology has a great potential for future exoplanet direct imaging missions., Comment: SPIE Optics + Photonics 2021

Transcriptional profiling has defined PDAC into distinct classical epithelial (E) or quasi-mesenchymal (QM) subtypes, and these subtypes exist on a continuum of interconverting cell states. This epithelial to mesenchymal transition (EMT) plasticity is thought to be important for the metastatic dissemination and intrinsic resistance to chemotherapy. Our prior work identified TAK1 as an important driver of EMT in mouse circulating tumor cells, and we had identified a compound (5z)-7-oxozeaenol (Oxo) as a small molecule inhibitor of TAK1. Here, we have expanded upon this prior work by evaluating Oxo in 3 human patient derived PDAC cell lines as an EMT plasticity inhibitor. Treatment with Oxo induced a shift of all PDAC cell lines towards an E phenotype in both E and QM PDAC cell lines using RNA-seq and quantitative RNA in situ hybridization. This was associated with increased FFX sensitivity, loss of migratory abilities of all cell lines by Boyden chamber, and decreased invasion in matrigel. Interestingly, genetic TAK1 knockout via CRISPR/Cas9 in these PDAC cell lines showed different effects from Oxo. We found that TAK1 knockout decreased E PDAC cell line migratory behavior and increased FFX sensitivity, but QM PDAC cell lines were not affected by TAK1 knockout. This suggests that QM PDAC cell lines have additional signaling cascades that compensate for TAK1 loss and that Oxo effects across PDAC cell lines are not solely TAK1 dependent. Using phospho-proteomics, in FFX treated PDAC cell lines, we have identified potential candidate pathways in RNA splicing and viral processes that may be additional processes affected by Oxo that merits further investigation. Altogether, we have identified Oxo as a potential broad EMT plasticity inhibitor across PDAC subtypes, and there is a potential role of selective TAK1 inhibitors for E PDAC tumors. Citation Format: Eunae You, Ildiko Phillips E. Phillips, Richard Y. Ebright, Niyati Desai, Michael J. Raabe, Evan R. Lang, Linda T. Nieman, Soroush Hajizadeh, Johannes Kreuzer, Wilhelm Haas, David T. Ting. 5z-7-oxozeaenol as an epithelial mesenchymal transition plasticity inhibitor in PDAC [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A066.

Although reprogramming of cellular metabolism is a hallmark of cancer, little is known about how metabolic reprogramming contributes to early stages of transformation. Here, we show that the histone deacetylase SIRT6 regulates tumor initiation during intestinal cancer by controlling glucose metabolism. Loss of SIRT6 results in an increase in the number of intestinal stem cells (ISCs), which translates into enhanced tumor initiating potential in APCmin mice. By tracking down the connection between glucose metabolism and tumor initiation, we find a metabolic compartmentalization within the intestinal epithelium and adenomas, where a rare population of cells exhibit features of Warburg-like metabolism characterized by high pyruvate dehydrogenase kinase (PDK) activity. Our results show that these cells are quiescent cells expressing +4 ISCs and enteroendocrine markers. Active glycolysis in these cells suppresses ROS accumulation and enhances their stem cell and tumorigenic potential. Our studies reveal that aerobic glycolysis represents a heterogeneous feature of cancer, and indicate that this metabolic adaptation can occur in non-dividing cells, suggesting a role for the Warburg effect beyond biomass production in tumors.

Immunoglobulin 4-related disease (IgG4-RD) is a multisystem disease, characterised by tumefactive lesions and a swift response to immunosuppressive therapy. Although elevated serum and tissue IgG4 are characteristic, T cells appear to be the primary driver of this immunologically mediated disease. The overarching goal was to examine the role of immunomodulatory cells in IgG4-RD.Biopsies from patients with IgG4-RD (n = 39) and mimics of this disease (n = 78) were evaluated for IgG4, IgG, CD8, programmed cell death ligand 1 (PD-L1) and a subset (n = 18) evaluated for CD4, purine rich box 1 (PU.1), forkhead box protein 3 (FoxP3), PD-L1, programmed cell death 1 (PD-1), indoleamine 2,3-dioxygenase 1 (IDO1) and lymphocyte-activation gene 3 (LAG3). Data pertaining to demographics and laboratory findings at baseline evaluation was extracted from electronic medical records. When compared to mimics, IgG4-RD showed increased numbers of PD-L1- (P = 0.0001), PD-1- (P = 0.001), IDO1- (P = 0.03), LAG3- (P = 0.04) and FoxP3- (P = 0.04)-positive immune cells. The PD-L1-positive cells were enriched within aggregates of CD4 and CD8-positive T cells. Thirty-one of 39 (80%) IgG4-RD cases showed greater than five PD-L1-positive cells per high-power field (HPF), while four of 78 (5%) mimics of this disease exceeded this cut-point. In IgG4-RD, PD-L1-positive macrophages correlated with PD-1- (P = 0.002), LAG3- (P = 0.001) and IDO1-positive cells (P = 0.001); a-positive correlation was also noted between IgG4/IgG ratio and PD-L1-, PD-1- and IDO1-positive cells.IgG4-RD shows expansion of mechanisms that maintain peripheral tolerance. The spatial and temporal relationship between T cells and the PD-L1-PD-1 axis and the up-regulation of multiple immunomodulatory proteins suggests that these immunoregulatory mechanisms play a significant role in IgG4-RD.

Tandem satellite repeats account for 3% of the human genome. One of them, Human Satellite II (HSATII), is highly expressed in several epithelial cancers and cancer cell lines. Here we report an acute induction of HSATII RNA in human cells infected with two herpes viruses. We show that human cytomegalovirus (HCMV) IE1 and IE2 proteins cooperate to induce HSATII RNA affecting several aspects of the HCMV replication cycle, viral titers and infected-cell processes. HSATII RNA expression in tissue from two chronic HCMV colitis patients correlates with the strength of CMV antigen staining. Thus, endogenous HSATII RNA synthesis after herpesvirus infections appears to have functionally important consequences for viral replication and may provide a novel insight into viral pathogenesis. The HSATII induction seen in both infected and cancer cells suggests possible convergence upon common HSATII-based regulatory mechanisms in these seemingly disparate diseases., The human genome includes a large amount of repetitive sequence, such as human satellite II (HSATII), but their function remains largely unknown. Here, Nogalski et al. show that herpesvirus infection induces HSATII RNA expression, which in turn affects virus replication and cell motility.

Background: Congenital infection of SARS-CoV-2 appears to be exceptionally rare despite many cases of COVID-19 during pregnancy. Robust proof of placental infection requires demonstration of viral localization within placental tissue. Only 2 of the few cases of possible vertical transmission have demonstrated placental infection. None have shown placental expression of the ACE2 or TMPRSS2 protein, both required for viral infection. Methods: We examined 19 COVID-19 exposed placentas for histopathologic findings, and for expression of ACE2, and TMPRSS2 by immunohistochemistry. Direct placental SARS-CoV-2 expression was studied by two methods - nucleocapsid protein expression by immunohistochemistry, and RNA expression by in-situ hybridization. Findings: ACE2 membranous expression in the syncytiotrophoblast of the chorionic villi is predominantly in a polarized pattern with expression highest on the stromal side of the trophoblast. In addition, cytotrophoblast and extravillous trophoblast express ACE2. No ACE2 expression was detected in villous stroma, Hofbauer cells, or endothelial cells. TMPRSS2 expression was only present weakly in the villous endothelium and rarely in the ST. In 2 of 19 cases SARS-CoV-2 RNA was present in the placenta focally in the syncytiotrophoblast and cytotrophoblast. There was no characteristic histopathology present in our cases including the 2 placental infections. Interpretation: We found that the placenta is capable of being infected but that this event is rare. We propose one explanation could be the polarized expression of ACE2 away from the maternal blood and pronounced paucity of TMPRSS2 expression in trophoblast. Funding Statement: Vickery-Colvin Award from the Department of Pathology, Massachusetts General Hospital and Advanced Cell Diagnostics. Research reported in this publication was partially supported by the Career Development Program in Substance Use and Addiction Medicine of the National Institutes of Health under award number K12DA043490. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Health. Declaration of Interests: Disclosures: D.T.T. has received consulting fees from Merrimack Pharmaceuticals, Ventana Roche, Foundation Medicine, Inc., and EMD Millipore Sigma, which are not related to this work. D.T.T. is a founder and has equity in PanTher Therapeutics and TellBio, Inc., which is not related to this work. D.T.T. is co-founder and own equity in ROME Therapeutics, which is not related to this work. Parts of this work were supported by ACD-Biotechne (N.D., V.D., D.T.T.). Dr. Ting’s interests were reviewed and are managed by Mass General Brigham in accordance with their conflict of interest policies. M.M.-K. has received consulting fees rom H3 Biomedicine and AstraZeneca and institutional grant support from Novartis, all of which are not related to this work. D.J.R. received royalties from UpToDate and Cambridge University Press as an author, not related to this work. All other authors declare no competing interests. Ethics Approval Statement: Institutional review board approval (IRB2020P001116 and IRB2020P001001) and Research support agreement (RSA2020A005296).

Early detection strategies for pancreatic ductal adenocarcinoma are needed. This article describes a high‐sensitivity platform for detection of epithelial cells shed from preneoplastic lesions at a high risk of becoming malignant and a combination of technologies that could be used for early detection of disease in high‐risk patients., Background. Recent work has demonstrated early shedding of circulating epithelial cells (CECs) from premalignant intraductal papillary mucinous neoplasms (IPMNs). However, the potential use of CECs as a “liquid biopsy” for patients with IPMNs has been limited by antigen dependence of CEC isolation devices and the lack of robust detection biomarkers across CEC phenotypes. Materials and Methods. We utilized a negative depletion microfluidic platform to purify CECs from contaminating leukocytes and coupled this platform with immunofluorescence, RNA in situ hybridization, and RNA sequencing (RNA‐seq) detection and enumeration. Results. Using established protein (EpCAM, cytokeratins) and novel noncoding RNA (HSATII, cytokeratins) biomarkers, we detected CECs in 88% of patients bearing IPMN lesions. RNA‐seq analysis for MUC genes confirm the likely origin of these CECs from pancreatic lesions. Conclusion. Our findings increase the sensitivity of detection of these cells and therefore could have clinical implications for cancer risk stratification. Implications for Practice. This work describes a high‐sensitivity platform for detection of epithelial cells shed from preneoplastic lesions at high risk of malignant transformation. Further research efforts are underway to define the transcriptional programs that might allow discrimination between circulating cells released from tumors that will become malignant and cells released from tumors that will not. After further refinement, this combination of technologies could be deployed for monitoring and early detection of patients at high risk for developing new or recurrent pancreatic malignancies.

Single-cell technologies have described heterogeneity across tissues, but the spatial distribution and forces that drive single-cell phenotypes have not been well defined. Combining single-cell RNA and protein analytics in studying the role of stromal cancer-associated fibroblasts (CAFs) in modulating heterogeneity in pancreatic cancer (pancreatic ductal adenocarcinoma [PDAC]) model systems, we have identified significant single-cell population shifts toward invasive epithelial-to-mesenchymal transition (EMT) and proliferative (PRO) phenotypes linked with mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling. Using high-content digital imaging of RNA in situ hybridization in 195 PDAC tumors, we quantified these EMT and PRO subpopulations in 319,626 individual cancer cells that can be classified within the context of distinct tumor gland "units." Tumor gland typing provided an additional layer of intratumoral heterogeneity that was associated with differences in stromal abundance and clinical outcomes. This demonstrates the impact of the stroma in shaping tumor architecture by altering inherent patterns of tumor glands in human PDAC.

The advantages of molecular testing for accurate diagnosis and optimal therapeutic management of patients infected with microorganisms is well established, yet many test methods are not feasible in underdeveloped or resource-limited settings. The disparity in testing methods is controversial from ethical, financial, and scientific perspectives. Over the years, philanthropic funding for early and accurate diagnosis has helped curtail the spread of disease and improved overall survival, yet infections caused by microorganisms are still the leading cause of death in the resource-limited parts of the world. Fortunately, there are promising new tests that are inexpensive, portable, easy-to-use, and practical at even small, rural, and remote health centers. This review describes the potential problems encountered in the routine implementation and performance of molecular assays in resource-poor settings and discusses the adaptations and alternative methodologies that are routinely used in most microbiology laboratories in such settings.

The lack of consensus on clinically relevant molecular subtypes by gene expression in high-grade serous ovarian carcinoma (HGSOC) creates a barrier to subtype-based clinical investigation for the development of targeted therapies. We previously discovered aberrant expression of noncoding repeat RNAs across epithelial cancers including ovarian (Ting, Science 2011), which can invoke innate immune responses in tumors (Chiappinelli, Cell 2015). Epigenetic alterations and loss of tumor suppressor function, especially p53, can derepress endogenous repetitive elements in cancer. Further, BRCA1 deficiency induces satellite repeat RNAs, which promote genomic instability via interacting with the BRCA1 complex (Zhu, Mol Cell 2018), highlighting their potential significance in HGSOC. Here we aim to comprehensively define the as yet uncharacterized “repeatome” in HGSOC to refine molecular subtypes and identify novel biomarkers and therapeutic targets. We developed a Total RNASeq platform and novel computational pipelines to quantify the repeatome (Solovyov, Cell Rep 2018). Results from RNASeq are validated in cell lines and human tumors with RNA in situ hybridization (RNA-ISH) using probes to specific repeat RNAs, and combined IHC is performed to quantify intratumoral immune subpopulations. Whole-exome sequencing is performed to link the repeatome with somatic mutations. To obtain a global landscape of the ovarian cancer repeatome, Total RNASeq was performed on 32 patient-derived ovarian cancer cell lines, 11 HGSOC PDX, and 11 additional ovarian cancer cell lines. We detect abundant repeat RNA expression from all major subclasses including retrotransposons, endogenous retroviruses, and satellites. HGSOC are enriched for satellite repeats compared with other cancers, most notably in BRCA-mutant HGSOC. Human satellite II (HSATII), a cancer-specific satellite, is strongly upregulated in HGSOC compared with fallopian tube epithelial cells and displays highly variable expression across different models. RNA-ISH for HSATII on human ovarian cancer tissue microarrays confirms the abundance and variation of HSATII. Additionally, the repeatome is altered in HGSOC following exposure to in vitro chemotherapy and epigenetic agents with distinct patterns of expression linked to specific agents. Additional RNAseq analysis using hierarchical clustering and principal component analysis are being used to understand the relationship of repeatome expression patterns with coding gene expression, somatic mutations, in vitro drug sensitivity, and clinical outcomes. Digital image analysis of repeat RNA-ISH expression and immune cell infiltrate quantitation will determine the link between tumor cell repeat RNA levels and the responding immune tumor microenvironment. Overall, these studies will define the undiscovered repeatome in HGSOC and provide a foundation for discovery of novel biomarkers and potential therapeutic strategies, particularly those to increase efficacy of immunotherapies. Citation Format: Rebecca L. Porter, Anna Szabolcs, Niyati Desai, Vishal Thapar, Raghav Mohan, Alexander Solovyov, David Pepin, Joyce Liu, Benjamin Greenbaum, David T. Ting. Repeatome profiling in high-grade serous ovarian cancer reveals abundant repeat noncoding RNA expression [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A66.

Single cell technologies have described heterogeneity across tissues, but the spatial distribution and forces that drive single cell phenotypes have not been well defined. Combining single cell RNA and protein analytics in pancreatic cancer (PDAC) model systems, we demonstrated the role of stromal fibroblasts in shaping PDAC single cell heterogeneity towards invasive (EMT) and proliferative (PRO) phenotypes. Using highcontent digital imaging of RNA in situ hybridization in 195 tumors, we observed these EMT and PRO subpopulations in 319,626 individual cancer cells. Interestingly, we found these EMT and PRO subpopulations form distinct tumor gland “units” associated with differences in stromal abundance and patient survival. This demonstrates the impact of the stroma in shaping tumor architecture by altering inherent patterns of tumor glands in human PDAC.

Circulating signals of drug resistance Cancer drugs often lose their effectiveness because tumors acquire genetic changes that confer drug resistance. Ideally, patients would be switched to a different drug before tumor growth resumes, but this requires early knowledge of how resistance arose. Miyamoto et al. have developed a non-invasive method to spot resistance by sequencing RNA transcripts in single circulating tumor cells (CTCs) (see the Perspective by Nanus and Giannakakou). For example, in prostate cancer patients, drug resistance was triggered by activation of the Wnt signaling pathway. But CTCs are rare and fragile, and the technology needs further development before it is used in clinical practice. Science , this issue p. 1351 ; see also p. 1283

Metastasis-competent circulating tumour cells (CTCs) experience oxidative stress in the bloodstream, but their survival mechanisms are not well defined. Here, comparing single-cell RNA-Seq profiles of CTCs from breast, prostate and lung cancers, we observe consistent induction of β-globin (HBB), but not its partner α-globin (HBA). The tumour-specific origin of HBB is confirmed by sequence polymorphisms within human xenograft-derived CTCs in mouse models. Increased intracellular reactive oxygen species (ROS) in cultured breast CTCs triggers HBB induction, mediated through the transcriptional regulator KLF4. Depletion of HBB in CTC-derived cultures has minimal effects on primary tumour growth, but it greatly increases apoptosis following ROS exposure, and dramatically reduces CTC-derived lung metastases. These effects are reversed by the anti-oxidant N-Acetyl Cysteine. Conversely, overexpression of HBB is sufficient to suppress intracellular ROS within CTCs. Altogether, these observations suggest that β-globin is selectively deregulated in cancer cells, mediating a cytoprotective effect during blood-borne metastasis., Circulating tumour cells contribute to metastatic spread. Here the authors find that beta-chain of haemoglobin is overexpressed in those cells and protects them from oxidative stress, prolonging their survival in circulation and thereby increasing the likelihood of metastasis formation.

Although human tumours are shaped by the genetic evolution of cancer cells, evidence also suggests that they display hierarchies related to developmental pathways and epigenetic programs in which cancer stem cells (CSCs) can drive tumour growth and give rise to differentiated progeny1. Yet, unbiased evidence for CSCs in solid human malignancies remains elusive. Here we profile 4,347 single cells from six IDH1 or IDH2 mutant human oligodendrogliomas by RNA sequencing (RNA-seq) and reconstruct their developmental programs from genome-wide expression signatures. We infer that most cancer cells are differentiated along two specialized glial programs, whereas a rare subpopulation of cells is undifferentiated and associated with a neural stem cell expression program. Cells with expression signatures for proliferation are highly enriched in this rare subpopulation, consistent with a model in which CSCs are primarily responsible for fuelling the growth of oligodendroglioma in humans. Analysis of copy number variation (CNV) shows that distinct CNV sub-clones within tumours display similar cellular hierarchies, suggesting that the architecture of oligodendroglioma is primarily dictated by developmental programs. Subclonal point mutation analysis supports a similar model, although a full phylogenetic tree would be required to definitively determine the effect of genetic evolution on the inferred hierarchies. Our single-cell analyses provide insight into the cellular architecture of oligodendrogliomas at single-cell resolution and support the cancer stem cell model, with substantial implications for disease management.

Cryptosporidium spp. is under recognized as an important pathogen causing diarrhea in children and HIV-infected individuals with associated high morbidity and mortality. In endemic areas, most symptomatic infections are in childhood and in immunocompromised adults. The immune status of the host plays a critical role in determining the severity of cryptosporidiosis. Infection is self-limited in immunocompetent hosts, but can be severe and persistent in the immunocompromised such as AIDS patients or malnourished children. Cryptosporidiosis in developing countries is a major cause of acute and persistent diarrhea in children and is associated with subsequent impairment in growth, physical fitness, and cognitive function. Despite recognition of the importance of immune status, the correlates of protective immunity in cryptosporidiosis in humans are poorly understood, and treatment modalities are limited.

Identification of candidate metastasis genes has traditionally resulted from comparison of primary and metastatic tumor specimens. However, Circulating Tumor Cells (CTCs) contain metastatic precursors that are present transiently in the bloodstream and their analysis may reveal additional pathways that are induced for a limited time, as they invade and survive within the vasculature. By comparing transcriptome profiles of CTCs from breast, prostate and lung cancers with their primary tumor of origin, we observed consistent and significant induction of the β-globin gene (HBB) within CTCs. In contrast, expression of α-globin, its binding partner within hematopoietic cells, is not coordinately upregulated. The tumor-specific origin of HBB was further confirmed by analysis of human xenografts-derived CTCs in mice, where human-specific HBB polymorphisms are readily distinguishable in the murine background. In cultured cancer cells, we further demonstrate that induction of HBB is triggered by exposure to reactive oxygen species (ROS), and we identify KLF-family transcriptional regulators that mediate this effect. To investigate the function of aberrant β-globin expression within CTCs, we performed shRNA-mediated knockdown of HBB in breast CTC-derived cultures. Cells with depleted HBB expression displayed elevated intracellular ROS levels, increased sensitivity to hydrogen peroxide, and impaired metastatic potential in mouse models. Taken together, these observations suggest that β-globin, a component of functional hemoglobin in red blood cells, is deregulated in disseminated tumor cells, where it may function as a ROS scavenger, reducing oxidative stress and facilitating cancer metastasis. Citation Format: Yu Zheng, David T. Miyamoto, Ben S. Wittner, James P. Sullivan, Nicola Aceto, Nicole Vincent Jordan, Min Yu, Nezihi Murat Karabacak, Valentine Comaills, Robert Morris, Rushil Desai, Niyati Desai, Erin Emmons, Richard J. Lee, Chin-Lee Wu, Lecia V. Sequist, Wilhelm Haas, David T. Ting, Mehmet Toner, Sridhar Ramaswamy, Shyamala Maheswaran, Daniel A. Haber. Induction of β-globin protects circulating tumor cells from oxidative stress during dissemination. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2679.

Purpose: The National Cancer Institute's (NCI) Center to Reduce Cancer Health Disparities (CRCHD) has been working to strengthen community outreach capacity through Community Health Educators (CHEs) of the NCI National Outreach Network (NON). The NON CHEs, based at academic and cancer centers across the country, strengthen NCI's ability to develop and disseminate culturally appropriate, evidence-based cancer information that is tailored to the specific needs and expectations of underserved communities. Over the past five years, NON CHEs have developed, adapted, and tested community education and outreach interventions that effectively reach underserved communities to address local and regional cancer health disparity issues. Methods: A subset of NON CHEs have focused on outreach and educational interventions to reduce disparities specifically among Latino populations at high risk of cancer or cancer survivors in community settings from inner cities to rural areas. In 2010, these CHEs, based at more than 12 cancer centers and academic institutions across the country, established a working group to support one another. The NON CHE Latino Working Group shares best practices for disseminating culturally sensitive, evidence-based cancer prevention, screening, and survivorship information tailored to the specific needs of their respective Latino communities. The Latino Working Group's interventions and campaigns targeted breast, cervical, colorectal, prostate, and lung cancers, and also explored and addressed the key issue of research participation by the Latino community. The group utilized various evidence-based programs, such as the Cancer 101 curriculum and the inflatable colon. The NON CHE Latino Working Group conducted needs assessments, gathered qualitative and quantitative data to inform the adaptation of interventions, implemented and evaluated community education and outreach projects, and published findings from their work. They developed multiple educational materials, including one in collaboration with the NCI's Geographical Management of Cancer Health Disparities Program (GMaP) Region 4 program: The Clinical Trials Outreach for Latinos (CTOL): Program Replication Manual. Preliminary Results: We will present outcomes from the NON CHE Latino Working Group's diverse projects including needs assessment and intervention testing results, review feedback from focus groups conducted in the United States and Puerto Rico, discuss the success of various evidence-based programs put into practice, share information on health education classes focusing on diet and nutrition, and the community's receptivity to participate in research. We will present data collected on over 5,000 Latinos who participated in the different NON CHE programs. The NON CHE Latino WG will also discuss lessons learned, challenges and best practices. Conclusion: Although Latinos have lower incidence rates of certain cancers, they also suffer from higher mortality rates. Latino communities at risk of developing cancer and affected by cancer are often marginalized due to multiple factors and barriers, including inadequate health education, language barriers, lack of access to healthcare, and mistaken cultural beliefs about cancer. Latinos often do not engage in regular screening procedures resulting in worse health outcomes when compared to the Non-Hispanic White population. Major framework efforts, such as the established NCI/CRCHD NON CHE program, have a major impact in reducing cancer health disparities among this underserved population by offering culturally sensitive interventions that improve delivery of and/or promote practices in cancer education, prevention, screening, treatment and survivorship services. Citation Format: Sandra L. San Miguel, Alelí Ayala-Marín, Katherine J. Briant, Niyati Desai, Leticia Gatus, Diego Gómez-Aristizabal, Evelyn González, Martha E. González, Natalia I. Heredia, Nadia Lazo, Rosa Ortiz, Lizette Rangel, Yonaira Rivera, Janet Sanchez, Mayra Serrano, Rose A. Treviño Whitaker. National Outreach Network Community Health Educators: An effective framework to reduce cancer health disparities among Latinos. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr A31.

Pancreatic adenocarcinoma (PDAC) is a lethal disease with a rising incidence, expected to become the second cause of cancer death in the next 5 years. Despite advances in novel therapies for other malignancies, PDAC has remained a highly chemorefractory disease leading to inevitable disease recurrence in the vast majority of patients. The identification of the cellular mechanisms of chemoresistance unique to PDAC is of particular importance. Our prior work using single cell RNA-sequencing in pancreatic circulating tumor cells (CTCs) identified 878 genes enriched in CTCs compared to matched primary tumor cells. CTCs are thought to be enriched for cells with high metastatic potential, which we predict to have intrinsic chemoresistant behavior. We hypothesized that CTC enriched genes may identify a subpopulation of cells resistant to chemotherapy that could be used as a biomarker and novel therapeutic target. The neuroendocrine marker SV2 was found in both mouse and human pancreatic CTCs and was selected given its presence on cell membranes, providing a potential therapeutic target. We evaluated a panel of patient derived cell lines and identified an SV2 positive subpopulation comprising ∼1% of cells in 3 of 5 cell lines. Notably, only differentiated epithelioid PDAC cell lines contained an SV2 subpopulation, while this population is not present in poorly differentiated quasi-mesenchymal cell lines. We performed RNA-ISH for SV2 isoforms in an annotated PDAC tissue microarray demonstrating positive staining in 77% of samples (245/317). Interestingly, SV2 positivity was often focally positive in a subpopulation of cells at the basolateral region of ductal adenocarcinoma and was found in more differentiated morphology (69% well/moderate vs 31% poor/undifferentiated; p = 0.047). The presence of SV2 cells was associated with an improved disease free survival (HR: 0.49 p = 0.009) and overall survival (HR: 0.54 p = 0.018) even after correction in multivariate analysis. Analysis in both cell lines and tissue revealed SV2 positive cells were also found to have higher levels of the ALDH1 stem cell marker (Pearson coefficient = 0.92 in tissue). Initial in vivo treatment of human orthotopic xenografts with gemcitabine, reveal an enrichment of SV2 positive cells pointing to their potential role as the recalcitrant cancer cells treated with cytotoxic chemotherapy population. Together, this is consistent with prior work demonstrating classical epithelial PDAC has improved survival compared to quasi-mesenchymal tumors. However, almost all patients with classical epithelial PDAC will die from recurrent disease, and we have now shown SV2 as a potential marker that identifies the chemoresistant pancreatic cancer cells in this patient population. Ultimately, a more detailed characterization of the SV2 subpopulation in classical PDAC will help us develop novel targeted therapies to overcome chemoresistance. Citation Format: Daniela Dias-Santos, Matteo Ligorio, Kshitij Arora, Vishal Thapar, Olivia C. MacKenzie, Srinjoy Sil, Niyati Desai, Vikram Deshpande, Miguel N. Rivera, Cristina R. Ferrone, David T. Ting. Molecular subtyping of pancreatic adenocarcinoma identifies SV2 positive subpopulation in classical PDAC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 609. doi:10.1158/1538-7445.AM2015-609

We examined the differentiation potential of an adult liver stem cell line (WB F344) in a cardiac microenvironment, ex vivo. WB F344 cells were established from a single cloned nonparenchymal epithelial cell isolated from a normal male adult rat liver. Genetically modified, WB F344 cells that express beta-galactosidase and green fluorescent protein or only beta-galactosidase were co-cultured with dissociated rat or mouse neonatal cardiac cells. After 4 to 14 days, WB F344-derived cardiomyocytes expressed cardiac-specific proteins and exhibited myofibrils, sarcomeres, and a nascent sarcoplasmic reticulum. Further, rhythmically beating WB F344-derived cardiomyocytes displayed calcium transients. Fluorescent recovery after photobleaching demonstrated that WB F344-derived cardiomyocytes were coupled with adjacent neonatal cardiomyocytes and other WB F344-derived cardiomyocytes. Fluorescence in situ hybridization experiments suggested that fusion between WB F344 cells and neonatal mouse cardiomyocytes did not take place. Collectively, these results support the conclusion that these adult-derived liver stem cells respond to signals generated in a cardiac microenvironment ex vivo acquiring a cardiomyocyte phenotype and function. The identification ex vivo of microenvironmental signals that appear to cross germ layer and species specificities should prove valuable in understanding the molecular basis of adult stem cell differentiation and phenotypic plasticity.

Background: Traditionally, clinical teaching pertaining to history taking skills from patient with low back pain is taught to postgraduate physiotherapy students. However, history obtained by various students may differ and hence there is need for an instrument that can be used to obtain standard history. Objectives: Primary objective of this study was to find out the utility of investigator developed checklist in low back pain history taking by Postgraduate Physiotherapy students. Secondary objective of this study was to find out students perception about this checklist. Methods: Postgraduate students were asked to take focused history about low back pain from a standardised patient and their performance was recorded with the 28 items checklist. After this, they were provided a copy of checklist to read and then they were requested to re-obtain the history. Results: An average score without reading the checklist was 17/28 and after reading the checklist and this score was 22/28 when they re-obtained the history. 100% of students liked this way of obtaining the history from low back pain patients and 17% of students felt that this will increase paper work dependency and will limit creative thinking. Conclusion: The use of checklist may serve as a valuable tool in low back pain history taking skills.

Lgr5(+) intestinal stem cells (ISCs) drive epithelial self-renewal, and their immediate progeny-intestinal bipotential progenitors-produce absorptive and secretory lineages via lateral inhibition. To define features of early transit from the ISC compartment, we used a microfluidics approach to measure selected stem- and lineage-specific transcripts in single Lgr5(+) cells. We identified two distinct cell populations, one that expresses known ISC markers and a second, abundant population that simultaneously expresses markers of stem and mature absorptive and secretory cells. Single-molecule mRNA in situ hybridization and immunofluorescence verified expression of lineage-restricted genes in a subset of Lgr5(+) cells in vivo. Transcriptional network analysis revealed that one group of Lgr5(+) cells arises from the other and displays characteristics expected of bipotential progenitors, including activation of Notch ligand and cell-cycle-inhibitor genes. These findings define the earliest steps in ISC differentiation and reveal multilineage gene priming as a fundamental property of the process.