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Figure S4 shows unsupervised clustering of PAM50 genes examined in all cells of patient 2

There is a need to individualize assays for tumor molecular phenotyping, given variations in the differentiation status of tumor and normal tissues in different patients. To address this, we performed single-cell genomics of breast tumors and adjacent normal cells propagated for a short duration under growth conditions that enable epithelial reprogramming. Cells analyzed were either unselected for a specific subpopulation or phenotypically defined as undifferentiated and highly clonogenic ALDH+/CD49f+/EpCAM+ luminal progenitors, which express both basal cell and luminal cell–enriched genes. We analyzed 420 tumor cells and 284 adjacent normal cells for expression of 93 genes that included a PAM50-intrinsic subtype classifier and stemness-related genes. ALDH+/CD49f+/EpCAM+ tumor and normal cells clustered differently compared with unselected tumor and normal cells. PAM50 gene-set analyses of ALDH+/CD49f+/EpCAM+ populations efficiently identified major and minor clones of tumor cells, with the major clone resembling clinical parameters of the tumor. Similarly, a stemness-associated gene set identified clones with divergent stemness pathway activation within the same tumor. This refined expression profiling technique distinguished genes truly deregulated in cancer from genes that identify cellular precursors of tumors. Collectively, the assays presented here enable more precise identification of cancer-deregulated genes, allow for early identification of therapeutically targetable tumor cell subpopulations, and ultimately provide a refinement of precision therapeutics for cancer treatment. Cancer Res; 77(10); 2759–69. ©2017 AACR.

Gene expression patterns in tumor and normal cells of three patients with statistical analyses

Despite significant advances in deciphering the molecular landscape of acute myeloid leukaemia (AML), therapeutic outcomes of this haematological malignancy have only modestly improved over the past decades. Drug resistance and disease recurrence almost invariably occur, highlighting the need for a deeper understanding of these processes. While low O2 compartments, such as bone marrow (BM) niches, are well‐recognized hosts of drug‐resistant leukaemic cells, standard in vitro studies are routinely performed under supra‐physiologic (21% O2, ambient air) conditions, which limits clinical translatability. We hereby identify molecular pathways enriched in AML cells that survive acute challenges with classic or targeted therapeutic agents. Experiments took into account variations in O2 tension encountered by leukaemic cells in clinical settings. Integrated RNA and protein profiles revealed that lipid biosynthesis, and particularly the cholesterol biogenesis branch, is a particularly therapy‐induced vulnerability in AML cells under low O2 states. We also demonstrate that the impact of the cytotoxic agent cytarabine is selectively enhanced by a high‐potency statin. The cholesterol biosynthesis programme is amenable to additional translational opportunities within the expanding AML therapeutic landscape. Our findings support the further investigation of higher‐potency statin (eg rosuvastatin)–based combination therapies to enhance targeting residual AML cells that reside in low O2 environments.

PURPOSE People aging with long-term physical disabilities (PAwLTPD) are aging at an accelerated rate beginning in middle-age. They face age-related challenges in conjunction with their existing disabilities; thus, maintaining independence as they age is often difficult. The aim of this systematic review was to examine the effectiveness of rehabilitation interventions for middle-aged PAwLTPD to participate independently in the home and community. MATERIALS AND METHODS We searched four databases - MEDLINE, CINAHL, Web of Science, and EMBASE - for studies published from January 2005 to December 2020. Information from included studies was extracted using a critical appraisal form. Studies were categorized based on common themes, assigned level of evidence, and assessed for risk of bias. RESULTS Fourteen articles were included. Common themes derived were fall risk reduction, functional capacity, community mobility, and function within the home. The strongest evidence supports wheelchair skills training programs (WSTPs) among manual wheelchair users and targeted paretic limb exercise post-stroke. Moderate evidence supports exercise and multicomponent interventions for those with multiple sclerosis, adaptive strategy training and WSTPs to improve satisfaction with mobility for power wheelchair users, and home modifications/assistive technology for mobility-impaired individuals. CONCLUSION Interventions with strong and moderate evidence should be routinely offered for middle-aged PAwLTPD. Future research should focus on developing evidence-based interventions for middle-aged PAwLTPD.IMPLICATIONS FOR REHABILITATIONMiddle-aged PAwLTPD face the same aging-related challenges as people without disabilities but will experience additional difficulties due to compounding effects of long-term health conditions and aging.Current effective interventions to promote participation for middle-aged PAwLTPD have been measured over a wide range of outcomes, and many interventions should be used by clinicians on a case-by-case basis.Wheelchair skills training was found to have the strongest evidence and is recommended for use with middle-aged PAwLTPD who use manual and power wheelchairs.

The mental health impact of the COVID-19 pandemic may be greater than that of the viral infection. This impact is likely greater for disadvantaged groups such as people with long-term physical disabilities (PwLTPD). This cross-sectional study used a multiple linear regression model to examine factors associated with depressive symptoms and to understand their relative importance during the onset of the COVID-19 pandemic. The model explained 58% of the variance in depressive symptoms. Anxiety was the most important predictor, accounting for 8% of variance. Higher levels of anxiety, loneliness, and financial stress; prior diagnosis of depression; and non-Black race predicted a higher level of depressive symptoms in PwLTPD during the onset of the COVID-19 pandemic. Occupational therapy practitioners should strategically acknowledge predictors of depression that cannot be modified and actively address those that can be modified through evidence-based interventions to improve depressive symptoms in PwLTPD.

Tamm–Horsfall protein (THP), also known as uromodulin, is a kidney-specific protein produced by cells of the thick ascending limb of the loop of Henle. Although predominantly secreted apically into the urine, where it becomes highly polymerized, THP is also released basolaterally, toward the interstitium and circulation, to inhibit tubular inflammatory signaling. Whether, through this latter route, THP can also regulate the function of renal interstitial mononuclear phagocytes (MPCs) remains unclear, however. Here, we show that THP is primarily in a monomeric form in human serum. Compared with wild-type mice, THP(−/−) mice had markedly fewer MPCs in the kidney. A nonpolymerizing, truncated form of THP stimulated the proliferation of human macrophage cells in culture and partially restored the number of kidney MPCs when administered to THP(−/−) mice. Furthermore, resident renal MPCs had impaired phagocytic activity in the absence of THP. After ischemia-reperfusion injury, THP(−/−) mice, compared with wild-type mice, exhibited aggravated injury and an impaired transition of renal macrophages toward an M2 healing phenotype. However, treatment of THP(−/−) mice with truncated THP after ischemia-reperfusion injury mitigated the worsening of AKI. Taken together, our data suggest that interstitial THP positively regulates mononuclear phagocyte number, plasticity, and phagocytic activity. In addition to the effect of THP on the epithelium and granulopoiesis, this new immunomodulatory role could explain the protection conferred by THP during AKI.

Background Inflammation is the most prevalent and widespread histological finding in the human prostate, and associates with the development and progression of benign prostatic hyperplasia and prostate cancer. Several factors have been hypothesized to cause inflammation, yet the role each may play in the etiology of prostatic inflammation remains unclear. This study examined the possibility that the common protozoan parasite Toxoplasma gondii induces prostatic inflammation and reactive hyperplasia in a mouse model. Methods Male mice were infected systemically with T. gondii parasites and prostatic inflammation was scored based on severity and focality of infiltrating leukocytes and epithelial hyperplasia. We characterized inflammatory cells with flow cytometry and the resulting epithelial proliferation with bromodeoxyuridine (BrdU) incorporation. Results We found that T. gondii infects the mouse prostate within the first 14 days of infection and can establish parasite cysts that persist for at least 60 days. T. gondii infection induces a substantial and chronic inflammatory reaction in the mouse prostate characterized by monocytic and lymphocytic inflammatory infiltrate. T. gondii-induced inflammation results in reactive hyperplasia, involving basal and luminal epithelial proliferation, and the exhibition of proliferative inflammatory microglandular hyperplasia in inflamed mouse prostates. Conclusions This study identifies the common parasite T. gondii as a new trigger of prostatic inflammation, which we used to develop a novel mouse model of prostatic inflammation. This is the first report that T. gondii chronically encysts and induces chronic inflammation within the prostate of any species. Furthermore, T. gondii-induced prostatic inflammation persists and progresses without genetic manipulation in mice, offering a powerful new mouse model for the study of chronic prostatic inflammation and microglandular hyperplasia.

Enediyne natural products are a class of compounds that were recognized for their potential as chemotherapeutic agents many years ago, but found to be highly cytotoxic due to their propensity for low thermal activation. Bergman cyclization of the enediyne moiety produces a diradical intermediate, and may subsequently induce DNA damage and account for the extreme cytotoxicity. While difficulties in controlling the thermal cyclization reaction have limited the clinical use of cyclic enediynes, we have previously shown that enediyne activity, and thus toxicity at physiological temperatures can be modulated by metallation of acyclic enediynes. Furthermore, the cytotoxicity of “metalloenediynes” can be potentiated by hyperthermia. In this study, we characterized a suite of novel metallated enediyne motifs that usually induced little or no cytotoxicity when two different human cancer cell lines were treated with the compounds at 37°C, but showed a significant enhancement of cytotoxicity after cells were exposed to moderate hyperthermia during drug treatment. Cultured U-1 melanoma or MDA-231 breast cancer cells were treated with various concentrations of Cu, Fe and Zn complexes of the enediyne (Z)-N,N′-bis[1-pyridyl-2-yl-meth-(E)-ylidene]octa-4-ene-2,6-diyne-1,8-diamine (PyED) and clonogenic survival was assessed to determine the effects of the drugs at 37°C and 42.5°C. Toxicity at 37°C varied for each compound, but hyperthermia potentiated the cytotoxicity of each compound in both cell lines. Cytotoxicity was concentration-, time- and temperature-dependent. Heating cells during drug treatment resulted in enhanced apoptosis, but the role of cell cycle perturbation in the response of the cells to the drugs was less clear. Lastly, we showed that hyperthermia enhanced the number of DNA double-strand breaks (DSBs) induced by the compounds, and inhibited their repair after drug treatment. Thus, thermal enhancement of cytotoxicity may be due, at least in part, to the propensity of the enediyne moiety to induce DSBs, and/or a reduction in DSB repair efficiency. We propose that “tuning” of metalloenediyne toxicity through better-controlled reactivity could have potential clinical utility, since we envision that such compounds could be administered systemically as relatively non-toxic agents, but cytotoxicity could be enhanced in, and confined to a tumor volume when subjected to localized heating.

Background Up to 25% of acute stroke patients first note symptoms upon awakening. We hypothesized that patients awaking with stroke symptoms may be safely treated with intravenous alteplase (IV tPA) using non-contrast head CT (NCHCT), if they meet all other standard criteria. Methods The SAfety of Intravenous thromboLytics in stroke ON awakening (SAIL ON) was a prospective, open-label, single treatment arm, pilot safety trial of standard dose IV tPA in patients who presented with stroke symptoms within 0–4.5 hours of awakening. From January 30, 2013, to September 1, 2015, twenty consecutive wakeup stroke patients selected by NCHCT were enrolled. The primary outcome was symptomatic intracerebral hemorrhage (sICH) in the first 36 hours. Secondary outcomes included NIH stroke scale (NIHSS) at 24 hours; and modified Rankin Score (mRS), NIHSS, and Barthel index at 90 days. Results The average age was 65 years (range 47–83); 40% were women; 50% were African American. The average NIHSS was 6 (range 4–11). The average time from wake-up to IV tPA was 205 minutes (range 114–270). The average time from last known well to IV tPA was 580 minutes (range 353–876). The median mRS at 90 days was 1 (range 0–5). No patients had sICH; two of 20 (10%) had asymptomatic ICH on routine post IV tPA brain imaging. Conclusions Administration of IV tPA was feasible and may be safe in wakeup stroke patients presenting within 4.5 hours from awakening, screened with NCHCT. An adequately powered randomized clinical trial is needed. Clinical trial registration ClinicalTrials.gov NCT01643902.

There is a need to individualize assays for tumor molecular phenotyping, given variations in the differentiation status of tumor and normal tissues in different patients. To address this, we performed single-cell genomics of breast tumors and adjacent normal cells propagated for a short duration under growth conditions that enable epithelial reprogramming. Cells analyzed were either unselected for a specific subpopulation or phenotypically defined as undifferentiated and highly clonogenic ALDH+/CD49f+/EpCAM+ luminal progenitors, which express both basal cell and luminal cell–enriched genes. We analyzed 420 tumor cells and 284 adjacent normal cells for expression of 93 genes that included a PAM50-intrinsic subtype classifier and stemness-related genes. ALDH+/CD49f+/EpCAM+ tumor and normal cells clustered differently compared with unselected tumor and normal cells. PAM50 gene-set analyses of ALDH+/CD49f+/EpCAM+ populations efficiently identified major and minor clones of tumor cells, with the major clone resembling clinical parameters of the tumor. Similarly, a stemness-associated gene set identified clones with divergent stemness pathway activation within the same tumor. This refined expression profiling technique distinguished genes truly deregulated in cancer from genes that identify cellular precursors of tumors. Collectively, the assays presented here enable more precise identification of cancer-deregulated genes, allow for early identification of therapeutically targetable tumor cell subpopulations, and ultimately provide a refinement of precision therapeutics for cancer treatment. Cancer Res; 77(10); 2759–69. ©2017 AACR.

Discovery of inter-individual functional variations in gene regulatory elements and the observation that tumor and normal tissues of the same organ are in different differentiation states necessitate rethinking of gene expression based subclassification/characterization of tumors. To address this issue, we performed single cell genomics of breast tumors and adjacent-normal cells propagated using epithelial reprogramming growth conditions for a short duration. Epithelial cells analyzed were either unselected for specific subpopulation or phenotypically defined undifferentiated ALDH+/CD49f+/EpCAM+ luminal progenitors present in the normal breast, which express both basal and luminal-enriched genes. Expression of 93 genes that included PAM50 intrinsic subtype classifier and stemness-related genes was analyzed in 420 tumor and 284 adjacent-normal cells. ALDH+/CD49f+/EpCAM+ tumor and normal cells clustered differently compared to unselected tumor and normal cells. PAM50 genes-set analyses of ALDH+/CD49f+/EpCAM+ populations efficiently identified major and minor clones of tumor cells with the major clone resembling clinical parameters of the tumor. Similarly, stemness-associated gene set identified clones with divergent stemness pathway activation within the same patient sample. This refined technique distinguished genes truly deregulated in cancer from genes that identify cellular precursors of tumors. Collectively, assays presented here may enable precise identification of genes deregulated in cancer, early identification of therapeutically-targetable minor population of tumor cells, and eventually to refinement of precision therapeutics. Citation Format: Harikrishna Nakshatri, Manjushree Anjanappa, Angelo Cardoso, Lijun Cheng, Safa Mohamad, Andrea Gunawan, Susan Rice, Yan Dong, Lang Li, Edward Srour. Refining breast cancer characterization through single-cell analysis of ex vivo reprogrammed tumor and adjacent normal cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1753. doi:10.1158/1538-7445.AM2017-1753