Age-friendly communities during the time of COVID-19: a model for rapid community response.

With the COVID-19 epidemic disproportionately impacting older adults, cities across the United States (U.S.) and the world scrambled to meet the needs of their older residents. Members of the World Health Organization's Age-Friendly Communities (AFCs) network rely on cross-system community collaborations and resident voices to create age-friendly social, built, and service environments. These key elements of AFCs place them in a unique position to quickly identify needs of older residents, launch short-term targeted interventions, and support integration of new programs into existing systems for post-crisis sustainability. This essay discusses how one age-friendly community applied key tenets of the Centers for Disease Control's rapid response team model to meet the immediate, short-term needs of older residents for social connection, food, personal protective equipment (PPE), emergency preparedness, and technology utilization. Sustainability of the rapid response interventions was supported through the relationships and structures created by the AFC.

Guidelines to contain disease outbreaks are helpful when responding to outcomes of outbreaks.Age-friendly communities core values align with the tenants of disaster response.Age-friendly communities are well positioned to respond to the consequences of COVID-19.

Authentication Analysis of MT-4 Cells Distributed by the National Institutes of Health AIDS Reagent Program.

The MT-4 human T-cell line expresses HTLV-1 Tax and is permissive for replication of an HIV-1 gp41 mutant lacking the cytoplasmic tail. MT-4 cells (lot 150048), distributed by the NIH AIDS Reagent Program (NIH-ARP), were found to be Tax deficient and unable to host replication of the gp41-truncated HIV-1 mutant. These findings, together with short tandem repeat profiling, established that lot 150048 are not bona fide MT-4 cells.

Virus-Mediated Alterations in miRNA Factors and Degradation of Viral miRNAs by MCPIP1.

Kaposi's sarcoma-associated herpesvirus (KSHV), the causative agent of Kaposi's sarcoma, encodes 25 mature viral miRNAs. MCP-1-induced protein-1 (MCPIP1), a critical regulator of immune homeostasis, has been shown to suppress miRNA biosynthesis via cleavage of precursor miRNAs through its RNase domain. We demonstrate that MCPIP1 can directly cleave KSHV and EBV precursor miRNAs and that MCPIP1 expression is repressed following de novo KSHV infection. In addition, repression with siRNAs to MCPIP1 in KSHV-infected cells increased IL-6 and KSHV miRNA expression, supporting a role for MCPIP1 in IL-6 and KSHV miRNA regulation. We also provide evidence that KSHV miRNAs repress MCPIP1 expression by targeting the 3'UTR of MCPIP1. Conversely, expression of essential miRNA biogenesis components Dicer and TRBP is increased following latent KSHV infection. We propose that KSHV infection inhibits a negative regulator of miRNA biogenesis (MCPIP1) and up-regulates critical miRNA processing components to evade host mechanisms that inhibit expression of viral miRNAs. KSHV-mediated alterations in miRNA biogenesis represent a novel mechanism by which KSHV interacts with its host and a new mechanism for the regulation of viral miRNA expression.

Kaposi's Sarcoma-Associated Herpesvirus MicroRNAs Target GADD45B To Protect Infected Cells from Cell Cycle Arrest and Apoptosis.

Kaposi's sarcoma is one of the most common malignancies in HIV-infected individuals. The responsible agent, Kaposi's sarcoma-associated herpesvirus (KSHV; HHV8), expresses multiple microRNAs (miRNAs), but the targets and functions of these miRNAs are not completely understood. After infection in primary endothelial cells with KSHV, growth arrest DNA damage-inducible gene 45 beta (GADD45B) is one of the most repressed genes using genomic expression profiling. GADD45B was also repressed in mRNA expression profiling experiments when KSHV miRNAs were introduced to uninfected cells. We hypothesized that KSHV miRNAs target human GADD45B to protect cells from consequences of DNA damage, which can be triggered by viral infection. Expression of GADD45B protein is induced by the p53 activator, Nutlin-3, and KSHV miRNA-K9 inhibits this induction. In addition, Nutlin-3 increased apoptosis and cell cycle arrest based on flow cytometry assays. KSHV miR-K9 protected primary endothelial cells from apoptosis and cell cycle arrest following Nutlin-3 treatment. Similar protective phenotypes were seen for targeting GADD45B with short interfering RNAs (siRNAs), as with miR-K9. KSHV miR-K9 also decreased the protein levels of cleaved caspase-3, cleaved caspase-7, and cleaved poly(ADP-ribose) polymerase (PARP). In B lymphocytes latently infected with KSHV, specific inhibitors of KSHV miR-K9 led to increased GADD45B expression and apoptosis, indicating that miR-K9 is important for reducing apoptosis in infected cells. Furthermore, ectopic expression of GADD45B in KSHV-infected cells promoted apoptosis. Together, these results identify a new miRNA target and demonstrate that KSHV miRNAs are important for protecting infected cells from DNA damage responses. IMPORTANCE: Kaposi's sarcoma-associated herpesvirus is a leading cause of cancers in individuals with AIDS. Promoting survival of infected cells is essential for maintaining viral infections. A virus needs to combat various cellular defense mechanisms designed to eradicate the viral infection. One such response can include DNA damage response factors, which can promote an arrest in cell growth and trigger cell death. We used a new approach to search for human genes repressed by small nucleic acids (microRNAs) expressed by a gammaherpesvirus (KSHV), which identified a gene called GADD45B as a target of microRNAs. Repression of GADD45B, which is expressed in response to DNA damage, benefited survival of infected cells in response to a DNA damage response. This information could be used to design new treatments for herpesvirus infections.

Kaposi's sarcoma-associated herpesvirus microRNAs repress breakpoint cluster region protein expression, enhance Rac1 activity, and increase in vitro angiogenesis.

UNLABELLED: MicroRNAs (miRNAs) are small, ∼ 22-nucleotide-long RNAs that regulate gene expression posttranscriptionally. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 12 pre-miRNAs during latency, and the functional significance of these microRNAs during KSHV infection and their cellular targets have been emerging recently. Using a previously reported microarray profiling analysis, we identified breakpoint cluster region mRNA (Bcr) as a cellular target of the KSHV miRNA miR-K12-6-5p (miR-K6-5). Bcr protein levels were repressed in human umbilical vein endothelial cells (HUVECs) upon transfection with miR-K6-5 and during KSHV infection. Luciferase assays wherein the Bcr 3' untranslated region (UTR) was cloned downstream of a luciferase reporter showed repression in the presence of miR-K6-5, and mutation of one of the two predicted miR-K6-5 binding sites relieved this repression. Furthermore, inhibition or deletion of miR-K6-5 in KSHV-infected cells showed increased Bcr protein levels. Together, these results show that Bcr is a direct target of the KSHV miRNA miR-K6-5. To understand the functional significance of Bcr knockdown in the context of KSHV-associated disease, we hypothesized that the knockdown of Bcr, a negative regulator of Rac1, might enhance Rac1-mediated angiogenesis. We found that HUVECs transfected with miR-K6-5 had increased Rac1-GTP levels and tube formation compared to HUVECs transfected with control miRNAs. Knockdown of Bcr in latently KSHV-infected BCBL-1 cells increased the levels of viral RTA, suggesting that Bcr repression by KSHV might aid lytic reactivation. Together, our results reveal a new function for both KSHV miRNAs and Bcr in KSHV infection and suggest that KSHV miRNAs, in part, promote angiogenesis and lytic reactivation. IMPORTANCE: Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) infection is linked to multiple human cancers and lymphomas. KSHV encodes small nucleic acids (microRNAs) that can repress the expression of specific human genes, the biological functions of which are still emerging. This report uses a variety of approaches to show that a KSHV microRNA represses the expression of the human gene called breakpoint cluster region (Bcr). Repression of Bcr correlated with the activation of a protein previously shown to cause KS-like lesions in mice (Rac1), an increase in KS-associated phenotypes (tube formation in endothelial cells and vascular endothelial growth factor [VEGF] synthesis), and modification of the life cycle of the virus (lytic replication). Our results suggest that KSHV microRNAs suppress host proteins and contribute to KS-associated pathogenesis.

Proteomic screening of human targets of viral microRNAs reveals functions associated with immune evasion and angiogenesis.

Kaposi's sarcoma (KS) is caused by infection with Kaposi's sarcoma-associated herpesvirus (KSHV). The virus expresses unique microRNAs (miRNAs), but the targets and functions of these miRNAs are not completely understood. In order to identify human targets of viral miRNAs, we measured protein expression changes caused by multiple KSHV miRNAs using pulsed stable labeling with amino acids in cell culture (pSILAC) in primary endothelial cells. This led to the identification of multiple human genes that are repressed at the protein level, but not at the miRNA level. Further analysis also identified that KSHV miRNAs can modulate activity or expression of upstream regulatory factors, resulting in suppressed activation of a protein involved in leukocyte recruitment (ICAM1) following lysophosphatidic acid treatment, as well as up-regulation of a pro-angiogenic protein (HIF1α), and up-regulation of a protein involved in stimulating angiogenesis (HMOX1). This study aids in our understanding of miRNA mechanisms of repression and miRNA contributions to viral pathogenesis.

Seminar: Extracellular Vesicles as Mediators of Environmental Stress in Human Disease.

BACKGROUND: Extracellular vesicles (EVs), membrane-bound particles containing a variety of RNA types, DNA, proteins, and other macromolecules, are now appreciated as an important means of communication between cells and tissues, both in normal cellular physiology and as a potential indicator of cellular stress, environmental exposures, and early disease pathogenesis. Extracellular signaling through EVs is a growing field of research for understanding fundamental mechanisms of health and disease and for the potential for biomarker discovery and therapy development. EVs are also known to play important roles in mediating the effects of exposure to environmental stress. OBJECTIVES: This seminar addresses the application of new tools and approaches for EV research, developed in part through the National Institutes of Health (NIH) Extracellular RNA Communication Program, and reflects presentations and discussions from a workshop held 27-28 September 2021 by the National Institute of Environmental Health Sciences (NIEHS) and the National Center for Advancing Translational Sciences (NCATS) on "Extracellular Vesicles, Exosomes, and Cell-Cell Signaling in Response to Environmental Stress." The panel of experts discussed current research on EVs and environmental exposures, highlighted recent advances in EV isolation and characterization, and considered research gaps and opportunities toward identifying and characterizing the roles for EVs in environmentally related diseases, as well as the current challenges and opportunities in this field. DISCUSSION: The authors discuss the application of new experimental models, particularly organ-on-chip (OOC) systems and in vitro approaches and how these have the potential to extend findings in population-based studies of EVs in exposure-related diseases. Given the complex challenges of identifying cell-specific EVs related to environmental exposures, as well as the general heterogeneity and variability in EVs in blood and other accessible biological samples, there is a critical need for rigorous reporting of experimental methods and validation studies. The authors note that these efforts, combined with cross-disciplinary approaches, would ensure that future research efforts in environmental health studies on EV biomarkers are rigorous and reproducible. https://doi.org/10.1289/EHP12980.

Global mapping of binding sites for Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network analysis.

The Nrf2 (nuclear factor E2 p45-related factor 2) transcription factor responds to diverse oxidative and electrophilic environmental stresses by circumventing repression by Keap1, translocating to the nucleus, and activating cytoprotective genes. Nrf2 responses provide protection against chemical carcinogenesis, chronic inflammation, neurodegeneration, emphysema, asthma and sepsis in murine models. Nrf2 regulates the expression of a plethora of genes that detoxify oxidants and electrophiles and repair or remove damaged macromolecules, such as through proteasomal processing. However, many direct targets of Nrf2 remain undefined. Here, mouse embryonic fibroblasts (MEF) with either constitutive nuclear accumulation (Keap1(-/-)) or depletion (Nrf2(-/-)) of Nrf2 were utilized to perform chromatin-immunoprecipitation with parallel sequencing (ChIP-Seq) and global transcription profiling. This unique Nrf2 ChIP-Seq dataset is highly enriched for Nrf2-binding motifs. Integrating ChIP-Seq and microarray analyses, we identified 645 basal and 654 inducible direct targets of Nrf2, with 244 genes at the intersection. Modulated pathways in stress response and cell proliferation distinguish the inducible and basal programs. Results were confirmed in an in vivo stress model of cigarette smoke-exposed mice. This study reveals global circuitry of the Nrf2 stress response emphasizing Nrf2 as a central node in cell survival response.

Pivoting Novel Exosome-Based Technologies for the Detection of SARS-CoV-2.

The National Institutes of Health (NIH) launched the Rapid Acceleration of Diagnostics (RADx) initiative to meet the needs for COVID-19 diagnostic and surveillance testing, and to speed its innovation in the development, commercialization, and implementation of new technologies and approaches. The RADx Radical (RADx-Rad) initiative is one component of the NIH RADx program which focuses on the development of new or non-traditional applications of existing approaches, to enhance their usability, accessibility, and/or accuracy for the detection of SARS-CoV-2. Exosomes are a subpopulation of extracellular vesicles (EVs) 30-140 nm in size, that are critical in cell-to-cell communication. The SARS-CoV-2 virus has similar physical and molecular properties as exosomes. Therefore, the novel tools and technologies that are currently in development for the isolation and detection of exosomes, may prove to be invaluable in screening for SARS-CoV-2 viral infection. Here, we describe how novel exosome-based technologies are being pivoted for the detection of SARS-CoV-2 and/or the diagnosis of COVID-19. Considerations for these technologies as they move toward clinical validation and commercially viable diagnostics is discussed along with their future potential. Ultimately, the technologies in development under the NIH RADx-Rad exosome-based non-traditional technologies toward multi-parametric and integrated approaches for SARS-CoV-2 program represent a significant advancement in diagnostic technology, and, due to a broad focus on the biophysical and biochemical properties of nanoparticles, the technologies have the potential to be further pivoted as tools for future infectious agents.

Extracellular RNAs as potential biomarkers for cancer.

The discovery that all cells secrete extracellular vesicles (EVs) to shuttle proteins and nucleic acids to recipient cells suggested they play an important role in intercellular communication. EVs are widely distributed in many body fluids, including blood, cerebrospinal fluid, urine and saliva. Exosomes are nano-sized EVs of endosomal origin that regulate many pathophysiological processes including immune responses, inflammation, tumour growth, and infection. Healthy individuals release exosomes with a cargo of different RNA, DNA, and protein contents into the circulation, which can be measured non-invasively as biomarkers of healthy and diseased states. Cancer-derived exosomes carry a unique set of DNA, RNA, protein and lipid reflecting the stage of tumour progression, and may serve as diagnostic and prognostic biomarkers for various cancers. However, many gaps in knowledge and technical challenges in EVs and extracellular RNA (exRNA) biology, such as mechanisms of EV biogenesis and uptake, exRNA cargo selection, and exRNA detection remain. The NIH Common Fund-supported exRNA Communication Consortium was launched in 2013 to address major scientific challenges in this field. This review focuses on scientific highlights in biomarker discovery of exosome-based exRNA in cancer and its possible clinical application as cancer biomarkers.

DAMGO-induced expression of chemokines and chemokine receptors: the role of TGF-beta1.

Studies from a number of laboratories suggest that modulation of cytokine expression plays an integral role in the immunomodulatory activity of opioids. Previously, our laboratory reported that activation of the mu-opioid receptor induced the expression of CCL2, CCL5, and CXCL10, as well as CCR5 and CXCR4. Previous work has also suggested the possibility that TGF-beta may participate in the opioid-induced regulation of immune competence, and in the present study, we set out to determine the role of this cytokine in the control of chemokine and chemokine receptor expression. We found that D-ala(2),N-Me-Phe(4)-Gly-ol(5)enkephalin (DAMGO), a highly selective mu-opioid agonist, induced the expression of TGF-beta1 expression at the protein and mRNA levels. In turn, the addition of TGF-beta1 was found to induce CCL5 and CXCR4 expression but not CCL2, CXCL10, or CCR5. Further analysis showed that pretreatment with neutralizing anti-TGF-beta1 blocked the ability of DAMGO to induce CCL5 or CXCR4. Similarly, pretreatment with cycloheximide prevented CCL5 or CXCR4 mRNA expression, consistent with the observation that DAMGO induction of chemokine and chemokine receptor expression requires newly synthesized TGF-beta1 protein. These results describe a common molecular basis for the activation of chemokine and chemokine receptor expression and may permit the development of strategies to inhibit certain undesirable immunological properties of micro-opioid agonists such as morphine and heroin.

KSHV MicroRNAs Repress Tropomyosin 1 and Increase Anchorage-Independent Growth and Endothelial Tube Formation.

Kaposi's sarcoma (KS) is characterized by highly vascularized spindle-cell tumors induced after infection of endothelial cells by Kaposi's sarcoma-associated herpesvirus (KSHV). In KS tumors, KSHV expresses only a few latent proteins together with 12 pre-microRNAs. Previous microarray and proteomic studies predicted that multiple splice variants of the tumor suppressor protein tropomyosin 1 (TPM1) were targets of KSHV microRNAs. Here we show that at least two microRNAs of KSHV, miR-K2 and miR-K5, repress protein levels of specific isoforms of TPM1. We identified a functional miR-K5 binding site in the 3' untranslated region (UTR) of one TPM1 isoform. Furthermore, the inhibition or loss of miR-K2 or miR-K5 restores expression of TPM1 in KSHV-infected cells. TPM1 protein levels were also repressed in KSHV-infected clinical samples compared to uninfected samples. Functionally, miR-K2 increases viability of unanchored human umbilical vein endothelial cells (HUVEC) by inhibiting anoikis (apoptosis after cell detachment), enhances tube formation of HUVECs, and enhances VEGFA expression. Taken together, KSHV miR-K2 and miR-K5 may facilitate KSHV pathogenesis.

Transcription factor NRF2 regulates miR-1 and miR-206 to drive tumorigenesis.

The mechanisms by which deregulated nuclear factor erythroid-2-related factor 2 (NRF2) and kelch-like ECH-associated protein 1 (KEAP1) signaling promote cellular proliferation and tumorigenesis are poorly understood. Using an integrated genomics and ¹³C-based targeted tracer fate association (TTFA) study, we found that NRF2 regulates miR-1 and miR-206 to direct carbon flux toward the pentose phosphate pathway (PPP) and the tricarboxylic acid (TCA) cycle, reprogramming glucose metabolism. Sustained activation of NRF2 signaling in cancer cells attenuated miR-1 and miR-206 expression, leading to enhanced expression of PPP genes. Conversely, overexpression of miR-1 and miR-206 decreased the expression of metabolic genes and dramatically impaired NADPH production, ribose synthesis, and in vivo tumor growth in mice. Loss of NRF2 decreased the expression of the redox-sensitive histone deacetylase, HDAC4, resulting in increased expression of miR-1 and miR-206, and not only inhibiting PPP expression and activity but functioning as a regulatory feedback loop that repressed HDAC4 expression. In primary tumor samples, the expression of miR-1 and miR-206 was inversely correlated with PPP gene expression, and increased expression of NRF2-dependent genes was associated with poor prognosis. Our results demonstrate that microRNA-dependent (miRNA-dependent) regulation of the PPP via NRF2 and HDAC4 represents a novel link between miRNA regulation, glucose metabolism, and ROS homeostasis in cancer cells.

Opioid-induced chemokine expression requires NF-κB activity: the role of PKCζ.

Opioid receptor agonists induce broad immunomodulatory activity, which substantially alters host defense and the inflammatory response. Previous studies have shown that the MOR selective agonist DAMGO has the capacity to increase the expression of the proinflammatory chemokines CCL2, CCL5, and CXCL10 in human PBMCs. NF-κB is a transcription factor that plays a pivotal role in innate and adaptive immune responses. We report that NF-κB is a vital player in the DAMGO-induced, MOR-mediated regulation of chemokine expression. Results show that NF-κB inhibitors prevent the induction of CCL2 expression in response to DAMGO administration and that the NF-κB subunit, p65, is phosphorylated at serine residues 311 and 536 in response to MOR activation. Furthermore, we demonstrate that PKCζ is phosphorylated following DAMGO-induced MOR activation, and this kinase is essential for NF-κB activation as well as CCL2 expression and transcriptional activity. Finally, ChIP analysis shows that DAMGO administration induces binding of p65 to the enhancer region of the CCL2 promoter. These data are consistent with the notion that MOR activation promotes a proinflammatory response, which involves NF-κB activation. Our results also suggest a significant and novel role for PKCζ as an essential participant in the MOR-mediated regulation of proinflammatory chemokine expression.

Expression of ABCG2 (BCRP) is regulated by Nrf2 in cancer cells that confers side population and chemoresistance phenotype.

ATP-binding cassette, subfamily G, member 2 (ABCG2) is expressed in both normal and cancer cells and plays a crucial role in side population (SP) formation and efflux of xenobiotics and drugs. Nrf2, a redox-sensing transcription factor, on constitutive activation in non-small-cell lung cancer cells upregulates a wide spectrum of genes involved in redox balance, glutathione metabolism, and drug detoxification, which contribute to chemoresistance and tumorigenicity. This study examined the mechanism underlying Nrf2-dependent expression of ABCG2 and its role in the multidrug resistance phenotype. In silico analysis of the 5'-promoter flanking region of ABCG2 identified an antioxidant response element (ARE) at -431 to -420 bp. A detailed promoter analysis using luciferase reporter assays showed that ARE at -431 to -420 bp is critical for the Nrf2-mediated expression in lung cancer cells. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays revealed that Nrf2 interacts with the ABCG2 ARE element at -431 to -420 bp in vitro and in vivo. Disruption of Nrf2 expression in lung and prostate cancer cells, by short hairpin RNA, attenuated the expression of ABCG2 transcript and protein, and dramatically reduced the SP fraction in Nrf2-depleted cancer cells. Moreover, depleted levels of ABCG2 in these Nrf2 knockdown cells sensitized them to mitoxantrone and topotecan, two chemotherapy drugs detoxified mainly by ABCG2. As expected, overexpression of Nrf2 cDNA in lung epithelial cells led to an increase in ABCG2 expression and a 2-fold higher SP fraction. Thus, Nrf2-mediated regulation of ABCG2 expression maintains the SP fraction and confers chemoresistance.