Decreasing the intrinsically disordered protein α-synuclein levels by targeting its structured mRNA with a ribonuclease-targeting chimera.

α-Synuclein is an important drug target for the treatment of Parkinson's disease (PD), but it is an intrinsically disordered protein lacking typical small-molecule binding pockets. In contrast, the encoding SNCA mRNA has regions of ordered structure in its 5' untranslated region (UTR). Here, we present an integrated approach to identify small molecules that bind this structured region and inhibit α-synuclein translation. A drug-like, RNA-focused compound collection was studied for binding to the 5' UTR of SNCA mRNA, affording Synucleozid-2.0, a drug-like small molecule that decreases α-synuclein levels by inhibiting ribosomes from assembling onto SNCA mRNA. This RNA-binding small molecule was converted into a ribonuclease-targeting chimera (RiboTAC) to degrade cellular SNCA mRNA. RNA-seq and proteomics studies demonstrated that the RiboTAC (Syn-RiboTAC) selectively degraded SNCA mRNA to reduce its protein levels, affording a fivefold enhancement of cytoprotective effects as compared to Synucleozid-2.0. As observed in many diseases, transcriptome-wide changes in RNA expression are observed in PD. Syn-RiboTAC also rescued the expression of ~50% of genes that were abnormally expressed in dopaminergic neurons differentiated from PD patient-derived iPSCs. These studies demonstrate that the druggability of the proteome can be expanded greatly by targeting the encoding mRNAs with both small molecule binders and RiboTAC degraders.

miR-182 targeting reprograms tumor-associated macrophages and limits breast cancer progression.

The protumor roles of alternatively activated (M2) tumor-associated macrophages (TAMs) have been well established, and macrophage reprogramming is an important therapeutic goal. However, the mechanisms of TAM polarization remain incompletely understood, and effective strategies for macrophage targeting are lacking. Here, we show that miR-182 in macrophages mediates tumor-induced M2 polarization and can be targeted for therapeutic macrophage reprogramming. Constitutive miR-182 knockout in host mice and conditional knockout in macrophages impair M2-like TAMs and breast tumor development. Targeted depletion of macrophages in mice blocks the effect of miR-182 deficiency in tumor progression while reconstitution of miR-182-expressing macrophages promotes tumor growth. Mechanistically, cancer cells induce miR-182 expression in macrophages by TGFß signaling, and miR-182 directly suppresses TLR4, leading to NFκb inactivation and M2 polarization of TAMs. Importantly, therapeutic delivery of antagomiR-182 with cationized mannan-modified extracellular vesicles effectively targets macrophages, leading to miR-182 inhibition, macrophage reprogramming, and tumor suppression in multiple breast cancer models of mice. Overall, our findings reveal a crucial TGFß/miR-182/TLR4 axis for TAM polarization and provide rationale for RNA-based therapeutics of TAM targeting in cancer.

Reversibly Reactive Affinity Selection-Mass Spectrometry Enables Identification of Covalent Peptide Binders.

Covalent peptide binders have found applications as activity-based probes and as irreversible therapeutic inhibitors. Currently, there is no rapid, label-free, and tunable affinity selection platform to enrich covalent reactive peptide binders from synthetic libraries. We address this challenge by developing a reversibly reactive affinity selection platform termed ReAct-ASMS enabled by tandem high-resolution mass spectrometry (MS/MS) to identify covalent peptide binders to native protein targets. It uses mixed disulfide-containing peptides to build reversible peptide-protein conjugates that can enrich for covalent variants, which can be sequenced by MS/MS after reduction. Using this platform, we identified covalent peptide binders against two oncoproteins, human papillomavirus 16 early protein 6 (HPV16 E6) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 protein (Pin1). The resulting peptide binders efficiently and selectively cross-link Cys58 of E6 at 37 °C and Cys113 of Pin1 at room temperature, respectively. ReAct-ASMS enables the identification of highly selective covalent peptide binders for diverse molecular targets, introducing an applicable platform to assist preclinical therapeutic development pipelines.

Service Provider Perspectives on Advance Care Planning Use in Rural Dementia Patients and Caregivers: A Qualitative Study.

Advanced care planning (ACP) utilization remains very limited in rural communities compared to urban areas. ACP earlier in the disease trajectory is particularly important for people with dementia (PWD) due to its progressive nature affecting their decision-making ability. Considering the well-documented benefits of ACP in improving the quality of end-of-life (EOL) care, the rural vs. urban disparity may indicate poorer EOL quality for rural PWD. This study aimed to explore barriers and current resources for ACP of PWD from the perspectives of health or social service providers serving rural communities. Using a qualitative approach, semi-structured face-to-face interviews were conducted with 11 health or social service professionals serving older adults and their caregivers in rural Alabama. Thematic analysis revealed three major barriers: (1) lack of knowledge, (2) psychosocial barriers, and (3) limited access to healthcare. Participants also showed misconception that a lawyer or a notary is required for ACP. Two themes arose in the participants' recommendations to address the barriers: (1) providing ACP-relevant information and (2) addressing psychosocial stressors about ACP. This study highlighted an urgent need for social policy in ACP education for caregivers and service providers in rural settings.

Logotherapy-Based Interventions for Chinese Family Caregivers of Older Adults with Dementia Through Online Groups: A Mixed-Methods Study.

Chinese family caregivers of people with dementia (PWD) can suffer from physical and psychological burden. This study aimed to examine the effects of logotherapy-based interventions on Chinese family caregivers of older adults with dementia to decrease caregiver burden. This mixed-methods study used a pre-experimental design with pre-posttests and semi-structured interviews. A purposive sample of 13 family caregivers from a suburban district in Shanghai was enrolled with (1) caregiver burden and (2) access and capability to use smart devices. Participants received eight online group logotherapy sessions with a focus on hope and meaning construction. Participants completed the Zarit Burden Interview, a 22-item measure of caregiver burden, before and after the intervention, and a 30-min semi-structured interview post-intervention. From the quantitative data, dementia caregivers reported severe caregiving burdens at the baseline (M = 54.77, SD = 9.33). Caregiver burden significantly decreased after the logotherapy-based intervention (M = 52.15, SD = 8.80, p < .001). Two themes pertaining to participants' experiences in intervention emerged from the qualitative data: (1) improved attitudes toward suffering, and (2) enhanced sense of meaning in life and hope. The cultural relevance of logotherapy to Chinese familism and Confucianism may further enhance its feasibility in the Chinese context.

A general fragment-based approach to identify and optimize bioactive ligands targeting RNA.

RNAs have important functions that are dictated by their structure. Indeed, small molecules that interact with RNA structures can perturb function, serving as chemical probes and lead medicines. Here we describe the development of a fragment-based approach to discover and optimize bioactive small molecules targeting RNA. We extended the target validation method chemical cross-linking and isolation by pull-down (Chem-CLIP) to identify and map the binding sites of low molecular weight fragments that engage RNA or Chem-CLIP fragment mapping (Chem-CLIP-Frag-Map). Using Chem-CLIP-Frag-Map, we identified several fragments that bind the precursor to oncogenic microRNA-21 (pre-miR-21). Assembly of these fragments provided a specific bioactive compound with improved potency that inhibits pre-miR-21 processing, reducing mature miR-21 levels. The compound exerted selective effects on the transcriptome and selectively mitigated a miR-21-associated invasive phenotype in triple-negative breast cancer cells. The Chem-CLIP-Frag-Map approach should prove general to expedite the identification and optimization of small molecules that bind RNA targets.

Translation of the intrinsically disordered protein α-synuclein is inhibited by a small molecule targeting its structured mRNA.

Many proteins are refractory to targeting because they lack small-molecule binding pockets. An alternative to drugging these proteins directly is to target the messenger (m)RNA that encodes them, thereby reducing protein levels. We describe such an approach for the difficult-to-target protein α-synuclein encoded by the SNCA gene. Multiplication of the SNCA gene locus causes dominantly inherited Parkinson's disease (PD), and α-synuclein protein aggregates in Lewy bodies and Lewy neurites in sporadic PD. Thus, reducing the expression of α-synuclein protein is expected to have therapeutic value. Fortuitously, the SNCA mRNA has a structured iron-responsive element (IRE) in its 5' untranslated region (5' UTR) that controls its translation. Using sequence-based design, we discovered small molecules that target the IRE structure and inhibit SNCA translation in cells, the most potent of which is named Synucleozid. Both in vitro and cellular profiling studies showed Synucleozid directly targets the α-synuclein mRNA 5' UTR at the designed site. Mechanistic studies revealed that Synucleozid reduces α-synuclein protein levels by decreasing the amount of SNCA mRNA loaded into polysomes, mechanistically providing a cytoprotective effect in cells. Proteome- and transcriptome-wide studies showed that the compound's selectivity makes Synucleozid suitable for further development. Importantly, transcriptome-wide analysis of mRNAs that encode intrinsically disordered proteins revealed that each has structured regions that could be targeted with small molecules. These findings demonstrate the potential for targeting undruggable proteins at the level of their coding mRNAs. This approach, as applied to SNCA, is a promising disease-modifying therapeutic strategy for PD and other α-synucleinopathies.

Branched Multimeric Peptides as Affinity Reagents for the Detection of α-Klotho Protein.

α-Klotho, an aging-related protein found in the kidney, parathyroid gland, and choroid plexus, acts as an essential co-receptor with the fibroblast growth factor 23 receptor complex to regulate serum phosphate and vitamin D levels. Decreased levels of α-Klotho are a hallmark of age-associated diseases. Detecting or labeling α-Klotho in biological milieu has long been a challenge, however, hampering the understanding of its role. Here, we developed branched peptides by single-shot parallel automated fast-flow synthesis that recognize α-Klotho with improved affinity relative to their monomeric versions. These peptides were further shown to selectively label Klotho for live imaging in kidney cells. Our results demonstrate that automated flow technology enables rapid synthesis of complex peptide architectures, showing promise for future detection of α-Klotho in physiological settings.

Serum and glucocorticoid-regulated kinase 1 regulates transforming growth factor ß1-connective tissue growth factor pathway in chronic rhinosinusitis.

PURPOSE: Serum/glucocorticoid-regulated kinase 1 (SGK1) has been identified as a crucial regulator in fibrotic disorders. Herein, we explored SGK1 role in tissue remodeling of chronic rhinosinusitis (CRS). METHODS: Lentivirus was employed to generate an SGK1-overexpressing human bronchial epithelial cell (16HBE) line. To screen SGK1 downstream genes, RNA sequencing was performed on SGK1-overexpressing and control cell lines. To determine protein and gene expression levels, immunohistochemistry, western blotting, and quantitative real-time polymerase chain reaction were employed. Correlation analysis was performed using mRNA expression levels of SGK1, transforming growth factor ß1 (TGF-ß1), and connective tissue growth factor (CTGF) derived from CRS mucosal tissue and GEO database. Gene set enrichment analysis was conducted using gene sets from Molecular Signatures Database. The severity of symptoms in CRS patients was assessed using the 22-Item Sinonasal Outcome Test. RESULTS: SGK1 overexpression significantly increased the expression of connective tissue growth factor (CTGF) in 16HBE cells (P < 0.01). Consistently, CTGF protein level was considerably greater in mucosal tissue of CRS without nasal polyps (CRSsNP) than in CRS with nasal polyps (CRSwNP) (P < 0.05) or in control subjects (P < 0.01). TGF-ß1 protein level was higher in mucosal tissue of CRSsNP patients than in CRSwNP patients (P < 0.001) or in the control group (P < 0.01). mRNA levels of SGK1 and CTGF (P < 0.05, r = 0.668; P = 0.001, r = 0.630), TGF-ß1 and CTGF (P < 0.05, r = 0.560; P < 0.05, r = 0.420), as well as SGK1 and TGF-ß1(P < 0.05, r = 0.612; P < 0.05, r = 0.524) were significantly correlated in CRS mucosal tissue and GSE36830 dataset, respectively. TGF-ß1-induced upregulated genes were significantly enriched in SGK1 overexpression group. In vitro assays, TGF-ß1 promoted SGK1 and CTGF expression in a concentration- and time-dependent manner. Administrating an SGK1 inhibitor, GSK650394, significantly inhibited TGF-ß1-induced CTGF expression in 16HBE and dispersed primary nasal polyp cells. CONCLUSIONS: TGF-ß1 stimulation significantly increases SGK1 and CTGF expression. By regulating TGF-ß1-CTGF pathway, SGK1 may participate in tissue remodeling in the pathological mechanism of CRS.

Reprogramming of Protein-Targeted Small-Molecule Medicines to RNA by Ribonuclease Recruitment.

Reprogramming known medicines for a novel target with activity and selectivity over the canonical target is challenging. By studying the binding interactions between RNA folds and known small-molecule medicines and mining the resultant dataset across human RNAs, we identified that Dovitinib, a receptor tyrosine kinase (RTK) inhibitor, binds the precursor to microRNA-21 (pre-miR-21). Dovitinib was rationally reprogrammed for pre-miR-21 by using it as an RNA recognition element in a chimeric compound that also recruits RNase L to induce the RNA's catalytic degradation. By enhancing the inherent RNA-targeting activity and decreasing potency against canonical RTK protein targets in cells, the chimera shifted selectivity for pre-miR-21 by 2500-fold, alleviating disease progression in mouse models of triple-negative breast cancer and Alport Syndrome, both caused by miR-21 overexpression. Thus, targeted degradation can dramatically improve selectivity even across different biomolecules, i.e., protein versus RNA.

COVID-19 Family Support Team: Providing Person and Family Centered Care During the COVID-19 Pandemic.

The arrival of COVID-19 brought about many disruptions to our world and communities. The hospital visitation restrictions were one of the more, if not most, challenging aspects of psychosocial care for patients and families. It was difficult to anticipate the emotional toll that visitation restrictions would take on patients, families, and staff. Once hospital visitation restrictions were in place, new strategies for patient/family connection and team communication had to be established. The Palliative Care team at a large, urban, Midwestern academic medical center created an interprofessional Family Support Team in the spring of 2020 to address the psychosocial needs of the families of critically ill COVID-19 patients.

Design, Optimization, and Study of Small Molecules That Target Tau Pre-mRNA and Affect Splicing.

Approximately 95% of human genes are alternatively spliced, and aberrant splicing events can cause disease. One pre-mRNA that is alternatively spliced and linked to neurodegenerative diseases is tau (microtubule-associated protein tau), which can cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) and can contribute to Alzheimer's disease. Here, we describe the design of structure-specific lead small molecules that directly target tau pre-mRNA from sequence. This was followed by hit expansion and analogue synthesis to further improve upon these initial lead molecules. The emergent compounds were assessed for functional activity in a battery of assays, including binding assays and an assay that mimics molecular recognition of tau pre-mRNA by a U1 small nuclear ribonucleoprotein (snRNP) splicing factor. Compounds that emerged from these studies had enhanced potency and selectivity for the target RNA relative to the initial hits, while also having significantly improved drug-like properties. The compounds are shown to directly target tau pre-mRNA in cells, via chemical cross-linking and isolation by pull-down target profiling, and to rescue disease-relevant splicing of tau pre-mRNA in a variety of cellular systems, including primary neurons. More broadly, this study shows that lead, structure-specific compounds can be designed from sequence and then further optimized for their physicochemical properties while at the same time enhancing their activity.

Using Genome Sequence to Enable the Design of Medicines and Chemical Probes.

Rapid progress in genome sequencing technology has put us firmly into a postgenomic era. A key challenge in biomedical research is harnessing genome sequence to fulfill the promise of personalized medicine. This Review describes how genome sequencing has enabled the identification of disease-causing biomolecules and how these data have been converted into chemical probes of function, preclinical lead modalities, and ultimately U.S. Food and Drug Administration (FDA)-approved drugs. In particular, we focus on the use of oligonucleotide-based modalities to target disease-causing RNAs; small molecules that target DNA, RNA, or protein; the rational repurposing of known therapeutic modalities; and the advantages of pharmacogenetics. Lastly, we discuss the remaining challenges and opportunities in the direct utilization of genome sequence to enable design of medicines.

Development of bioorthogonal reactions and their applications in bioconjugation.

Biomolecule labeling using chemical probes with specific biological activities has played important roles for the elucidation of complicated biological processes. Selective bioconjugation strategies are highly-demanded in the construction of various small-molecule probes to explore complex biological systems. Bioorthogonal reactions that undergo fast and selective ligation under bio-compatible conditions have found diverse applications in the development of new bioconjugation strategies. The development of new bioorthogonal reactions in the past decade has been summarized with comments on their potentials as bioconjugation method in the construction of various biological probes for investigating their target biomolecules. For the applications of bioorthogonal reactions in the site-selective biomolecule conjugation, examples have been presented on the bioconjugation of protein, glycan, nucleic acids and lipids.

Previous Experience in Medical Decision Making and Advance Care Planning Conversations: Findings From a Nationwide Cross-Sectional Survey.

Background: Despite the extensive literature on facilitating advance care planning (ACP) with patients with serious illnesses, opportunities to include surrogates or family caregivers are overlooked. The research objective was to examine whether previous medical decision-making involvement for a loved one is associated with one's own ACP conversations with family and the potential mediating effect of knowledge of a loved one's end-of-life treatment preferences. Methods: This study employed a cross-sectional design using data from the 2016 Kaiser Family Foundation/The Economist Four-Country Survey on Aging and End-of-Life Medical Care. The sample included 627 US adults who completed the survey and were involved in making medical decisions for a loved one in the past. Multiple binary logistic regression and linear regression models were established for mediation analyses. Results: Participants in our nationally derived sample were largely confident in their knowledge of a deceased loved one's end-of-life treatment preferences. 66.8% of the sample had ACP conversations with family. The involvement in a loved one's medical decision making was significantly associated with higher odds of having ACP conversations with family (OR = 1.93, P = .01), but this relationship was significantly mediated by knowledge of one's end-of-life treatment preferences (b = .31, Boot CI = .12-.49). Conclusions: Previous experience in making medical decisions for a loved one may facilitate one's own ACP behavior through knowing a loved one's end-of-life treatment preferences. Clinicians in end-of-life settings are uniquely positioned to engage family members who were involved in medical decision-making for others before in ACP.

Spanish Translation and Dissemination of EMPOWER Materials to Address Barriers to Pain Management at the End of Life.

Introduction: The Effective Management of Pain by Overcoming Worries to Enable Relief (EMPOWER) intervention is an evidence-supported approach for addressing barriers to pain management (e.g., patient/family concerns about addiction) at the end of life. Such barriers appear more pronounced among Spanish-speaking individuals. This study aimed to (1) translate EMPOWER materials into Spanish, (2) disseminate materials to hospices with ≥25% Hispanic patients, and (3) survey hospices about the use and usefulness of materials. Methods: We back translated EMPOWER materials with harmonization, then disseminated materials to 242 hospices. Thereafter, we used a semistructured survey to assess use and usefulness of EMPOWER materials using univariate statistics and content analysis. Results: Thirty-eight hospice representatives responded (participation rate = 15.7%). Respondents were primarily non-White (55.3%) and Hispanic (60.5%). Nealy half (47.4%) were nurses. A majority (81.6%) indicated they currently employ ≥1 full-time English-Spanish bilingual team member. Among those who reported receiving the EMPOWER materials (n = 29), 58.6% indicated they-or another staff member-used them with patients or families. Using a single-item rating (0 = not useful to 10 = very useful), respondents evaluated the English EMPOWER materials' usefulness as 7.6 (standard deviation [SD] = 1.4) and Spanish materials as 8.4 (SD = 1.4). Most (62.1%) indicated they would likely use EMPOWER materials in the future. Conclusion: Thematic findings suggest EMPOWER reinforces clinical education, promotes discussion about pain management, and helps address culturally specific barriers to care. EMPOWER appears to be a useful, easy to use, and promising intervention that can be implemented among both English- and Spanish-speaking populations.

Development of an α-Klotho Recognizing High-Affinity Peptide Probe from In-Solution Enrichment.

The kidney, parathyroid gland, and choroid plexus express the aging-related transmembrane protein α-Klotho, a coreceptor of the fibroblast growth factor 23 (FGF23) receptor complex. Reduced α-Klotho levels are correlated with chronic kidney disease and other age-related diseases, wherein they are released from membranes into circulation. Klotho's potential physiological action as a hormone is of current scientific interest. Part of the challenges associated with advancing these studies, however, has been the long-standing difficulty in detecting soluble α-Klotho in biofluids. Here, we describe the discovery of peptides that recognize α-Klotho with high affinity and selectivity by applying in-solution size-exclusion-based affinity selection-mass spectrometry (AS-MS). After two rounds of AS-MS and subsequent N-terminal modifications, the peptides improved their binding affinity to α-Klotho by approximately 2300-fold compared to the reported starting peptide Pep-10, previously designed based on the C-terminal region of FGF23. The lead peptide binders were shown to enrich α-Klotho from cell lysates and to label α-Klotho in kidney cells. Our results further support the utility of in-solution, label-free AS-MS protocols to discover peptide-based binders to target proteins of interest with high affinity and selectivity, resulting in functional probes for biological studies.

High performance GaN-based LEDs on patterned sapphire substrate with patterned composite SiO2/Al2O3 passivation layers and TiO2/Al2O3 DBR backside reflector.

GaN-based light-emitting diodes (LEDs) on patterned sapphire substrate (PSS) with patterned composite SiO(2)/Al(2)O(3) passivation layers and TiO(2)/Al(2)O(3) distributed Bragg reflector (DBR) backside reflector have been proposed and fabricated. Highly passivated Al(2)O(3) layer deposited on indium tin oxide (ITO) layer with excellent uniformity and quality has been achieved with atomic layer deposition (ALD) technology. With a 60 mA current injection, an enhancement of 21.6%, 59.7%, and 63.4% in the light output power (LOP) at 460 nm wavelength was realized for the LED with the patterned composite SiO(2)/Al(2)O(3) passivation layers, the LED with the patterned composite SiO(2)/Al(2)O(3) passivation layers and Ag mirror + 3-pair TiO(2)/SiO(2) DBR backside reflector, and the LED with the patterned composite SiO(2)/Al(2)O(3) passivation layer and Ag mirror + 3-pair ALD-grown TiO(2)/Al(2)O(3) DBR backside reflector as compared with the conventional LED only with a single SiO(2) passivation layer, respectively.

When Living Wills go Missing: Associations With Hospice Use and Hospital Death Using National Longitudinal Data.

Despite documented benefits of advance care planning (ACP), understandings about the impact of advance directives (AD)-critical steps in the ACP process-remain limited. As a type of AD, living wills (LWs) are often misplaced or forgotten about. This study explores the prevalence of missing LWs among older adults in the U.S. and its association with in-hospital death and hospice care use. Analyses are based on 692 participants who responded to LW completion questions in the 2016 Core and 2018 wave of the Health and Retirement Study Nearly a fifth of American older adults age 65+ had an LW that went missing. Stepwise logistic regression results show that completed LWs confirmed by both individuals and their proxies were associated with increased hospice use and fewer hospital deaths. Individuals whose LWs went missing still had better end-of-life care than those who did not have an LW.

Perceived Barriers and Social Cultural Factors Associated With Advance Care Planning Conversations Among Chinese American Older Adults.

Despite the well-documented benefits of advance care planning (ACP), persistent racial and ethnic disparities continue to exist in ACP engagement. Guided by a social ecological model, this study examined perceived barriers and sociocultural factors associated with informal ACP conversations among Chinese American older adults. A purposive sample of 281 community-dwelling older Chinese Americans aged 55 years or older in Arizona and Maryland completed a survey in 2018. Hierarchical logistic regression models were conducted. There were 26.5% of participants who engaged in advance care planning. Lower perceived barriers and sociocultural factors (i.e., length of stay in the U.S. and English language proficiency) were positively associated with ACP conversations. Social support had a significant moderation effect. Findings highlighted the importance of language services and social support in facilitating ACP discussions among older Chinese immigrants. Effective strategies are needed to reduce the barriers to ACP at various levels for older Chinese American populations.