An Evaluation of the Implementation of the Women, Infants, and Children (WIC) Farmers Market Nutrition Program at Four Chicago WIC Sites.

The Farmers Market Nutrition Program (FMNP), a program of the U.S. Department of Agriculture (USDA), provides coupons to individuals/families enrolled in the Special Supplemental Nutrition program for Women, Infants, and Children (WIC) to purchase fresh produce from approved farmers markets. While some studies suggest FMNP may improve nutrition for WIC clients, there is limited research on program implementation in practice. A mixed-methods equitable evaluation framework was utilized to: (1) better understand the FMNP in practice at four WIC clinics on Chicago's west and southwest sides that serve predominately Black and Latinx families; (2) detail facilitators and barriers to participation in the FMNP; and (3) describe the potential impact on nutrition. In this manuscript, we present qualitative findings from Aim 1. We identified six steps for how the FMNP has been implemented in our study setting and opportunities to improve the implementation of the program. Findings suggest that well-defined and consistent guidelines on: (1) how to seek state approval for farmers markets; and (2) coupon distribution and redemption, are necessary to optimize utilization. Future research should explore the impact of newly offered electronic coupons on redemption rates and fresh fruit and vegetable purchasing behaviors.

Implementation of an Evidence-Based Intervention with Safety Net Clinics to Improve Mammography Appointment Adherence Among Underserved Women.

The Peace of Mind Program is an evidence-based intervention to improve mammography appointment adherence in underserved women. The aim of this study was to assess effectiveness of the intervention and implementation of the intervention in safety net clinics. The intervention was implemented through a non-randomized stepped wedge cluster hybrid study design with 19 Federally Qualified Health Centers and charity care clinics within the Greater Houston area. A multivariable generalized estimating equation logistic regression was conducted to examine mammography appointment adherence. A survey assessing Consolidated Framework for Implementation Research constructs was also conducted with clinic staff prior to adoption and eight weeks post implementation. One-sided t-tests were conducted to analyze mean score changes between the surveys. A total of 4402 women (baseline period = 2078; intervention period = 2324) were included in the final regression analysis. Women in the intervention period were more likely to attend or reschedule their mammography appointment (OR = 1.30; p < 0.01) than those in the baseline period receiving usual care. Women who completed the intervention were more likely to attend or reschedule their mammography appointment than those who did not complete the intervention (OR = 1.62; p < 0.01). The mammography appointment no-show rates for those in the baseline period, in the intervention period, and who completed the intervention were, respectively, 22%, 19%, and 15%. A total of 15 clinics prior to adoption and eight clinics completed the survey at 8 weeks post implementation A statistically significant mean score decrease was observed in Inner Setting and in two Inner Setting CFIR constructs, Culture-Effort, and Implementation Climate. While the intervention improved mammography appointment adherence, there are opportunities to further integrate Consolidated Framework for Implementation Research constructs. Trial registration: Clinical trials registration number: NCT02296177.

Development, implementation, and evaluation of Teach Back curriculum for community health workers.

Teach Back is a commonly used communication method to improve patient understanding and retention of health information. The method has been shown to be effective in improving patient and healthcare system outcomes, including patient health literacy and hospital readmissions. Community health workers (CHWs) are frontline healthcare workers who can help address patient health and social needs associated with hospital readmissions. However, a gap exists in Teach Back curricula and training methods reflecting the scope of work for CHWs. The objective of this training was to provide CHWs with didactic information and skill building practice curriculum focused on the integration of Teach Back into clinical patient interactions, care coordination, and follow-up support. A multidisciplinary team of academic and clinical partners at a large academic health university developed, implemented, and evaluated a 3-week pilot Teach Back training with CHWs through a quality improvement approach. The CHWs reported overall satisfaction with the training and instructors. The academic clinical partnership allowed the training to be tailored to the daily clinical workflow as reflected in the CHWs agreement that the training was relevant and practical. With the repeated exposure to Teach Back each week, the CHWs also reported an increase in confidence and conviction in using Teach Back. Additional implementation and evaluation of the training curriculum for CHWs is needed to gain further insights into Teach Back and training best practices and translation into practice.

Young mice administered adult doses of AAV5-hFVIII-SQ achieve therapeutic factor VIII expression into adulthood.

Valoctocogene roxaparvovec (AAV5-hFVIII-SQ) gene transfer provided reduced bleeding for adult clinical trial participants with severe hemophilia A. However, pediatric outcomes are unknown. Using a mouse model of hemophilia A, we investigated the effect of vector dose and age at treatment on transgene production and persistence. We dosed AAV5-hFVIII-SQ to neonatal and adult mice based on body weight or at a fixed dose and assessed human factor VIII-SQ variant (hFVIII-SQ) expression through 16 weeks. AAV5-hFVIII-SQ dosed per body weight in neonatal mice did not result in meaningful plasma hFVIII-SQ protein levels in adulthood. When treated with the same total vector genomes per mouse as adult mice, neonates maintained hFVIII-SQ expression into adulthood, although plasma levels were 3- to 4-fold lower versus mice dosed as adults. Mice <1 week old initially exhibited high hFVIII-SQ plasma levels and maintained meaningful levels into adulthood, despite a partial decline potentially due to age-related body mass and blood volume increases. Spatial transduction patterns differed between mice dosed as neonates versus adults. No features of hepatotoxicity or endoplasmic reticulum stress were observed with dosing at any age. These data suggest that young mice require the same total vector genomes as adult mice to sustain hFVIII-SQ plasma levels.

Development of Training Curriculum to Improve Patient Communication Skills and Social Support Among Community Health Workers.

BACKGROUND: Effective provider communication skills are important for patient decision-making and understanding, particularly for those with low health literacy. A gap exists in training methods and curriculum for community health workers (CHWs). Brief description of activity: Through a clinical and academic partnership, pilot training curriculum focused on patient communication skills was developed to align with CHW scope of work. IMPLEMENTATION: The curriculum was implemented in three 2-hour training sessions over WebEx with seven state-certified CHWs. The goal was for CHWs to understand the key elements and application of active listening, Teach Back, and action planning in a clinical setting. The sessions included didactic and skills practice modules for each skill. RESULTS: A survey was distributed to CHWs to evaluate knowledge, skills, and attitudes and reactions to training methods, instructors, and relevance using the Kirkpatrick's evaluation model (Reaction and Learning). Although CHWs agreed that they had actively participated in the training and that the instructors were well-prepared, there was less agreement that the course was relevant. CHWs reported an increase in understanding of active listening and action planning, capability of using Teach Back and providing social support, and ability to teach, whereas a decrease was reported in the capability to use action planning. When probed about training relevance, CHWs felt action listening and Teach Back were relevant, but that action planning was not relevant to their responsibilities. This gap in responsibilities was also acknowledged by the clinical leadership. LESSONS LEARNED: The training allowed the CHWs to build on subsequent skills from previous sessions and to discuss struggles. A need for tools for integrating the skills in the clinical workflow were requested by CHWs and clinical leadership. These tools offer the opportunity to tailor future trainings on communication skills or patient scenarios. Future trainings should include CHWs to provide insight into scope of work. [HLRP: Health Literacy Research and Practice. 2022;6(2):e142-e150.] Plain Language Summary: It is important for community health workers to communicate with patients so that patients can understand information and make their own decisions. There is not enough known about the best way to train CHWs in patient communication. This training was created to help CHWs use three patient communication skills in their clinic.

Predicting health-related social needs in Medicaid and Medicare populations using machine learning.

Providers currently rely on universal screening to identify health-related social needs (HRSNs). Predicting HRSNs using EHR and community-level data could be more efficient and less resource intensive. Using machine learning models, we evaluated the predictive performance of HRSN status from EHR and community-level social determinants of health (SDOH) data for Medicare and Medicaid beneficiaries participating in the Accountable Health Communities Model. We hypothesized that Medicaid insurance coverage would predict HRSN status. All models significantly outperformed the baseline Medicaid hypothesis. AUCs ranged from 0.59 to 0.68. The top performance (AUC = 0.68 CI 0.66-0.70) was achieved by the "any HRSNs" outcome, which is the most useful for screening prioritization. Community-level SDOH features had lower predictive performance than EHR features. Machine learning models can be used to prioritize patients for screening. However, screening only patients identified by our current model(s) would miss many patients. Future studies are warranted to optimize prediction of HRSNs.

Association of Social Needs and Healthcare Utilization Among Medicare and Medicaid Beneficiaries in the Accountable Health Communities Model.

BACKGROUND: Integration of health-related social needs (HRSNs) data into clinical care is recognized as a driver for improving healthcare. However, few published studies on HRSNs and their impact are available. CMS sought to fill this gap through the Accountable Health Communities (AHC) Model, a national RCT of HRSN screening, referral, and navigation. Data from the AHC Model could significantly advance the field of HRSN screening and intervention in the USA. OBJECTIVE: To present data from the Greater Houston AHC (GH-AHC) Model site on HRSN frequency and the association between HRSNs, sociodemographic factors, and self-reported ED utilization using a cross-sectional design. Analyses included descriptive statistics and multinomial logistic regression. PARTICIPANTS (OR PATIENTS OR SUBJECTS): All community-dwelling Medicare, Medicaid, or dually covered beneficiaries at participating GH-AHC clinical delivery sites were eligible. MAIN MEASURES: Self-reported ED utilization in the previous 12 months served as the outcome; demographic characteristics including race, ethnicity, age, sex, income, education level, number of people living in the household, and insurance type were treated as covariates. HRSNs included food insecurity, housing instability, transportation, difficulty paying utility bills, and interpersonal safety. Clinical delivery site type was used as the clustering variable. KEY RESULTS: Food insecurity was the most common HRSN identified (38.7%) followed by housing instability (29.0%), transportation (28.0%), and difficulty paying utility bills (26.7%). Interpersonal safety was excluded due to low prevalence. More than half of the beneficiaries (56.9%) reported at least one of the four HRSNs. After controlling for covariates, having multiple co-occurring HRSNs was strongly associated with increased risk of two or more ED visits (OR 1.8-9.47 for two to four needs, respectively; p < 0.001). Beneficiaries with four needs were at almost 10 times higher risk of frequent ED utilization (p < 0.001). CONCLUSIONS: To our knowledge, this is only the second published study to report screening data from the AHC Model. Future research focused on the impact of multiple co-occurring needs on health outcomes is warranted.

Barriers and facilitators to implementation of the Accountable Health Communities (AHC) Model: Findings from a between-site qualitative assessment of implementation strategies.

Introduction: A multitude of HRSN interventions are undergoing testing in the U.S., with the CMS Accountable Health Communities (AHC) Model as the largest. HRSN interventions typically include screening for social needs, referral to community resources, and patient navigation to ensure needs are met. There is currently a paucity of evidence on implementation of HRSN interventions. The Consolidated Framework for Implementation Research (CFIR) is a determinant framework widely used to plan and assess implementation. To the authors knowledge, there are no published studies assessing CFIR constructs for HRSN intervention implementation in the U.S. In the Assessment step of the Strengthening Peer AHC Navigation (SPAN) model, a between-site qualitative assessment methodology was used to examine implementation within and between AHC bridge organizations (BOs) within six ERIC implementation strategies identified by the authors based on AHC Model requirements. Objective: Our aim was to identify and present between-site barriers and facilitators to AHC Model implementation strategies. Design: A multi-site qualitative analysis methodology was used. CFIR determinants were linked to six Expert Recommendations for Implementing Change (ERIC) strategies: staff training, identify and prepare champions, facilitation, community resource engagement (alignment through advisory boards and working groups), data systems, and quality monitoring and assurance. Interviews were analyzed using thematic content analysis in NVivo 12 (QSR International). Setting: Five health-related bridge organizations participating in the AHC Model. Results: Fifty-eight interviews were completed with 34 staff and 24 patients or patient proxies. Facilitators were identified across five of the six ERIC strategies. Barriers were identified across all six. While organizations found the AHC Model compatible and facilitators to implementation included previous experience, meeting patient needs and resources, and leadership engagement and support, a number of barriers presented challenges to implementation. Issues with adequate staff training, staff skills to resolve HRSN, including patient communication and boundary spanning, setting staff goals, beneficiary caseloads and measurement of progress, data infrastructure (including EHR), available resources to implement and differences in perceptions between clinical delivery site (CDS), and CSP of how to measure and resolve HRSN. Conclusions and relevance: The conduct of a pre-implementation readiness assessment benefited from identifying CFIR determinants linked to various ERIC implementation strategies.

Ultra-sensitive AAV capsid detection by immunocapture-based qPCR following factor VIII gene transfer.

Adeno-associated virus (AAV)-based gene therapy vectors are replication-incompetent and thus pose minimal risk for horizontal transmission or release into the environment. In studies with AAV5-FVIII-SQ (valoctocogene roxaparvovec), an investigational gene therapy for hemophilia A, residual vector DNA was detectable in blood, secreta, and excreta, but it remained unclear how long structurally intact AAV5 vector capsids were present. Since a comprehensive assessment of vector shedding is required by regulatory agencies, we developed a new method (termed iqPCR) that utilizes capsid-directed immunocapture followed by qPCR amplification of encapsidated DNA. The limit of detection for AAV5 vector capsids was 1.17E+04 and 2.33E+04 vg/mL in plasma and semen, respectively. Acceptable precision, accuracy, selectivity, and specificity were verified; up to 1.00E+09 vg/mL non-encapsidated vector DNA showed no interference. Anti-AAV5 antibody plasma concentrations above 141 ng/mL decreased AAV5 capsid quantification, suggesting that iqPCR mainly detects free capsids and not those complexed with antibodies. In a clinical study, AAV5-FVIII-SQ capsids were found in plasma and semen but became undetectable within nine weeks after dose administration. Hence, iqPCR monitors the presence and shedding kinetics of intact vector capsids following AAV gene therapy and informs the potential risk for horizontal transmission.

Stakeholder Engagement in Adoption, Implementation, and Sustainment of an Evidence-Based Intervention to Increase Mammography Adherence Among Low-Income Women.

Multi-level organizational stakeholder engagement plays an important role across the research process in a clinical setting. Stakeholders provide organizational specific adaptions in evidence-based interventions to ensure effective adoption, implementation, and sustainability. Stakeholder engagement strategies involve building mutual trust, providing clear communication, and seeking feedback. Using constructs from the Consolidated Framework for Implementation Research and The International Association for Public Participation spectrum, a conceptual framework was created to guide stakeholder engagement in an evidence-based intervention to increase mammography appointment adherence in underserved and low-income women. A document review was used to explore the alignment of the conceptual framework with intervention activities and stakeholder engagement strategies. The results indicate an alignment with the conceptual framework constructs and a real-world application of stakeholder engagement in a mammography evidence-based intervention. The conceptual framework and stakeholder engagement strategies can be applied across a range of community-based cancer programs and interventions, organizations, and clinical settings.

Adoption of an Evidence-Based Intervention for Mammography Screening Adherence in Safety Net Clinics.

Through an academic-community partnership, an evidence-based intervention to reduce mammography appointment no-show rates in underserved women was expanded to safety net clinics. The partnership implemented four strategies to improve the adoption and scale-up of evidence-based interventions with Federally Qualified Health Centers and charity care clinics: (1) an outreach email blast targeting the community partner member clinics to increase program awareness, (2) an adoption video encouraging enrollment in the program, (3) an outreach webinar educating the community partner member clinics about the program, encouraging enrollment and outlining adoption steps, and (4) an adoption survey adapted from Consolidated Framework for Implementation Research constructs from the Cancer Prevention and Control Research Network for cancer control interventions with Federally Qualified Health Centers. The development of academic-community partnerships can lead to successful adoption of evidence-based interventions particularly in safety net clinics.

Monitoring cell-mediated immune responses in AAV gene therapy clinical trials using a validated IFN-γ ELISpot method.

Adeno-associated virus (AAV)-based gene therapies have recently shown promise as a novel treatment for hereditary diseases. Due to the viral origin of the vector capsid, however, cellular immune response may be elicited that could eliminate transduced target cells. To monitor cellular immune responses in clinical trials, we optimized and bioanalytically validated a sensitive, robust, and reliable interferon-γ (IFN-γ) enzyme-linked immunospot (ELISpot) assay. For method performance validation, human peripheral blood mononuclear cells (PBMCs) were stimulated with peptides derived from AAV5 capsid proteins and the encoded transgene product, human blood clotting factor VIII (FVIII), in addition to positive controls, such as peptides from the 65-kDa phosphoprotein of cytomegalovirus. We statistically assessed the limit of detection and confirmatory cutpoint, evaluated precision and linearity, and confirmed specificity using HIV peptides. Robustness parameter ranges and sample stability periods were established. The validated IFN-γ ELISpot assay was then implemented in an AAV5-FVIII gene therapy clinical trial. Cellular immune responses against the AAV5 capsid were observed in most participants as soon as 2 weeks following dose administration; only limited responses against the transgene product were detected. These data underscore the value of using validated methods for monitoring cellular immunity in AAV gene therapy trials.

Developing and Evaluating a Quality Improvement Intervention to Facilitate Patient Navigation in the Accountable Health Communities Model.

Introduction: The Accountable Health Communities (AHC) Model was designed to address the health-related social needs of Centers for Medicare & Medicaid Services beneficiaries. Bridge organizations across the AHC Model have identified lack of technical assistance and peer planning as potential barriers to Model success, particularly around patient navigation. The technical assistance and peer planning literature lacks an organizing, conceptual framework, but implementation science frameworks could serve as useful guides. The Strengthening Peer AHC Navigation (SPAN) research protocol seeks to fill this gap and will apply three implementation science frameworks, Consolidated Framework for Implementation Research, Intervention Mapping, and the Expert Recommendations for Implementing Change compilation, to develop a multi-level quality improvement intervention and evaluate the impact of peer planning on Model outcomes. The aims of the SPAN study are to implement and evaluate a novel multi-level quality improvement intervention to improve AHC implementation and navigation milestones through structured peer planning and to provide successful technical assistance for the AHC Model. Methods and Analysis: The quality improvement intervention is outlined in four Tasks: (1) Assessment - to conduct an assessment of each bridge organization's current implementation, needs, and readiness in AHC Model navigation activities; (2) Planning - to engage in a peer planning approach to build capacity for AHC Model navigation activities; (3) Implementation with technical assistance - Co-creation of a quality improvement protocol for AHC Model navigation activities; and (4) Evaluation - measure the impact of the peer planning and technical assistance approach. Alongside the development and implementation of the quality improvement intervention, this protocol describes a mixed method, convergent parallel study design which will be used to evaluate whether the quality improvement intervention will lead to better outcomes. Tasks will be replicated with five bridge organizations participating in the AHC Model. Discussion: This research protocol provides a framework that can be used to conduct structured peer planning with technical assistance for social needs programs. This study will provide data on both implementation and outcomes which eventually may impact healthcare cost and utilization.

The Impact of Pre-existing Immunity on the Non-clinical Pharmacodynamics of AAV5-Based Gene Therapy.

Adeno-associated virus (AAV)-based vectors are widely used for gene therapy, but the effect of pre-existing antibodies resulting from exposure to wild-type AAV is unclear. In addition, other poorly defined plasma factors could inhibit AAV vector transduction where antibodies are not detected. To better define the relationship between various forms of pre-existing AAV immunity and gene transfer, we studied valoctocogene roxaparvovec (BMN 270) in cynomolgus monkeys with varying pre-dose levels of neutralizing anti-AAV antibodies and non-antibody transduction inhibitors. BMN 270 is an AAV5-based vector for treating hemophilia A that encodes human B domain-deleted factor VIII (FVIII-SQ). After infusion of BMN 270 (6.0 × 1013 vg/kg) into animals with pre-existing anti-AAV5 antibodies, there was a mean decrease in maximal FVIII-SQ plasma concentration (Cmax) and AUC of 74.8% and 66.9%, respectively, compared with non-immune control animals, and vector genomes in the liver were reduced. In contrast, animals with only non-antibody transduction inhibitors showed FVIII-SQ plasma concentrations and liver vector copies comparable with those of controls. These results demonstrate that animals without AAV5 antibodies are likely responders to AAV5 gene therapy, regardless of other inhibiting plasma factors. The biological threshold for tolerable AAV5 antibody levels varied between individual animals and should be evaluated further in clinical studies.

Integration-independent Transgenic Huntington Disease Fragment Mouse Models Reveal Distinct Phenotypes and Life Span in Vivo.

The cascade of events that lead to cognitive decline, motor deficits, and psychiatric symptoms in patients with Huntington disease (HD) is triggered by a polyglutamine expansion in the N-terminal region of the huntingtin (HTT) protein. A significant mechanism in HD is the generation of mutant HTT fragments, which are generally more toxic than the full-length HTT. The protein fragments observed in human HD tissue and mouse models of HD are formed by proteolysis or aberrant splicing of HTT. To systematically investigate the relative contribution of the various HTT protein proteolysis events observed in vivo, we generated transgenic mouse models of HD representing five distinct proteolysis fragments ending at amino acids 171, 463, 536, 552, and 586 with a polyglutamine length of 148. All lines contain a single integration at the ROSA26 locus, with expression of the fragments driven by the chicken ß-actin promoter at nearly identical levels. The transgenic mice N171-Q148 and N552-Q148 display significantly accelerated phenotypes and a shortened life span when compared with N463-Q148, N536-Q148, and N586-Q148 transgenic mice. We hypothesized that the accelerated phenotype was due to altered HTT protein interactions/complexes that accumulate with age. We found evidence for altered HTT complexes in caspase-2 fragment transgenic mice (N552-Q148) and a stronger interaction with the endogenous HTT protein. These findings correlate with an altered HTT molecular complex and distinct proteins in the HTT interactome set identified by mass spectrometry. In particular, we identified HSP90AA1 (HSP86) as a potential modulator of the distinct neurotoxicity of the caspase-2 fragment mice (N552-Q148) when compared with the caspase-6 transgenic mice (N586-Q148).

A genome-scale RNA-interference screen identifies RRAS signaling as a pathologic feature of Huntington's disease.

A genome-scale RNAi screen was performed in a mammalian cell-based assay to identify modifiers of mutant huntingtin toxicity. Ontology analysis of suppressor data identified processes previously implicated in Huntington's disease, including proteolysis, glutamate excitotoxicity, and mitochondrial dysfunction. In addition to established mechanisms, the screen identified multiple components of the RRAS signaling pathway as loss-of-function suppressors of mutant huntingtin toxicity in human and mouse cell models. Loss-of-function in orthologous RRAS pathway members also suppressed motor dysfunction in a Drosophila model of Huntington's disease. Abnormal activation of RRAS and a down-stream effector, RAF1, was observed in cellular models and a mouse model of Huntington's disease. We also observe co-localization of RRAS and mutant huntingtin in cells and in mouse striatum, suggesting that activation of R-Ras may occur through protein interaction. These data indicate that mutant huntingtin exerts a pathogenic effect on this pathway that can be corrected at multiple intervention points including RRAS, FNTA/B, PIN1, and PLK1. Consistent with these results, chemical inhibition of farnesyltransferase can also suppress mutant huntingtin toxicity. These data suggest that pharmacological inhibition of RRAS signaling may confer therapeutic benefit in Huntington's disease.

Caspase-6 activity in a BACHD mouse modulates steady-state levels of mutant huntingtin protein but is not necessary for production of a 586 amino acid proteolytic fragment.

Huntington's disease (HD) is caused by a mutation in the huntingtin (htt) gene encoding an expansion of glutamine repeats at the N terminus of the Htt protein. Proteolysis of Htt has been identified as a critical pathological event in HD models. In particular, it has been postulated that proteolysis of Htt at the putative caspase-6 cleavage site (at amino acid Asp-586) plays a critical role in disease progression and pathogenesis. However, whether caspase-6 is indeed the essential enzyme that cleaves Htt at this site in vivo has not been determined. To evaluate, we crossed the BACHD mouse model with a caspase-6 knock-out mouse (Casp6(-/-)). Western blot and immunocytochemistry confirmed the lack of caspase-6 protein in Casp6(-/-) mice, regardless of HD genotype. We predicted the Casp6(-/-) mouse would have reduced levels of caspase-6 Htt fragments and increased levels of full-length Htt protein. In contrast, we found a significant reduction of full-length mutant Htt (mHtt) and fragments in the striatum of BACHD Casp6(-/-) mice. Importantly, we detected the presence of Htt fragments consistent with cleavage at amino acid Asp-586 of Htt in the BACHD Casp6(-/-) mouse, indicating that caspase-6 activity cannot fully account for the generation of the Htt 586 fragment in vivo. Our data are not consistent with the hypothesis that caspase-6 activity is critical in generating a potentially toxic 586 aa Htt fragment in vivo. However, our studies do suggest a role for caspase-6 activity in clearance pathways for mHtt protein.

Identification and evaluation of small molecule pan-caspase inhibitors in Huntington's disease models.

Huntington's Disease (HD) is characterized by a mutation in the huntingtin (Htt) gene encoding an expansion of glutamine repeats on the N terminus of the Htt protein. Numerous studies have identified Htt proteolysis as a critical pathological event in HD postmortem human tissue and mouse HD models, and proteases known as caspases have emerged as attractive HD therapeutic targets. We report the use of the substrate activity screening method against caspase-3 and -6 to identify three novel, pan-caspase inhibitors that block proteolysis of Htt at caspase-3 and -6 cleavage sites. In HD models these irreversible inhibitors suppressed Hdh(111Q/111Q)-mediated toxicity and rescued rat striatal and cortical neurons from cell death. In this study, the identified nonpeptidic caspase inhibitors were used to confirm the role of caspase-mediated Htt proteolysis in HD. These results further implicate caspases as promising targets for HD therapeutic development.

Matrix metalloproteinases are modifiers of huntingtin proteolysis and toxicity in Huntington's disease.

Proteolytic cleavage of huntingtin (Htt) is known to be a key event in the pathogenesis of Huntington's disease (HD). Our understanding of proteolytic processing of Htt has thus far focused on the protease families-caspases and calpains. Identifying critical proteases involved in Htt proteolysis and toxicity using an unbiased approach has not been reported. To accomplish this, we designed a high-throughput western blot-based screen to examine the generation of the smallest N-terminal polyglutamine-containing Htt fragment. We screened 514 siRNAs targeting the repertoire of human protease genes. This screen identified 11 proteases that, when inhibited, reduced Htt fragment accumulation. Three of these belonged to the matrix metalloproteinase (MMP) family. One family member, MMP-10, directly cleaves Htt and prevents cell death when knocked down in striatal Hdh(111Q/111Q) cells. Correspondingly, MMPs are activated in HD mouse models, and loss of function of Drosophila homologs of MMPs suppresses Htt-induced neuronal dysfunction in vivo.

Testing the validity of microwave-interfaced, in situ Raman spectroscopy as a tool for kinetic studies.

Raman spectroscopy in conjunction with microwave heating is a convenient and robust tool for monitoring organic reactions from a qualitative perspective. Its validity as a method for obtaining quantitative data is shown. Activation enthalpies for the synthesis of a range of substituted chalcones are determined and the results compared with well-established previous data and reactivity trends for this aldol condensation. The methodology is used for obtaining previously unreported pK(a) data for substituted acetophenones.