Translating policy to practice: Theory-based formative research to improve EC OTC access and messaging in Italy

Background: Italy\u27s 2015 emergency contraception (EC) policy has increased access and reduced some barriers for women to obtain EC. EC is now available over-the-counter for individuals 18 years and older; however, women living in Italy continue to face knowledge and access barriers. Conscientious objection, where providers and pharmacists refuse to prescribe or dispense EC due to personal beliefs, further complicates access and dissemination. Objective: The purpose of the current paper is to understand EC knowledge, attitudes, and behaviors among women living in Italy. Additionally, a secondary purpose is to explore the impact of the 2015 EC policy. Methods: Thirty in-person interviews were conducted among women living in or around Florence, aged 18 to 50 years, and using the Italian healthcare system at the time of study enrollment. Researchers used an expanded grounded theory approach to understand women\u27s experiences with EC with diffusion of innovations serving as a conceptual lens for data analysis. HyperRESEARCH, a data management system, assisted with open and axial coding and theme development. Results: Women described low observability of the 2015 policy, expressing surprise regarding increased EC availability. Participants suggested increased messaging in strategic locations to overcome this barrier. Participants held both positive and negative attitudes toward EC. While some perceived the relative advantage of EC compared with unintended pregnancy, others expressed concerns about irresponsibility and EC safety. Finally, conscientious objection impacted healthcare access, despite participant desire for autonomous EC decision-making, suggesting support for increased EC access despite provider barriers. Discussion: Findings offer practical recommendations to guide EC messaging in Italy to increase women\u27s knowledge and to empower women’s access. Additionally, opportunities for communication strategies and access campaigns to improve attitudes and increase knowledge and uptake of over-the-counter EC are discussed

CHIP-Dependent Regulation of the Actin Cytoskeleton is Linked to Neuronal Cell Membrane Integrity

Summary: CHIP is an E3-ubiquitin ligase that contributes to healthy aging and has been characterized as neuroprotective. To elucidate dominant CHIP-dependent changes in protein steady-state levels in a patient-derived human neuronal model, CHIP function was ablated using gene-editing and an unbiased proteomic analysis conducted to compare knock-out and wild-type isogenic induced pluripotent stem cell (iPSC)-derived cortical neurons. Rather than a broad effect on protein homeostasis, loss of CHIP function impacted on a focused cohort of proteins from actin cytoskeleton signaling and membrane integrity networks. In support of the proteomics, CHIP knockout cells had enhanced sensitivity to induced membrane damage. We conclude that the major readout of CHIP function in cortical neurons derived from iPSC of a patient with elevate α-synuclein, Parkinson's disease and dementia, is the modulation of substrates involved in maintaining cellular “health”. Thus, regulation of the actin cytoskeletal and membrane integrity likely contributes to the neuroprotective function(s) of CHIP

Emergent Role of IFITM1/3 towards Splicing Factor (SRSF1) and Antigen-Presenting Molecule (HLA-B) in Cervical Cancer

The IFITM restriction factors play a role in cancer cell progression through undefined mechanisms. We investigate new protein–protein interactions for IFITM1/3 in the context of cancer that would shed some light on how IFITM1/3 attenuate the expression of targeted proteins such as HLA-B. SBP-tagged IFITM1 protein was used to identify an association of IFITM1 protein with the SRSF1 splicing factor and transporter of mRNA to the ribosome. Using in situ proximity ligation assays, we confirmed a predominant cytosolic protein–protein association for SRSF1 and IFITM1/3. Accordingly, IFITM1/3 interacted with HLA-B mRNA in response to IFNγ stimulation using RNA–protein proximity ligation assays. In addition, RT-qPCR assays in IFITM1/IFITM3 null cells and wt-SiHa cells indicated that HLA-B gene expression at the mRNA level does not account for lowered HLA-B protein synthesis in response to IFNγ. Complementary, shotgun RNA sequencing did not show major transcript differences between IFITM1/IFITM3 null cells and wt-SiHa cells. Furthermore, ribosome profiling using sucrose gradient sedimentation identified a reduction in 80S ribosomal fraction an IFITM1/IFITM3 null cells compared to wild type. It was partially reverted by IFITM1/3 complementation. Our data link IFITM1/3 proteins to HLA-B mRNA and SRSF1 and, all together, our results begin to elucidate how IFITM1/3 catalyze the synthesis of target proteins. IFITMs are widely studied for their role in inhibiting viruses, and multiple studies have associated IFITMs with cancer progression. Our study has identified new proteins associated with IFITMs which support their role in mediating protein expression; a pivotal function that is highly relevant for viral infection and cancer progression. Our results suggest that IFITM1/3 affect the expression of targeted proteins; among them, we identified HLA-B. Changes in HLA-B expression could impact the presentation and recognition of oncogenic antigens on the cell surface by cytotoxic T cells and, ultimately, limit tumor cell eradication. In addition, the role of IFITMs in mediating protein abundance is relevant, as it has the potential for regulating the expression of viral and oncogenic proteins

Alterations in erythrocyte fatty acid composition in preclinical Alzheimer\u27s disease

Brain and blood fatty acids (FA) are altered in Alzheimer’s disease and cognitively impaired individuals, however, FA alterations in the preclinical phase, prior to cognitive impairment have not been investigated previously. The current study therefore evaluated erythrocyte FA in cognitively normal elderly participants aged 65 – 90 years via trans-methylation followed by gas chromatography. The neocortical beta-amyloid load (NAL) measured via positron emission tomography (PET) using ligand 18F-Florbetaben, was employed to categorise participants as low NAL (standard uptake value ratio; SUVR \u3c 1.35, N = 65) and high NAL or preclinical AD (SUVR ≥ 1.35, N = 35) wherein, linear models were employed to compare FA compositions between the two groups. Increased arachidonic acid (AA, p \u3c 0.05) and decreased docosapentaenoic acid (DPA, p \u3c 0.05) were observed in high NAL. To differentiate low from high NAL, the area under the curve (AUC) generated from a ‘base model’ comprising age, gender, APOEε4 and education (AUC = 0.794) was outperformed by base model + AA:DPA (AUC = 0.836). Our findings suggest that specific alterations in erythrocyte FA composition occur very early in the disease pathogenic trajectory, prior to cognitive impairment. As erythrocyte FA levels are reflective of tissue FA, these alterations may provide insight into the pathogenic mechanism(s) of the disease and may highlight potential early diagnostic markers and therapeutic targets

Families’ perceptions of and experiences related to a pediatric weight management program.

Objective: To examine parents' and children's perceptions of and experiences related to a Parents as Agents of Change (PAC) intervention for managing pediatric obesity. Methods: Ten families were recruited from a PAC intervention. Participants were interviewed before (10 adults and 9 children), during (9 adults and 8 children), and after (8 adults) the intervention. Results: Before the intervention, families reported goals to increase physical activity, plan and eat healthier meals, reduce screen time, and lose weight. During the intervention, families described different approaches to making behavior changes depending on who assumed responsibility (parent, child, or shared responsibility). After the intervention, group setting, goal setting, and portion size activities were viewed positively. Suggestions for improvement included engaging children and reducing intervention length. Conclusions and Implications: Practitioners delivering PAC interventions should discuss families' goals and concerns, and who is responsible for making lifestyle changes. Practical activities are valuable. The length of interventions and engagement of children should be considere

Rearrangement of Mitochondrial Pyruvate Dehydrogenase Subunit Dihydrolipoamide Dehydrogenase Protein-Protein Interactions by the MDM2 Ligand Nutlin-3.

Drugs targeting MDM2's hydrophobic pocket activate p53. However, these agents act allosterically and have agonist effects on MDM2's protein interaction landscape. Dominant p53-independent MDM2-drug responsive-binding proteins have not been stratified. We used as a variable the differential expression of MDM2 protein as a function of cell density to identify Nutlin-3 responsive MDM2-binding proteins that are perturbed independent of cell density using SWATH-MS. Dihydrolipoamide dehydrogenase, the E3 subunit of the mitochondrial pyruvate dehydrogenase complex, was one of two Nutlin-3 perturbed proteins identified fours hour posttreatment at two cell densities. Immunoblotting confirmed that dihydrolipoamide dehydrogenase was induced by Nutlin-3. Depletion of MDM2 using siRNA also elevated dihydrolipoamide dehydrogenase in Nutlin-3 treated cells. Mitotracker confirmed that Nutlin-3 inhibits mitochondrial activity. Enrichment of mitochondria using TOM22+ immunobeads and TMT labeling defined key changes in the mitochondrial proteome after Nutlin-3 treatment. Proximity ligation identified rearrangements of cellular protein–protein complexes in situ. In response to Nutlin-3, a reduction of dihydrolipoamide dehydrogenase/dihydrolipoamide acetyltransferase protein complexes highlighted a disruption of the pyruvate dehydrogenase complex. This coincides with an increase in MDM2/dihydrolipoamide dehydrogenase complexes in the nucleus that was further enhanced by the nuclear export inhibitor Leptomycin B. The data suggest one therapeutic impact of MDM2 drugs might be on the early perturbation of specific protein–protein interactions within the mitochondria. This methodology forms a blueprint for biomarker discovery that can identify rearrangements of MDM2 protein–protein complexes in drug-treated cells

Identification of novel interferon responsive protein partners of human leukocyte antigen A (HLA-A) using cross-linking mass spectrometry (CLMS) approach

The interferon signalling system elicits a robust cytokine response against a wide range of environmental pathogenic and internal pathological signals, leading to induction of a subset of interferon-induced proteins. We applied DSS (disuccinimidyl suberate) mediated cross-linking mass spectrometry (CLMS) to capture novel protein–protein interactions within the realm of interferon induced proteins. In addition to the expected interferon-induced proteins, we identified novel inter- and intra-molecular cross-linked adducts for the canonical interferon induced proteins, such as MX1, USP18, OAS3, and STAT1. We focused on orthogonal validation of a cohort of novel interferon-induced protein networks formed by the HLA-A protein (H2BFS-HLA-A-HMGA1) using co-immunoprecipitation assay, and further investigated them by molecular dynamics simulation. Conformational dynamics of the simulated protein complexes revealed several interaction sites that mirrored the interactions identified in the CLMS findings. Together, we showcase a proof-of-principle CLMS study to identify novel interferon-induced signaling complexes and anticipate broader use of CLMS to identify novel protein interaction dynamics within the tumour microenvironment