A microphysiological system for studying barrier health of live tissues in real time.

Epithelial cells create barriers that protect many different components in the body from their external environment. Increased gut barrier permeability (leaky gut) has been linked to several chronic inflammatory diseases. Understanding the cause of leaky gut and effective interventions are elusive due to the lack of tools that maintain tissue's physiological environment while elucidating cellular functions under various stimuli ex vivo. Here we present a microphysiological system that records real-time barrier permeability of mouse colon in a physiological environment over extended durations. The system includes a microfluidic chamber; media composition that preserves microbiome and creates necessary oxygen gradients across the barrier; and integrated sensor electrodes for acquiring transepithelial electrical resistance (TEER). Our results demonstrate that the system can maintain tissue viability for up to 72 h. The TEER sensors can distinguish levels of barrier permeability when treated with collagenase and low pH media and detect different thickness in the tissue explant.

HBeeID: a molecular tool that identifies honey bee subspecies from different geographic populations.

BACKGROUND: Honey bees are the principal commercial pollinators. Along with other arthropods, they are increasingly under threat from anthropogenic factors such as the incursion of invasive honey bee subspecies, pathogens and parasites. Better tools are needed to identify bee subspecies. Genomic data for economic and ecologically important organisms is increasing, but in its basic form its practical application to address ecological problems is limited. RESULTS: We introduce HBeeID a means to identify honey bees. The tool utilizes a knowledge-based network and diagnostic SNPs identified by discriminant analysis of principle components and hierarchical agglomerative clustering. Tests of HBeeID showed that it identifies African, Americas-Africanized, Asian, and European honey bees with a high degree of certainty even when samples lack the full 272 SNPs of HBeeID. Its prediction capacity decreases with highly admixed samples. CONCLUSION: HBeeID is a high-resolution genomic, SNP based tool, that can be used to identify honey bees and screen species that are invasive. Its flexible design allows for future improvements via sample data additions from other localities.

Health and Health Care Use of American Indian/Alaska Native Women Veterans: A Scoping Review.

BACKGROUND: American Indian/Alaska Native (AI/AN) women serve in the U.S. military, use Veterans Health Administration (VA) health care, and reside in rural areas at the highest rates compared with other women veterans. However, little is known about their unique health care needs, access, and health care use. OBJECTIVE: We assessed the existing literature on the health and health care use of U.S. AI/AN women veterans. METHODS: Online databases were searched to identify studies. Study characteristics extracted included health care topic, study design, overall sample size and number of AI/AN women veterans, and funding source. We screened 1,508 publications for inclusion; 28 publications were ultimately retained. RESULTS: Health care access and use were the most common health care research topics (39%), followed by mental health (36%) and physical health (25%). Few studies considered the impact of rurality. Most studies found significant differences between AI/AN women veterans and other women veterans or AI/AN men veterans. Publication dates ranged from 1998 to 2023, with 71% published after 2010. The majority of studies (75%) were secondary analyses of extant health care data. More than three-quarters of studies (82%) were funded federally (e.g., VA). Many studies were based on VA administrative data, resulting in a gap in knowledge regarding AI/AN women veterans who are not eligible for, or choose not to use, VA health care. CONCLUSION: Research to inform the health and health care of AI/AN women veterans is limited, especially in terms of known AI/AN and women veterans' prevalent health concerns (e.g., diabetes, hypertension), women's health and reproduction, and how AI/AN women veterans access, use, and confront barriers to health care. Moreover, there is scarce research specific to cultural, tribal, and regional factors that likely affect access and use of particular health care systems or that can affect perspectives on illness that impact long-term treatment adherence and patient outcomes.

The hydrophobicity of the CARD8 N-terminus tunes inflammasome activation.

Mounting evidence indicates that proteotoxic stress is a primary activator of the CARD8 inflammasome, but the complete array of signals that control this inflammasome have not yet been established. Notably, we recently discovered that several hydrophobic radical-trapping antioxidants (RTAs), including JSH-23, potentiate CARD8 inflammasome activation through an unknown mechanism. Here, we report that these RTAs directly alkylate several cysteine residues in the N-terminal disordered region of CARD8. These hydrophobic modifications destabilize the repressive CARD8 N-terminal fragment and accelerate its proteasome-mediated degradation, thereby releasing the inflammatory CARD8 C-terminal fragment from autoinhibition. Consistently, we also found that unrelated (non-RTA) hydrophobic electrophiles as well as genetic mutation of the CARD8 cysteine residues to isoleucines similarly potentiate inflammasome activation. Overall, our results not only provide further evidence that protein folding stress is a key CARD8 inflammasome-activating signal, but also indicate that the N-terminal cysteines can play key roles in tuning the response to this stress.

What's Gender Got to Do With It: Accounting for Differences in Incident Guideline Discordant Prescribing for PTSD Among Women and Men Veterans.

Objectives: Women veterans are more likely than men veterans to receive medications that Department of Veterans Affairs clinical practice guidelines recommend against to treat posttraumatic stress disorder (PTSD). To understand this difference, we examined potential confounders in incident prescribing of guideline discordant medications (GDMs) in veterans with PTSD.Methods: Veterans receiving care for PTSD during 2020 were identified using Veterans Health Administration administrative data. PTSD diagnosis was established by the presence of at least 1 ICD-10 coded outpatient encounter or inpatient hospitalization during the calendar year 2020. Incident GDM prescribing was assessed during 2021, including benzodiazepines, antipsychotics, select anticonvulsants, and select antidepressants. Log-binomial regression was used to estimate the difference in risk for GDM initiation between men and women, adjusted for patient, prescriber, and facility-level covariates, and to identify key confounding variables.Results: Of 704,699 veterans with PTSD, 16.9% of women and 10.1% of men initiated a GDM, an increased risk of 67% for women [relative risk (RR) = 1.67; 95% CI, 1.65-1.70]. After adjustment, the gender difference decreased to 1.22 (95% CI, 1.20-1.24) in a fully specified model. Three key confounding variables were identified: bipolar disorder (RR = 1.60; 95% CI, 1.57-1.63), age (<40 years: RR = 1.20 [1.18-1.22]; 40-54 years: RR = 1.13 [1.11-1.16]; ≥65 years: RR = 0.64 [0.62-0.65]), and count of distinct psychiatric medications prescribed in the prior year (RR = 1.14; 1.13-1.14).Conclusions: Women veterans with PTSD were 67% more likely to initiate a GDM, where more than half of this effect was explained by bipolar disorder, age, and prior psychiatric medication. After adjustment, women veterans remained at 22% greater risk for an incident GDM, suggesting that other factors remain unidentified and warrant further investigation.

A Microphysiological System for Studying Barrier Health of Live Tissues in Real Time.

Epithelial cells create barriers that protect many different components in the body from their external environment. The gut in particular carries bacteria and other infectious agents. A healthy gut epithelial barrier prevents unwanted substances from accessing the underlying lamina propria while maintaining the ability to digest and absorb nutrients. Increased gut barrier permeability, better known as leaky gut, has been linked to several chronic inflammatory diseases. Yet understanding the cause of leaky gut and developing effective interventions are still elusive due to the lack of tools to maintain tissue's physiological environment while elucidating cellular functions under various stimuli ex vivo. This paper presents a microphysiological system capable of recording real-time barrier permeability of mouse gut tissues in a realistic physiological environment over extended durations. Key components of the microphysiological system include a microfluidic chamber designed to hold the live tissue explant and create a sufficient microphysiological environment to maintain tissue viability; proper media composition that preserves a microbiome and creates necessary oxygen gradients across the barrier; integrated sensor electrodes and supporting electronics for acquiring and calculating transepithelial electrical resistance (TEER); and a scalable system architecture to allow multiple chambers running in parallel for increased throughput. The experimental results demonstrate that the system can maintain tissue viability for up to 72 hours. The results also show that the custom-built and integrated TEER sensors are sufficiently sensitive to distinguish differing levels of barrier permeability when treated with collagenase and low pH media compared to control. Permeability variations in tissue explants from different positions in the intestinal tract were also investigated using TEER revealing their disparities in permeability. Finally, the results also quantitatively determine the effect of the muscle layer on total epithelial resistance.

The interaction between NLRP1 and oxidized TRX1 involves a transient disulfide bond.

NLRP1 is an innate immune receptor that detects pathogen-associated signals, assembles into a multiprotein structure called an inflammasome, and triggers a proinflammatory form of cell death called pyroptosis. We previously discovered that the oxidized, but not the reduced, form of thioredoxin-1 directly binds to NLRP1 and represses inflammasome formation. However, the molecular basis for NLRP1's selective association with only the oxidized form of TRX1 has not yet been established. Here, we leveraged AlphaFold-Multimer, site-directed mutagenesis, thiol-trapping experiments, and mass spectrometry to reveal that a specific cysteine residue (C427 in humans) on NLRP1 forms a transient disulfide bond with oxidized TRX1. Overall, this work demonstrates how NLRP1 monitors the cellular redox state, further illuminating an unexpected connection between the intracellular redox potential and the innate immune system.

The impact of patient-provider relationships on choosing between VA and VA-purchased care: A qualitative study of health care decision-making among rural veterans.

PURPOSE: Since the Choice Act in 2014, many Veterans have had greater options for seeking Veteran Affairs (VA)-purchased care in the community. We investigated factors that influence rural Veterans' decisions regarding where to seek care. METHODS: We utilized semi-structured telephone interviews to query Veterans living in rural or highly rural areas of Midwestern states about their health care options, preferences, and experiences. Interviews were recorded and transcribed, thematically coded, and deductively analyzed using a socioecological approach. FINDINGS: Forty rural Veterans (20 men/20 women) ages 28-76 years completed interviews in 2019. We found that rural Veterans often spoke about their relationships and interactions with providers as an important factor in deciding where to seek care. They expressed three socioecological qualities of patient-provider relationships that affected their decisions: (1) personal level-rural Veterans traveled longer distances for more compatible patient-provider relationships; (2) interpersonal level-they sought stable patient-provider relationships that encouraged familiarity, trust, and communication; and (3) organizational level-they emphasized shared identities and expertise that fostered a sense of belonging with their provider. Participants also described how impersonal interactions, status differences, and staff turnover impacted their choice of provider and were disruptive to patient-provider relationships. CONCLUSIONS: Rural Veterans' interview responses suggest exploring innovative ways to measure socioecological dimensions (i.e., personal, interpersonal, and organizational) of access-related decisions and patient-provider relationships to better understand health care barriers and needs. Such measures align with the VA's Whole Health approach that emphasizes person-centered care and the value of social relationships to Veterans' health.

Retraction of "Identification of Purine-Scaffold Small-Molecule Inhibitors of Stat3 Activation by QSAR Studies".

[This retracts the article DOI: 10.1021/ml100224d.].

The interaction between NLRP1 and oxidized TRX1 involves a transient disulfide bond.

NLRP1 is an innate immune receptor that detects pathogen-associated signals, assembles into a multiprotein structure called an inflammasome, and triggers a proinflammatory form of cell death called pyroptosis. We previously discovered that the oxidized, but not the reduced, form of thioredoxin-1 directly binds to NLRP1 and represses inflammasome formation. However, the molecular basis for NLRP1's selective association with only the oxidized form of TRX1 has not yet been established. Here, we leveraged Alphafold-Multimer, site-directed mutagenesis, thiol-trapping experiments, and mass spectrometry to reveal that a specific cysteine residue (C427 in humans) on NLRP1 forms a transient disulfide bond with oxidized TRX1. Overall, this work demonstrates how NLRP1 monitors the cellular redox state, further illuminating an unexpected connection between the intracellular redox potential and the innate immune system.

Optimized M24B Aminopeptidase Inhibitors for CARD8 Inflammasome Activation.

Inflammasomes are innate immune signaling platforms that trigger pyroptotic cell death. NLRP1 and CARD8 are related human inflammasomes that detect similar danger signals, but NLRP1 has a higher activation threshold and triggers a more inflammatory form of pyroptosis. Both sense the accumulation of intracellular peptides with Xaa-Pro N-termini, but Xaa-Pro peptides on their own without a second danger signal only activate the CARD8 inflammasome. We recently reported that a dual inhibitor of the Xaa-Pro-cleaving M24B aminopeptidases PEPD and XPNPEP1 called CQ31 selectively activates the CARD8 inflammasome by inducing the build-up of Xaa-Pro peptides. Here, we performed structure-activity relationship studies on CQ31 to develop the optimized dual PEPD/XPNPEP1 inhibitor CQ80 that more effectively induces CARD8 inflammasome activation. We anticipate that CQ80 will become a valuable tool to study the basic biology and therapeutic potential of selective CARD8 inflammasome activation.

The NLRP1 and CARD8 inflammasomes detect reductive stress.

The danger signals that activate the related nucleotide-binding domain leucine-rich repeat pyrin domain-containing 1 (NLRP1) and caspase activation and recruitment domain-containing 8 (CARD8) inflammasomes have not been fully established. We recently reported that the oxidized form of TRX1 binds to NLRP1 and represses inflammasome activation. These findings suggested that intracellular reductive stress, which would reduce oxidized TRX1 and thereby abrogate the NLRP1-TRX1 interaction, is an NLRP1 inflammasome-activating danger signal. However, no agents that induce reductive stress were known to test this premise. Here, we identify and characterize several radical-trapping antioxidants, including JSH-23, that induce reductive stress. We show that these compounds accelerate the proteasome-mediated degradation of the repressive N-terminal fragments of both NLRP1 and CARD8, releasing the inflammasome-forming C-terminal fragments from autoinhibition. Overall, this work validates chemical probes that induce reductive stress and establishes reductive stress as a danger signal sensed by both the NLRP1 and CARD8 inflammasomes.

Protein folding stress potentiates NLRP1 and CARD8 inflammasome activation.

NLRP1 and CARD8 are related pattern-recognition receptors (PRRs) that detect intracellular danger signals and form inflammasomes. Both undergo autoproteolysis, generating N-terminal (NT) and C-terminal (CT) fragments. The proteasome-mediated degradation of the NT releases the CT from autoinhibition, but the stimuli that trigger NT degradation have not been fully elucidated. Here, we show that several distinct agents that interfere with protein folding, including aminopeptidase inhibitors, chaperone inhibitors, and inducers of the unfolded protein response, accelerate NT degradation. However, these agents alone do not trigger inflammasome formation because the released CT fragments are physically sequestered by the serine dipeptidase DPP9. We show that DPP9-binding ligands must also be present to disrupt these complexes and allow the CT fragments to oligomerize into inflammasomes. Overall, these results indicate that NLRP1 and CARD8 detect a specific perturbation that induces both protein folding stress and DPP9 ligand accumulation.

The impact of local staging of prostate cancer determined on MRI or DRE at time of radical prostatectomy on progression-free survival: A Will Rogers phenomenon.

INTRODUCTION: We aimed to test whether the current practice of using mpMRI stage might lead to a Will Rogers phenomenon with a stage migration compared to DRE in men undergoing radical prostatectomy. MATERIAL AND METHODS: A total of 572 consecutive patients who underwent radical prostatectomy at a single institution (2007-2017) were included. Clinical stage using digital rectal examination was determined on table by the operating surgeon; mpMRI and pathological stage were recorded after tumor board review. Progression-free survival (PFS) was defined as no rising PSA, no adjuvant/salvage treatment, and no metastases or mortality. PFS was compared between groups and a model incorporating mpMRI into the EAU risk groups was created. RESULTS: Median age was 63 years (IQR 58.5-67) and median PSA was 8.9 ng/ml (IQR 6.5-13.2). Using DRE stage, 20% were NCCN low risk, 43% were intermediate, and 37% high. Median follow-up was 48 months (IQR 22-73). Estimated PFS at 1, 3, and 5 years was 75%, 59%, and 54%, respectively. When comparing PFS between DRE and mpMRI stages, patients deemed T1 (P < 0.01) or T3 (P = 0.03) by mpMRI showed better outcomes than patients staged T1 or T3 by DRE. On univariable analysis lower risk for failure was seen for MRI T1 disease (HR 0.10 95%, CI 0.01-0.73, P = 0.02) or MRI T3 (HR 0.70, CI 0.51-0.97, P = 0.03). On multivariable analysis, only MRI T1 remained a significant predictor (HR 0.08, 95% CI 0.01-0.59, P = 0.01). The subsequent, modified EAU risk model using both DRE and mpMRI performed significantly better than the DRE model. CONCLUSION: PFS based on mpMRI is not the same as DRE staging. Current risk groups which use DRE should be used with caution in whom local stage is based on mpMRI. Our modified EAU-risk categories can provide greater accuracy.

A framework for addressing Alzheimer's disease: Without a frame, the work has no aim.

Confronting Alzheimer's disease (AD) involves patients, healthcare professionals, supportive services, caregivers, and government agencies interacting along a continuum from initial awareness to diagnosis, treatment, support, and care. This complex scope presents a challenge for health system transformation supporting individuals at risk for, or diagnosed with, AD. The AD systems preparedness framework was developed to help health systems identify specific opportunities to implement and evaluate focused improvement programs. The framework is purposely flexible to permit local adaptation across different health systems and countries. Health systems can develop solutions tailored to system-specific priorities considered within the context of the overall framework. Example metric concepts and initiatives are provided for each of ten areas of focus. Examples of funded projects focusing on screening and early detection are provided. It is our hope that stakeholders utilize the common framework to generate and share additional implementation evidence to benefit individuals with AD.

Oxidized thioredoxin-1 restrains the NLRP1 inflammasome.

The danger signals that activate the NLRP1 inflammasome have not been established. Here, we report that the oxidized, but not the reduced, form of thioredoxin-1 (TRX1) binds to NLRP1. We found that oxidized TRX1 associates with the NACHT-LRR region of NLRP1 in an ATP-dependent process, forming a stable complex that restrains inflammasome activation. Consistent with these findings, patient-derived and ATPase-inactivating mutations in the NACHT-LRR region that cause hyperactive inflammasome formation interfere with TRX1 binding. Overall, this work strongly suggests that reductive stress, the cellular perturbation that will eliminate oxidized TRX1 and abrogate the TRX1-NLRP1 interaction, is a danger signal that activates the NLRP1 inflammasome.

A ubiquitin-independent proteasome pathway controls activation of the CARD8 inflammasome.

CARD8 is a pattern-recognition receptor that forms a caspase-1-activating inflammasome. CARD8 undergoes constitutive autoproteolysis, generating an N-terminal (NT) fragment with a disordered region and a ZU5 domain and a C-terminal (CT) fragment with UPA and CARD domains. Dipeptidyl peptidase 8 and dipeptidyl peptidase 9 inhibitors, including Val-boroPro, accelerate the degradation of the NT fragment via a poorly characterized proteasome-mediated pathway, thereby releasing the inflammatory CT fragment from autoinhibition. Here, we show that the core 20S proteasome, which degrades disordered and misfolded proteins independent of ubiquitin modification, controls activation of the CARD8 inflammasome. In unstressed cells, we discovered that the 20S proteasome degrades just the NT disordered region, leaving behind the folded ZU5, UPA, and CARD domains to act as an inhibitor of inflammasome assembly. However, in Val-boroPro-stressed cells, we show the 20S proteasome degrades the entire NT fragment, perhaps due to ZU5 domain unfolding, freeing the CT fragment from autoinhibition. Taken together, these results show that the susceptibility of the CARD8 NT domain to 20S proteasome-mediated degradation controls inflammasome activation.

M24B aminopeptidase inhibitors selectively activate the CARD8 inflammasome.

Inflammasomes are multiprotein complexes that sense intracellular danger signals and induce pyroptosis. CARD8 and NLRP1 are related inflammasomes that are repressed by the enzymatic activities and protein structures of the dipeptidyl peptidases 8 and 9 (DPP8/9). Potent DPP8/9 inhibitors such as Val-boroPro (VbP) activate both NLRP1 and CARD8, but chemical probes that selectively activate only one have not been identified. Here we report a small molecule called CQ31 that selectively activates CARD8. CQ31 inhibits the M24B aminopeptidases prolidase (PEPD) and Xaa-Pro aminopeptidase 1 (XPNPEP1), leading to the accumulation of proline-containing peptides that inhibit DPP8/9 and thereby activate CARD8. NLRP1 is distinct from CARD8 in that it directly contacts DPP8/9's active site; these proline-containing peptides, unlike VbP, do not disrupt this repressive interaction and thus do not activate NLRP1. We expect that CQ31 will now become a valuable tool to study CARD8 biology.

Focal therapy compared to radical prostatectomy for non-metastatic prostate cancer: a propensity score-matched study.

INTRODUCTION: Focal therapy (FT) ablates areas of prostate cancer rather than treating the whole gland. We compared oncological outcomes of FT to radical prostatectomy (RP). METHODS: Using prospective multicentre databases of 761 FT and 572 RP cases (November/2005-September/2018), patients with PSA < 20 ng/ml, Gleason

Activation of the CARD8 Inflammasome Requires a Disordered Region.

Several cytosolic pattern-recognition receptors (PRRs) form multiprotein complexes called canonical inflammasomes in response to intracellular danger signals. Canonical inflammasomes recruit and activate caspase-1 (CASP1), which in turn cleaves and activates inflammatory cytokines and gasdermin D (GSDMD), inducing pyroptotic cell death. Inhibitors of the dipeptidyl peptidases DPP8 and DPP9 (DPP8/9) activate both the human NLRP1 and CARD8 inflammasomes. NLRP1 and CARD8 have different N-terminal regions but have similar C-terminal regions that undergo autoproteolysis to generate two non-covalently associated fragments. Here, we show that DPP8/9 inhibition activates a proteasomal degradation pathway that targets disordered and misfolded proteins for destruction. CARD8's N terminus contains a disordered region of ∼160 amino acids that is recognized and destroyed by this degradation pathway, thereby freeing its C-terminal fragment to activate CASP1 and induce pyroptosis. Thus, CARD8 serves as an alarm to signal the activation of a degradation pathway for disordered and misfolded proteins.