Neuroprotective amyloid ß N-terminal peptides differentially alter human α7- and α7ß2-nicotinic acetylcholine (nACh) receptor single-channel properties.

BACKGROUND AND PURPOSE: Oligomeric amyloid ß 1-42 (oAß1-42) exhibits agonist-like action at human α7- and α7ß2-containing nicotinic receptors. The N-terminal amyloid ß1-15 fragment (N-Aß fragment) modulates presynaptic calcium and enhances hippocampal-based synaptic plasticity via α7-containing nicotinic receptors. Further, the N-Aß fragment and its core sequence, the N-amyloid-beta core hexapeptide (N-Aßcore), protect against oAß1-42-associated synapto- and neurotoxicity. Here, we investigated how oAß1-42, the N-Aß fragment, and the N-Aßcore regulate the single-channel properties of α7- and α7ß2-nicotinic receptors. EXPERIMENTAL APPROACH: Single-channel recordings measured the impact of acetylcholine, oAß1-42, the N-Aß fragment, and the N-Aßcore on the unitary properties of human α7- and α7ß2-containing nicotinic receptors expressed in nicotinic-null SH-EP1 cells. Molecular dynamics simulations identified potential sites of interaction between the N-Aß fragment and orthosteric α7+/α7- and α7+/ß2- nicotinic receptor binding interfaces. KEY RESULTS: The N-Aß fragment and N-Aßcore induced α7- and α7ß2-nicotinic receptor single-channel openings. Relative to acetylcholine, oAß1-42 preferentially enhanced α7ß2-nicotinic receptor single-channel open probability and open-dwell times. Co-application with the N-Aßcore neutralized these effects. Further, administration of the N-Aß fragment alone, or in combination with acetylcholine or oAß1-42, selectively enhanced α7-nicotinic receptor open probability and open-dwell times (compared to acetylcholine or oAß1-42). CONCLUSIONS AND IMPLICATIONS: Amyloid-beta peptides demonstrate functional diversity in regulating α7- and α7ß2-nicotinic receptor function, with implications for a wide range of nicotinic receptor-mediated functions in Alzheimer's disease. The effects of these peptides on α7- and/or α7ß2-nicotinic receptors revealed complex interactions with these subtypes, providing novel insights into the neuroprotective actions of amyloid ß-derived fragments against the toxic effects of oAß1-42.

Evaluation of an enhanced depression and anxiety screening with targeted pharmacist intervention.

BACKGROUND: Depression is a major source of morbidity but often goes undiagnosed. Broader screening is recommended, and pharmacists could contribute. OBJECTIVES: This study aimed to assess the feasibility of community pharmacy depression and anxiety screening and describe the medication-related problems (MRPs) identified, pharmacist interventions, and provider responses for high-risk patients. METHODS: This pilot was conducted between October 2022 and January 2023 at an independently owned community pharmacy in the Midwest United States. Patients aged 18-45 years with ready prescriptions were identified through weekly reports, and tags were placed on prescription bags. A convenience sample of patients fluent in English were offered the Patient Health Questionnaire (PHQ2) and Generalized Anxiety Disorder (GAD2), with follow-up PHQ9 and GAD7 for at-risk individuals. High-risk individuals met with the pharmacist for consultation and recommendations were discussed. Descriptive statistics were calculated for participant demographics, questionnaire responses, MRPs, and provider responses. Patient profiles were examined 2 months after the workup to identify medication changes. RESULTS: A total of 29 patients volunteered to be screened for anxiety and depression; of these, 41% scored in the high-risk category for depression or anxiety and met with the pharmacist for the consultation. The pharmacist identified multiple MRPs. The most common was the need for additional therapy and inadequate dosages. Patients were reluctant for the pharmacist to follow up with their prescriber and were unreachable for telephone follow-up. Profiles reviewed 2 months after assessment showed half of the at-risk patients had one or more mental health medication changes. CONCLUSION: Community pharmacists may have a role in the screening and management of patient mental health, although there were challenges with screening uptake and follow-up. The pharmacist identified multiple MRPs for this high-risk group for which greater routine monitoring and follow-up may be beneficial. More work seems needed to engage both patients and prescribers.

Genome sequence of the lytic bacteriophage Alucard, a cluster EE actinophage.

Bacteriophage Alucard is a lytic phage isolated from the soil collected in southern Maine on Microbacterium foliorum NRRL B-24224. Alucard has siphovirus morphology with a 17,363-bp genome encoding 25 putative genes. Based on gene content similarity to actinobacteriophages, Alucard is assigned to cluster EE.

Early childhood language gains, kindergarten readiness, and Grade 3 reading achievement.

In this preregistered study, we used latent change score models to address two research aims: (1) whether preschool-aged children's language gains, over a year of early childhood education, were associated with later performance on state-mandated, literacy-focused kindergarten readiness and Grade 3 reading achievement assessments, and (2) whether gains in language, a more complex skill, predicted these outcomes after controlling for more basic emergent literacy skills. There were 724 participating children (mean = 57 months; 51% male; 76% White, 12% Black, 6% multiple races, and 5% Hispanic or Latino). We found that language gains significantly predicted kindergarten readiness when estimated in isolation (effect = 0.24 SDs, p < .001), but not when gains in letter knowledge and phonological awareness were also included.

Chemical remodeling of a cellular chaperone to target the active state of mutant KRAS.

The discovery of small-molecule inhibitors requires suitable binding pockets on protein surfaces. Proteins that lack this feature are considered undruggable and require innovative strategies for therapeutic targeting. KRAS is the most frequently activated oncogene in cancer, and the active state of mutant KRAS is such a recalcitrant target. We designed a natural product-inspired small molecule that remodels the surface of cyclophilin A (CYPA) to create a neomorphic interface with high affinity and selectivity for the active state of KRASG12C (in which glycine-12 is mutated to cysteine). The resulting CYPA:drug:KRASG12C tricomplex inactivated oncogenic signaling and led to tumor regressions in multiple human cancer models. This inhibitory strategy can be used to target additional KRAS mutants and other undruggable cancer drivers. Tricomplex inhibitors that selectively target active KRASG12C or multiple RAS mutants are in clinical trials now (NCT05462717 and NCT05379985).

Carbon isotope fractionation by an ancestral rubisco suggests that biological proxies for CO2 through geologic time should be reevaluated.

The history of Earth's carbon cycle reflects trends in atmospheric composition convolved with the evolution of photosynthesis. Fortunately, key parts of the carbon cycle have been recorded in the carbon isotope ratios of sedimentary rocks. The dominant model used to interpret this record as a proxy for ancient atmospheric CO2 is based on carbon isotope fractionations of modern photoautotrophs, and longstanding questions remain about how their evolution might have impacted the record. Therefore, we measured both biomass (εp) and enzymatic (εRubisco) carbon isotope fractionations of a cyanobacterial strain (Synechococcus elongatus PCC 7942) solely expressing a putative ancestral Form 1B rubisco dating to ≫1 Ga. This strain, nicknamed ANC, grows in ambient pCO2 and displays larger εp values than WT, despite having a much smaller εRubisco (17.23 ± 0.61‰ vs. 25.18 ± 0.31‰, respectively). Surprisingly, ANC εp exceeded ANC εRubisco in all conditions tested, contradicting prevailing models of cyanobacterial carbon isotope fractionation. Such models can be rectified by introducing additional isotopic fractionation associated with powered inorganic carbon uptake mechanisms present in Cyanobacteria, but this amendment hinders the ability to accurately estimate historical pCO2 from geological data. Understanding the evolution of rubisco and the CO2 concentrating mechanism is therefore critical for interpreting the carbon isotope record, and fluctuations in the record may reflect the evolving efficiency of carbon fixing metabolisms in addition to changes in atmospheric CO2.

The neuroprotective N-terminal amyloid-ß core hexapeptide reverses reactive gliosis and gliotoxicity in Alzheimer's disease pathology models.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by accumulation of extracellular amyloid beta (Aß) and intracellular neurofibrillary tangles, leading to chronic activation of astrocytes and microglia and persistent neuroinflammation. Aß-linked activation of microglia and astrocytes leads to increased intracellular calcium and production of proinflammatory cytokines, impacting the progression of neurodegeneration. An N-terminal Aß fragment (Aß1-15) and a shorter hexapeptide core sequence within the N-Aß fragment (N-Aßcore: Aß10-15) have previously been shown to protect against Aß-induced mitochondrial dysfunction, oxidative stress and apoptosis in neurons and rescue synaptic and spatial memory deficits in an APP/PSEN1 mouse model. Here, we hypothesized that the N-Aß fragment and N-Aßcore are protective against Aß-induced gliotoxicity, promoting a neuroprotective environment and potentially alleviating the characteristically persistent neuroinflammation present in AD. METHODS: We treated ex vivo organotypic brain slice cultures from an aged familial AD mouse model, 5xFAD, with the N-Aßcore and used immunocytochemistry to assess the impact on astrogliosis and microgliosis and alterations in synaptophysin-positive puncta engulfed by microglia. Isolated neuron/glia cultures, mixed glial cultures or a microglial cell line were treated with oligomeric human Aß at concentrations mimicking the pathogenic concentrations (µM) observed in AD in the absence or presence of the non-toxic N-terminal Aß fragments. Resultant changes in synaptic density, gliosis, oxidative stress, mitochondrial dysfunction, apoptosis, and the expression and release of proinflammatory markers were then determined. RESULTS: We demonstrate that the N-terminal Aß fragments mitigated the phenotypic switch leading to astrogliosis and microgliosis induced by pathological concentrations of Aß in mixed glial cultures and organotypic brain slice cultures from the transgenic 5xFAD mouse model, while protecting against Aß-induced oxidative stress, mitochondrial dysfunction and apoptosis in isolated astrocytes and microglia. Moreover, the addition of the N-Aßcore attenuated the expression and release of proinflammatory mediators in microglial cells activated by Aß and rescued microglia-mediated loss of synaptic elements induced by pathological levels of Aß. CONCLUSIONS: Together, these findings indicate the protective functions of the N-terminal Aß fragments extend to reactive gliosis and gliotoxicity induced by Aß, by preventing or reversing glial reactive states indicative of neuroinflammation and synaptic loss central to AD pathogenesis.

Bile salt hydrolase in non-enterotoxigenic Bacteroides potentiates colorectal cancer.

Bile salt hydrolase (BSH) in Bacteroides is considered a potential drug target for obesity-related metabolic diseases, but its involvement in colon tumorigenesis has not been explored. BSH-expressing Bacteroides is found at high abundance in the stools of colorectal cancer (CRC) patients  with overweight and in the feces of a high-fat diet (HFD)-induced CRC mouse model. Colonization of B. fragilis 638R, a strain with low BSH activity, overexpressing a recombinant bsh gene from B. fragilis NCTC9343 strain, results in increased unconjugated bile acids in the colon and accelerated progression of CRC under HFD treatment. In the presence of high BSH activity, the resultant elevation of unconjugated deoxycholic acid and lithocholic acid activates the G-protein-coupled bile acid receptor, resulting in increased ß-catenin-regulated chemokine (C-C motif) ligand 28 (CCL28) expression in colon tumors. Activation of the ß-catenin/CCL28 axis leads to elevated intra-tumoral immunosuppressive CD25+FOXP3+ Treg cells. Blockade of the ß-catenin/CCL28 axis releases the immunosuppression to enhance the intra-tumoral anti-tumor response, which decreases CRC progression under HFD treatment. Pharmacological inhibition of BSH reduces HFD-accelerated CRC progression, coincident with suppression of the ß-catenin/CCL28 pathway. These findings provide insights into the pro-carcinogenetic role of Bacteroides in obesity-related CRC progression and characterize BSH as a potential target for CRC prevention and treatment.

Hepatitis C screening in a community pharmacy setting: Patient perspective.

BACKGROUND: Hepatitis C virus (HCV) is an infection of the liver, which contributes to over 15,000 deaths in the United States annually. When treated, HCV has a 90% or greater cure rate, however testing for HCV remains low. OBJECTIVES: To assess patient perspectives on HCV screenings in the community pharmacy setting including awareness of screening, willingness to be screened, barriers to screening, and willingness to pay for HCV screening. METHODS: This study used a cross-sectional survey design. The surveys were distributed by staff at an independent community pharmacy participating in an HCV screening initiative through the state department of public health. Eligible patients were born between 1945 and 1965. Descriptive statistics were calculated for survey variables. Open-ended responses were analyzed for additional context. RESULTS: Fifty-seven surveys were returned and analyzed. The majority of the respondents were White (94%), female (56%), and had some college education (26%). Only 7% were aware that a finger-stick point-of-care test was available and 67% were unaware of the Centers for Disease Control and Prevention (CDC) recommendation for testing. The most frequently reported barrier or hesitation to screening was the patient not thinking they were at risk (29%) followed by uncertainty about cost (14%). Over half of respondents (63%) were either somewhat interested or very interested in testing in a community pharmacy, however, the majority (71%) were not willing to pay or only willing to pay less than $20. CONCLUSIONS: Survey respondents were largely unaware of the recommendations and availability of finger-stick HCV screenings at community pharmacies but many were willing to be tested if low-cost. Providing patient education on the importance of HCV screenings and CDC recommendations may bolster interest in screening.

Community Pharmacist and Family Medicine Collaboration for Pre-Visit Planning for Shared Patients Receiving Chronic Care Management Services.

Background: Pre-visit planning entails completing necessary tasks prior to clinic appointments. Community pharmacists (CPs) have unique knowledge about patients' medication use but do not routinely provide drug therapy reviews before clinic visits. Objectives: (1) Create and implement a business partnership between a CP and family medicine clinic (FMC) for CP provision of pre-visit medication reviews, and (2) describe the billing experience for shared patients in the FMC chronic care management (CCM) program. Methods: A prospective 8-month study in one community pharmacy and FMC in Iowa. Eligible patients were enrolled in the clinic CCM program and received their prescriptions at the CP. CPs were granted access to the clinic electronic health record (EHR), performed medication reviews, and recorded drug therapy recommendations (DTRs) in the clinic EHR. FMC physicians reviewed CP DTRs before the patient encounter. Time tracking software in the EHR recorded CP and FMC time performing CCM services. CCM revenue was prorated between parties. FMC physicians completed a survey about their experience. Results: Overall, there were 129 CP reviews performed for 95 patients. These reviews resulted in 169 DTRs and 76% were accepted by the physician. There were 71 CCM claims billed and CCM revenue was $3596 ($1796 FMC, $1800 CP). More than 90% of physicians (N = 11) indicated they reviewed CP DTRs before the patient encounter and agreed they were helpful to their practice. Conclusion: CPs completed pre-visit medication reviews and made accepted medication therapy recommendations. CCM billing provided a mechanism for CPs to receive revenue for their services.

Workshop-based learning and networking: a scalable model for research capacity strengthening in low- and middle-income countries.

Science education and research have the potential to drive profound change in low- and middle-income countries (LMICs) through encouraging innovation, attracting industry, and creating job opportunities. However, in LMICs, research capacity is often limited, and acquisition of funding and access to state-of-the-art technologies is challenging. The Alliance for Global Health and Science (the Alliance) was founded as a partnership between the University of California, Berkeley (USA) and Makerere University (Uganda), with the goal of strengthening Makerere University's capacity for bioscience research. The flagship program of the Alliance partnership is the MU/UCB Biosciences Training Program, an in-country, hands-on workshop model that trains a large number of students from Makerere University in infectious disease and molecular biology research. This approach nucleates training of larger and more diverse groups of students, development of mentoring and bi-directional research partnerships, and support of the local economy. Here, we describe the project, its conception, implementation, challenges, and outcomes of bioscience research workshops. We aim to provide a blueprint for workshop implementation, and create a valuable resource for bioscience research capacity strengthening in LMICs.

Anti-Neuroinflammatory Effects of a Semi-Synthetic Isoorientin-Based Glycogen Synthase Kinase-3ß Inhibitor in Lipopolysaccharide-Activated Microglial Cells.

Neuroinflammation contributes to the pathogenesis of several neurodegenerative disorders. Glycogen synthase kinase-3ß (GSK-3ß) regulates the release of proinflammatory cytokines and promotes inflammatory responses in immune cells. Microglia are the resident mononuclear immune cells of the central nervous system. Here, we investigated the anti-neuroinflammatory effects of (2S,3S,4R,5R,6S)-6-(2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chromen-6-yl)-3,4,5-trihydroxy-N-((S)-1,1,1-trifluoropropan-2-yl)tetrahydro-2H-pyran-2-carboxamide (TFGF-18), a semisynthetic GSK-3ß inhibitor, in lipopolysaccharide (LPS) activation of spontaneously immortalized SIM-A9 microglial cells and of mouse cortical microglia. TFGF-18 at 2.5 µM concentration inhibited LPS-induced production of nitric oxide by 56.3% and the proinflammatory cytokines TNF-α and IL-1ß by 28.3 and 59.2% in SIM-A9 cells, respectively, relative to the LPS treatment control group. Pretreatment of mouse primary microglial cells with TFGF-18 at 2.5 µM concentration led to a reduction of 58.7% in TNF-α+ microglial cells at 24 h post-LPS stimulation. The migration of LPS-activated SIM-A9 cells was also reduced by 26.7% with pretreatment of TFGF-18 in a scratch assay. Analyses of signaling pathways demonstrated that TFGF-18 led to the suppression of LPS-induced GSK-3ß activation and p65/NF-κB activity. Furthermore, the co-culture of SIM-A9 with SH-SY5Y neuroblastoma cells showed the suppression of TFGF-18 to microglia-mediated neurotoxicity in vitro. The findings indicate strong inhibitory effects of TFGF-18 on LPS-induced microglia activation via regulation of GSK-3ß and downstream p65/NF-κB signaling. The results suggest a potential role of TFGF-18 in neuroprotection via its anti-neuroinflammatory effect.

The encapsulin from Thermotoga maritima is a flavoprotein with a symmetry matched ferritin-like cargo protein.

Bacterial nanocompartments, also known as encapsulins, are an emerging class of protein-based 'organelles' found in bacteria and archaea. Encapsulins are virus-like icosahedral particles comprising a ~ 25-50 nm shell surrounding a specific cargo enzyme. Compartmentalization is thought to create a unique chemical environment to facilitate catalysis and isolate toxic intermediates. Many questions regarding nanocompartment structure-function remain unanswered, including how shell symmetry dictates cargo loading and to what extent the shell facilitates enzymatic activity. Here, we explore these questions using the model Thermotoga maritima nanocompartment known to encapsulate a redox-active ferritin-like protein. Biochemical analysis revealed the encapsulin shell to possess a flavin binding site located at the interface between capsomere subunits, suggesting the shell may play a direct and active role in the function of the encapsulated cargo. Furthermore, we used cryo-EM to show that cargo proteins use a form of symmetry-matching to facilitate encapsulation and define stoichiometry. In the case of the Thermotoga maritima encapsulin, the decameric cargo protein with fivefold symmetry preferentially binds to the pentameric-axis of the icosahedral shell. Taken together, these observations suggest the shell is not simply a passive barrier-it also plays a significant role in the structure and function of the cargo enzyme.

A nanocompartment system contributes to defense against oxidative stress in Mycobacterium tuberculosis.

Encapsulin nanocompartments are an emerging class of prokaryotic protein-based organelle consisting of an encapsulin protein shell that encloses a protein cargo. Genes encoding nanocompartments are widespread in bacteria and archaea, and recent works have characterized the biochemical function of several cargo enzymes. However, the importance of these organelles to host physiology is poorly understood. Here, we report that the human pathogen Mycobacterium tuberculosis (Mtb) produces a nanocompartment that contains the dye-decolorizing peroxidase DyP. We show that this nanocompartment is important for the ability of Mtb to resist oxidative stress in low pH environments, including during infection of host cells and upon treatment with a clinically relevant antibiotic. Our findings are the first to implicate a nanocompartment in bacterial pathogenesis and reveal a new mechanism that Mtb uses to combat oxidative stress.

The aryl hydrocarbon receptor activates ceramide biosynthesis in mice contributing to hepatic lipogenesis.

Aryl hydrocarbon receptor (AHR) activation via 2,3,7,8-tetrachlorodibenzofuran (TCDF) induces the accumulation of hepatic lipids. Here we report that AHR activation by TCDF (24  µg/kg body weight given orally for five days) induced significant elevation of hepatic lipids including ceramides in mice, was associated with increased expression of key ceramide biosynthetic genes, and increased activity of their respective enzymes. Results from chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA) and cell-based reporter luciferase assays indicated that AHR directly activated the serine palmitoyltransferase long chain base subunit 2 (Sptlc2, encodes serine palmitoyltransferase 2 (SPT2)) gene whose product catalyzes the initial rate-limiting step in de novo sphingolipid biosynthesis. Hepatic ceramide accumulation was further confirmed by mass spectrometry-based lipidomics. Taken together, our results revealed that AHR activation results in the up-regulation of Sptlc2, leading to ceramide accumulation, thus promoting lipogenesis, which can induce hepatic lipid accumulation.

Conditional Process Analysis for Nursing Education Researchers.

Nurse faculty need methodologically rigorous yet accessible techniques to evaluate the impact of their teaching strategies and build a strong body of evidence in nursing education science. Conditional process analysis is one such technique that allows researchers to include multiple variables in a single model to disentangle complex influences that instructional processes, student characteristics, and classroom variables have on learning outcomes. Researchers are encouraged to consider conditional process analysis to test and explain the effects of their educational strategies. Practical tools and resources are described that make conditional process analysis feasible for faculty with a basic understanding of statistical methods. [J Nurs Educ. 2020;60(5):249-251.].

Discovery and characterization of a novel family of prokaryotic nanocompartments involved in sulfur metabolism.

Prokaryotic nanocompartments, also known as encapsulins, are a recently discovered proteinaceous organelle-like compartment in prokaryotes that compartmentalize cargo enzymes. While initial studies have begun to elucidate the structure and physiological roles of encapsulins, bioinformatic evidence suggests that a great diversity of encapsulin nanocompartments remains unexplored. Here, we describe a novel encapsulin in the freshwater cyanobacterium Synechococcus elongatus PCC 7942. This nanocompartment is upregulated upon sulfate starvation and encapsulates a cysteine desulfurase enzyme via an N-terminal targeting sequence. Using cryo-electron microscopy, we have determined the structure of the nanocompartment complex to 2.2 Å resolution. Lastly, biochemical characterization of the complex demonstrated that the activity of the cysteine desulfurase is enhanced upon encapsulation. Taken together, our discovery, structural analysis, and enzymatic characterization of this prokaryotic nanocompartment provide a foundation for future studies seeking to understand the physiological role of this encapsulin in various bacteria.

The Neuroprotective Beta Amyloid Hexapeptide Core Reverses Deficits in Synaptic Plasticity in the 5xFAD APP/PS1 Mouse Model.

Alzheimer's disease (AD) is the most common cause of dementia in the aging population. Evidence implicates elevated soluble oligomeric Aß as one of the primary triggers during the prodromic phase leading to AD, effected largely via hyperphosphorylation of the microtubule-associated protein tau. At low, physiological levels (pM-nM), however, oligomeric Aß has been found to regulate synaptic plasticity as a neuromodulator. Through mutational analysis, we found a core hexapeptide sequence within the N-terminal domain of Aß (N-Aßcore) accounting for its physiological activity, and subsequently found that the N-Aßcore peptide is neuroprotective. Here, we characterized the neuroprotective potential of the N-Aßcore against dysfunction of synaptic plasticity assessed in ex vivo hippocampal slices from 5xFAD APP/PS1 mice, specifically hippocampal long-term potentiation (LTP) and long-term depression (LTD). The N-Aßcore was shown to reverse impairment in synaptic plasticity in hippocampal slices from 5xFAD APP/PS1 model mice, both for LTP and LTD. The reversal by the N-Aßcore correlated with alleviation of downregulation of hippocampal AMPA-type glutamate receptors in preparations from 5xFAD mice. The action of the N-Aßcore depended upon a critical di-histidine sequence and involved the phosphoinositide-3 (PI3) kinase pathway via mTOR (mammalian target of rapamycin). Together, the present findings indicate that the non-toxic N-Aßcore hexapeptide is not only neuroprotective at the cellular level but is able to reverse synaptic dysfunction in AD-like models, specifically alterations in synaptic plasticity.