Loss-of-function of ALDH3B2 transdifferentiates human pancreatic duct cells into beta-like cells.

Replenishment of pancreatic beta cells is a key to the cure for diabetes. Beta cells regeneration is achieved predominantly by self-replication especially in rodents, but it was also shown that pancreatic duct cells can transdifferentiate into beta cells. How pancreatic duct cells undergo transdifferentiated and whether we could manipulate the transdifferentiation to replenish beta cell mass is not well understood. Using a genome-wide CRISPR screen, we discovered that loss-of-function of ALDH3B2 is sufficient to transdifferentiate human pancreatic duct cells into functional beta-like cells. The transdifferentiated cells have significant increase in beta cell marker genes expression, secrete insulin in response to glucose, and reduce blood glucose when transplanted into diabetic mice. Our study identifies a novel gene that could potentially be targeted in human pancreatic duct cells to replenish beta cell mass for diabetes therapy.

Fine particulate matter infiltration at Western Montana residences during wildfire season.

BACKGROUND/AIMS: Wildfire air pollution is a growing public health concern as wildfires increase in size, intensity, and duration in the United States. The public is often encouraged to stay indoors during wildfire smoke events to reduce exposure. However, there is limited information on how much wildfire smoke infiltrates indoors at residences and what household/behavioral characteristics contribute to higher infiltration. We assessed fine particulate matter (PM2.5) infiltration into Western Montana residences during wildfire season. METHODS: We measured continuous outdoor and indoor PM2.5 concentrations from July-October 2022 at 20 residences in Western Montana during wildfire season using low-cost PM2.5 sensors. We used paired outdoor/indoor PM2.5 data from each household to calculate infiltration efficiency (Finf; range 0-1; higher values indicate more outdoor PM2.5 infiltration to the indoor environment) using previously validated methods. Analyses were conducted for all households combined and for various household subgroups. RESULTS: Median (25th percentile, 75th percentile) daily outdoor PM2.5 at the households was 3.7 µg/m3 (2.1, 7.1) during the entire study period and 29.0 µg/m3 (19.0, 49.4) during a 2-week period in September impacted by wildfire smoke. Median daily indoor PM2.5 at the households was 2.5 µg/m3 (1.3, 5.5) overall and 10.4 µg/m3 (5.6, 21.0) during the wildfire period. Overall Finf was 0.34 (95 % Confidence Interval [95%CI]: 0.33, 0.35) with lower values during the wildfire period (0.32; 95%CI: 0.28, 0.36) versus non-wildfire period (0.39; 95%CI: 0.37, 0.42). Indoor PM2.5 concentrations and Finf varied substantially across household subgroups such as household income, age of the home, presence of air conditioning units, and use of portable air cleaners. CONCLUSIONS: Indoor PM2.5 was substantially higher during wildfire-impacted periods versus the rest of the study. Indoor PM2.5 and Finf were highly variable across households. Our results highlight potentially modifiable behaviors and characteristics that can be used in targeted intervention strategies.

Protective Renalase Deficiency in ß-Cells Shapes Immune Metabolism and Function in Autoimmune Diabetes.

Type 1 diabetes (T1D) is caused by the immune-mediated loss of pancreatic ß-cells that produce insulin. The latest advances in stem cell (SC) ß-cell differentiation methods have made a cell replacement therapy for T1D feasible. However, recurring autoimmunity would rapidly destroy transplanted SC ß-cells. A promising strategy to overcome immune rejection is to genetically engineer SC ß-cells. We previously identified Renalase (Rnls) as a novel target for ß-cell protection. Here we show that Rnls deletion endows ß-cells with the capacity to modulate the metabolism and function of immune cells within the local graft microenvironment. We used flow cytometry and single-cell RNA sequencing to characterize ß-cell graft-infiltrating immune cells in a mouse model for T1D. Loss of Rnls within transplanted ß-cells affected both the composition and the transcriptional profile of infiltrating immune cells in favor of an anti-inflammatory profile with decreased antigen-presenting capacity. We propose that changes in ß-cell metabolism mediate local immune regulation and that this feature could be exploited for therapeutic goals. ARTICLE HIGHLIGHTS: Protective Renalase (Rnls) deficiency impacts ß-cell metabolism. Rnls-deficient ß-cell grafts do not exclude immune infiltration. Rnls deficiency in transplanted ß-cells broadly modifies local immune function. Immune cell in Rnls mutant ß-cell grafts adopt a noninflammatory phenotype.

Activity of Fatty Acid Biosynthesis Initiating Ketosynthase FabH with Acetyl/Malonyl-oxa/aza(dethia)CoAs.

Fatty acid and polyketide biosynthetic enzymes exploit the reactivity of acyl- and malonyl-thioesters for catalysis. A prime example is FabH, which initiates fatty acid biosynthesis in many bacteria and plants. FabH performs an acyltransferase reaction with acetyl-CoA to generate an acetyl-S-FabH acyl-enzyme intermediate and subsequent decarboxylative Claisen-condensation with a malonyl-thioester carried by an acyl carrier protein (ACP). We envision that crystal structures of FabH with substrate analogues can provide insight into the conformational changes and enzyme/substrate interactions underpinning the distinct reactions. Here, we synthesize acetyl/malonyl-CoA analogues with esters or amides in place of the thioester and characterize their stability and behavior as Escherichia coli FabH substrates or inhibitors to inform structural studies. We also characterize the analogues with mutant FabH C112Q that mimics the acyl-enzyme intermediate allowing dissection of the decarboxylation reaction. The acetyl- and malonyl-oxa(dethia)CoA analogues undergo extremely slow hydrolysis in the presence of FabH or the C112Q mutant. Decarboxylation of malonyl-oxa(dethia)CoA by FabH or C112Q mutant was not detected. The amide analogues were completely stable to enzyme activity. In enzyme assays with acetyl-CoA and malonyl-CoA (rather than malonyl-ACP) as substrates, acetyl-oxa(dethia)CoA is surprisingly slightly activating, while acetyl-aza(dethia)CoA is a moderate inhibitor. The malonyl-oxa/aza(dethia)CoAs are inhibitors with Ki's near the Km of malonyl-CoA. For comparison, we determine the FabH catalyzed decomposition rates for acetyl/malonyl-CoA, revealing some fundamental catalytic traits of FabH, including hysteresis for malonyl-CoA decarboxylation. The stability and inhibitory properties of the substrate analogues make them promising for structure-function studies to reveal fatty acid and polyketide enzyme/substrate interactions.

Bioluminescent Monitoring of Graft Survival in an Adoptive Transfer Model of Autoimmune Diabetes in Mice.

Type 1 diabetes is characterized by the autoimmune destruction of the insulin-producing beta cells of the pancreas. A promising treatment for this disease is the transplantation of stem cell-derived beta cells. Genetic modifications, however, may be necessary to protect the transplanted cells from persistent autoimmunity. Diabetic mouse models are a useful tool for the preliminary evaluation of strategies to protect transplanted cells from autoimmune attack. Described here is a minimally invasive method for transplanting and imaging cell grafts in an adoptive transfer model of diabetes in mice. In this protocol, cells from the murine pancreatic beta cell line NIT-1 expressing the firefly luciferase transgene luc2 are transplanted subcutaneously into immunodeficient non-obese diabetic (NOD)-severe combined immunodeficient (scid) mice. These mice are simultaneously injected intravenously with splenocytes from spontaneously diabetic NOD mice to transfer autoimmunity. The grafts are imaged at regular intervals via non-invasive bioluminescent imaging to monitor the cell survival. The survival of mutant cells is compared to that of control cells transplanted into the same mouse.

Results of Engineering, Primary Care, Oncology Collaborative Regarding a Survey of Primary Care on a Re-Engineered Survivorship Care Plan.

Survivorship care plans (SCPs) may facilitate cancer survivorship care shared between oncologists and primary care, particularly for patients more likely to receive care across healthcare systems such as rural patients. However, limited research has addressed primary care clinicians' information or workflow needs with regard to SCPs. This study's objective was to assess primary care clinicians' perceived usefulness with a re-engineered SCP previously developed by applying engineering approaches and informed by primary care preferences. An emailed survey of primary care clinicians assessed perceived usefulness with the re-engineered SCP. Clinicians were recruited across the USA from primary care practice-based research networks (PBRNs) with high concentrations of rural practices. Over 90% of respondents (n = 111) agreed that (1) the re-engineered SCP was useful (n = 95) and (2) they would want to receive a similar SCP (n = 93). The majority demonstrated high agreement regarding the SCP's relevance, understandability, content, and ability to help provide better survivorship care. Perceived usefulness was consistent between rural and non-rural clinicians. Suggested improvements involved decreased length, addition of a bulleted list, and electronic health record integration. Results indicate that the majority of primary care clinicians perceive the re-engineered SCP as useful. However, primary care clinicians indicated continued barriers despite end-user specific alterations. Future research should investigate additional strategies to support primary care survivorship-related workload, provide essential SCP content, and improve survivorship care delivery.

Defining the role of the polyasparagine repeat domain of the S. cerevisiae transcription factor Azf1p.

Across eukaryotes, homopolymeric repeats of amino acids are enriched in regulatory proteins such as transcription factors and chromatin remodelers. These domains play important roles in signaling, binding, prion formation, and functional phase separation. Azf1p is a prion-forming yeast transcription factor that contains two homorepeat domains, a polyglutamine and a polyasparagine domain. In this work, we report a new phenotype for Azf1p and identify a large set of genes that are regulated by Azf1p during growth in glucose. We show that the polyasparagine (polyN) domain plays a subtle role in transcription but is dispensable for Azf1p localization and prion formation. Genes upregulated upon deletion of the polyN domain are enriched in functions related to carbon metabolism and storage. This domain may therefore be a useful target for engineering yeast strains for fermentation applications and small molecule production. We also report that both the polyasparagine and polyglutamine domains vary in length across strains of S. cerevisiae and propose a model for how this variation may impact protein function.

Contractions of the C-Terminal Domain of Saccharomyces cerevisiae Rpb1p Are Mediated by Rad5p.

The C-terminal domain (CTD) is an essential domain of the largest subunit of RNA polymerase II, Rpb1p, and is composed of 26 tandem repeats of a seven-amino acid sequence, YSPTSPS. Despite being an essential domain within an essential gene, we have previously demonstrated that the CTD coding region is genetically unstable. Furthermore, yeast with a truncated or mutated CTD sequence are capable of promoting spontaneous genetic expansion or contraction of this coding region to improve fitness. We investigated the mechanism by which the CTD contracts using a tet-off reporter system for RPB1 to monitor genetic instability within the CTD coding region. We report that contractions require the post-replication repair factor Rad5p but, unlike expansions, not the homologous recombination factors Rad51p and Rad52p Sequence analysis of contraction events reveals that deleted regions are flanked by microhomologies. We also find that G-quadruplex forming sequences predicted by the QGRS Mapper are enriched on the noncoding strand of the CTD compared to the body of RPB1 Formation of G-quadruplexes in the CTD coding region could block the replication fork, necessitating post-replication repair. We propose that contractions of the CTD result when microhomologies misalign during Rad5p-dependent template switching via fork reversal.

Abuse, cancer and sexual dysfunction in women: A potentially vicious cycle.

More than 30% of women have a history of abuse. Women with cancer may be at substantially increased risk for abuse, but this issue is rarely discussed in the course of oncology care. Women with a history of abuse who present for cancer care commonly have a high prevalence of co-morbid illness. Sexual dysfunction, a highly prevalent but under-recognized condition among women of all ages, is also more common among both women with a history of abuse and women with cancer. Although common after cancer, sexual dysfunction, like abuse, can be stigmatizing and often goes undiagnosed and untreated. This review first examines the literature for evidence of a relationship between any history of abuse and cancer among women, addressing two questions: 1) How does abuse promote or create risk for developing cancer? 2) How does cancer increase a woman's susceptibility to abuse? We then examine evidence for a relationship between abuse and female sexual dysfunction, followed by an investigation of the complex relationship between all three factors: abuse, sexual dysfunction and cancer. The literature is limited by a lack of harmonization of measures across studies, retrospective designs, and small and idiosyncratic samples. Despite these limitations, it is imperative that providers integrate the knowledge of this complex relationship into the care of women with cancer.

Does risk perception affect behaviour and exposure? A pilot study amongst asbestos workers.

It is hypothesised that there is a causal association between workers' perceptions of the risks from handling hazardous materials, their behaviour while working and their consequent exposure. This has been investigated in a small group of workers carrying out remedial work on amosite insulating boards and similar products. Risk perception was first assessed using a questionnaire and then the workers' behaviour was recorded alongside task-based measurements of fibre exposure. There was a clear association of higher cumulative exposures when workers used power tools compared to manual methods (about seven times higher). Careful bagging was shown to reduce exposures by a smaller margin, (approximately half). Workers whose perception of the risks was poorer were found to be more likely to use power tools to remove the asbestos containing material. However, fibre exposure was not found to be directly associated with risk perception. Further work is necessary to clarify the validity of the original hypothesis.