Engineered nanoparticles enable deep proteomics studies at scale by leveraging tunable nano-bio interactions.

SignificanceDeep profiling of the plasma proteome at scale has been a challenge for traditional approaches. We achieve superior performance across the dimensions of precision, depth, and throughput using a panel of surface-functionalized superparamagnetic nanoparticles in comparison to conventional workflows for deep proteomics interrogation. Our automated workflow leverages competitive nanoparticle-protein binding equilibria that quantitatively compress the large dynamic range of proteomes to an accessible scale. Using machine learning, we dissect the contribution of individual physicochemical properties of nanoparticles to the composition of protein coronas. Our results suggest that nanoparticle functionalization can be tailored to protein sets. This work demonstrates the feasibility of deep, precise, unbiased plasma proteomics at a scale compatible with large-scale genomics enabling multiomic studies.

Evaluation of Encequidar as An Intestinal P-gp and BCRP Specific Inhibitor to Assess the Role of Intestinal P-gp and BCRP in Drug-Drug Interactions.

PURPOSE: The differences between intestinal and systemic (hepatic and renal) P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) roles in drug disposition are difficult to define. Accordingly, we characterized Encequidar (ECD) as an intestinal P-gp and BCRP specific inhibitor to evaluate their role in drug disposition. METHODS: We assessed the in vitro and in vivo inhibition potential of ECD towards human and animal P-gp and BCRP. RESULTS: ECD is a potent inhibitor with a high degree of selectivity in inhibiting human P-gp (hP-gp) over human BCRP (hBCRP) (IC50s of 0.0058 ± 0.0006 vs. > 10 µM, respectively). In contrast, ECD is a potent inhibitor of rat and cynomolgus monkey BCRP (IC50 ranged from 0.059 to 0.18 µM). While the AUC of IV paclitaxel (PTX) was significantly increased by elacridar (ELD) (P < 0.05) but not ECD in rats (15 mg/kg; PO) (2.55- vs. 0.93-fold), that of PO PTX was significantly elevated to a similar extent between the inhibitors (39.5- vs. 33.5-fold). Similarly, the AUC of PO sulfasalazine (SFZ) was dramatically increased by ELD and ECD (16.6- vs. 3.04-fold) although that of IV SFZ was not significantly affected by ELD and ECD in rats (1.18- vs. 1.06-fold). Finally, a comparable ECD-induced increase of the AUC of PO talinolol in cynomolgus monkeys was observed compared with ELD (2.14- vs. 2.12-fold). CONCLUSIONS: ECD may allow an in-depth appraisal of the role of intestinal efflux transporter(s) in drug disposition in animals and humans through local intestinal drug interactions.

The crystal structures of a chloride-pumping microbial rhodopsin and its proton-pumping mutant illuminate proton transfer determinants.

Microbial rhodopsins are versatile and ubiquitous retinal-binding proteins that function as light-driven ion pumps, light-gated ion channels, and photosensors, with potential utility as optogenetic tools for altering membrane potential in target cells. Insights from crystal structures have been central for understanding proton, sodium, and chloride transport mechanisms of microbial rhodopsins. Two of three known groups of anion pumps, the archaeal halorhodopsins (HRs) and bacterial chloride-pumping rhodopsins, have been structurally characterized. Here we report the structure of a representative of a recently discovered third group consisting of cyanobacterial chloride and sulfate ion-pumping rhodopsins, the Mastigocladopsis repens rhodopsin (MastR). Chloride-pumping MastR contains in its ion transport pathway a unique Thr-Ser-Asp (TSD) motif, which is involved in the binding of a chloride ion. The structure reveals that the chloride-binding mode is more similar to HRs than chloride-pumping rhodopsins, but the overall structure most closely resembles bacteriorhodopsin (BR), an archaeal proton pump. The MastR structure shows a trimer arrangement reminiscent of BR-like proton pumps and shows features at the extracellular side more similar to BR than the other chloride pumps. We further solved the structure of the MastR-T74D mutant, which contains a single amino acid replacement in the TSD motif. We provide insights into why this point mutation can convert the MastR chloride pump into a proton pump but cannot in HRs. Our study points at the importance of precise coordination and exact location of the water molecule in the active center of proton pumps, which serves as a bridge for the key proton transfer.

Early Intracellular Trafficking of Granulibacter bethesdensis in Human Macrophages.

Granulibacter bethesdensis is a Gram-negative bacterium that infects patients with chronic granulomatous disease (CGD), a primary immunodeficiency marked by a defect in NOX2, the phagocyte NADPH oxidase. Previous studies have shown that NOX2 is essential for killing of G. bethesdensis by neutrophils and monocytes and that the bacteriostatic activity of monocyte-derived macrophages (MDM) requires NOX2 and gamma interferon (IFN-γ) pretreatment. To determine whether G. bethesdensis evades phagolysosomal killing, a host defense pathway intact in both normal and CGD MDM, or whether it occupies a distinct intracellular niche in CGD MDM, we assessed the trafficking patterns of this organism. We observed colocalization of G. bethesdensis with an early endosome antigen 1 (EEA1)-positive compartment, followed by colocalization with lysosome-associated membrane protein 1 (LAMP1)-positive and LysoTracker-positive late phagosomes; these characteristics were similar in both normal and CGD MDM. Despite localization to acidified late phagosomes, viable G. bethesdensis cells were recovered from viable MDM in numbers greater than in the initial input up to 6 days after infection. G. bethesdensis remains, and in some cases appears to divide, within a membrane-bound compartment for the entire 6-day time course. These findings indicate that this organism resists both oxygen-dependent and oxygen-independent phagolysosomal antimicrobial systems of human macrophages.

An AAVS1-targeted minigene platform for correction of iPSCs from all five types of chronic granulomatous disease.

There are five genetic forms of chronic granulomatous disease (CGD), resulting from mutations in any of five subunits of phagocyte oxidase, an enzyme complex in neutrophils, monocytes, and macrophages that produces microbicidal reactive oxygen species. We generated induced pluripotent stem cells (iPSCs) from peripheral blood CD34(+) hematopoietic stem cells of patients with each of five CGD genotypes. We used zinc finger nuclease (ZFN) targeting the AAVS1 safe harbor site together with CGD genotype-specific minigene plasmids with flanking AAVS1 sequence to target correction of iPSC representing each form of CGD. We achieved targeted insertion with constitutive expression of desired oxidase subunit in 70-80% of selected iPSC clones. Neutrophils and macrophages differentiated from corrected CGD iPSCs demonstrated restored oxidase activity and antimicrobial function against CGD bacterial pathogens Staphylococcus aureus and Granulibacter bethesdensis. Using a standard platform that combines iPSC generation from peripheral blood CD34(+) cells and ZFN mediated AAVS1 safe harbor minigene targeting, we demonstrate efficient generation of genetically corrected iPSCs using an identical approach for all five genetic forms of CGD. This safe harbor minigene targeting platform is broadly applicable to a wide range of inherited single gene metabolic disorders.

Simultaneous Host-Pathogen Transcriptome Analysis during Granulibacter bethesdensis Infection of Neutrophils from Healthy Subjects and Patients with Chronic Granulomatous Disease.

Polymorphonuclear leukocytes (PMN) from patients with chronic granulomatous disease (CGD) fail to produce microbicidal concentrations of reactive oxygen species (ROS) due to mutations in NOX2. Patients with CGD suffer from severe, life-threatening infections and inflammatory complications. Granulibacter bethesdensis is an emerging Gram-negative pathogen in CGD that resists killing by PMN of CGD patients (CGD PMN) and inhibits PMN apoptosis through unknown mechanisms. Microarray analysis was used to study mRNA expression in PMN from healthy subjects (normal PMN) and CGD PMN during incubation with G. bethesdensis and, simultaneously, in G. bethesdensis with normal and CGD PMN. We detected upregulation of antiapoptotic genes (e.g., XIAP and GADD45B) and downregulation of proapoptotic genes (e.g., CASP8 and APAF1) in infected PMN. Transcript and protein levels of inflammation- and immunity-related genes were also altered. Upon interaction with PMN, G. bethesdensis altered the expression of ROS resistance genes in the presence of normal but not CGD PMN. Levels of bacterial stress response genes, including the ClpB gene, increased during phagocytosis by both normal and CGD PMN demonstrating responses to oxygen-independent PMN antimicrobial systems. Antisense knockdown demonstrated that ClpB is dispensable for extracellular growth but is essential for bacterial resistance to both normal and CGD PMN. Metabolic adaptation of Granulibacter growth in PMN included the upregulation of pyruvate dehydrogenase. Pharmacological inhibition of pyruvate dehydrogenase by triphenylbismuthdichloride was lethal to Granulibacter. This study expands knowledge of microbial pathogenesis of Granulibacter in cells from permissive (CGD) and nonpermissive (normal) hosts and identifies potentially druggable microbial factors, such as pyruvate dehydrogenase and ClpB, to help combat this antibiotic-resistant pathogen.

Transmembrane peptides as unique tools to demonstrate the in vivo action of a cross-class GPCR heterocomplex.

Angiotensin (ANGII) and secretin (SCT) share overlapping, interdependent osmoregulatory functions in brain, where SCT peptide/receptor function is required for ANGII action, yet the molecular basis is unknown. Since receptors for these peptides (AT1aR, SCTR) are coexpressed in osmoregulatory centers, a possible mechanism is formation of a cross-class receptor heterocomplex. Here, we demonstrate such a complex and its functional importance to modulate signaling. Association of AT1aR with SCTR reduced ability of SCT to stimulate cyclic adenosine monophosphate (cAMP), with signaling augmented in presence of ANGII or constitutively active AT1aR. Several transmembrane (TM) peptides of these receptors were able to affect their conformation within complexes, reducing receptor BRET signals. AT1aR TM1 affected only formation and activity of the heterocomplex, without effect on homomers of either receptor, and reduced SCT-stimulated cAMP responses in cells expressing both receptors. This peptide was active in vivo by injection into mouse lateral ventricle, thereby suppressing water-drinking behavior after hyperosmotic shock, similar to SCTR knockouts. This supports the interpretation that active conformation of AT1aR is a key modulator of cAMP responses induced by SCT stimulation of SCTR. The SCTR/AT1aR complex is physiologically important, providing differential signaling to SCT in settings of hyperosmolality or food intake, modulated by differences in levels of ANGII.

Acute Effects of a Spinach Extract Rich in Thylakoids on Satiety: A Randomized Controlled Crossover Trial.

OBJECTIVE: By retarding fat digestion, thylakoids, the internal photosynthetic membrane system of green plants, promote the release of satiety hormones. This study examined the effect of consuming a single dose of concentrated extract of thylakoids from spinach on satiety, food intake, lipids, and glucose compared to a placebo. DESIGN: Sixty overweight and obese individuals enrolled in a double-blind randomized crossover study consumed the spinach extract or placebo in random order at least a week apart. Blood was drawn for assessments of lipids and glucose before a standard breakfast meal, followed 4 hours later by a 5 g dose of the extract and a standard lunch. Visual analog scales were administered before lunch and at intervals until an ad libitum pizza dinner served 4 hours later. Two hours after lunch a second blood draw was conducted. Mixed models were used to analyze response changes. RESULTS: Compared to placebo, consuming the spinach extract reduced hunger (p < 0.01) and longing for food over 2 hours (p < 0.01) and increased postprandial plasma glucose concentrations (p < 0.01). There were no differences in plasma lipids and energy intake at dinner, but males showed a trend toward decreased energy intake (p = 0.08). CONCLUSIONS: At this dose, the spinach extract containing thylakoids increases satiety over a 2-hour period compared to a placebo. Thylakoid consumption may influence gender-specific food cravings.

Persistence of the bacterial pathogen Granulibacter bethesdensis in chronic granulomatous disease monocytes and macrophages lacking a functional NADPH oxidase.

Granulibacter bethesdensis is a Gram-negative pathogen in patients with chronic granulomatous disease (CGD), a deficiency in the phagocyte NADPH oxidase. Repeated isolation of genetically identical strains from the same patient over years, and prolonged waxing and waning seropositivity in some subjects, raises the possibility of long-term persistence. G. bethesdensis resists killing by serum, CGD polymorphonuclear leukocytes (PMN), and antimicrobial peptides, indicating resistance to nonoxidative killing mechanisms. Although G. bethesdensis extends the survival of PMN, persistent intracellular bacterial survival might rely on longer-lived macrophages and their precursor monocytes. Therefore, we examined phagocytic killing by primary human monocytes and monocyte-derived macrophages (MDM). Cells from both normal and CGD subjects internalized G. bethesdensis similarly. G. bethesdensis stimulated superoxide production in normal monocytes, but to a lesser degree than in normal PMN. Normal but not CGD monocytes and MDM killed G. bethesdensis and required in vitro treatment with IFN-γ to maintain this killing effect. Although in vitro IFN-γ did not enhance G. bethesdensis killing in CGD monocytes, it restricted growth in proportion to CGD PMN residual superoxide production, providing a potential method to identify patients responsive to IFN-γ therapy. In IFN-γ-treated CGD MDM, G. bethesdensis persisted for the duration of the study (7 d) without decreasing viability of the host cells. These results indicate that G. bethesdensis is highly resistant to oxygen-independent microbicides of myeloid cells, requires an intact NADPH oxidase for clearance, and can persist long-term in CGD mononuclear phagocytes, most likely relating to the persistence of this microorganism in infected CGD patients.

Dicer function is required in the metanephric mesenchyme for early kidney development.

MicroRNAs (miRNAs) are small, noncoding regulatory RNAs that act as posttranscriptional repressors by binding to the 3'-untranslated region (3'-UTR) of target genes. They require processing by Dicer, an RNase III enzyme, to become mature regulatory RNAs. Previous work from our laboratory revealed critical roles for miRNAs in nephron progenitors at midgestation (Ho J, Pandey P, Schatton T, Sims-Lucas S, Khalid M, Frank MH, Hartwig S, Kreidberg JA. J Am Soc Nephrol 22: 1053-1063, 2011). To interrogate roles for miRNAs in the early metanephric mesenchyme, which gives rise to nephron progenitors as well as the renal stroma during kidney development, we conditionally ablated Dicer function in this lineage. Despite normal ureteric bud outgrowth and condensation of the metanephric mesenchyme to form nephron progenitors, early loss of miRNAs in the metanephric mesenchyme resulted in severe renal dysgenesis. Nephron progenitors are initially correctly specified in the mutant kidneys, with normal expression of several transcription factors known to be critical in progenitors, including Six2, Pax2, Sall1, and Wt1. However, there is premature loss of the nephron progenitor marker Cited1, marked apoptosis, and increased expression of the proapoptotic protein Bim shortly after the initial inductive events in early kidney development. Subsequently, there is a failure in ureteric bud branching and nephron progenitor differentiation. Taken together, our data demonstrate a previously undetermined requirement for miRNAs during early kidney organogenesis and indicate a crucial role for miRNAs in regulating the survival of this lineage.

Metabolites Associated with Polygenic Risk of Breast Cancer.

While hundreds of germline genetic variants have been associated with breast cancer risk, the mechanisms underlying the impacts of most of these variants on breast cancer remain uncertain. Metabolomics may offer valuable insights into the mechanisms underlying genetic risks of breast cancer. Among 143 cancer-free female participants, we used linear regression analyses to explore associations between the genetic risk of breast cancer, as determined by a previously developed polygenic risk score (PRS) that included 266 single-nucleotide polymorphisms (SNPs), and 223 measures of metabolites obtained from blood samples using nuclear magnetic resonance (NMR). A false discovery rate of 10% was applied to account for multiple comparisons. PRS was statistically significantly associated with 45 metabolite measures. These were primarily measures of very low-density lipoproteins (VLDLs) and high-density lipoproteins (HDLs), including triglycerides, cholesterol, and phospholipids. For example, the strongest effect was observed with the percent ratio of medium VLDL triglycerides to total lipids (0.53 unit increase in mean-standardized ln-transformed percent ratio per unit increase in PRS; q = 0.1). While larger-scale studies are needed to confirm these results, this exploratory study presents biologically plausible findings that are consistent with previously reported associations between lipids and breast cancer risk. If confirmed, these lipids could be targeted for lifestyle and pharmaceutical interventions among women at increased genetic risk of breast cancer.

Secretome profiling reveals acute changes in oxidative stress, brain homeostasis, and coagulation following short-duration spaceflight.

As spaceflight becomes more common with commercial crews, blood-based measures of crew health can guide both astronaut biomedicine and countermeasures. By profiling plasma proteins, metabolites, and extracellular vesicles/particles (EVPs) from the SpaceX Inspiration4 crew, we generated "spaceflight secretome profiles," which showed significant differences in coagulation, oxidative stress, and brain-enriched proteins. While >93% of differentially abundant proteins (DAPs) in vesicles and metabolites recovered within six months, the majority (73%) of plasma DAPs were still perturbed post-flight. Moreover, these proteomic alterations correlated better with peripheral blood mononuclear cells than whole blood, suggesting that immune cells contribute more DAPs than erythrocytes. Finally, to discern possible mechanisms leading to brain-enriched protein detection and blood-brain barrier (BBB) disruption, we examined protein changes in dissected brains of spaceflight mice, which showed increases in PECAM-1, a marker of BBB integrity. These data highlight how even short-duration spaceflight can disrupt human and murine physiology and identify spaceflight biomarkers that can guide countermeasure development.

RFWD3-Mdm2 ubiquitin ligase complex positively regulates p53 stability in response to DNA damage.

In unstressed cells, the tumor suppressor p53 is maintained at low levels by ubiquitin-mediated proteolysis mainly through Mdm2. In response to DNA damage, p53 is stabilized and becomes activated to turn on transcriptional programs that are essential for cell cycle arrest and apoptosis. Activation of p53 leads to accumulation of Mdm2 protein, a direct transcriptional target of p53. It is not understood how p53 is protected from degradation when Mdm2 is up-regulated. Here we report that p53 stabilization in the late phase after ionizing radiation correlates with active ubiquitination. We found that an E3 ubiquitin ligase RFWD3 (RNF201/FLJ10520) forms a complex with Mdm2 and p53 to synergistically ubiquitinate p53 and is required to stabilize p53 in the late response to DNA damage. This process is regulated by the DNA damage checkpoint, because RFWD3 is phosphorylated by ATM/ATR kinases and the phosphorylation mutant fails to stimulate p53 ubiquitination. In vitro experiments suggest that RFWD3 is a p53 E3 ubiquitin ligase and that RFWD3-Mdm2 complex restricts the polyubiquitination of p53 by Mdm2. Our study identifies RFWD3 as a positive regulator of p53 stability when the G(1) cell cycle checkpoint is activated and provides an explanation for how p53 is protected from degradation in the presence of high levels of Mdm2.

Effect of mobile interventions with nicotine replacement therapy sampling on long-term smoking cessation in community smokers: A pragmatic randomized clinical trial.

INTRODUCTION: Mobile interventions enable personalized behavioral support that could improve smoking cessation (SC) in smokers ready to quit. Scalable interventions, including unmotivated smokers, are needed. We evaluated the effect of personalized behavioral support through mobile interventions plus nicotine replacement therapy sampling (NRT-S) on SC in Hong Kong community smokers. METHODS: A total of 664 adult daily cigarette smokers (74.4% male, 51.7% not ready to quit in 30 days) were proactively recruited from smoking hotspots and individually randomized (1:1) to the intervention and control groups (each, n=332). Both groups received brief advice and active referral to SC services. The intervention group received 1-week NRT-S at baseline and 12-week personalized behavioral support through SC advisor-delivered Instant Messaging (IM) and a fully automated chatbot. The control group received regular text messages regarding general health at a similar frequency. Primary outcomes were carbon monoxide-validated smoking abstinence at 6 and 12 months post-treatment initiation. Secondary outcomes included self-reported 7-day point-prevalence and 24-week continuous abstinence, quit attempts, smoking reduction, and SC service use at 6 and 12 months. RESULTS: By intention-to-treat, the intervention group did not significantly increase validated abstinence at 6 months (3.9% vs 3.0%, OR=1.31; 95% CI: 0.57-3.04) and 12 months (5.4% vs 4.5%, OR=1.21; 95% CI: 0.60-2.45), as were self-reported 7-day point-prevalence abstinence, smoking reduction, and SC service use at 6 and 12 months. More participants in the intervention than control group made a quit attempt by 6 months (47.0% vs 38.0%, OR=1.45; 95% CI: 1.06-1.97). Intervention engagement rates were low, but engagement in IM alone or combined with chatbot showed higher abstinence at 6 months (adjusted odds ratios, AORs=4.71 and 8.95, both p<0.05). CONCLUSIONS: Personalized behavioral support through mobile interventions plus NRT-S did not significantly improve abstinence in community smokers compared to text only messaging. The suboptimal intervention engagement needs to be addressed in future studies. TRIAL REGISTRATION: ClinicalTrials.gov NCT04001972.

RING finger and WD repeat domain 3 (RFWD3) associates with replication protein A (RPA) and facilitates RPA-mediated DNA damage response.

DNA damage response is crucial for maintaining genomic integrity and preventing cancer by coordinating the activation of checkpoints and the repair of damaged DNA. Central to DNA damage response are the two checkpoint kinases ATM and ATR that phosphorylate a wide range of substrates. RING finger and WD repeat domain 3 (RFWD3) was initially identified as a substrate of ATM/ATR from a proteomic screen. Subsequent studies showed that RFWD3 is an E3 ubiquitin ligase that ubiquitinates p53 in vitro and positively regulates p53 levels in response to DNA damage. We report here that RFWD3 associates with replication protein A (RPA), a single-stranded DNA-binding protein that plays essential roles in DNA replication, recombination, and repair. Binding of RPA to single-stranded DNA (ssDNA), which is generated by DNA damage and repair, is essential for the recruitment of DNA repair factors to damaged sites and the activation of checkpoint signaling. We show that RFWD3 is physically associated with RPA and rapidly localizes to sites of DNA damage in a RPA-dependent manner. In vitro experiments suggest that the C terminus of RFWD3, which encompass the coiled-coil domain and the WD40 domain, is necessary for binding to RPA. Furthermore, DNA damage-induced phosphorylation of RPA and RFWD3 is dependent upon each other. Consequently, loss of RFWD3 results in the persistent foci of DNA damage marker γH2AX and the repair protein Rad51 in damaged cells. These findings suggest that RFWD3 is recruited to sites of DNA damage and facilitates RPA-mediated DNA damage signaling and repair.

Innate immunity against Granulibacter bethesdensis, an emerging gram-negative bacterial pathogen.

Acetic acid bacteria were previously considered nonpathogenic in humans. However, over the past decade, five genera of Acetobacteraceae have been isolated from patients with inborn or iatrogenic immunodeficiencies. Here, we describe the first studies of the interactions of the human innate immune system with a member of this bacterial family, Granulibacter bethesdensis, an emerging pathogen in patients with chronic granulomatous disease (CGD). Efficient phagocytosis of G. bethesdensis by normal and CGD polymorphonuclear leukocytes (CGD PMN) required heat-labile serum components (e.g., C3), and binding of C3 and C9 to G. bethesdensis was detected by immunoblotting. However, this organism survived in human serum concentrations of ≥90%, indicating a high degree of serum resistance. Consistent with the clinical host tropism of G. bethesdensis, CGD PMN were unable to kill this organism, while normal PMN, in the presence of serum, reduced the number of CFU by about 50% after a 24-h coculture. This finding, together with the observations that G. bethesdensis was sensitive to H(2)O(2) but resistant to LL-37, a human cationic antimicrobial peptide, suggests an inherent resistance to O(2)-independent killing. Interestingly, 10 to 100 times greater numbers of G. bethesdensis were required to achieve the same level of reactive oxygen species (ROS) production induced by Escherichia coli in normal PMN. In addition to the relative inability of the organism to elicit production of PMN ROS, G. bethesdensis inhibited both constitutive and FAS-induced PMN apoptosis. These properties of reduced PMN activation and resistance to nonoxidative killing mechanisms likely play an important role in G. bethesdensis pathogenesis.

Insignificant effect of secretin in rodent models of polycystic kidney and liver disease.

Polycystic kidney (PKD) and liver (PLD) diseases cause significant morbidity and mortality. A large body of evidence indicates that cyclic AMP plays an important role in their pathogenesis. Clinical trials of drugs that reduce cyclic AMP levels in target tissues are now in progress. Secretin may contribute to adenylyl cyclase-dependent urinary concentration and is a major agonist of adenylyl cyclase in cholangiocytes. To investigate the role of secretin in PKD and PLD, we have studied the expression of secretin and the secretin receptor in rodent models orthologous to autosomal recessive (PCK rat) and dominant (Pkd2(-/WS25) mouse) PKD; the effects of exogenous secretin administration to PCK rats, PCK rats lacking circulating vasopressin (PCK(di/di)), and Pkd2(-/WS25) mice; and the impact of a nonfunctional secretin receptor on disease development in Pkd2(-/WS25):SCTR(-/-) double mutants. Renal and hepatic secretin and secretin receptor mRNA and plasma secretin were increased in both models, and secretin receptor protein was increased in the kidneys and liver of Pkd2(-/WS25) mice. However, exogenous secretin administered subcutaneously via osmotic pumps had minimal or negligible effects and the absence of a functional secretin receptor had no influence on the severity of PKD or PLD. Therefore, it is unlikely that by itself secretin plays a significant role in the pathogenesis of PKD and/or PLD.

Secretin as a neurohypophysial factor regulating body water homeostasis.

Hypothalamic magnocellular neurons express either one of the neurohypophysial hormones, vasopressin or oxytocin, along with different neuropeptides or neuromodulators. Axonal terminals of these neurons are generally accepted to release solely the two hormones but not others into the circulation. Here, we show that secretin, originally isolated from upper intestinal mucosal extract, is present throughout the hypothalamo-neurohypophysial axis and that it is released from the posterior pituitary under plasma hyperosmolality conditions. In the hypothalamus, it stimulates vasopressin expression and release. Considering these findings together with our previous findings that show a direct effect of secretin on renal water reabsorption, we propose here that secretin works at multiple levels in the hypothalamus, pituitary, and kidney to regulate water homeostasis. Findings presented here challenge previous understanding regarding the neurohypophysis and could provide new concepts in treating disorders related to osmoregulation.

Agreement between self-report and administrative health data on occurrence of non-cancer chronic disease among participants of the BC generations project.

Population-based studies of non-cancer chronic disease often rely on self-reported data for disease diagnosis, which may be incomplete, unreliable and suffer from bias. Recently, the British Columbia Generations Project (BCGP; n = 29,736) linked self-reported chronic disease history data to a Chronic Disease Registry (CDR) that applied algorithms to administrative health data to ascertain diagnoses of multiple chronic diseases in the Province of British Columbia. For the 10 diseases captured by both self-report and the CDR, including asthma, chronic obstructive pulmonary disease (COPD), diabetes, hypertension, multiple sclerosis, myocardial infarction, osteoarthritis, osteoporosis, rheumatoid arthritis, and stroke, we calculated Cohen's kappa coefficient to examine concordance of chronic disease status (i.e., ever/never diagnosed) between the data sources. Using CDR data as the gold standard, we also calculated sensitivity, specificity, and positive-predictive value (PPV) for self-reported chronic disease occurrence. The prevalence of each chronic disease was similar across both data sources. Substantial levels of concordance (0.66-0.73) and moderate to high sensitivities (0.64-0.92), specificities (0.98-0.99) and PPVs (0.55-0.84) were observed for diabetes, hypertension, multiple sclerosis, and myocardial infarction. We did observe degree of concordance to vary by age, sex, body mass index (BMI), health perception, and ethnicity across most of the chronic diseases that were evaluated. While administrative health data are imperfect, they are less likely to suffer from bias, making them a reasonable gold standard. Our results demonstrate that for at least some chronic diseases, self-report may be a reasonable method for case ascertainment. However, characteristics of the study population will likely have impacts on the quality of the data.

The BC Generations Project as a Tumor Tissue Resource for Cancer Research.

Population-based cohort studies can be a resource for tumor specimens, annotated with demographic, lifestyle, and health history data, that support innovative studies of cancer. Our aim was to establish and test a process for accessing tumor samples, held at pathology laboratories around British Columbia (BC), for participants of the BC Generations Project (BCGP). Through the BC Cancer Registry, we identified pathology reports for 1100 (93%) of the 1180 incident solid cancer cases diagnosed in BCGP as of 2019. Using manually abstracted data from the reports, we successfully retrieved 183 (92%) of the 200 formalin-fixed, paraffin-embedded (FFPE) blocks (breast, lung, bladder, and pancreas cancer cases) that we requested from pathology laboratories. No important differences in retrieval rates by cancer site, sample location (Greater Vancouver vs. Outside Greater Vancouver), sample type (biopsy vs. excision) or year of diagnosis were identified. A text mining solution recently implemented by the Registry will allow us to automate the process for data abstraction and should capture pathology reports for 100% of all newly diagnosed BCGP cancer cases moving forward. This will further enhance the utility of BCGP as a high-quality tumor tissue research resource.