In 2016, Rose Lamont and Tana Fishman were the first patient-clinician dyad from outside North America to attend the North American Primary Care Research Group (NAPCRG) Patient and Clinician Engagement Program workshop. They returned to New Zealand inspired and formed the Pacific People’s Health Advisory Group and a Pacific practice-based research network (PBRN). They are guided by the principles of co-design, and the Samoan research framework fa’afaletui, which emphasizes a collective approach and importance of reciprocity and relationships. Their collective inquiry aims to reduce health inequalities experienced by Pacific people in South Auckland. Their community group members and PBRN are generating research questions being answered by university-based graduate students. When they embarked, they knew not the direction in which they headed. With guidance, their community members and clinicians have led the way. By giving everyone a say in where they are going and how they get there, they are modeling what they wish to achieve—an egalitarian approach which decreases disparities for Pacific people.

PURPOSE

Operational failures are system-level errors in the supply of information, equipment, and materials to health care personnel. We aimed to review and synthesize the research literature to determine how operational failures in primary care affect the work of primary care physicians.

METHODS

We conducted a critical interpretive synthesis. We searched 7 databases for papers published in English from database inception until October 2017 for primary research of any design that addressed problems interfering with primary care physicians’ work. All potentially eligible titles/abstracts were screened by 1 reviewer; 30% were subject to second screening. We conducted an iterative critique, analysis, and synthesis of included studies.

RESULTS

Our search retrieved 8,544 unique citations. Though no paper explicitly referred to "operational failures," we identified 95 papers that conformed to our general definition. The included studies show a gap between what physicians perceived they should be doing and what they were doing, which was strongly linked to operational failures—including those relating to technology, information, and coordination—over which physicians often had limited control. Operational failures actively configured physicians’ work by requiring significant compensatory labor to deliver the goals of care. This labor was typically unaccounted for in scheduling or reward systems and had adverse consequences for physician and patient experience.

CONCLUSIONS

Primary care physicians’ efforts to compensate for suboptimal work systems are often concealed, risking an incomplete picture of the work they do and problems they routinely face. Future research must identify which operational failures are highest impact and tractable to improvement.

PURPOSE

Anticholinergic burden (ACB), the cumulative effect of anticholinergic medications, is associated with adverse outcomes in older people but is less studied in middle-aged populations. Numerous scales exist to quantify ACB. The aims of this study were to quantify ACB in a large cohort using the 10 most common anticholinergic scales, to assess the association of each scale with adverse outcomes, and to assess overlap in populations identified by each scale.

METHODS

We performed a longitudinal analysis of the UK Biobank community cohort (502,538 participants, baseline age: 37-73 years, median years of follow-up: 6.2). The ACB was calculated at baseline using 10 scales. Baseline data were linked to national mortality register records and hospital episode statistics. The primary outcome was a composite of all-cause mortality and major adverse cardiovascular event (MACE). Secondary outcomes were all-cause mortality, MACE, hospital admission for fall/fracture, and hospital admission with dementia/delirium. Cox proportional hazards models (hazard ratio [HR], 95% CI) quantified associations between ACB scales and outcomes adjusted for age, sex, socioeconomic status, body mass index, smoking status, alcohol use, physical activity, and morbidity count.

RESULTS

Anticholinergic medication use varied from 8% to 17.6% depending on the scale used. For the primary outcome, ACB was significantly associated with all-cause mortality/MACE for each scale. The Anticholinergic Drug Scale was most strongly associated with mortality/MACE (HR = 1.12; 95% CI, 1.11-1.14 per 1-point increase in score). The ACB was significantly associated with all secondary outcomes. The Anticholinergic Effect on Cognition scale was most strongly associated with dementia/delirium (HR = 1.45; 95% CI, 1.3-1.61 per 1-point increase).

CONCLUSIONS

The ACB was associated with adverse outcomes in a middle- to older-aged population. Populations identified and effect size differed between scales. Scale choice influenced the population identified as potentially requiring reduction in ACB in clinical practice or intervention trials.

PURPOSE

We aimed to evaluate the efficacy and safety of use of the Fasting Algorithm for Singaporeans with Type 2 Diabetes (FAST) during Ramadan.

METHODS

We performed a prospective, multicenter, randomized controlled trial. The inclusion criteria were age ≥21 years, baseline glycated hemoglobin (HbA_1c) level ≤9.5%, and intention to fast for ≥10 days during Ramadan. Exclusion criteria included baseline estimated glomerular filtration rate <30 mL/min, diabetes-related hospitalization, and short-term corticosteroid therapy. Participants were randomized to intervention (use of FAST) or control (usual care without FAST) groups. Efficacy outcomes were HbA_1c level and fasting blood glucose and postprandial glucose changes, and the safety outcome was incidence of major or minor hypoglycemia during the Ramadan period. Glycemic variability and diabetes distress were also investigated. Linear mixed models were constructed to assess changes.

RESULTS

A total of 97 participants were randomized (intervention: n = 46, control: n = 51). The HbA_1c improvement during Ramadan was 4 times greater in the intervention group (–0.4%) than in the control group (–0.1%) (P = .049). The mean fasting blood glucose level decreased in the intervention group (–3.6 mg/dL) and increased in the control group (+20.9 mg/dL) (P = .034). The mean postprandial glucose level showed greater improvement in the intervention group (–16.4 mg/dL) compared to the control group (–2.3 mg/dL). There were more minor hypoglycemic events based on self-monitered blood glucose readings in the control group (intervention: 4, control: 6; P = .744). Glycemic variability was not significantly different between the 2 groups (P = .284). No between-group differences in diabetes distress were observed (P = .479).

CONCLUSIONS

Our findings emphasize the importance of efficacious, safe, and culturally tailored epistemic tools for diabetes management.

PURPOSE

Most real-world studies on anticoagulants have been based on health insurance databases or performed in secondary care. The aim of this study was to compare safety and effectiveness between patients treated with vitamin K antagonists (VKAs) and patients treated with direct oral anticoagulants (DOACs) in a general practice setting.

METHODS

The CACAO study (Comparison of Accidents and their Circumstances with Oral Anticoagulants) is a multicenter prospective cohort study conducted among ambulatory patients taking an oral anticoagulant. Participants were patients from the study’s cross-sectional phase receiving oral anticoagulants because of nonvalvular atrial fibrillation, for secondary prevention of venous thromboembolism, or both. They were followed as usual for 1 year by their general practitioners, who collected data on changes in therapy, thromboembolic events, bleeding, and deaths. All events were adjudicated by an independent committee. We used a propensity score and a Cox regression model to derive hazard ratios.

RESULTS

Between April and December 2014, a total of 3,082 patients were included. At 1 year, 42 patients (1.7%) had experienced an arterial or venous event; 151 (6.1%) had experienced bleeding, including 47 (1.9%) who experienced major bleeding; and 105 (4.1%) had died. There was no significant difference between the VKA and DOAC groups regarding arterial or venous events, or major bleeding. The VKA group had a lower risk of overall bleeding (hazard ratio = 0.65; 95% CI, 0.43-0.98) but twice the risk of death (hazard ratio = 1.98; 95% CI, 1.15-3.42).

CONCLUSIONS

VKAs and DOACs had fairly similar safety and effectiveness in general practice. The substantially higher incidence of deaths with VKAs is consistent with known data from health insurance databases and calls for further research to understand its cause.

PURPOSE

General practitioners (GPs) are part of the US physician workforce, but little is known about who they are, what they do, and how they differ from family physicians (FPs). We describe self-identified GPs and compare them with board-certified FPs.

METHODS

Analysis of data on 102,604 Doctor of Medicine and Doctor of Osteopathy physicians in direct patient care in the United States in 2016, who identify themselves as GPs or FPs. The study used linking databases (American Medical Association Masterfile, American Board of Family Medicine [ABFM], Area Health Resource File, Medicare Public Use File) to examine personal, professional, and practice characteristics.

RESULTS

Of the physicians identified, 6,661 self-designated as GPs and 95,943 self-designated as FPs. Of the self-designated GPs, 116 had been ABFM certified and were excluded from the study. Of the remaining 102,488 physicians, those who self-designated as GPs but were never ABFM certified constituted the GP group (n = 6,545, 6%). Self-designated FPs that were ABFM certified made up the FP group (n = 79,449, 78%). The remaining self-designated FPs not ABFM certified constituted the uncertified group (n = 16,494, 16%). GPs differed from FPs in every characteristic examined. Compared with FPs, GPs are more likely to be older, male, Doctors of Osteopathy, graduates of non-US medical schools, and have no family medicine residency training. GPs practice location is similar to FPs, but GPs are less likely to participate in Medicare or to work in hospitals.

CONCLUSIONS

GPs in the United States are a varied group that differ from FPs. Researchers, educators, and policy makers should not lump GPs together with FPs in data collection, analysis, and reporting.

PURPOSE

Online programs may help to engage patients in advance care planning in outpatient settings. We sought to implement an online advance care planning program, PREPARE (Prepare for Your Care; http://www.prepareforyourcare.org), at home and evaluate the changes in advance care planning engagement among patients attending outpatient clinics.

METHODS

We undertook a prospective before-and-after study in 15 primary care clinics and 2 outpatient cancer centers in Canada. Patients were aged 50 years or older (primary care) or 18 years or older (cancer care) and free of cognitive impairment. They used the PREPARE website over 6 weeks, with reminders sent at 2 or 4 weeks. We used the 55-item Advance Care Planning Engagement Survey, which measures behavior change processes (knowledge, contemplation, self-efficacy, readiness) on 5-point scales and actions relating to substitute decision makers, quality of life, flexibility for the decision maker, and asking doctors questions on an overall scale from 0 to 21; higher scores indicate greater engagement.

RESULTS

In total, 315 patients were screened and 172 enrolled, of whom 75% completed the study (mean age = 65.6 years, 51% female, 35% had cancer). The mean behavior change process score was 2.9 (SD 0.8) at baseline and 3.5 (SD 0.8) at follow-up (mean change = 0.6; 95% CI, 0.49-0.73); the mean action measure score was 4.0 (SD 4.9) at baseline and 5.2 (SD 5.4) at follow-up (mean change = 1.2; 95% CI, 0.54-1.77). The effect size was moderate (0.75) for the former and small (0.23) for the latter. Findings were similar in both primary care and cancer care populations.

CONCLUSIONS

Implementation of the online PREPARE program in primary care and cancer care clinics increased advance care planning engagement among patients.

Congenital anomalies of the kidneys and urinary tracts (CAKUT) are disorders caused by defects in the development of the kidneys and their outflow tracts. The formation of the kidneys begins at week 3 and nephrogenesis continues until week 36, therefore, the kidneys and outflow tracts are susceptible to environmental risk factors that perturb development throughout gestation. Many genes have been implicated in kidney and outflow tract development, and mutations have been identified in patients with CAKUT. In severe cases of CAKUT, when the kidneys do not form, the fetus will not survive. However, in less severe cases, the baby can survive with combined kidney and outflow tract defects or they may only be identified in adulthood. In this review, we will cover the clinical presentation of CAKUT, its epidemiology, and its long-term outcomes. We will then discuss risk factors for CAKUT, including genetic and environmental contributions. Although severe CAKUT is rare, low nephron number is a much more common disorder with its effect on kidney function increasingly apparent as a person ages. Low nephron number appears to arise by the same mechanisms as CAKUT, but it differs in terms of the magnitude of the insult and the timing of when it occurs during gestation. By understanding the causes of CAKUT and low nephron number, we can begin to identify preventive treatments and establish clinical guidelines for how these patients should be followed.

Background and objectives

Oxidative stress is a hallmark and mediator of CKD. Diminished antioxidant defenses are thought to be partly responsible. However, there is currently no way to prospectively assess antioxidant defenses in humans. Tin protoporphyrin (SnPP) induces mild, transient oxidant stress in mice, triggering increased expression of select antioxidant proteins (e.g., heme oxygenase 1 [HO-1], NAD[P]H dehydrogenase [quinone] 1 [NQO1], ferritin, p21). Hence, we tested the hypothesis that SnPP can also variably increase these proteins in humans and can thus serve as a pharmacologic "stress test" for gauging gene responsiveness and antioxidant reserves.

Design, setting, participants, & measurements

A total of 18 healthy volunteers and 24 participants with stage 3 CKD (n=12; eGFR 30–59 ml/min per 1.73 m²) or stage 4 CKD (n=12; eGFR 15–29 ml/min per 1.73 m²) were injected once with SnPP (9, 27, or 90 mg). Plasma and/or urinary antioxidant proteins were measured at baseline and for up to 4 days post-SnPP dosing. Kidney safety was gauged by serial measurements of BUN, creatinine, eGFR, albuminuria, and four urinary AKI biomarkers (kidney injury molecule 1, neutrophil gelatinase-associated lipocalin, cystatin C, and N-acetyl glucosaminidase).

Results

Plasma HO-1, ferritin, p21, and NQO1 were all elevated at baseline in CKD participants. Plasma HO-1 and urine NQO1 levels each inversely correlated with eGFR (r=–0.85 to –0.95). All four proteins manifested statistically significant dose- and time-dependent elevations after SnPP injection. However, marked intersubject differences were observed. p21 responses to high-dose SnPP and HO-1 responses to low-dose SnPP were significantly suppressed in participants with CKD versus healthy volunteers. SnPP was well tolerated by all participants, and no evidence of nephrotoxicity was observed.

Conclusions

SnPP can be safely administered and, after its injection, the resulting changes in plasma HO-1, NQO1, ferritin, and p21 concentrations can provide information as to antioxidant gene responsiveness/reserves in subjects with and without kidney disease.

Clinical Trial registry name and registration number

A Study with RBT-1, in Healthy Volunteers and Subjects with Stage 3–4 Chronic Kidney Disease, NCT0363002 and NCT03893799

Metabolic reprogramming is critical for the polarization and function of tumor-associated macrophages (TAM) and hepatocarcinogenesis, but how this reprogramming occurs is unknown. Here, we showed that receptor-interacting protein kinase 3 (RIPK3), a central factor in necroptosis, is downregulated in hepatocellular carcinoma (HCC)–associated macrophages, which correlated with tumorigenesis and enhanced the accumulation and polarization of M2 TAMs. Mechanistically, RIPK3 deficiency in TAMs reduced reactive oxygen species and significantly inhibited caspase1-mediated cleavage of PPAR. These effects enabled PPAR activation and facilitated fatty acid metabolism, including fatty acid oxidation (FAO), and induced M2 polarization in the tumor microenvironment. RIPK3 upregulation or FAO blockade reversed the immunosuppressive activity of TAMs and dampened HCC tumorigenesis. Our findings provide molecular basis for the regulation of RIPK3-mediated, lipid metabolic reprogramming of TAMs, thus highlighting a potential strategy for targeting the immunometabolism of HCC.

Vaccinia virus (VACV) is a double-stranded DNA virus that devotes a large portion of its 200 kbp genome to suppressing and manipulating the immune response of its host. Here, we investigated how targeted removal of immunomodulatory genes from the VACV genome impacted immune cells in the tumor microenvironment with the intention of improving the therapeutic efficacy of VACV in breast cancer. We performed a head-to-head comparison of six mutant oncolytic VACVs, each harboring deletions in genes that modulate different cellular pathways, such as nucleotide metabolism, apoptosis, inflammation, and chemokine and interferon signaling. We found that even minor changes to the VACV genome can impact the immune cell compartment in the tumor microenvironment. Viral genome modifications had the capacity to alter lymphocytic and myeloid cell compositions in tumors and spleens, PD-1 expression, and the percentages of virus-targeted and tumor-targeted CD8⁺ T cells. We observed that while some gene deletions improved responses in the nonimmunogenic 4T1 tumor model, very little therapeutic improvement was seen in the immunogenic HER2/neu TuBo model with the various genome modifications. We observed that the most promising candidate genes for deletion were those that interfere with interferon signaling. Collectively, this research helped focus attention on the pathways that modulate the immune response in the context of VACV oncolytic virotherapy. They also suggest that the greatest benefits to be obtained with these treatments may not always be seen in "hot tumors."

Fnr is a transcriptional regulator that controls the expression of a variety of genes in response to oxygen limitation in bacteria. Genome sequencing revealed four genes (fnr1, fnr3, fnr5, and fnr7) coding for Fnr proteins in Paenibacillus polymyxa WLY78. Fnr1 and Fnr3 showed more similarity to each other than to Fnr5 and Fnr7. Also, Fnr1 and Fnr3 exhibited high similarity with Bacillus cereus Fnr and Bacillus subtilis Fnr in sequence and structures. Both the aerobically purified His-tagged Fnr1 and His-tagged Fnr3 in Escherichia coli could bind to the specific DNA promoter. Deletion analysis showed that the four fnr genes, especially fnr1 and fnr3, have significant impacts on growth and nitrogenase activity. Single deletion of fnr1 or fnr3 led to a 50% reduction in nitrogenase activity, and double deletion of fnr1 and fnr3 resulted to a 90% reduction in activity. Genome-wide transcription analysis showed that Fnr1 and Fnr3 indirectly activated expression of nif (nitrogen fixation) genes and Fe transport genes under anaerobic conditions. Fnr1 and Fnr3 inhibited expression of the genes involved in the aerobic respiratory chain and activated expression of genes responsible for anaerobic electron acceptor genes.

IMPORTANCE The members of the nitrogen-fixing Paenibacillus spp. have great potential to be used as a bacterial fertilizer in agriculture. However, the functions of the fnr gene(s) in nitrogen fixation and other metabolisms in Paenibacillus spp. are not known. Here, we found that in P. polymyxa WLY78, Fnr1 and Fnr3 were responsible for regulation of numerous genes in response to changes in oxygen levels, but Fnr5 and Fnr7 exhibited little effect. Fnr1 and Fnr3 indirectly or directly regulated many types of important metabolism, such as nitrogen fixation, Fe uptake, respiration, and electron transport. This study not only reveals the function of the fnr genes of P. polymyxa WLY78 in nitrogen fixation and other metabolisms but also will provide insight into the evolution and regulatory mechanisms of fnr in Paenibacillus.

This study investigated the effects of long-term soil fertilization on the composition and potential for phosphorus (P) and nitrogen (N) cycling of bacterial communities associated with hyphae of the P-solubilizing fungus Penicillium canescens. Using a baiting approach, hyphosphere bacterial communities were recovered from three soils that had received long-term amendment in the field with mineral or mineral plus organic fertilizers. P. canescens hyphae recruited bacterial communities with a decreased diversity and an increased abundance of Proteobacteria relative to what was observed in soil communities. As core bacterial taxa, Delftia and Pseudomonas spp. were present in all hyphosphere samples irrespective of soil fertilization. However, the type of fertilization showed significant impacts on the diversity, composition, and distinctive taxa/operational taxonomic units (OTUs) of hyphosphere communities. The soil factors P (Olsen method), exchangeable Mg, exchangeable K, and pH were important for shaping soil and hyphosphere bacterial community compositions. An increased relative abundance of organic P metabolism genes was found in hyphosphere communities from soil that had not received P fertilizers, which could indicate P limitation near the fungal hyphae. Additionally, P. canescens hyphae recruited bacterial communities with a higher abundance of N fixation genes than found in soil communities, which might imply a role of hyphosphere communities for fungal N nutrition. Furthermore, the relative abundances of denitrification genes were greater in several hyphosphere communities, indicating an at least partly anoxic microenvironment with a high carbon-to-N ratio around the hyphae. In conclusion, soil fertilization legacy shapes P. canescens hyphosphere microbiomes and their functional potential related to P and N cycling.

IMPORTANCE P-solubilizing Penicillium strains are introduced as biofertilizers to agricultural soils to improve plant P nutrition. Currently, little is known about the ecology of these biofertilizers, including their interactions with other soil microorganisms. This study shows that communities dominated by Betaproteobacteria and Gammaproteobacteria colonize P. canescens hyphae in soil and that the compositions of these communities depend on the soil conditions. The potential of these communities for N and organic P cycling is generally higher than that of soil communities. The high potential for organic P metabolism might complement the ability of the fungus to solubilize inorganic P, and it points to the hyphosphere as a hot spot for P metabolism. Furthermore, the high potential for N fixation could indicate that P. canescens recruits bacteria that are able to improve its N nutrition. Hence, this community study identifies functional groups relevant for the future optimization of next-generation biofertilizer consortia for applications in soil.

Random transposon mutagenesis is a powerful and unbiased genetic approach to answer fundamental biological questions. Here, we introduce an improved mariner-based transposon system with enhanced stability during propagation and versatile applications in mutagenesis. We used a low-copy-number plasmid as a transposon delivery vehicle, which affords a lower frequency of unintended recombination during vector construction and propagation in Escherichia coli. We generated a variety of transposons allowing for gene disruption or artificial overexpression, each in combination with one of four different antibiotic resistance markers. In addition, we provide transposons that will report gene/protein expression due to transcriptional or translational coupling. We believe that the TnFLX system will help enhance the flexibility of future transposon modification and application in Bacillus and other organisms.

IMPORTANCE The stability of transposase-encoding vectors during cloning and propagation is crucial for the reliable application of transposons. Here, we increased the stability of the mariner delivery vehicle in E. coli. Moreover, the TnFLX transposon system will improve the application of forward genetic methods with an increased number of antibiotic resistance markers and the ability to generate unbiased green fluorescent protein (GFP) fusions to report on protein translation and subcellular localization.

Dietary protein residue can result in microbial generation of various toxic metabolites in the gut, such as ammonia. A prebiotic is "a substrate that is selectively utilised by host microorganisms conferring a health benefit" (G. R. Gibson, R. Hutkins, M. E. Sanders, S. L. Prescott, et al., Nat Rev Gastroenterol Hepatol 14:491–502, 2017, https://doi.org/10.1038/nrgastro.2017.75). Prebiotics are carbohydrates that may have the potential to reverse the harmful effects of gut bacterial protein fermentation. Three-stage continuous colonic model systems were inoculated with fecal samples from omnivore and vegetarian volunteers. Casein (equivalent to 105 g protein consumption per day) was used within the systems as a protein source. Two different doses of inulin-type fructans (Synergy1) were later added (equivalent to 10 g per day in vivo and 15 g per day) to assess whether this influenced protein fermentation. Bacteria were enumerated by fluorescence in situ hybridization with flow cytometry. Metabolites from bacterial fermentation (short-chain fatty acid [SCFA], ammonia, phenol, indole, and p-cresol) were monitored to further analyze proteolysis and the prebiotic effect. A significantly higher number of bifidobacteria was observed with the addition of inulin together with reduction of Desulfovibrio spp. Furthermore, metabolites from protein fermentation, such as branched-chain fatty acids (BCFA) and ammonia, were significantly lowered with Synergy1. Production of p-cresol varied among donors, as we recognized four high producing models and two low producing models. Prebiotic addition reduced its production only in vegetarian high p-cresol producers.

IMPORTANCE Dietary protein levels are generally higher in Western populations than in the world average. We challenged three-stage continuous colonic model systems containing high protein levels and confirmed the production of potentially harmful metabolites from proteolysis, especially replicates of the transverse and distal colon. Fermentations of proteins with a prebiotic supplementation resulted in a change in the human gut microbiota and inhibited the production of some proteolytic metabolites. Moreover, we observed both bacterial and metabolic differences between fecal bacteria from omnivore donors and vegetarian donors. Proteins with prebiotic supplementation showed higher Bacteroides spp. and inhibited Clostridium cluster IX in omnivore models, while in vegetarian modes, Clostridium cluster IX was higher and Bacteroides spp. lower with high protein plus prebiotic supplementation. Synergy1 addition inhibited p-cresol production in vegetarian high p-cresol-producing models while the inhibitory effect was not seen in omnivore models.

Vibralactone, a hybrid compound derived from phenols and a prenyl group, is a strong pancreatic lipase inhibitor with a rare fused bicyclic β-lactone skeleton. Recently, a researcher reported a vibralactone derivative (compound C1) that caused inhibition of pancreatic lipase with a half-maximal inhibitory concentration of 14 nM determined by structure-based optimization, suggesting a potential candidate as a new antiobesity treatment. In the present study, we sought to identify the main gene encoding prenyltransferase in Stereum vibrans, which is responsible for the prenylation of phenol leading to vibralactone synthesis. Two RNA silencing transformants of the identified gene (vib-PT) were obtained through Agrobacterium tumefaciens-mediated transformation. Compared to wild-type strains, the transformants showed a decrease in vib-PT expression ranging from 11.0 to 56.0% at 5, 10, and 15 days in reverse transcription-quantitative PCR analysis, along with a reduction in primary vibralactone production of 37 to 64% at 15 and 21 days, respectively, as determined using ultra-high-performance liquid chromatography-mass spectrometry analysis. A soluble and enzymatically active fusion Vib-PT protein was obtained by expressing vib-PT in Escherichia coli, and the enzyme’s optimal reaction conditions and catalytic efficiency (K_m/k_cat) were determined. In vitro experiments established that Vib-PT catalyzed the C-prenylation at C-3 of 4-hydroxy-benzaldehyde and the O-prenylation at the 4-hydroxy of 4-hydroxy-benzenemethanol in the presence of dimethylallyl diphosphate. Moreover, Vib-PT shows promiscuity toward aromatic compounds and prenyl donors.

IMPORTANCE Vibralactone is a lead compound with a novel skeleton structure that shows strong inhibitory activity against pancreatic lipase. Vibralactone is not encoded by the genome directly but rather is synthesized from phenol, followed by prenylation and other enzyme reactions. Here, we used an RNA silencing approach to identify and characterize a prenyltransferase in a basidiomycete species that is responsible for the synthesis of vibralactone. The identified gene, vib-PT, was expressed in Escherichia coli to obtain a soluble and enzymatically active fusion Vib-PT protein. In vitro characterization of the enzyme demonstrated the catalytic mechanism of prenylation and broad substrate range for different aromatic acceptors and prenyl donors. These characteristics highlight the possibility of Vib-PT to generate prenylated derivatives of aromatics and other compounds as improved bioactive agents or potential prodrugs.

A longstanding awareness in generating resistance to common antimicrobial therapies by Gram-negative bacteria has made them a major threat to global health. The application of antimicrobial peptides as a therapeutic agent would be a great opportunity to combat bacterial diseases. Here, we introduce a new antimicrobial peptide (~8.3 kDa) from probiotic strain Lactobacillus acidophilus ATCC 4356, designated acidocin 4356 (ACD). This multifunctional peptide exerts its anti-infective ability against Pseudomonas aeruginosa through an inhibitory action on virulence factors, bacterial killing, and biofilm degradation. Reliable performance over tough physiological conditions and low hemolytic activity confirmed a new hope for the therapeutic setting. Antibacterial kinetic studies using flow cytometry technique showed that the ACD activity is related to the change in permeability of the membrane. The results obtained from molecular dynamic (MD) simulation were perfectly suited to the experimental data of ACD behavior. The structure-function relationship of this natural compound, along with the results of transmission electron microscopy analysis and MD simulation, confirmed the ability of the ACD aimed at enhancing bacterial membrane perturbation. The peptide was effective in the treatment of P. aeruginosa infection in mouse model. The results support the therapeutic potential of ACD for the treatment of Pseudomonas infections.

IMPORTANCE Multidrug-resistant bacteria are a major threat to global health, and the Pseudomonas bacterium with the ability to form biofilms is considered one of the main causative agents of nosocomial infections. Traditional antibiotics have failed because of increased resistance. Thus, finding new biocompatible antibacterial drugs is essential. Antimicrobial peptides are produced by various organisms as a natural defense mechanism against pathogens, inspiring the possible design of the next generation of antibiotics. In this study, a new antimicrobial peptide was isolated from Lactobacillus acidophilus ATCC 4356, counteracting both biofilm and planktonic cells of Pseudomonas aeruginosa. A detailed investigation was then conducted concerning the functional mechanism of this peptide by using fluorescence techniques, electron microscopy, and in silico methods. The antibacterial and antibiofilm properties of this peptide may be important in the treatment of Pseudomonas infections.

In Lysobacter enzymogenes OH11, RpfB1 and RpfB2 were predicted to encode acyl coenzyme A (CoA) ligases. RpfB1 is located in the Rpf gene cluster. Interestingly, we found an RpfB1 homolog (RpfB2) outside this canonical gene cluster, and nothing is known about its functionality or mechanism. Here, we report that rpfB1 and rpfB2 can functionally replace EcFadD in the Escherichia coli fadD mutant JW1794. RpfB activates long-chain fatty acids (n-C_16:0 and n-C_18:0) for the corresponding fatty acyl-CoA ligase (FCL) activity in vitro, and Glu-361 plays critical roles in the catalytic mechanism of RpfB1 and RpfB2. Deletion of rpfB1 and rpfB2 resulted in significantly increased heat-stable antifungal factor (HSAF) production, and overexpression of rpfB1 or rpfB2 completely suppressed HSAF production. Deletion of rpfB1 and rpfB2 resulted in increased L. enzymogenes diffusible signaling factor 3 (LeDSF3) synthesis in L. enzymogenes. Overall, our results showed that changes in intracellular free fatty acid levels significantly altered HSAF production. Our report shows that intracellular free fatty acids are required for HSAF production and that RpfB affects HSAF production via FCL activity. The global transcriptional regulator Clp directly regulated the expression of rpfB1 and rpfB2. In conclusion, these findings reveal new roles of RpfB in antibiotic biosynthesis in L. enzymogenes.

IMPORTANCE Understanding the biosynthetic and regulatory mechanisms of heat-stable antifungal factor (HSAF) could improve the yield in Lysobacter enzymogenes. Here, we report that RpfB1 and RpfB2 encode acyl coenzyme A (CoA) ligases. Our research shows that RpfB1 and RpfB2 affect free fatty acid metabolism via fatty acyl-CoA ligase (FCL) activity to reduce the substrate for HSAF synthesis and, thereby, block HSAF production in L. enzymogenes. Furthermore, these findings reveal new roles for the fatty acyl-CoA ligases RpfB1 and RpfB2 in antibiotic biosynthesis in L. enzymogenes. Importantly, the novelty of this work is the finding that RpfB2 lies outside the Rpf gene cluster and plays a key role in HSAF production, which has not been reported in other diffusible signaling factor (DSF)/Rpf-producing bacteria.

The N-acetylglucosaminidase LytB of Streptococcus pneumoniae is involved in nasopharyngeal colonization and is responsible for cell separation at the end of cell division; thus, lytB mutants form long chains of cells. This paper reports the construction and properties of a defective pneumococcal mutant producing an inactive LytB protein (LytB_E585A). It is shown that an enzymatically active LytB is required for in vitro biofilm formation, as lytB mutants (either lytB or producing the inactive LytB_E585A) are incapable of forming substantial biofilms, despite that extracellular DNA is present in the biofilm matrix. Adding small amounts (0.5 to 2.0 μg/ml) of exogenous LytB or some LytB constructs restored the biofilm-forming capacity of lytB mutants to wild-type levels. The LytB_E585A mutant formed biofilm more rapidly than lytB mutants in the presence of LytB. This suggests that the mutant protein acted in a structural role, likely through the formation of complexes with extracellular DNA. The chain-dispersing capacity of LytB allowed the separation of daughter cells, presumably facilitating the formation of microcolonies and, finally, of biofilms. A role for the possible involvement of LytB in the synthesis of the extracellular polysaccharide component of the biofilm matrix is also discussed.

IMPORTANCE It has been previously accepted that biofilm formation in S. pneumoniae must be a multigenic trait because the mutation of a single gene has led to only to partial inhibition of biofilm production. In the present study, however, evidence that the N-acetylglucosaminidase LytB is crucial in biofilm formation is provided. Despite the presence of extracellular DNA, strains either deficient in LytB or producing a defective LytB enzyme formed only shallow biofilms.

Deep-subsurface hot brines in northwest Poland, extracted through boreholes reaching 1.6 and 2.6 km below the ground surface, were microbiologically investigated using culture-independent and culture-dependent methods. The high-throughput sequencing of 16S rRNA gene amplicons showed a very low diversity of bacterial communities, which were dominated by phyla Proteobacteria and Firmicutes. Bacterial genera potentially involved in sulfur oxidation and nitrate reduction (Halothiobacillus and Methylobacterium) prevailed in both waters over the sulfate reducers ("Candidatus Desulforudis" and Desulfotomaculum). Only one archaeal taxon, affiliated with the order Thermoplasmatales, was detected in analyzed samples. Bacterial isolates obtained from these deep hot brines were closely related to Bacillus paralicheniformis based on the 16S rRNA sequence similarity. However, genomic and physiological analyses made for one of the isolates, Bacillus paralicheniformis strain TS6, revealed the existence of more diverse metabolic pathways than those of its moderate-temperature counterpart. These specific traits may be associated with the ecological adaptations to the extreme habitat, which suggest that some lineages of B. paralicheniformis are halothermophilic.

IMPORTANCE Deep-subsurface aquifers, buried thousands of meters down the Earth’s crust, belong to the most underexplored microbial habitats. Although a few studies revealed the existence of microbial life at the depths, the knowledge about the microbial life in the deep hydrosphere is still scarce due to the limited access to such environments. Studying the subsurface microbiome provides unique information on microbial diversity, community structure, and geomicrobiological processes occurring under extreme conditions of the deep subsurface. Our study shows that low-diversity microbial assemblages in subsurface hot brines were dominated by the bacteria involved in biogeochemical cycles of sulfur and nitrogen. Based on genomic and physiological analyses, we found that the Bacillus paralicheniformis isolate obtained from the brine under study differed from the mesophilic species in the presence of specific adaptations to harsh environmental conditions. We indicate that some lineages of B. paralicheniformis are halothermophilic, which was not previously reported.

Bacteria accumulate small, organic compounds called compatible solutes via uptake from the environment or biosynthesis from available precursors to maintain the turgor pressure of the cell in response to osmotic stress. The halophile Vibrio parahaemolyticus has biosynthesis pathways for the compatible solutes ectoine (encoded by ectABC-asp_ect) and glycine betaine (encoded by betIBA-proXWV), four betaine-carnitine-choline transporters (encoded by bccT1 to bccT4), and a second ProU transporter (encoded by proVWX). All of these systems are osmotically inducible with the exception of bccT2. Previously, it was shown that CosR, a MarR-type regulator, was a direct repressor of ectABC-asp_ect in Vibrio species. In this study, we investigated whether CosR has a broader role in the osmotic stress response. Expression analyses demonstrated that betIBA-proXWV, bccT1, bccT3, bccT4, and proVWX are repressed in low salinity. Examination of an in-frame cosR deletion mutant showed that expression of these systems is derepressed in the mutant at low salinity compared with the wild type. DNA binding assays demonstrated that purified CosR binds directly to the regulatory region of both biosynthesis systems and four transporters. In Escherichia coli green fluorescent protein (GFP) reporter assays, we demonstrated that CosR directly represses transcription of betIBA-proXWV, bccT3, and proVWX. Similar to Vibrio harveyi, we showed betIBA-proXWV was directly activated by the quorum-sensing LuxR homolog OpaR, suggesting a conserved mechanism of regulation among Vibrio species. Phylogenetic analysis demonstrated that CosR is ancestral to the Vibrionaceae family, and bioinformatics analysis showed widespread distribution among Gammaproteobacteria in general. Incidentally, in Aliivibrio fischeri, Aliivibrio finisterrensis, Aliivibrio sifiae, and Aliivibrio wodanis, an unrelated MarR-type regulator gene named ectR was clustered with ectABC-asp, which suggests the presence of another novel ectoine biosynthesis regulator. Overall, these data show that CosR is a global regulator of osmotic stress response that is widespread among bacteria.

IMPORTANCE Vibrio parahaemolyticus can accumulate compatible solutes via biosynthesis and transport, which allow the cell to survive in high salinity conditions. There is little need for compatible solutes under low salinity conditions, and biosynthesis and transporter systems need to be repressed. However, the mechanism(s) of this repression is not known. In this study, we showed that CosR played a major role in the regulation of multiple compatible solute systems. Phylogenetic analysis showed that CosR is present in all members of the Vibrionaceae family as well as numerous Gammaproteobacteria. Collectively, these data establish CosR as a global regulator of the osmotic stress response that is widespread in bacteria, controlling many more systems than previously demonstrated.

Biofilm formation by Pseudomonas aeruginosa contributes to its survival on surfaces and represents a major clinical threat because of the increased tolerance of biofilms to disinfecting agents. This study aimed to investigate the efficacy of 405-nm light-emitting diode (LED) illumination in eliminating P. aeruginosa biofilms formed on stainless steel coupons under different temperatures. Time-dependent killing assays using planktonic and biofilm cells were used to determine the antimicrobial and antibiofilm activities of LED illumination. We also evaluated the effects of LED illumination on the disinfectant susceptibility, biofilm structure, extracellular polymeric substance (EPS) structure and composition, and biofilm-related gene expression of P. aeruginosa biofilm cells. Results showed that the abundance of planktonic P. aeruginosa cells was reduced by 0.88, 0.53, and 0.85 log CFU/ml following LED treatment for 2 h compared with untreated controls at 4, 10, and 25°C, respectively. For cells in biofilms, significant reductions (1.73, 1.59, and 1.68 log CFU/cm²) were observed following LED illumination for 2 h at 4, 10, and 25°C, respectively. Moreover, illuminated P. aeruginosa biofilm cells were more sensitive to benzalkonium chloride or chlorhexidine than untreated cells. Scanning electron microscopy and confocal laser scanning microscopic observation indicated that both the biofilm structure and EPS structure were disrupted by LED illumination. Further, reverse transcription-quantitative PCR revealed that LED illumination downregulated the transcription of several genes associated with biofilm formation. These findings suggest that LED illumination has the potential to be developed as an alternative method for prevention and control of P. aeruginosa biofilm contamination.

IMPORTANCE Pseudomonas aeruginosa can form biofilms on medical implants, industrial equipment, and domestic surfaces, contributing to high morbidity and mortality rates. This study examined the antibiofilm activity of 405-nm light-emitting diode (LED) illumination against mature biofilms formed on stainless steel coupons. We found that the disinfectant susceptibility, biofilm structure, and extracellular polymeric substance structure and composition were disrupted by LED illumination. We then investigated the transcription of several critical P. aeruginosa biofilm-related genes and analyzed the effect of illumination temperature on the above characteristics. Our results confirmed that LED illumination could be developed into an effective and safe method to counter P. aeruginosa biofilm contamination. Further research will be focused on the efficacy and application of LED illumination for elimination of complicated biofilms in the environment.

The response to iron limitation of the Gram-positive soil bacterium Corynebacterium glutamicum was analyzed with respect to secreted metabolites, the transcriptome, and the proteome. During growth in glucose minimal medium, iron limitation caused a shift from lactate to pyruvate as the major secreted organic acid complemented by l-alanine and 2-oxoglutarate. Transcriptome and proteome analyses revealed that a pronounced iron starvation response governed by the transcriptional regulators DtxR and RipA was detectable in the late, but not in the early, exponential-growth phase. A link between iron starvation and thiamine pyrophosphate (TPP) biosynthesis was uncovered by the strong upregulation of thiC. As phosphomethylpyrimidine synthase (ThiC) contains an iron-sulfur cluster, limiting activities of the TPP-dependent pyruvate–2-oxoglutarate dehydrogenase supercomplex probably cause the excretion of pyruvate and 2-oxoglutarate. In line with this explanation, thiamine supplementation could strongly diminish the secretion of these acids. The upregulation of thiC and other genes involved in thiamine biosynthesis and transport is presumably due to TPP riboswitches present at the 5' end of the corresponding operons. The results obtained in this study provide new insights into iron homeostasis in C. glutamicum and demonstrate that the metabolic consequences of iron limitation can be due to the iron dependency of coenzyme biosynthesis.

IMPORTANCE Iron is an essential element for most organisms but causes problems due to poor solubility under oxic conditions and due to toxicity by catalyzing the formation of reactive oxygen species (ROS). Therefore, bacteria have evolved complex regulatory networks for iron homeostasis aiming at a sufficient iron supply while minimizing ROS formation. In our study, the responses of the actinobacterium Corynebacterium glutamicum to iron limitation were analyzed, resulting in a detailed view on the processes involved in iron homeostasis in this model organism. In particular, we provide evidence that iron limitation causes TPP deficiency, presumably due to insufficient activity of the iron-dependent phosphomethylpyrimidine synthase (ThiC). TPP deficiency was deduced from the upregulation of genes controlled by a TPP riboswitch and secretion of metabolites caused by insufficient activity of the TPP-dependent enzymes pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase. To our knowledge, the link between iron starvation and thiamine synthesis has not been elaborated previously.

Background:

Historically, some chiropractors have been critical of vaccination, and this has been the subject of recent media attention in Canada. We explored the association between media attention and public dissemination of vaccination information on Canadian chiropractors’ websites.

Methods:

In 2016, we identified all Canadian chiropractors’ websites that provided information on vaccination by extracting details from the regulatory college website for each province using the search engine on their "find a chiropractor" page. We assessed the quality of information using the Web Resource Rating Tool (scores range from 0% [worst] to 100% [best]), determined whether vaccination was portrayed in a positive, neutral or negative manner, and conducted thematic analysis of vaccination content. We revisited all identified websites in 2019 to explore for changes to posted vaccination material.

Results:

In July 2016, of 3733 chiropractic websites identified, 94 unique websites provided information on vaccination: 59 (63%) gave negative messaging, 19 (20%) were neutral and 16 (17%) were positive. The quality of vaccination content on the websites was generally poor, with a median Web Resource Rating Tool score of 19%. We identified 4 main themes: there are alternatives to vaccination, vaccines are harmful, evidence regarding vaccination and health policy regarding vaccination. From 2012 to 2016, there was 1 Canadian newspaper story concerning antivaccination statements by chiropractors, whereas 51 news articles were published on this topic between 2017 and 2019. In April 2019, 45 (48%) of the 94 websites we had identified in 2016 had removed all vaccination content or had been discontinued.

Interpretation:

In 2016, a minority of Canadian chiropractors provided vaccination information on their websites, the majority of which portrayed vaccination negatively. After substantial national media attention, about half of all vaccination material on chiropractors’ websites was removed within several years.

Background:

It is unclear how patient-reported access to primary care differs by physician payment model and participation in team-based care. We examined the association between timely and after-hours access to primary care and physician payment model and participation in team-based care, and sought to assess how access varied by patient characteristics.

Methods:

We conducted a cross-sectional analysis of adult (age ≥ 16 yr) Ontarians who responded to the Ontario Health Care Experience Survey between January 2013 and September 2015, reported having a primary care provider and agreed to have their responses linked to health administrative data. Access measures included the proportion of respondents who reported same-day or next-day access when sick, satisfaction with time to appointment when sick, telephone access and knowledge of an after-hours clinic. We tested the association between practice model and measures of access using logistic regression after stratifying for rurality.

Results:

A total of 33 665 respondents met our inclusion criteria. In big cities, respondents in team and nonteam capitation models were less likely to report same-day or next-day access when sick than respondents in enhanced fee-for-service models (team capitation 43%, adjusted odds ratio [OR] 0.88, 95% confidence interval [CI] 0.79–0.98; nonteam capitation 39%, adjusted OR 0.78, 95% CI 0.70–0.87; enhanced fee-for-service 46% [reference]). Respondents in team and nonteam capitation models were more likely than those in enhanced fee-for-service models to report that their provider had an after-hours clinic (team capitation 59%, adjusted OR 2.59, 95% CI 2.39–2.81; nonteam capitation 51%, adjusted OR 1.90, 95% CI 1.76–2.04; enhanced fee-for service 34% [reference]). Patterns were similar for respondents in small towns. There was minimal to no difference by model for satisfaction with time to appointment or telephone access.

Interpretation:

In our setting, there was an association between some types of access to primary care and physician payment model and team-based care, but the direction was not consistent. Different measures of timely access are needed to understand health care system performance.

Background:

Incentive payments for chronic diseases in British Columbia were intended to support primary care physicians in providing more comprehensive care, but research shows that not all physicians bill incentives and not all eligible patients have them billed on their behalf. We investigated patient and physician characteristics associated with billing incentives for chronic diseases in BC.

Methods:

We conducted a retrospective cohort analysis using linked administrative health data to examine community-based primary care physicians and patients with eligible chronic conditions in BC during 2010–2013. Descriptive analyses of patients and physicians compared 3 groups: no incentives in any of the 4 years, incentives in all 4 years, and incentives in any of the study years. We used hierarchical logistic regression models to identify the patient- and physician-level characteristics associated with billing incentives.

Results:

Of 428 770 eligible patients, 142 475 (33.2%) had an incentive billed on their behalf in all 4 years, and 152 686 (35.6%) never did. Of 3936 physicians, 2625 (66.7%) billed at least 1 incentive in each of the 4 years, and 740 (18.8%) billed no incentives during the study period. The strongest predictors of having an incentive billed were the number of physician contacts a patient had (odds ratio [OR] for > 48 contacts 134.77, 95% confidence interval [CI] 112.27–161.78) and whether a physician had a large number of patients in his or her practice for whom incentives were billed (OR 42.38 [95% CI 34.55–52.00] for quartile 4 v. quartile 1).

Interpretation:

The findings suggest that primary care physicians bill incentives for patients based on whom they see most often rather than using a population health management approach to their practice.

Background:

Drugs are the fastest growing cost in the Canadian health care system, owing to the increasing number of high-cost drugs. The objective of this study was to examine the characteristics of high–drug-cost beneficiaries of public drug plans across Canada relative to other beneficiaries.

Methods:

We conducted a cross-sectional study among public drug plan beneficiaries residing in all provinces except Quebec. We used the Canadian Institute for Health Information’s National Prescription Drug Utilization Information System to identify all drugs dispensed to beneficiaries of public drug programs in 2016/17. We stratified the cohort into 2 groups: high–drug-cost beneficiaries (top 5% of beneficiaries based on annual costs) and other beneficiaries (remaining 95%). For each group, we reported total drug costs, prevalence of high-cost claims (> $1000), median number of drugs, proportion of beneficiaries aged 65 or more, the 10 most costly reimbursed medications and the 10 medications most commonly reimbursed. We reported estimates overall and by province.

Results:

High–drug-cost beneficiaries accounted for nearly half (46.5%) of annual spending, with an average annual spend of $14 610 per beneficiary, compared to $1570 among other beneficiaries. The median number of drugs dispensed was higher among high–drug-cost beneficiaries than among other beneficiaries (13 [interquartile range (IQR) 7–19] v. 8 [IQR 4–13]), and a much larger proportion of high–drug-cost beneficiaries than other beneficiaries received at least 1 high-cost claim (40.9% v. 0.6%). Long-term medications were the most commonly used medications for both groups, whereas biologics and antivirals were the most costly medications for high–drug-cost beneficiaries.

Interpretation:

High–drug-cost beneficiaries were characterized by the use of expensive medications and polypharmacy relative to other beneficiaries. Interventions and policies to help reduce spending need to consider both of these factors.

Background:

Deaths from respiratory tract infections (RTIs) in children are preventable through timely access to public health and medical interventions. We aimed to assess whether socioeconomic disparities in mortality related to pediatric RTI persisted after accounting for health status at birth.

Methods:

We compared the prevalence of and risk factors for RTI-related death in singletons aged 28 days to 4 years across Ontario (Canada), Scotland and England (jurisdictions with universal health care) using linked administrative data for 2003–2013. We estimated rates of RTI-related mortality for children living in deprived areas and those born to teenage girls; we estimated both crude rates and those adjusted for health status at birth.

Results:

A total of 1 299 240 (Ontario), 547 556 (Scotland) and 3 910 401 (England) children were included in the study. Across all jurisdictions, children born in the most deprived areas experienced the highest rates of RTI-related mortality. After adjustment for high-risk chronic conditions and prematurity, we observed differences in mortality according to area-level deprivation in Ontario and England but not in Scotland. In Ontario, teenage motherhood was also an independent risk factor for RTI-related mortality.

Interpretation:

Socioeconomic disparities played a substantial role in child mortality related to RTI in all 3 jurisdictions. Context-specific investigations around the mechanisms of this increased risk and development of programs to address socioeconomic disparities are needed.

Background:

Female physicians have been shown to receive fewer awards from medical societies than their male colleagues. We examined the sex distribution of recipients of Canadian residency association awards.

Methods:

We conducted a retrospective observational study of the sex of staff and resident physician recipients of resident-selected awards from provincial and national residency associations using data from 2000–2018. We classified awards into professionalism, advocacy and wellness awards, and education and teaching awards based on award names and descriptions, and compared the proportion of male and female recipients in these categories.

Results:

We identified 314 recipients of staff physician awards and 129 recipients of resident physician awards. Male staff and resident physicians had higher odds of receiving awards than their female counterparts (odds ratio [OR] 1.45, 95% confidence interval [CI] 1.13–1.89 and OR 1.70, 95% CI 1.18–2.46, respectively). There was a reduction in the odds of male residents’ receiving an award over the study period (OR 0.94, 95% CI 0.90–0.98). Male physicians had higher odds of receiving education and teaching awards than female physicians as staff but not as residents (OR 3.21, 95% CI 1.72–5.95 and OR 1.96, 95% CI 0.84–4.60, respectively).

Interpretation:

Male staff and resident physicians in Canada had higher odds of receiving awards from provincial and national residency associations between 2000 and 2018 than their female counterparts. Given this disparity, it would be prudent for organizations that distribute awards to physicians, residents and medical students to examine their nomination criteria and processes for potential bias.

Whether low plasma 25-hydroxyvitamin D concentrations cause osteoporotic fractures is unclear. We tested the hypothesis that low plasma 25-hydroxyvitamin D concentrations are associated with increased risk of osteoporotic fractures using a Mendelian randomization analysis.We genotyped 116 335 randomly chosen white Danish persons aged 20–100 years in 2 population-based cohort studies for plasma 25-hydroxyvitamin D decreasing genotypes in CYP2R1 (rs117913124 and rs12794714), DHCR7 (rs7944926 and rs11234027), GEMIN2 (rs2277458), and HAL (rs3819817); 35 833 had information on plasma 25-hydroxyvitamin D. We assessed risk of total, osteoporotic, and anatomically localized fractures from 1981 through 2017. Information on fractures and vital status was obtained from nationwide registries.During up to 36 years of follow-up, we observed 17 820 total fractures, 10 861 osteoporotic fractures, and 3472 fractures of hip or femur. Compared with individuals with 25-hydroxyvitamin D ≥ 50nmol/L, multivariable adjusted hazard ratios (95% CIs) for total fractures were 1.03 (0.97–1.09) for individuals with 25–49.9 nmol/L, 1.19 (1.10–1.28) for individuals with 12.5–24.9 nmol/L, and 1.39 (1.21–1.60) for individuals with 25-hydroxyvitamin D < 12.5 nmol/L. Corresponding hazard ratios were 1.07 (1.00–1.15), 1.25 (1.13–1.37), and 1.49 (1.25–1.77) for osteoporotic fractures and 1.09 (0.98–1.22), 1.37 (1.18–1.57), and 1.41 (1.09–1.81) for fractures of hip or femur, respectively. Hazard ratios per 1 increase in vitamin D allele score, corresponding to 3.0% (approximately 1.6 nmol/L) lower 25-hydroxyvitamin D concentrations, were 0.99 (0.98–1.00) for total fractures, 0.99 (0.97–1.00) for osteoporotic fractures, and 0.98 (0.95–1.00) for fractures of hip or femur.Low plasma 25-hydroxyvitamin D concentrations were associated with osteoporotic fractures; however, Mendelian randomization analysis provided no evidence supporting a causal role for vitamin D in the risk for osteoporotic fractures.

The aim of this study was to assess the association of high-sensitivity cardiac troponin (hs-cTnT) and other cardiac, kidney, hyperglycemia, and inflammatory biomarkers with peripheral neuropathy (PN) in a community-based population.We conducted a cross-sectional analysis of 3056 black and white participants in the Atherosclerosis Risk in Communities (ARIC) study who underwent standardized monofilament PN testing and had measures of cardiac function (hs-cTnT, N-terminal pro–B-type natriuretic peptide [NT-proBNP], and growth differentiation factor 15 [GDF15]), kidney function (serum creatinine, cystatin C, β-2 microglobulin, urine albumin-to-creatinine ratio), hyperglycemia (fasting glucose, hemoglobin A_1c [Hb A_1c], fructosamine, glycated albumin, 1,5-anhydroglucitol), and inflammation (C-reactive protein) assessed at visit 6 (2016–2017; age 71–94 years). We used logistic regression to assess the associations of these biomarkers (modeled in diabetes-specific tertiles) with PN in older adults with and without diabetes after adjusting for traditional risk factors.In total, 33.5% of participants had PN (37.3% with diabetes and 31.9% without diabetes). There was an independent association of hs-cTnT with PN regardless of diabetes status (diabetes T3 vs. T1: odds ratio [OR], 2.15 [95% CI, 1.44–3.22]; no diabetes: OR, 2.31 [95%CI, 1.76–3.03]; P = 0.72 for interaction). Among participants without diabetes, there were also significant associations of NT-proBNP (OR, 1.40 [95% CI, 1.08–1.81]) and urine albumin-to-creatinine ratio (OR, 1.55 [95% CI, 1.22–1.97]) with PN. Associations of hyperglycemia biomarkers including Hb A_1c (OR, 1.76 [95% CI, 1.22–2.54]), fructosamine (OR, 1.71 [95% CI, 1.19–2.46]), and glycated albumin (OR, 1.45 [95% CI, 1.03–2.03]) with PN were significant only among participants with diabetes.Overall, hs-cTnT appears to be a global marker of end organ damage, including PN. Laboratory biomarkers may be able to help us identify those individuals with PN.

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Recognizing that guideline-directed histologic grading of endomyocardial biopsy tissue samples for rejection surveillance has limited diagnostic accuracy, quantitative, in situ characterization was performed of several important immune cell types in a retrospective cohort of clinical endomyocardial tissue samples. Differences between cases were identified and were grouped by histologic grade versus clinical rejection trajectory, with significantly increased programmed death ligand 1+, forkhead box P3+, and cluster of differentiation 68+ cells suppressed in clinically evident rejections, especially cases with marked clinical-histologic discordance. Programmed death ligand 1+, forkhead box P3+, and cluster of differentiation 68+ cell proportions are also significantly higher in "never-rejection" when compared with "future-rejection." These findings suggest that in situ immune modulators regulate the severity of cardiac allograft rejection.