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Background Infections with extended-spectrum β-lactamase (ESBL)-producing pathogens present treatment challenges with higher costs of care. Hospitalized patients (pts) with ESBL infections often require outpatient antimicrobial therapy (OPAT) following discharge. An Infectious Disease (ID) physician can facilitate optimal management in both the inpatient and outpatient setting, particularly when a POIC is an option. The increase of ESBL incidence led us to evaluate management and outcomes of genitourinary (GU) and intra-abdominal infections (IAI), the top two ESBL diagnoses treated in POICs. Methods Records were queried for pts hospitalized with ESBL infection and discharged to a POIC between 2019 and 2020. Primary outcome was completion of OPAT. Non-successful completion was defined as premature discontinuation of OPAT including hospital readmission. Adverse events (AEs) and re-treatment within 30 days were captured. Results A total of 112 pts (mean age: 59±17 years, 50% female) from 14 POICs were identified (93 GU, 19 IAI). Of these, 51% had their first ESBL infection and 52% were community-acquired prior to hospitalization. A total of 38% presented with concomitant bacteremia. ESBL E. coli was the predominant Gram-negative pathogen in both infection types (Table 1). Mean hospital LOS was 6±4 days (5±2 days GU, 11±6 days IAI) followed by 13±9 days of OPAT (12±7 days GU, 16±11 days IAI). Carbapenems were most frequently used as treatment (89% ertapenem, 7% meropenem). Overall, 106 pts (95%) successfully completed OPAT. Six pts (5%) discontinued due to hospital readmission. AEs were reported in 15 pts (13%), the majority of which were mild. Re-treatment in the POIC within 30 days post OPAT completion occurred in 6 GU pts. None were lost to follow-up. Table 1 summarizes primary outcome by clinical characteristics. Both GU and IAI pts had 95% successful OPAT completion. Table 1.Successful and non-successful completion of OPAT in patients with ESBL infections Conclusion Outpatient treatment of ESBL infections in POICs post-hospitalization showed high completion rates with low occurrence of readmissions and re-treatments. Consistent with a rise in ESBL infections, our incidence of community-acquired ESBL infections prior to hospitalization were high at 52%. Once ESBL infections were identified in the hospital, the ID physicians with a POIC rapidly facilitated discharge to OPAT. Disclosures Ramesh V. Nathan, MD, FIDSA, Eli Lilly & Co.: Grant/Research Support Brian S. Metzger, MD, MPH, AbbVie: Advisor/Consultant|Cumberland: Advisor/Consultant Lucinda J. Van Anglen, PharmD, Merck & Co.: Grant/Research Support|Paratek: Grant/Research Support.

ObjectivesThis double-blind randomized controlled trial investigated raising serum 25-hydroxyvitamin D (25D) with extended-release calcifediol (ERC) on time to symptom resolution in patients with mild to moderate COVID-19.MethodsCOVID-19 outpatients received oral ERC (300 mcg on days 1-3 and 60 mcg on days 4-27) or placebo (NCT04551911). Symptoms were self-reported daily. Primary end points were raising 25D to ≥50 ng/mL and decreasing resolution time for five aggregated symptoms (three respiratory).ResultsIn all, 171 patients were randomized, 160 treated and 134 (65 ERC, 69 placebo) retained. The average age was 43 y (range 18-71), 59% were women. The mean baseline 25D was 37 ± 1 (SE) ng/mL. In the full analysis set (FAS), 81% of patients in the ERC group achieved 25D levels of ≥50 ng/mL versus 15% in the placebo group (P < 0.0001). In the per-protocol (PP) population, mean 25D increased with ERC to 82 ± 4 (SE) ng/mL (P < 0.0001) by day 7; the placebo group trended lower. Symptom resolution time was unchanged in the FAS by ERC (hazard ratio [HR], 0.983; 95% confidence interval [CI], 0.695-1.390; P=0.922). In the PP population, respiratory symptoms resolved 4 d faster when 25D was elevated above baseline level at both days 7 and 14 (median 6.5 versus 10.5 d; HR, 1.372; 95% CI, 0.945-1.991; P=0.0962; Wilcoxon P=0.0386). Symptoms resolved in both treatment groups to a similar extent by study end. Safety concerns including hypercalcemia were absent with ERC treatment.ConclusionERC safely raised serum 25D to ≥50 ng/mL in outpatients with COVID-19, possibly accelerating resolution of respiratory symptoms and mitigating the risk for pneumonia. These findings warrant further study.

ImportanceThe benefit of vitamin D treatment for coronavirus disease 2019 (COVID-19) remains unclear.ObjectiveTo investigate the effect of raising serum total 25-hydroxyvitamin D (25D) to 50-100 ng/mL with oral extended-release calcifediol (ERC) on time to symptom resolution in mild to moderate COVID-19.Design, Setting, and ParticipantsA multicenter, randomized, double-blind, placebo-controlled study evaluated treatment of 160 outpatients with COVID-19 diagnosed between November 2020 and October 2021.InterventionsPatients were treated for 4 weeks with ERC (30 mcg/capsule; 300 mcg on Days 1-3 and 60 mcg on Days 4-27) or placebo.Outcome MeasuresPrimary endpoints were raising serum 25D to ≥50 ng/mL at Day 14 and resolution time for five aggregated symptoms. Secondary endpoints included resolution time for aggregated and individual symptoms as a function of serum 25D and changes in clinical biomarkers.Results171 subjects randomized, 160 treated and 134 (65 ERC and 69 placebo) retained. Average age was 43 (range: 18-71); 59% female, 92% White, 80% Hispanic, 7% African-American, 1% Other, 76% overweight, 40% obese, 26% comorbidities, mean baseline 25D of 37±1 (SE) ng/mL. ERC increased mean 25D to 82±4 ng/mL (pConclusions and RelevanceERC was effective in increasing serum 25D in outpatients with COVID-19, which may have accelerated resolution of respiratory symptoms suggesting mitigation of COVID-19 pneumonia risk, findings which warrant further study.

BackgroundRecent studies have identified vitamin D deficiency (serum 25-hydroxyvitamin D [25(OH)D] < 20 ng/L) as a potentially modifiable risk factor for prosthetic joint infection (PJI) in arthroplasty. The purpose of this study is to determine whether implementation of preoperative 25(OH)D repletion is cost-effective for reducing PJI following total knee arthroplasty (TKA).MethodsA cost estimation predictive model was generated to determine the utility of both selective and nonselective 25(OH)D repletion in primary TKA to prevent PJI. Input data on the incidence of 25(OH)D deficiency, relative complication rates, and costs of serum 25(OH)D repletion and 2-stage revision for PJI were derived from previously published literature identified using systematic review and publicly available data from Medicare reimbursement schedules. Mean, lower, and upper bounds of 1-year cost savings were computed for nonselective and selective repletion relative to no repletion.ResultsSelective preoperative 25(OH)D screening and repletion were projected to result in $1,504,857 (range, $215,084-$4,256,388) in cost savings per 10,000 cases. Nonselective 25(OH)D repletion was projected to result in $1,906,077 (range, $616,304-$4,657,608) in cost savings per 10,000 cases. With univariate adjustment, nonselective repletion is projected to be cost-effective in scenarios where revision for PJI costs ≥$10,636, incidence of deficiency is ≥1.1%, and when repletion has a relative risk reduction ≥4.2%.ConclusionThis predictive model supports the potential role of 25(OH)D repletion as a cost-effective mechanism of reducing PJI risk in TKA. Given the low cost of 25(OH)D repletion relative to serum laboratory testing, nonselective repletion appears to be more cost-effective than selective repletion. Further prospective investigation to assess this modifiable risk factor is warranted.

2020 marks 40 years of the publication of Urban Geography. The journal was established at about the same time as the American Association of Geographers (AAG) Urban Geography Specialty Group (UGSG)...

BackgroundAs the interest in manned spaceflight increases, so does the requirement to understand the transcriptomic mechanisms that underlay the detrimental physiological adaptations of skeletal muscle to microgravity. While microgravity-induced differential gene expression (DGE) has been extensively investigated, the contribution of differential alternative splicing (DAS) to the plasticity and functional status of the skeletal muscle transcriptome has not been studied in an animal model. Therefore, by evaluating both DGE and DAS across spaceflight, we set out to provide the first comprehensive characterization of the transcriptomic landscape of skeletal muscle during exposure to microgravity.MethodsRNA-sequencing, immunohistochemistry, and morphological analyses were conducted utilizing total RNA and tissue sections isolated from the gastrocnemius and quadriceps muscles of 30-week-old female BALB/c mice exposed to microgravity or ground control conditions for 9 weeks.ResultsIn response to microgravity, the skeletal muscle transcriptome was remodeled via both DGE and DAS. Importantly, while DGE showed variable gene network enrichment, DAS was enriched in structural and functional gene networks of skeletal muscle, resulting in the expression of alternatively spliced transcript isoforms that have been associated with the physiological changes to skeletal muscle in microgravity, including muscle atrophy and altered fiber type function. Finally, RNA-binding proteins, which are required for regulation of pre-mRNA splicing, were themselves differentially spliced but not differentially expressed, an upstream event that is speculated to account for the downstream splicing changes identified in target skeletal muscle genes.ConclusionsOur work serves as the first investigation of coordinate changes in DGE and DAS in large limb muscles across spaceflight. It opens up a new opportunity to understand (i) the molecular mechanisms by which splice variants of skeletal muscle genes regulate the physiological adaptations of skeletal muscle to microgravity and (ii) how small molecule splicing regulator therapies might thwart muscle atrophy and alterations to fiber type function during prolonged spaceflight.

Context Experimental studies suggest that vitamin D receptor signaling may benefit the gut microbiome. In humans, whether vitamin D supplementation directly alters the gut microbiome is not well studied. Objective To determine whether correcting vitamin D deficiency with cholecalciferol (vitamin D3, D3) or calcifediol (25-hydroxyvitamin D3, 25(OH)D3) changes gut microbiome composition. Methods 18 adults with vitamin D deficiency (25-hydroxyvitamin D [25(OH)D] Results Vitamin D3 and 25(OH)D3 groups were similar. After 8 weeks of vitamin D3, mean 25(OH)D and 24,25(OH)2D increased significantly, but 1,25(OH)2D did not (25(OH)D: 17.8-30.1 ng/mL, P = .002; 24,25(OH)2D: 1.1 to 2.7 ng/mL, P =0.003; 1,25(OH)2D: 49.5-53.0 pg/mL, P = .9). After 8 weeks of 25(OH)D3, mean 25(OH)D, 24,25(OH)2D, and 1,25(OH)2D increased significantly (25(OH)D: 16.7-50.6 ng/mL, P Conclusion In a small sample of healthy adults with vitamin D deficiency, restoration of vitamin D sufficiency with vitamin D3 or 25(OH)D3 did not lead to lasting changes in the fecal microbiota.

The vitamin D receptor is highly expressed in the gastrointestinal tract where it transacts gene expression. With current limited understanding of the interactions between the gut microbiome and vitamin D, we conduct a cross-sectional analysis of 567 older men quantifying serum vitamin D metabolites using LC-MSMS and defining stool sub-Operational Taxonomic Units from16S ribosomal RNA gene sequencing data. Faith’s Phylogenetic Diversity and non-redundant covariate analyses reveal that the serum 1,25(OH)2D level explains 5% of variance in α-diversity. In β-diversity analyses using unweighted UniFrac, 1,25(OH)2D is the strongest factor assessed, explaining 2% of variance. Random forest analyses identify 12 taxa, 11 in the phylum Firmicutes, eight of which are positively associated with either 1,25(OH)2D and/or the hormone-to-prohormone [1,25(OH)2D/25(OH)D] “activation ratio.” Men with higher levels of 1,25(OH)2D and higher activation ratios, but not 25(OH)D itself, are more likely to possess butyrate producing bacteria that are associated with better gut microbial health., Here, the authors investigate associations of vitamin D metabolites with gut microbiome in a cross-sectional analysis of 567 elderly men enrolled in the Osteoporotic Fractures in Men (MrOS) Study and find larger alpha-diversity correlates with high 1,25(OH)2D and high 24,25(OH)2D and higher ratios of activation and catabolism.

CCN1/Cyr61 is a dynamically expressed matricellular protein that serves regulatory functions in multiple tissues. Previous studies from our laboratory demonstrated that CCN1 regulates bone maintenance. Using an osteoblast and osteocyte conditional knockout mouse model (Ccn1OCN ), we found a significant decrease in trabecular and cortical bone mass in vivo, in part through suppression of Wnt signaling since the expression of the Wnt antagonist sclerostin (SOST) is increased in osteoblasts lacking CCN1. It has been established that parathyroid hormone (PTH) signaling also suppresses SOST expression in bone. We therefore investigated the interaction between CCN1 and PTH-mediated responses in this study. We find that loss of Ccn1 in osteoblasts leads to impaired responsiveness to anabolic intermittent PTH treatment in Ccn1OCN mice in vivo and in osteoblasts from these mice in vitro. Analysis of Ccn1OCN mice demonstrated a significant decrease in parathyroid hormone receptor-1 (PTH1R) expression in osteoblasts in vivo and in vitro. We investigated the regulatory role of a non-canonical integrin-binding domain of CCN1 because several studies indicate that specific integrins are critical to mechanotransduction, a PTH-dependent response, in bone. These data suggest that CCN1 regulates the expression of PTH1R through interaction with the αvβ3 and/or αvβ5 integrin complexes. Osteoblasts that express a mutant form of CCN1 that cannot interact with αvβ3/β5 integrin demonstrate a significant decrease in mRNA and protein expression of both PTH1R and αv integrin. Overall, these data suggest that the αvβ3/β5-binding domain of CCN1 is required to endow PTH signaling with anabolic activity in bone cells. © 2020 American Society for Bone and Mineral Research (ASBMR).

Overlapping geographical occurrence, history of traditional use, confusion in species identification, and morphological resemblances among various species are some considerations that necessitate the importance of qualitative analysis for efficient quality control and safer botanical products. This paper provides detailed morpho-anatomies of the leaves and stems of Tinospora cordifolia, Tinospora crispa, and Tinospora sinensis, and stems of Tinospora baenzigeri. Microscopy studies of the selected Tinospora species revealed key diagnostic features that can help distinguish the closely related species of Tinospora as well as to detect any adulteration or substitution in the raw materials. HPTLC profiles of the authenticated plant materials, as well as commercial products claiming to contain Tinospora, were compared to distinguish T. crispa from other closely related species and to establish an efficient method to assess the identity and quality of the products using qualified chemical markers. HPTLC chromatograms of both plant samples and dietary supplements were compared with six reference marker compounds. The analysis revealed that borapetoside B and C were useful to identify T. crispa while tinosineside A was found to be characteristic to authenticate the T. sinensis products.

Context Vitamin D deficiency disproportionately affects nonwhite individuals. Controversy persists over how to best restore low 25D levels, and how to best define vitamin D status [total (protein bound plus free) vs free 25D]. Objective To assess the effects of vitamin D3 (cholecalciferol, or D3) vs 25-hydroxyvitamin D3 (calcifediol, or 25D3) on total and free 25D in a multiethnic cohort of adults, and whether change in parathyroid hormone (PTH) is more strongly associated with total vs free 25D. Design Sixteen-week randomized controlled trial. Biochemistries at 0, 4, 8, and 16 weeks. Setting Academic medical center. Participants Thirty-five adults ≥18 years of age with 25D levels Intervention Sixty micrograms (2400 IU)/d of D3 or 20 μg/d of 25D3. Main outcome measures Total and free 25D, and PTH. Results Baseline total (16.2 ± 3.7 vs 17.0 ± 2.5 ng/mL; P = 0.4) and free (4.2 ± 0.8 vs 4.7 ± 1.0 pg/mL; P = 0.2) 25D were similar between D3 and 25D3 groups, respectively; 25D3 increased total (+25.5 vs +13.8 ng/mL; P = 0.001) and free (+6.6 vs +3.5 pg/mL; P = 0.03) 25D more than D3. By 4 weeks, 87.5% of 25D3 participants had total 25D levels ≥30 ng/mL, compared with 23.1% of D3 participants (P = 0.001). Change in PTH was associated with both total (P = 0.01) and free 25D (P = 0.04). Conclusions 25D3 increased total and free 25D levels more rapidly than D3, regardless of race/ethnicity. Free and total 25D were similarly associated with change in PTH.

Manual development of liquid chromatography tandem-mass spectrometry (LC-MS/MS) methods is a rate limiting step in analytical laboratories, particularly if several compounds have the same multiple reaction monitoring (MRM) transitions. This study describes the application of Multi-platform Unbiased optimisation of Spectrometry via Closed-Loop Experimentation (MUSCLE) software to automate the development of an LC-MS/MS method to measure multiple metabolites of vitamin D. Comparison with a manually developed method for the same compounds was used to evaluate the effectiveness of MUSCLE in improving method parameters. LC and MS parameter ranges were set up in MUSCLE, which optimised the method during a fully-automated 200 sample sequence. Visual scripts altered method parameters after each sample run while a closed-loop multi-objective optimisation approach identified optimum instrument parameters throughout the sequence to improve sensitivity and run time. The optimised sample run developed using MUSCLE shortened analysis time for 10 metabolites from 8.2 minutes to 6.2 minutes. This was achieved by increased initial methanol concentration in the mobile phase and an altered gradient that increased the in-run organic mobile phase. However, MS parameters could not be optimised further to improve analyte sensitivity over manual optimisation, although in most cases MUSCLE confirmed the manually optimised conditions. Comparison between each of the developed methods showed no significant analyte bias between methods. MUSCLE has been shown here to automate and improve the throughput of a multiple analyte vitamin D LC-MS/MS method. Utilisation of this software could be applied to industries requiring fast automated method development such as clinical and pharmaceutical laboratories.

Background New vancomycin (VAN) guidelines have been published, with recommendations that dosing in patients (pts) with methicillin-resistant S. aureus (MRSA) infections be guided by VAN area under the concentration-time curve from 0 to 24 h to MIC ratio (AUC24). The guidelines emphasize daily AUC24 values should be between 400-600 mg*h/L to maximize efficacy and minimize likelihood of acute kidney injury (AKI). Our physicians and clinical pharmacists currently use trough levels to manage outpatient VAN dosing with a general target of 15-20 mg/L. The current pharmacokinetic (PK) model provides an option for dosing using trough or calculated AUC24. Methods We identified pts receiving VAN for S. aureus infections (default MIC of 1 mg/mL) from 2018-2020. We conducted a PK evaluation of pts with ≥1 trough level and compared it to model predicted AUC24. Data collected included pt characteristics, VAN regimen, trough concentrations, PK evaluation, and AKI, defined as a 50% decrease in CrCL from baseline. A Bayesian PK model was used to calculate predicted dosing based upon trough concentrations (DoseMeRx®, Moorestown, NJ). Results 100 pts (mean age: 61±15 yrs, 62% male) from 6 OICs were included, with 82% treated for MRSA and 18% for methicillin-sensitive S. aureus infection. Mean initial dose of VAN in the OIC was 2.6±1 g/d in divided doses, most frequently every 12 hrs (68%). Median duration of outpatient therapy was 28 days [IQR, 16-36]. 69% received VAN in the hospital prior to the OIC. 100 pts had 239 trough levels with a corresponding PK analysis. Mean trough levels were 17.1±6.4 mg/L. Mean corresponding AUC24 was 498±98 mg*h/L. The relationship between trough and AUC24 is shown in Fig 1. 25 evaluations indicated an AUC24 < 400, with 8 (32%) resulting in a dose increase. 13 evaluations indicated AUC24 > 600, with 6 (46%) resulting in a subsequent dose decrease. 4 pts developed reversible AKI, all with AUC24 > 540. Use of AUC24 for dosing provided opportunities to adjust dosing in 38/239 evaluations (16%). Figure 1. Relationship between Trough and AUC24 (n=239) Conclusion This PK evaluation showed a correlation between trough levels and AUC24 with opportunities for VAN dose adjustment using AUC24, and to identify pts at risk for developing AKI. Dosing with AUC24 is particularly useful in the outpatient setting in which true trough evaluations can be difficult to obtain. Disclosures Brian S. Metzger, MD, MPH, Allergan (Speaker’s Bureau)Cumberland (Speaker’s Bureau)Melinta (Speaker’s Bureau) Richard L. Hengel, MD, Merck & Co. (Other Financial or Material Support, Grant Steering Committee Member) Kimberly A. Couch, PharmD, MA, FIDSA, FASHP, Allergan (Speaker’s Bureau) Lucinda J. Van Anglen, PharmD, Merck & Co. (Grant/Research Support)

Vitamin D has a long-established role in bone health. In the last two decades, there has been a dramatic resurgence in research interest in vitamin D due to studies that have shown its possible benefits for non-skeletal health. Underpinning the renewed interest in vitamin D was the identification of the vital role of intracrine or localized, tissue-specific, conversion of inactive pro-hormone 25-hydroxyvitamin D [25(OH)D] to active 1,25-dihydroxyvitamin D [1,25(OH)2D]. This intracrine mechanism is the likely driving force behind vitamin D action resulting in positive effects on human health. To fully capture the effect of this localized, tissue-specific conversion to 1,25(OH)2D, adequate 25(OH)D would be required. As such, low serum concentrations of 25(OH)D would compromise intracrine generation of 1,25(OH)2D within target tissues. Consistent with this is the observation that all adverse human health consequences of vitamin D deficiency are associated with a low serum 25(OH)D level and not with low 1,25(OH)2D concentrations. Thus, clinical investigators have sought to define what concentration of serum 25(OH)D constitutes adequate vitamin D status. However, since 25(OH)D is transported in serum bound primarily to vitamin D binding protein (DBP) and secondarily to albumin, is the total 25(OH)D (bound plus free) or the unbound free 25(OH)D the crucial determinant of the non-classical actions of vitamin D? While DBP-bound-25(OH)D is important for renal handling of 25(OH)D and endocrine synthesis of 1,25(OH)2D, how does DBP impact extra-renal synthesis of 1,25(OH)2D and subsequent 1,25(OH)2D actions? Are their pathophysiological contexts where total 25(OH)D and free 25(OH)D would diverge in value as a marker of vitamin D status? This review aims to introduce and discuss the concept of free 25(OH)D, the molecular biology and biochemistry of vitamin D and DBP that provides the context for free 25(OH)D, and surveys in vitro, animal, and human studies taking free 25(OH)D into consideration.

BACKGROUND AND OBJECTIVES: Youth with chronic physical conditions (CPCs) may be at greater risk for developing chronic mental health conditions (MHCs), and limitations in the ability to engage in developmentally appropriate activities may contribute to the risk of MHCs among youth with CPCs. We compared the risk of incident MHCs in youth with and without CPCs and explored whether activity limitations contribute to any such association. METHODS: The 2003–2014 Medical Expenditure Panel Survey provided a nationally representative cohort of 48 572 US youth aged 6 to 25 years. We calculated the 2-year cumulative incidence of MHCs overall and by baseline CPC status. Cox proportional hazard models were used to estimate the association between CPCs and incident MHCs, adjusting for sociodemographic characteristics. Stepwise models and the Sobel test evaluated activity limitations as a mediator of this relationship. RESULTS: The 2-year cumulative incidence of MHCs was 7.8% overall, 11.5% in youth with CPCs (14.7% of sample), and 7.1% in those without. The adjusted risk of incident MHCs was 51% greater (adjusted hazard ratio 1.51; 95% confidence interval 1.30–1.74) in youth with CPCs compared with those without. Activity limitations mediated 13.5% of this relationship (P < .001). CONCLUSIONS: This nationally representative cohort study supports the hypotheses that youth with CPCs have increased risk for MHCs and that activity limitations may play a role in MHC development. Youth with CPCs may benefit from services to bolster their ability to participate in developmentally important activities and to detect and treat new onset MHCs.

Vitamin D-deficiency has been linked to inflammatory diseases including rheumatoid arthritis (RA). Studies to date have focused on the impact of serum 25-hydroxyvitamin D3 (25(OH)D3), an inactive form of vitamin D, on RA disease activity and progression. However, anti-inflammatory actions of vitamin D are likely to be mediated at sites of RA disease, namely the inflamed joint, and may involve other vitamin D metabolites notably the active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)(2)D3). In the current study serum and synovial fluid samples from n=20 patients with persistent RA and n=7 patients with reactive arthritis (ReA) were analysed for multiple vitamin D metabolites. Serum data for RA and ReA patients were compared to healthy controls (HC). There was no significant difference between RA or ReA patients relative to HC for 25(OH)D3, 24,25(OH)(2)D3, 1,25(OH)(2)D3 or 25(OH)D2. However, 3-epi-25(OH)D3 was significantly lower in RA and ReA patients compared to HC (p

A Correction to this paper has been published: https://doi.org/10.1038/s41467-021-20933-x.

We examined the bidirectional impact of vitamin D on the composition and diversity of the gut microbiome in 567 MrOS men. Vitamin D metabolites were measured using LC-MSMS and stool sub-operational taxonomic units defined from 16S ribosomal RNA sequencing data using Deblur and Greengenes 13.8. Men’s mean serum level of 25(OH)D was in the sufficient range. Faith’s Phylogenetic Diversity and non-redundant covariate analyses revealed that 1,25(OH)2D explained 5% of variance in α-diversity; the other non-redundant covariates of site, race, recent antibiotic and antidepressant use explained another 6%. In β-diversity analyses using unweighted UniFrac, 1,25(OH)2D was the strongest factor assessed, explaining 2%. Random forest plot analyses identified 12 taxa, 6 in the phylum Firmicutes, positively associated with either 1,25(OH)2D and/or [1,25(OH)2D/25(OH)D] activation ratio. Higher levels of the active 1,25(OH)2D, but not 25(OH)D, were associated with butyrate producing bacteria. Men with favorable vitamin D activation profiles also had greater gut microbial diversity.

Traditionally recognized as an RNA splicing regulator, heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNPC1/C2) can also bind to double-stranded DNA and function in trans as a vitamin D response element (VDRE)-binding protein. As such, hnRNPC1/C2 may couple transcription induced by the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D) with subsequent RNA splicing. In MG63 osteoblastic cells, increased expression of the 1,25(OH)2D target gene CYP24A1 involved immunoprecipitation of hnRNPC1/C2 with CYP24A1 chromatin and RNA. Knockdown of hnRNPC1/C2 suppressed expression of CYP24A1, but also increased expression of an exon 10-skipped CYP24A1 splice variant; in a minigene model the latter was attenuated by a functional VDRE in the CYP24A1 promoter. In genome-wide analyses, knockdown of hnRNPC1/C2 resulted in 3500 differentially expressed genes and 2232 differentially spliced genes, with significant commonality between groups. 1,25(OH)2D induced 324 differentially expressed genes, with 187 also observed following hnRNPC1/C2 knockdown, and a further 168 unique to hnRNPC1/C2 knockdown. However, 1,25(OH)2D induced only 10 differentially spliced genes, with no overlap with differentially expressed genes. These data indicate that hnRNPC1/C2 binds to both DNA and RNA and influences both gene expression and RNA splicing, but these actions do not appear to be linked through 1,25(OH)2D-mediated induction of transcription.

This article provides an overview of the chronic care model (CCM), examines the evidence for its utility in pediatric and adolescent chronic illness, and discusses practical steps for improving chronic illness care in the pediatric medical home.Few studies have used the CCM as an improvement framework in pediatrics. However, in recent years, several quality improvement efforts based on the CCM have demonstrated improvement in important process measures or clinical outcomes in pediatric or adolescent obesity, inflammatory bowel disease, attention-deficit/hyperactivity disorder, depression, and asthma.The CCM is an improvement framework that has demonstrated success in improving the care of children and adolescents with chronic disease. More research is needed to identify priority conditions for improvement efforts, to better understand the mediators of health outcomes in pediatric chronic disease, and to rigorously demonstrate the effectiveness of new models of chronic illness care. The evidence to date suggests that the CCM may be useful in guiding the redesign of care delivery systems to improve the health outcomes of young people with chronic disease.

Vitamin D is hypothesized to suppress inflammation. We tested total and free vitamin D metabolites and their association with inflammatory markers. Interleukin-6 levels were lower with higher 25-hydroxyvitamin D. 1,25-dihydroxyvitamin D and free 25OHD associations mirrored those of 25OHD. However, associations for the two metabolites diverged for tumor necrosis factor alpha (TNF-α) soluble receptors. Vitamin D is hypothesized to suppress inflammation, and circulating 25-hydroxyvitamin D (25OHD) and inflammatory markers are inversely correlated. However, total serum 25OHD may not be the best indicator of biologically active vitamin D. We tested serum total 25OHD, total 1,25(OH)2D, vitamin D binding protein (DBP), and estimated free 25OHD and free 1,25(OH)2D associations with inflammatory markers serum interleukin-6 (IL-6), TNF-α and their soluble receptors, interleukin-10 (IL-10), and C-reactive protein (CRP) as continuous outcomes and the presence of ≥2 inflammatory markers in the highest quartile as a dichotomous outcome, in a random subcohort of 679 men in the Osteoporotic Fractures in Men (MrOS) study. IL-6 was lower in men with higher 25OHD (−0.23 μg/mL per standard deviation (SD) increase in 25OHD, 95 % confidence intervals (CI) −0.07 to −0.38 μg/mL) and with higher 1,25(OH)2D (−0.20 μg/mL, 95 % CI −0.0004 to −0.39 μg/mL); free D associations were slightly stronger. 25OHD and DBP, but not 1,25(OH)2D, were independently associated with IL-6. TNF-α soluble receptors were inversely associated with 1,25(OH)2D but positively associated with 25OHD, and each had independent effects. The strongest association with ≥2 inflammatory markers in the highest quartile was for free 1,25(OH)2D (odds ratios (OR) 0.70, 95 % CI 0.54 to 0.89 per SD increase in free 1,25(OH)2D). Associations of 1,25(OH)2D and free 25OHD with IL-6 mirrored those of 25OHD, suggesting that 1,25(OH)2D and free D do not improve upon 25OHD in population-based IL-6 studies. However, associations for the two metabolites diverged for TNF-α soluble receptor, warranting examination of both metabolites in studies of TNF-α and its antagonists.

Following infection, virulent mycobacteria persist and grow within the macrophage, suggesting that the intrinsic activation of an innate antimicrobial response is subverted by the intracellular pathogen. For Mycobacterium leprae, the intracellular bacterium that causes leprosy, the addition of exogenous innate or adaptive immune ligands to the infected monocytes/macrophages was required to detect a vitamin D-dependent antimicrobial activity. We investigated whether there is an intrinsic immune response to M. leprae in macrophages that is inhibited by the pathogen. Upon infection of monocytes with M. leprae, there was no upregulation of CYP27B1 nor its enzymatic activity converting the inactive prohormone form of vitamin D (25-hydroxyvitamin D) to the bioactive form (1,25α-dihydroxyvitamin D). Given that M. leprae-induced type I interferon (IFN) inhibited monocyte activation, we blocked the type I IFN receptor (IFNAR), revealing the intrinsic capacity of monocytes to recognize M. leprae and upregulate CYP27B1. Consistent with these in vitro studies, an inverse relationship between expression of CYP27B1 vs. type I IFN downstream gene OAS1 was detected in leprosy patient lesions, leading us to study cytokine-derived macrophages (MΦ) to model cellular responses at the site of disease. Infection of IL-15-derived MΦ, similar to MΦ in lesions from the self-limited form of leprosy, with M. leprae did not inhibit induction of the vitamin D antimicrobial pathway. In contrast, infection of IL-10-derived MΦ, similar to MΦ in lesions from patients with the progressive form of leprosy, resulted in induction of type I IFN and suppression of the vitamin D directed pathway. Importantly, blockade of the type I IFN response in infected IL-10 MΦ decreased M. leprae viability. These results indicate that M. leprae evades the intrinsic capacity of human monocytes/MΦ to activate the vitamin D-mediated antimicrobial pathway via the induction of type I IFN., Author summary Our macrophages are equipped with the ability to detect and kill invading pathogens, and yet, these cells of the innate immune system are still subject to infection by intracellular bacterium. In particular, mycobacterium, the type of intracellular bacteria responsible for diseases such as tuberculosis and leprosy, are very successful at establishing infection within macrophages. By studying Mycobacterium leprae, the etiological agent of leprosy, we describe an immune evasion mechanism whereby this bacterial pathogen utilizes our own antiviral immune response against the macrophage. Type I interferons (IFN) are a major part of our immune response to viral infections; however, this response will also suppress our ability to fight opportunistic bacterial infection. During infection of our macrophages, M. leprae induces an aberrant type I IFN response that subsequently suppresses our macrophage’s ability to activate the vitamin D-mediated antimicrobial pathway, a critical antimicrobial response for containment of mycobacterium. Thus, understanding how these pathogens can evade our immune response will be important for the development of new therapies against these chronic infections.

Inflammatory bowel diseases (IBD) such as ulcerative colitis and Crohn’s disease have been linked to vitamin D-deficiency. Using a dextran sodium sulphate (DSS)-induced model of IBD we have shown previously that mice raised on vitamin D-deficient diets from weaning have lower serum 25-hydroxyvitamin D (25OHD) levels and develop more severe colitis compared to vitamin D-sufficient counterparts. We have also shown in vitro that immune responses to 25OHD may depend on ‘free’ rather than total serum concentrations of 25OHD. To investigate the possible effects of free versus total 25OHD on anti-inflammatory immune responses in vivo we have studied DSS-induced colitis in wild type C57BL/6 mice raised from weaning on diets containing vitamin D2 (D2) or vitamin D3 (D3) only (both 1000 IU/kg feed). 25OHD2 has lower binding affinity for the vitamin D binding protein than 25OHD3 which results in higher levels of free 25OHD2 relative to free 25OHD3 in mice raised on a D2-only diet. Total serum 25OHD concentrations, measured by liquid chromatography-tandem mass spectrometry (LC–MS/MS), showed that D2 mice had significantly lower levels of 25OHD than D3 mice (6.85 ± 2.61 nmol/L vs. 49.16 ± 13.8 nmol/L for D2 and D3 respectively). Despite this, direct ELISA measurement showed no difference in free serum 25OHD levels between D2 and D3 mice (13.62 ± 2.26 pmol/L vs. 14.11 ± 2.24 pmol/L for D2 and D3 respectively). Analysis of DSS-induced colitis also showed no difference in weight loss or disease progression between D2 and D3 mice. These data indicate that despite D2-fed mice being vitamin D-deficient based on serum total 25OHD concentrations, these mice showed no evidence of increased inflammatory colitis disease relative to vitamin D-sufficient D3 mice. We therefore propose that free, rather than total serum 25OHD, may be a better marker of immune responses to vitamin D in vivo.

The purpose of this study was to determine the relative incidence of postoperative complications in 25-hydroxyvitamin D (25D)-deficient and -sufficient patients undergoing total knee arthroplasty (TKA). Patients who were either serum 25D deficient (25D International Classification of Diseases, Ninth Revision and Current Procedural Terminology codes. Risk-adjusted odds ratios (ORs) were calculated using multivariate logistic regression with age, sex, and Charlson Comorbidity Index as covariates. In total, 868 of 6593 patients who underwent TKA from 2007 to 2016 were 25D deficient, corresponding to a 13.2% prevalence rate. On adjustment for age, sex, and Charlson Comorbidity Index, 25D-deficient patients had a higher incidence of postoperative stiffness requiring manipulation under anesthesia (OR, 1.69; 95% confidence interval [CI], 1.39–2.04; P P =.001), and prosthesis explantation (OR, 2.97; 95% CI, 2.04–4.31; P P P P =.006). Thus, serum 25D levels below 20 ng/mL are associated with a higher incidence of postoperative complications and may be a perioperative modifiable risk factor in TKA. [ Orthopedics . 2018; 41(4):e489–e495.]

The California Institute for Regenerative Medicine (CIRM) Alpha Stem Cell Clinic (ASCC) Network was launched in 2015 to address a compelling unmet medical need for rigorous, FDA-regulated, stem cell-related clinical trials for patients with challenging, incurable diseases. Here, we describe our multi-center experiences addressing current and future challenges.

CONTEXT: The physiologic role of free 25-hydroxyvitamin D [25(OH)D] in humans is unclear. OBJECTIVE: To assess whether rise in total vs free 25(OH)D is associated with change in downstream biomarkers of 25(OH)D entry into target cells in kidney and parathyroid: 24,25-dihyroxyvitamin D [24,25(OH)(2)D] and PTH, respectively. DESIGN: 16-week randomized controlled trial. INTERVENTION: 60 μg (2400 IU)/d of D3 or 20 μg/d of 25(OH)D3. SETTING: Academic medical center. PARTICIPANTS: 35 adults age ≥18 years with 25(OH)D levels < 20 ng/mL. MAIN OUTCOME MEASURES: 24,25(OH)(2)D, 1,25-dihyroxyvitamin D [1,25(OH)(2)D] and PTH. RESULTS: At baseline, participants [D3 and 25(OH)D3 groups combined] were 35.1 ± 10.6 years. Mean total 25(OH)D, free 25(OH)D, 24,25(OH)(2)D, and PTH were 16.6 ng/mL, 4.6 pg/mL, 1.3 ng/mL, and 37.2 pg/mL, respectively. From 0 to 4 weeks, rise in only free 25(OH)D was associated with a concurrent 24,25(OH)(2)D increase [P = 0.03, adjusted for change in 1,25(OH)(2)D and supplementation regimen] and PTH decrease (P = 0.01, adjusted for change in calcium and supplementation regimen). Between 4 and 8 weeks, and again from 8 to 16 weeks, rises in free and total 25(OH)D were associated with 24,25(OH)(2)D increase; in contrast, rise in neither total nor free 25(OH)D was associated with PTH decrease during these time periods. CONCLUSIONS: Early rise in free 25(OH)D during treatment of vitamin D deficiency was more strongly associated with changes in biomarkers of 25(OH)D entry into target kidney and parathyroid cells, suggesting a physiologic role of free 25(OH)D in humans.

The aim of this chapter is to describe the mechanisms associated with the regulation and function of the vitamin D-activating enzyme, 25-hydoxyvitamin D (25(OH)D)-1α-hydroxylase (1α-OHase) in two distinct settings. The first sections of the chapter will detail the pathways and key regulatory factors associated with renal expresssion of 1α-OHase and how this supports circulating levels of the active hormonal form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D), and the endocrine actions of 1,25(OH)2D. The latter sections of the chapter will document how 1α-OHase is regulated outside the kidney. Numerous extrarenal sites have been reported to express 1α-OHase and the tissue-specific impact of this localized capacity to generate 1,25(OH)2D is documented in other chapters throughout the rest of the book. However, perhaps the most well-documented examples of extrarenal 1α-OHase expression and activity are the innate immune system and skin keratinocytes. The current chapter will therefore focus on these tissues to illustrate the specific pathways associated with the regulation of 1α-OHase outside the kidney. The overall aim of the chapter will be to describe the wide array of mechanisms that regulate synthesis of 1,25(OH)2D, the clinical implications of these mechanisms, and their translational potential.

Background Patients with serious bacterial infections (SBI), identified as bone and joint infections (BJI), bacteremia/endocarditis, and central nervous system (CNS) infections are frequently discharged on outpatient parenteral antimicrobial therapy (OPAT). They account for 48% of all infections treated in our network of Infectious Disease (ID) physician office infusion centers (POICs). Care for these patients poses risks and challenges to ensure safe and successful outcomes while avoiding hospitalizations. This study examines clinical outcomes and complications of our SBI patients receiving OPAT in ID POICs. Methods All patients were identified with SBI receiving OPAT in 2017 from 14 POICs. A group of 250 patients were randomly selected by incidence of diagnosis and a retrospective chart review performed. Demographics, treatment regimen, clinical outcomes, adverse drug reactions (ADRs) and unplanned hospitalizations during OPAT were collected. Clinical success was defined as clinical cure or improvement at completion of OPAT. Patients were included who were hospitalized for Results SBI patients included BJI (n = 175), bacteremia/endocarditis (n = 60) and CNS infections (n = 15) as described in Table 1. Successful clinical outcomes were reported in 224 patients (89.6%) after a mean duration of OPAT of 32±20 days. Of these, 15 patients (6.7%) were hospitalized during OPAT and returned to the POIC for a successful clinical outcome. Clinical success rates for BJI, bacteremia/endocarditis and CNS infections were 89.1%, 91.6% and 86.7%, respectively. The primary reason for nonfavorable outcomes was worsening of infection (15/26, 58%). Serious ADRs were reported in 12 patients (4.8%) with 6 (2.4%) leading to hospitalization. Unplanned hospitalizations during OPAT occurred in 33 patients (13.2%) with the majority (21/33, 64%) related to disease. ADRs and hospitalizations compare favorably to data previously reported. (Schmidt et al. OFID 16.4, 2017). Conclusion Patients with serious bacterial infections had high success rates when treated by an ID physician in infusion centers. Adverse events and unplanned hospitalization rates were low. Disclosures All authors: No reported disclosures.

Background A significant complication of prosthetic joint replacement is the development of a PJI. Therapy includes prolonged IV antibiotics (IVAB), usually delivered in the outpatient setting. Follow-up (FU) in this population can be difficult, particularly for the treating ID physician. We report our experience treating PJI in an ID POIC. Methods Retrospective chart review was conducted of patients (patients) with initial knee or hip PJI, who received ≥3 days of IVAB in 14 ID POICs from July 2015 to July 2017. Initial clinical success (ICS) was defined as no evidence of infection at the completion of outpatient parenteral antimicrobial therapy (OPAT), although continued oral antibiotics were allowed. Available FU patients were assessed at 6 months for recurrence and associated factors analyzed using χ2 and Fisher’s exact test. Results We evaluated 171 patients (122 knees, 49 hips) with a median age of 65 years (range 31–91) and 64% male. Infection occurred within 90 days of the implant in 40% (25% within 30 days). Prostheses were retained in 109 patients (64%). 91% were hospitalized prior to OPAT for 4.7 median days, with the remainder treated from the community. The median length of OPAT was 37 days (range: 8–77). Cultures were positive in 154 patients with 122 Staphylococcus spp. pathogens (43% MSSA, 43% CoNS, 13% MRSA) in 112 patients. Most commonly prescribed IVABs were vancomycin (41%) and cefazolin (37%). ICS was achieved in 163 patients (95%), independent of prosthesis removal, with 119 (73%) continuing oral antibiotics post OPAT. Eight patients did not complete OPAT. Six-month FU to the ID physician was available for 97/163 patients completing therapy (60%). Of these, recurrence occurred in 22 patients (23%) at a median of 2.1 months (range 0.2–6). Risk factors for recurrence are noted below. Oral rifampin use with IVAB was associated with a significantly lower rate of recurrence in patients with staphylococcal PJI (P = 0.004). Conclusion This real-world evaluation underscores the challenges of successful treatment of PJI. ICS was readily achieved (95%). High recurrence (23%) may be exaggerated by lower likelihood of FU in asymptomatic patients. Lack of improvement in 6-month cure with prosthesis removal merits additional inquiry. Although the group was small, adjunctive rifampin suggests improved outcomes in staphylococcal PJI. Disclosures B. S. Metzger, Allergan: Speaker’s Bureau, Speaker honorarium. L. J. Van Anglen, Merck & Co.: Investigator, Research grant.

Purpose In contrast to studies of adults, there are limited published data regarding palliative radiation therapy (RT) for children, and further study is greatly needed. Methods and materials We performed a retrospective review of all pediatric patients referred to our radiation oncology department over a 5-year span from January 1, 2007, to December 31, 2011. Results Of 244 total pediatric patients referred, a subset of 45 (18.4%) were treated specifically with palliative intent for a total of 83 courses of RT. Follow-up data until study closure or death were available for 98% of patients. The median survival after initiation of palliative RT was 6.5 months. Overall, 23% of the children were alive at last follow-up visit, and 77% were deceased. The prescribed RT was completed in 93% of courses; 7% of courses were discontinued because of clinical deterioration due to systemic disease progression. The overall symptom response rate (partial or complete) was 72%. Overall response rate by symptom was 80% for bone pain, 55% for dyspnea or chest pain, 58% for neurologic symptoms, 50% for bleeding, and 100% for liver pain or ascites. Response rates by histology were 100% for leukemias, 91% for neuroblastoma, 76% for Ewing sarcoma, 64% for rhabdomyosarcoma, 54% for osteosarcoma, and 50% for primary central nervous system neoplasms. For responders, the median time from RT initiation to response was 1 week. For 7% of patients, a repeat course of RT for the same site and symptom was performed. No patients experienced RTOG (Radiation Therapy Oncology Group) grade 3 or greater acute or late toxicities. Conclusions RT is a useful palliative tool for pediatric patients that merits continued use and further study.

The active form of vitamin D (1α,25-dihydroxyvitamin D, 1,25(OH)2D) exerts its genomic effects via binding to a nuclear high-affinity vitamin D receptor (VDR). Recent deep sequencing analysis of VDR binding locations across the complete genome has significantly expanded our understanding of the actions of vitamin D and VDR on gene transcription. However, these studies have also promoted appreciation of the extra-transcriptional impact of vitamin D on gene expression. It is now clear that vitamin D interacts with the epigenome via effects on DNA methylation, histone acetylation, and microRNA generation to maintain normal biological functions. There is also increasing evidence that vitamin D can influence pre-mRNA constitutive splicing and alternative splicing, although the mechanism for this remains unclear. Pre-mRNA splicing has long been thought to be a post-transcription RNA processing event, but current data indicate that this occurs co-transcriptionally. Several steroid hormones have been recognized to coordinately control gene transcription and pre-mRNA splicing through the recruitment of nuclear receptor co-regulators that can both control gene transcription and splicing. The current review will discuss this concept with specific reference to vitamin D, and the potential role of heterogeneous nuclear ribonucleoprotein C (hnRNPC), a nuclear factor with an established function in RNA splicing. hnRNPC, has been shown to be involved in the VDR transcriptional complex as a vitamin D-response element-binding protein (VDRE-BP), and may act as a coupling factor linking VDR-directed gene transcription with RNA splicing. In this way hnRNPC may provide an additional mechanism for the fine-tuning of vitamin D-regulated target gene expression. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

Human monocytes activated by toll-like receptor 2/1 ligand (TLR2/1L) show enhanced expression of the vitamin D receptor (VDR) and the vitamin D-activating enzyme 1α-hydroxylase (CYP27B1). The resulting intracrine conversion of precursor 25-hydroxyvitamin D3 (25OHD) to active 1,25-dihydroxyvitamin D (1,25(OH)2D) can stimulate expression of antibacterial cathelicidin (CAMP). To determine whether this response is functional in HIV-infected subjects (HIV+), serum from HIV+ subjects pre- and post-vitamin D supplementation was utilized in monocyte cultures with or without TLR2/1L. Expression of CYP27B1 and VDR was enhanced following treatment with TLR2/1L, although this effect was lower in HIV+ vs HIV- serum (p

BackgroundDespite recent advances, infection remains the most common etiology of arthroplasty failure. Recent work suggests that 25-hydroxyvitamin D (25D) deficiency correlates with the frequency of periprosthetic joint infection (PJI). We endeavored to examine whether 25D3 deficiency leads to increased bacterial burden in vivo in an established mouse model of PJI and, if so, whether this effect can be reversed by preoperative 25D3 supplementation.MethodsMice (lys-EGFP) possessing fluorescent neutrophils were fed a vitamin D3-sufficient (n = 20) or deficient (n = 40) diet for 6 weeks. A group of 25D3-deficient mice (n = 20) were "rescued" with 1 intraperitoneal dose of 25D3 at 3 days before surgery. A stainless steel implant was inserted into the knee joint and the joint space was inoculated with bioluminescent Staphylococcus aureus (1 × 10 colony forming units [CFUs]). In vivo imaging was used to monitor bacterial burden and neutrophil infiltration. Blood was drawn to confirm 25D3 levels 3 days before surgery and on postoperative days (PODs) 0 and 14. Mice were killed at POD 21, and CFUs were quantified after culture. Myeloperoxidase (MPO) and β-N-acetylglucosaminidase (NAG) were assayed to look at neutrophil infiltration and activated tissue macrophage recruitment, respectively.ResultsSerum values confirmed 25D3 deficiency and repletion of the 25D3-rescued group. Bacterial bioluminescence and neutrophil fluorescence were significantly greater (p < 0.05) in the 25D3-deficient group. CFU counts from the joint tissue and implant were also significantly greater in this group (p < 0.05). Rescue treatment significantly decreased bacterial burden and neutrophil infiltration (p < 0.05). Compared with the 25D3-sufficient and 25D3-rescued groups, MPO activity was higher (p < 0.02) and NAG activity was lower (p < 0.03) in the 25D3-deficient group.ConclusionsThis study demonstrated in vivo in a mouse model of PJI that (1) 25D3 deficiency results in increased bacterial burden and neutrophil infiltration, and (2) this effect can be reversed with preoperative repletion of 25D3.Clinical relevanceConsidering that >65% of patients undergoing arthroplasty have insufficient or low levels of total 25D and that 25D levels can be replenished with ease using a U.S. Food and Drug Administration (FDA)-approved, oral 25D3 product, 25D deficiency may be an important modifiable risk factor in humans undergoing joint replacement.

Activation of precursor 25-hydroxyvitamin D3 (25D) to hormonal 1,25-dihydroxyvitamin D3 (1,25D) is a pivotal step in vitamin D physiology, catalysed by the enzyme 25-hydroxyvitamin D-1α-hydroxylase (1α-hydroxylase). To establish new models for assessing the physiological importance of the 1α-hydroxylase-25D-axis, we used Danio rerio (zebrafish) to characterize expression and biological activity of the gene for 1α-hydroxylase (cyp27b1). Treatment of day 5 zebrafish larvae with inactive 25D (5–150 nM) or active 1,25D (0.1–10 nM) induced dose responsive expression (15–95-fold) of the vitamin D-target gene cyp24a1 relative to larvae treated with vehicle, suggesting the presence of Cyp27b1 activity. A full-length zebrafish cyp27b1 cDNA was then generated using RACE and RT-PCR methods. Sequencing of the resulting clone revealed an open reading frame encoding a protein of 505 amino acids with 54% identity to human CYP27B1. Transfection of a cyp27b1 expression vector into HKC-8, a human kidney proximal tubular epithelial cell line, enhanced intracrine metabolism of 25D to 1,25D resulting in greater than twofold induction of CYP24A1 mRNA expression and a 25-fold increase in 1,25D production compared to empty vector. These data indicate that we have cloned a functional zebrafish CYP27B1, representing a phylogenetically distant branch from mammals of this key enzyme in vitamin D metabolism. Further analysis of cyp27b1 expression and activity in zebrafish may provide new perspectives on the biological importance of 25D metabolism. Copyright © 2014 John Wiley & Sons, Ltd.

This article describes content of a weekly, semester-long professional development seminar for geography graduate students, introducing aspects of careers in academia, government, business and the non-profit worlds normally missing from master’s and Ph.D. programs, e.g., contemporary critiques of American higher education; planning a career; effective participation in professional meetings; preparing to teach; the world of scholarly publishing; obtaining research money; how colleges and universities function; tenure processes; research ethics; and work/life balance.

Cartilage injury represents one of the most significant clinical conditions. Implantation of expanded autologous chondrocytes from noninjured compartments of the joint is a typical strategy for repairing cartilage. However, two-dimensional culture causes dedifferentiation of chondrocytes, making them functionally inferior for cartilage repair. We hypothesized that functional exclusion of dedifferentiated chondrocytes can be achieved by the selective mapping of collagen molecules deposited by chondrogenic cells in a three-dimensional environment. Freshly isolated and in vitro expanded human fetal or adult articular chondrocytes were cultured in a thermoreversible hydrogel at density of 1 × 10(7) cells/mL for 24 h. Chondrocytes were released from the gel, stained with antibodies against collagen type 2 (COL II) or COL I or COL X and sorted by fluorescence activated cell sorting. Imaging flow cytometry, immunohistochemistry, quantitative polymerase chain reaction, and glycosaminoglycan (GAG) assays were performed to evaluate the differences between COL II domain forming and COL II domain-negative cells. Freshly dissected periarticular chondrocytes robustly formed domains that consisted of the extracellular matrix surrounding cells in the hydrogel as a capsule clearly detectable by imaging flow cytometry (ImageStream) and confocal microscopy. These domains were almost exclusively formed by COL II. In contrast to that, a significant percentage of freshly isolated growth plate pre-hypertrophic and hyperdrophic chondrocytes deposited matrix domains positive for COL II, COL I, and COL X. The proportion of the cells producing COL II domains decreased with the increased passage of in vitro expanded periarticular fetal or adult articular chondrocytes. Sorted COL II domain forming cells deposited much higher levels of COL II and GAGs in pellet assays than COL II domain-negative cells. COL II domain forming cells expressed chondrogenic genes at higher levels than negative cells. We report a novel method that allows separation of functionally active chondrogenic cells, which deposit high levels of COL II from functionally inferior dedifferentiated cells or hypertrophic chondrocytes producing COL X. This approach may significantly improve current strategies used for cartilage repair.

When bound to the vitamin D receptor (VDR), the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is a potent regulator of osteoblast transcription. Less clear is the impact of 1,25D on posttranscriptional events in osteoblasts, such as the generation and action of microRNAs (miRNAs). Microarray analysis using replicate (n = 3) primary cultures of human osteoblasts (HOBs) identified human miRNAs that were differentially regulated by >1.5-fold following treatment with 1,25D (10 nM, 6 hours), which included miRNAs 637 and 1228. Quantitative reverse transcription PCR analyses showed that the host gene for miR-1228, low-density lipoprotein receptor-related protein 1 (LRP1), was coinduced with miR-1228 in a dose-dependent fashion following treatment with 1,25D (0.1–10 nM, 6 hours). By contrast, the endogenous host gene for miR-637, death-associated protein kinase 3 (DAPK3), was transcriptionally repressed by following treatment with 1,25D. Analysis of two potential targets for miR-637 and miR-1228 in HOB, type IV collagen (COL4A1) and bone morphogenic protein 2 kinase (BMP2K), respectively, showed that 1,25D-mediates suppression of these targets via distinct mechanisms. In the case of miR-637, suppression of COL4A1 appears to occur via decreased levels of COL4A1 mRNA. By contrast, suppression of BMP2K by miR-1228 appears to occur by inhibition of protein translation. In mature HOBs, small interfering RNA (siRNA) inactivation of miR-1228 alone was sufficient to abrogate 1,25D-mediated downregulation of BMP2K protein expression. This was associated with suppression of prodifferentiation responses to 1,25D in HOB, as represented by parallel decrease in osteocalcin and alkaline phosphatase expression. These data show for the first time that the effects of 1,25D on human bone cells are not restricted to classical VDR-mediated transcriptional responses but also involve miRNA-directed posttranscriptional mechanisms.