Genetic analysis of osteoblast activity identifies Zbtb40 as a regulator of osteoblast activity and bone mass.

Osteoporosis is a genetic disease characterized by progressive reductions in bone mineral density (BMD) leading to an increased risk of fracture. Over the last decade, genome-wide association studies (GWASs) have identified over 1000 associations for BMD. However, as a phenotype BMD is challenging as bone is a multicellular tissue affected by both local and systemic physiology. Here, we focused on a single component of BMD, osteoblast-mediated bone formation in mice, and identified associations influencing osteoblast activity on mouse Chromosomes (Chrs) 1, 4, and 17. The locus on Chr. 4 was in an intergenic region between Wnt4 and Zbtb40, homologous to a locus for BMD in humans. We tested both Wnt4 and Zbtb40 for a role in osteoblast activity and BMD. Knockdown of Zbtb40, but not Wnt4, in osteoblasts drastically reduced mineralization. Additionally, loss-of-function mouse models for both genes exhibited reduced BMD. Our results highlight that investigating the genetic basis of in vitro osteoblast mineralization can be used to identify genes impacting bone formation and BMD.

Long-term Concentrations of Nitrogen Dioxide and Mortality: A Meta-analysis of Cohort Studies.

BACKGROUND: Concentrations of outdoor nitrogen dioxide (NO2) have been associated with increased mortality. Hazard ratios (HRs) from cohort studies are used to assess population health impact and burden. We undertook meta-analyses to derive concentration-response functions suitable for such evaluations and assessed their sensitivity to study selection based upon cohort characteristics. METHODS: We searched online databases and existing reviews for cohort studies published to October 2016 that reported HRs for NO2 and mortality. We calculated meta-analytic summary estimates using fixed/random-effects models. RESULTS: We identified 48 articles analyzing 28 cohorts. Meta-analysis of HRs found positive associations between NO2 and all cause (1.02 [95% confidence interval (CI): 1.01, 1.03]; prediction interval [PI]: [0.99, 1.06] per 10 µg/m increment in NO2), cardiovascular (1.03 [95% CI: 1.02, 1.05]; PI: [0.98, 1.08]), respiratory (1.03 [95% CI: 1.01, 1.05]; PI: [0.97, 1.10]), and lung cancer mortality (1.05 [95% CI: 1.02, 1.08]; PI: [0.94, 1.17]) with evidence of substantial heterogeneity between studies. In subgroup analysis, summary HRs varied by age at cohort entry, spatial resolution of pollution estimates, and adjustment for smoking and body mass index at the individual level; for some subgroups, the HR was close to unity, with lower confidence limits below 1. CONCLUSIONS: Given the many uncertainties inherent in the assessment of this evidence base and the sensitivity of health impact calculations to small changes in the magnitude of the HRs, calculation of the impact on health of policies to reduce long-term exposure to NO2 should use prediction intervals and report ranges of impact rather than focusing upon point estimates.

Mid-infrared supercontinuum generation from 1.6 to >11 µm using concatenated step-index fluoride and chalcogenide fibers.

We demonstrate an all-fiber supercontinuum source that generates a continuous spectrum from 1.6 µm to >11 µm with 417 mW on-time average power at 33% duty cycle. By utilizing a master oscillator power amplifier pump with three amplification stages and concatenating solid core ZBLAN, arsenic sulfide, and arsenic selenide fibers, we shift 1550 nm light to ∼4.5 µm, ∼6.5 µm, and >11 µm, respectively. With 69 mW past 7.5 µm, this source provides both high power and broad spectral expansion, while outputting a single fundamental mode.

Generation of near-diffraction-limited, high-power supercontinuum from 1.57 µm to 12 µm with cascaded fluoride and chalcogenide fibers.

We generate a supercontinuum (SC) spectrum ranging from 1.57 µm to 12 µm (20 dB bandwidth) with a soft glass fiber cascade consisting of ZrF4-BaF2-LaF3-AlF3-NaF fiber, As2S3 fiber, and As2Se3 fiber pumped by a nanosecond thulium master oscillator power amplifier system. The highest on-time average power generated is 417 mW at 33% duty cycle. We observe a near-diffraction-limit beam quality across the wavelength range from 3 µm to 12 µm, even though the As2Se3 fiber is multimode below 12 µm. Our study also shows that parameters of the As2Se3 fiber, such as numerical aperture, core size, and core/cladding composition, have significant effects on the long wavelength edge of the generated SC spectrum. Our results suggest that the high numerical aperture of 0.76 and low-loss As2Se3/GeAs2Se5 core/cladding material all contribute to broad SC generation in the long-wave infrared spectral region. Also, among our results, 10 µm core diameter selenide fiber yields the best spectral expansion, while the 12 µm core diameter selenide fiber yields the highest output power.

A humoral immune defect distinguishes the response to Staphylococcus aureus infections in mice with obesity and type 2 diabetes from that in mice with type 1 diabetes.

Obesity and diabetes are among the greatest risk factors for infection following total joint arthroplasty. However, the underlying mechanism of susceptibility is unclear. We compared orthopedic implant-associated Staphylococcus aureus infections in type 1 (T1D) versus type 2 (T2D) diabetic mouse models and in patients with S. aureus infections, focusing on the adaptive immune response. Mice were fed a high-fat diet to initiate obesity and T2D. T1D was initiated with streptozotocin. Mice were then given a trans-tibial implant that was precoated with bioluminescent Xen36 S. aureus. Although both mouse models of diabetes demonstrated worse infection severity than controls, infection in T2D mice was more severe, as indicated by increases in bioluminescence, S. aureus CFU in tissue, and death within the first 7 days. Furthermore, T2D mice had an impaired humoral immune response at day 14 with reduced total IgG, decreased S. aureus-specific IgG, and increased IgM. These changes were not present in T1D mice. Similarly, T2D patients and obese nondiabetics with active S. aureus infections had a blunted IgG response to S. aureus. In conclusion, we report the first evidence of a humoral immune deficit, possibly due to an immunoglobulin class switch defect, in obesity and T2D during exacerbated S. aureus infection which may contribute to the increased infection risk following arthroplasty in patients with T2D and obesity.

Monitoring tacrolimus toxicity following Paxlovid administration in a liver transplant patient.

Maintaining therapeutic plasma tacrolimus concentrations is essential for mitigating potential solid organ transplant rejection and preventing toxic adverse side effects. While patients can benefit greatly from tacrolimus therapy, co-administration of drugs such as Paxlovid (nirmatrelvir/ritonavir) place patients at serious risk for drug interactions and harm. Here we present a case of tacrolimus toxicity following Paxlovid administration in a liver transplant patient. Therapeutic drug monitoring was further complicated by a limited upper reportable threshold for tacrolimus testing and highlights the value of validating a higher limit to the clinical reportable range to improve tacrolimus monitoring and meet clinical needs in the setting of drug toxicity.

Analytical Performance Evaluation of Three Point-of-Care CBC Analyzers for Management of Clozapine Therapy in Ambulatory Psychiatry Clinics.

BACKGROUND: Clozapine is a first-line therapy and the only FDA-approved drug for patients with treatment-resistant schizophrenia (TRS). However, frequent measurement of absolute neutrophil count (ANC) is required to monitor for potential adverse severe neutropenia from clozapine therapy. We evaluated 3 point-of-care (POC) instruments that perform the complete blood count (CBC) with differential to assess their analytical performance and potential to meet the clinical need for clozapine therapy management. METHODS: A CBC with differential was performed on 104 residual whole blood specimens using 3 CBC analyzers (Sight OLO, PixCell HemoScreen, and Sysmex pocH-100i) to assess analytical precision, linearity, and accuracy vs the ADVIA 2120i and manual differential reference methods. Clinical concordance of ANC between POC devices and manual differential at medical decision points for mild, moderate, or severe neutropenia, and the threshold for clozapine therapy discontinuation (1.0 × 109/L) were determined. RESULTS: For CBC parameters, a CV ≤ 6.4% was observed on the OLO, CV ≤ 6.2% for the HemoScreen, and CV ≤ 5.1% with the pocH-100i. Each device accurately identified ANC with the greatest mean bias ±0.42 × 109/L using the pocH-100i vs manual differential. For results near the medical decision points (ANC <1.5 × 109/L), clinical concordance of ANC results was 55.6% for the OLO, 89.5% for the HemoScreen, and 82.4% for the pocH-100i. CONCLUSIONS: The HemoScreen device demonstrated the best clinical concordance in ANC values at medical decision thresholds for clozapine therapy management.

Ski inhibits TGF-ß/phospho-Smad3 signaling and accelerates hypertrophic differentiation in chondrocytes.

Since transforming growing factor-ß (TGF-ß)/Smad signaling inhibits chondrocyte maturation, endogenous negative regulators of TGF-ß signaling are likely also important regulators of the chondrocyte differentiation process. One such negative regulator, Ski, is an oncoprotein that is known to inhibit TGF-ß/Smad3 signaling via its interaction with phospho-Smad3 and recruitment of histone deacetylases (HDACs) to the DNA binding complex. Based on this, we hypothesized that Ski inhibits TGF-ß signaling and accelerates maturation in chondrocytes via recruitment of HDACs to transcriptional complexes containing Smads. We tested this hypothesis in chick upper sternal chondrocytes (USCs), where gain and loss of Ski expression experiments were performed. Over-expression of Ski not only reversed the inhibitory effect of TGF-ß on the expression of hypertrophic marker genes such as type X collagen (colX) and osteocalcin, it induced these genes basally as well. Conversely, knockdown of Ski by RNA interference led to a reduction of colX and osteocalcin expression under basal conditions. Furthermore, Ski blocked TGF-ß induction of cyclinD1 and caused a basal up-regulation of Runx2, consistent with the observed acceleration of hypertrophy. Regarding mechanism, not only does Ski associate with phospho-Smad2 and 3, but its association with phospho-Smad3 is required for recruitment of HDAC4 and 5. Implicating this recruitment of HDACs in the phenotypic effects of Ski in chondrocytes, the HDAC inhibitor SAHA reversed the up-regulation of colX and osteocalcin in Ski over-expressing cells. These results suggest that inhibition of TGF-ß signaling by Ski, which involves its association with phospho-Smad3 and recruitment of HDAC4 and 5, leads to accelerated chondrocyte differentiation.

Does outdoor air pollution induce new cases of asthma? Biological plausibility and evidence; a review.

It is widely accepted that air pollution can exacerbate asthma in those who already have the condition. What is less clear is whether air pollution can contribute to the initiation of new cases of asthma. Mechanistic evidence from toxicological studies, together with recent information on genes that predispose towards the development of asthma, suggests that this is biologically plausible, particularly in the light of the current understanding of asthma as a complex disease with a variety of phenotypes. The epidemiological evidence for associations between ambient levels of air pollutants and asthma prevalence at a whole community level is unconvincing; meta-analysis confirms a lack of association. In contrast, a meta-analysis of cohort studies found an association between asthma incidence and within-community variations in air pollution (largely traffic dominated). Similarly, a systematic review suggests an association of asthma prevalence with exposure to traffic, although only in those living very close to heavily trafficked roads carrying a lot of trucks. Based on this evidence, the U.K.'s Committee on the Medical Effects of Air Pollutants recently concluded that, overall, the evidence is consistent with the possibility that outdoor air pollution might play a role in causing asthma in susceptible individuals living very close to busy roads carrying a lot of truck traffic. Nonetheless, the effect on public health is unlikely to be large: air pollutants are likely to make only a small contribution, compared with other factors, in the development of asthma, and in only a small proportion of the population.

Transcriptome-wide association study and eQTL colocalization identify potentially causal genes responsible for human bone mineral density GWAS associations.

Genome-wide association studies (GWASs) for bone mineral density (BMD) in humans have identified over 1100 associations to date. However, identifying causal genes implicated by such studies has been challenging. Recent advances in the development of transcriptome reference datasets and computational approaches such as transcriptome-wide association studies (TWASs) and expression quantitative trait loci (eQTL) colocalization have proven to be informative in identifying putatively causal genes underlying GWAS associations. Here, we used TWAS/eQTL colocalization in conjunction with transcriptomic data from the Genotype-Tissue Expression (GTEx) project to identify potentially causal genes for the largest BMD GWAS performed to date. Using this approach, we identified 512 genes as significant using both TWAS and eQTL colocalization. This set of genes was enriched for regulators of BMD and members of bone relevant biological processes. To investigate the significance of our findings, we selected PPP6R3, the gene with the strongest support from our analysis which was not previously implicated in the regulation of BMD, for further investigation. We observed that Ppp6r3 deletion in mice decreased BMD. In this work, we provide an updated resource of putatively causal BMD genes and demonstrate that PPP6R3 is a putatively causal BMD GWAS gene. These data increase our understanding of the genetics of BMD and provide further evidence for the utility of combined TWAS/colocalization approaches in untangling the genetics of complex traits.

Surgical Induction of Posttraumatic Osteoarthritis in the Mouse.

Given the prevalence and the scope of the personal and societal burden of osteoarthritis (OA), investigators continue to be deeply interested in understanding the pathogenic basis of disease and developing novel disease modifying OA therapies. Because joint trauma/injury is considered a leading predisposing factor in the development of OA, and since posttraumatic OA is one of the most common forms of OA in general, large animal and rodent models of knee injury that accurately recapitulate the OA disease process have become increasingly widespread over the past decade. To enable study in the context of defined genetic backgrounds, investigative teams have developed standardized protocols for injuring the mouse knee that aim to induce a reproducible degenerative process both in terms of severity and temporal pacing of disease progression. The destabilization of the medial meniscus (DMM) is one of the most commonly employed surgical procedure in rodents that reproducibly models posttraumatic OA and allows for the study of disease progression from initiation to end-stage disease. The description provided here sets the stage for both inexperienced and established investigators to employ the DMM procedure, or other similar surgical destabilization methods, to initiate the development of posttraumatic OA in the mouse. Successful application of this method provides a preclinical platform to study the mechanisms driving the pathogenesis of posttraumatic OA and for testing therapeutic strategies to treat it.

IKKß-NF-κB signaling in adult chondrocytes promotes the onset of age-related osteoarthritis in mice.

Canonical nuclear factor κB (NF-κB) signaling mediated by homo- and heterodimers of the NF-κB subunits p65 (RELA) and p50 (NFKB1) is associated with age-related pathologies and with disease progression in posttraumatic models of osteoarthritis (OA). Here, we established that NF-κB signaling in articular chondrocytes increased with age, concomitant with the onset of spontaneous OA in wild-type mice. Chondrocyte-specific expression of a constitutively active form of inhibitor of κB kinase ß (IKKß) in young adult mice accelerated the onset of the OA-like phenotype observed in aging wild-type mice, including degenerative changes in the articular cartilage, synovium, and menisci. Both in vitro and in vivo, chondrocytes expressing activated IKKß had a proinflammatory secretory phenotype characterized by markers typically associated with the senescence-associated secretory phenotype (SASP). Expression of these factors was differentially regulated by p65, which contains a transactivation domain, and p50, which does not. Whereas the loss of p65 blocked the induction of genes encoding SASP factors in chondrogenic cells treated with interleukin-1ß (IL-1ß) in vitro, the loss of p50 enhanced the IL-1ß­induced expression of some SASP factors. The loss of p50 further exacerbated cartilage degeneration in mice with chondrocyte-specific IKKß activation. Overall, our data reveal that IKKß-mediated activation of p65 can promote OA onset and that p50 may limit cartilage degeneration in settings of joint inflammation including advanced age.

Pollution, Particles, and Dementia: A Hypothetical Causative Pathway.

Epidemiological studies of air pollution have shown associations between exposure to particles and dementia. The mechanism of this is unclear. As these seem unlikely in terms of the very small dose likely to reach the brain in usual Western urban circumstances, we extend our 1995 hypothetical explanation of the association of air pollution with cardiac deaths as a plausible alternative explanation of its associations with dementia. Since our original proposal, it has become apparent that inflammation may be carried by blood from organ to organ by biologic microparticles derived from cell membranes. These transmit inflammatory messages to endothelial cells throughout the body as part of a general defensive response to assumed bacterial infection; particulate air pollution has recently been shown to be associated with their release into the blood. We propose that episodic release of biologic microparticles from pollution-induced lung inflammation causes secondary inflammation in the blood-brain barrier and cerebral microbleeds, culminating over time in cognitive impairment. Ultimately, by incomplete repair and accumulation of amyloid, this increases the risk of Alzheimer's disease. Importantly, this mechanism may also explain the relationships of other inflammatory conditions and environmental factors with cognitive decline, and point to new opportunities to understand and prevent dementia.

Mouse Models and Online Resources for Functional Analysis of Osteoporosis Genome-Wide Association Studies.

Osteoporosis is a complex genetic disease in which the number of loci associated with the bone mineral density, a clinical risk factor for fracture, has increased at an exponential rate in the last decade. The identification of the causative variants and candidate genes underlying these loci has not been able to keep pace with the rate of locus discovery. A large number of tools and data resources have been built around the use of the mouse as model of human genetic disease. Herein, we describe resources available for functional validation of human Genome Wide Association Study (GWAS) loci using mouse models. We specifically focus on large-scale phenotyping efforts focused on bone relevant phenotypes and repositories of genotype-phenotype data that exist for transgenic and mutant mice, which can be readily mined as a first step toward more targeted efforts designed to deeply characterize the role of a gene in bone biology.

Evaluating the toxicity of airborne particulate matter and nanoparticles by measuring oxidative stress potential--a workshop report and consensus statement.

BACKGROUND: There is a strong need for laboratory in vitro test systems for the toxicity of airborne particulate matter and nanoparticles. The measurement of oxidative stress potential offers a promising way forward. OBJECTIVES: A workshop was convened involving leading workers from the field in order to review the available test methods and to generate a Consensus Statement. DISCUSSIONS: Workshop participants summarised their own research activities as well as discussion the relative merits of different test methods. CONCLUSIONS: In vitro test methods have an important role to play in the screening of toxicity in airborne particulate matter and nanoparticles. In vitro cell challenges were preferable to in vitro acellular systems but both have a potential major role to play and offer large cost advantages relative to human or animal inhalation studies and animal in vivo installation experiments. There remains a need to compare tests one with another on standardised samples and also to establish a correlation with the results of population-based epidemiology.

Characterization of expression and alternative splicing of the gene cadherin-like and PC esterase domain containing 1 (Cped1).

Cadherin-like and PC-esterase domain containing 1 (CPED1) is an uncharacterized gene with no known function. Human genome wide association studies (GWAS) for bone mineral density (BMD) have repeatedly identified a significant locus on Chromosome 7 that contains the gene CPED1, but it remains unclear if this gene could be causative. While an open reading frame for this gene has been predicted, there has been no systematic exploration of expression or alternate splicing for CPED1 in humans or mice.Using mouse models, we demonstrate that Cped1 is alternately spliced whereby transcripts are generated with exon 3 or exons 16 and 17 removed. In calvarial-derived pre-osteoblasts, Cped1 utilizes the predicted promoter upstream of exon 1 as well as alternate promoters upstream of exon 3 and exon 12.Lastly, we have determined that some transcripts terminate at the end of exon 10 and therefore do not contain the cadherin like and the PC esterase domains.Together, these data suggest that multiple protein products may be produced by this gene, with some products either lacking or containing both the predicted functional domains. Our data provide a framework upon which future functional studies will be built to understand the role of this gene in bone biology.

Daily oral consumption of hydrolyzed type 1 collagen is chondroprotective and anti-inflammatory in murine posttraumatic osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease for which there are no disease modifying therapies. Thus, strategies that offer chondroprotective or regenerative capability represent a critical unmet need. Recently, oral consumption of a hydrolyzed type 1 collagen (hCol1) preparation has been reported to reduce pain in human OA and support a positive influence on chondrocyte function. To evaluate the tissue and cellular basis for these effects, we examined the impact of orally administered hCol1 in a model of posttraumatic OA (PTOA). In addition to standard chow, male C57BL/6J mice were provided a daily oral dietary supplement of hCol1 and a meniscal-ligamentous injury was induced on the right knee. At various time points post-injury, hydroxyproline (hProline) assays were performed on blood samples to confirm hCol1 delivery, and joints were harvested for tissue and molecular analyses were performed, including histomorphometry, OARSI and synovial scoring, immunohistochemistry and mRNA expression studies. Confirming ingestion of the supplements, serum hProline levels were elevated in experimental mice administered hCol1. In the hCol1 supplemented mice, chondroprotective effects were observed in injured knee joints, with dose-dependent increases in cartilage area, chondrocyte number and proteoglycan matrix at 3 and 12 weeks post-injury. Preservation of cartilage and increased chondrocyte numbers correlated with reductions in MMP13 protein levels and apoptosis, respectively. Supplemented mice also displayed reduced synovial hyperplasia that paralleled a reduction in Tnf mRNA, suggesting an anti-inflammatory effect. These findings establish that in the context of murine knee PTOA, daily oral consumption of hCol1 is chondroprotective, anti-apoptotic in articular chondrocytes, and anti-inflammatory. While the underlying mechanism driving these effects is yet to be determined, these findings provide the first tissue and cellular level information explaining the already published evidence of symptom relief supported by hCol1 in human knee OA. These results suggest that oral consumption of hCol1 is disease modifying in the context of PTOA.