Renal TLR-7/TNF-α pathway as a potential female-specific mechanism in the pathogenesis of autoimmune-induced hypertension.

Hypertension is prevalent in patients with systemic lupus erythematosus (SLE). The goal of the current study is to track the pathogenesis of hypertension and renal injury in SLE, identify contributory mechanisms, and highlight differences in disease development among sexes. Mean arterial pressure was measured in conscious male and female SLE (NZBWF1) and control (NZW) mice at 34-35 wk of age using indwelling arterial catheters. Measures of renal injury, renal inflammation, and renal hemodynamics were used to monitor the potential contributors to latent sex differences. Both male and female SLE mice were hypertensive at 35 wk of age, and the hypertension was linked to renal injury in females, but not in males. A known contributor of renal pathology in SLE, Toll-like receptor (TLR)-7, and its downstream effector, the proinflammatory cytokine tumor necrosis factor (TNF)-α, were lower in male SLE mice than in females. Male SLE mice also had higher glomerular filtration rate (GFR) and lower renal vascular resistance (RVR) than females. Our data suggest that although hypertension in female SLE mice is associated with renal mechanisms, hypertension in male SLE mice may develop independent of renal changes. Future studies will continue to dissect sex-specific factors that should be considered when treating patients with hypertension with underlying chronic inflammation and/or autoimmunity.NEW & NOTEWORTHY There is a high prevalence of hypertension in male and female SLE; however, male SLE mice are hypertensive without renal involvement. The development of hypertension in female SLE mice is renocentric and strongly associated with injurious renal mechanisms like the TLR-7→TNF-α pathway. This clear difference in the pathogenesis among the sexes could have a significant impact on how we treat patients with hypertension with underlying chronic autoimmune/inflammatory diseases.

Replication study of previous migraine genome-wide association study findings in a Spanish sample of migraine with aura.

BACKGROUND: Migraine is a common disabling condition that affects approximately 15% of the population. Several genome-wide association studies have attempted to identify susceptibility variants involved in migraine, reporting several candidate loci for the disorder. METHODS: In order to replicate findings from previous genome-wide association studies, a case-control association study was performed. Twelve single nucleotide polymorphisms were genotyped in a Spanish sample of 512 migraine with aura patients and 535 migraine-free controls. RESULTS: Nominal associations were found for single nucleotide polymorphisms rs2651899 (within the PRDM16 gene), rs10166942 (near TRPM8), rs12134493 (close to TSPAN2) and rs10504861 (near MMP16) in our migraine with aura sample. CONCLUSIONS: Our study provides suggestive replication, in a Spanish migraine with aura sample, of four genome-wide association study findings previously reported in common migraine. However, larger sample sets should be explored to confirm our results.

Subchondral pre-solidified chitosan/blood implants elicit reproducible early osteochondral wound-repair responses including neutrophil and stromal cell chemotaxis, bone resorption and repair, enhanced repair tissue integration and delayed matrix deposition.

BACKGROUND: In this study we evaluated a novel approach to guide the bone marrow-driven articular cartilage repair response in skeletally aged rabbits. We hypothesized that dispersed chitosan particles implanted close to the bone marrow degrade in situ in a molecular mass-dependent manner, and attract more stromal cells to the site in aged rabbits compared to the blood clot in untreated controls. METHODS: Three microdrill hole defects, 1.4 mm diameter and 2 mm deep, were created in both knee trochlea of 30 month-old New Zealand White rabbits. Each of 3 isotonic chitosan solutions (150, 40, 10 kDa, 80% degree of deaceylation, with fluorescent chitosan tracer) was mixed with autologous rabbit whole blood, clotted with tissue factor to form cylindrical implants, and press-fit in drill holes in the left knee while contralateral holes received tissue factor or no treatment. At day 1 or day 21 post-operative, defects were analyzed by micro-computed tomography, histomorphometry and stereology for bone and soft tissue repair. RESULTS: All 3 implants filled the top of defects at day 1 and were partly degraded in situ at 21 days post-operative. All implants attracted neutrophils, osteoclasts and abundant bone marrow-derived stromal cells, stimulated bone resorption followed by new woven bone repair (bone remodeling) and promoted repair tissue-bone integration. 150 kDa chitosan implant was less degraded, and elicited more apoptotic neutrophils and bone resorption than 10 kDa chitosan implant. Drilled controls elicited a poorly integrated fibrous or fibrocartilaginous tissue. CONCLUSIONS: Pre-solidified implants elicit stromal cells and vigorous bone plate remodeling through a phase involving neutrophil chemotaxis. Pre-solidified chitosan implants are tunable by molecular mass, and could be beneficial for augmented marrow stimulation therapy if the recruited stromal cells can progress to bone and cartilage repair.

Chitosan rate of uptake in HEK293 cells is influenced by soluble versus microparticle state and enhanced by serum-induced cell metabolism and lactate-based media acidification.

UNLABELLED: Chitosan is a biocompatible polysaccharide composed of glucosamine and N-acetylglucosamine. The polymer has a unique behavior of fluctuating between soluble chains at pH 6 and insoluble microparticles at pH 7. The purpose of this study was to test the hypothesis that chitosan structure, solubility state, and serum influence the rate of cell uptake. Chitosans with 80% and 95% degree of deacetylation (medium and low viscosity) were tagged with rhodamine and analyzed for particle size, media solubility, and uptake by HEK293 epithelial cells using live confocal microscopy and flow cytometry. In media pH 7.4 with or without 10% serum, chitosans fully precipitated into 0.5 to 1.4 µm diameter microparticles with a slight negative charge. During 24 h of culture in serum-free medium, chitosan particles remained extracellular. In cultures with serum, particles were taken up into intracellular vesicles in a serum dose-dependent manner. Opsonization of chitosan with serum, or replacement of serum by epidermal growth factor (EGF) failed to mediate serum-free chitosan particle uptake. Serum stimulated cells to acidify the media, partly by lactate generation. Media acidified to pH 6.5 by 7 mM lactate maintained 50% of chitosan in the soluble fraction, and led to minor uniform serum-free uptake in small vesicles. CONCLUSION: Media acidification mediates minor in vitro uptake of non-biofouled soluble chitosan chains, while serum-biofouled insoluble chitosan microparticles require sustained serum exposure to generate energy required for macropinocytosis.

Reference equations using segmented regressions for impulse oscillometry in healthy subjects aged 2.7-90 years.

Background: Published reference equations for impulse oscillometry (IOS) usually encompass a specific age group but not the entire lifespan. This may lead to discordant predicted values when two or more non-coincident equations can be applied to the same person, or when a person moves from one equation to the next non-convergent equation as he or she gets older. Thus, our aim was to provide a single reference equation for each IOS variable that could be applied from infancy to old age. Methods: This was an ambispective cross-sectional study in healthy nonsmokers, most of whom lived in Mexico City, who underwent IOS according to international standards. A multivariate piecewise linear regression, also known as segmented regression, was used to obtain reference equations for each IOS variable. Results: In a population of 830 subjects (54.0% female) aged 2.7 to 90 years (54.8% children ≤12 years), segmented regression estimated two breakpoints for age in almost all IOS variables, except for R5-R20 in which only one breakpoint was detected. With this approach, multivariate regressions including sex, age, height and body mass index as independent variables were constructed, and coefficients for calculating predicted value, lower and upper limits of normal, percentage of predicted and z-score were obtained. Conclusions: Our study provides IOS reference equations that include the major determinants of lung function, i.e. sex, age, height and body mass index, that can be easily implemented for subjects of almost any age.

A replication study of a GWAS finding in migraine does not identify association in a Spanish case-control sample.

BACKGROUND: Migraine is a prevalent neurological disorder with a complex genetic background characterized by recurrent episodes of headache. The disease is subclassified into migraine with aura (MA) and migraine without aura (MO). Many association studies have been performed to date to identify genetic risk variants for common migraine, most of them focusing on selected candidate genes, with variable and often inconsistent results. Recently, a clinic-based genome-wide association study for migraine reported a functionally relevant risk variant (SNP rs1835740), involved in glutamate homeostasis, which showed a significant association with MA. We aimed to replicate this finding in a clinic-based study of a Spanish cohort with MA and MO patients. METHODS: We genotyped SNP rs1835740 in a Spanish sample of 1521 patients and 1379 screened controls and performed a case-control association study. CONCLUSION: No association was found between the assayed SNP and any of the clinical groups considered.

The cartilage-bone interface.

In the knee joint, the purpose of the cartilage-bone interface is to maintain structural integrity of the osteochondral unit during walking, kneeling, pivoting, and jumping--during which tensile, compressive, and shear forces are transmitted from the viscoelastic articular cartilage layer to the much stiffer mineralized end of the long bone. Mature articular cartilage is integrated with subchondral bone through a approximately 20 to approximately 250 microm thick layer of calcified cartilage. Inside the calcified cartilage layer, perpendicular chondrocyte-derived collagen type II fibers become structurally cemented to collagen type I osteoid deposited by osteoblasts. The mature mineralization front is delineated by a thin approximately 5 microm undulating tidemark structure that forms at the base of articular cartilage. Growth plate cartilage is anchored to epiphyseal bone, sometimes via a thin layer of calcified cartilage and tidemark, while the hypertrophic edge does not form a tidemark and undergoes continual vascular invasion and endochondral ossification (EO) until skeletal maturity upon which the growth plates are fully resorbed and replaced by bone. In this review, the formation of the cartilage-bone interface during skeletal development and cartilage repair, and its structure and composition are presented. Animal models and human anatomical studies show that the tidemark is a dynamic structure that forms within a purely collagen type II-positive and collagen type I-negative hyaline cartilage matrix. Cartilage repair strategies that elicit fibrocartilage, a mixture of collagen type I and type II, are predicted to show little tidemark/calcified cartilage regeneration and to develop a less stable repair tissue-bone interface. The tidemark can be regenerated through a bone marrow-driven growth process of EO near the articular surface.

SNP variants within the vanilloid TRPV1 and TRPV3 receptor genes are associated with migraine in the Spanish population.

The transient receptor potential (TRP) superfamily of non-selective cationic channels are involved in several processes plausibly relevant to migraine pathophysiology, including multimodal sensory and pain perception, central and peripheral sensitization, and regulation of calcium homeostasis. With the aim of identifying single nucleotide polymorphisms (SNPs) in TRP genes that may confer increased genetic susceptibility to migraine, we carried out a case-control genetic association study with replication, including a total of 1,040 cases and 1,037 controls. We genotyped 149 SNPs covering 14 TRP genes with known brain expression. The two-stage study comprised samples of 555 and 485 Spanish, Caucasian patients, selected according to the ICHD-II criteria for the diagnosis of migraine without aura (MO) or migraine with aura (MA). In the discovery sample, 19 SNPs in ten TRP genes showed nominal association (P < 0.05) with MO, MA, or overall migraine. In the replication sample, nominal association was confirmed for TRPV3 rs7217270 in MA and TRPV1 rs222741 in the overall migraine group. Risk haplotypes were identified for seven of the genes showing nominal association in the discovery set, but none of them was replicated. The present findings suggest that members of the vanilloid TRPV subfamily of receptors contribute to the genetic susceptibility to migraine in the Spanish population.

Chitosan coatings with distinct innate immune bioactivities differentially stimulate angiogenesis, osteogenesis and chondrogenesis in poly-caprolactone scaffolds with controlled interconnecting pore size.

This study tested whether osseous integration into poly (ε-caprolactone) (PCL) bioplastic scaffolds with fully-interconnecting 155 ± 8 µm pores is enhanced by an adhesive, non-inflammatory 99% degree of deacetylation (DDA) chitosan coating (99-PCL), or further incorporation of pro-inflammatory 83% DDA chitosan microparticles (83-99-PCL) to accelerate angiogenesis. New Zealand White rabbit osteochondral knee defects were press-fit with PCL, 99-PCL, 83-99-PCL, or allowed to bleed (drill-only). Between day 1 and 6 weeks of repair, drill-only defects repaired by endochondral ossification, with an 8-fold higher bone volume fraction (BVF) versus initial defects, compared to a 2-fold (99-PCL), 1.1-fold (PCL), or 0.4-fold (83-99-PCL) change in BVF. Hematoma innate immune cells swarmed to 83-99-PCL, elicited angiogenesis throughout the pores and induced slight bone resorption. PCL and 99-PCL pores were variably filled with cartilage or avascular mesenchyme near the bone plate, or angiogenic mesenchyme into which repairing trabecular bone infiltrated up to 1 mm deep. More repair cartilage covered the 99-PCL scaffold (65%) than PCL (18%) or 83-99-PCL (0%) (p < 0.005). We report the novel finding that non-inflammatory chitosan coatings promoted cartilage infiltration into and over a bioplastic scaffold, and were compatible with trabecular bone integration. This study also revealed that in vitro osteogenesis assays have limited ability to predict osseous integration into porous scaffolds, because (1) in vivo, woven bone integrates from the leading edge of regenerating trabecular bone and not from mesenchymal cells adhering to scaffold surfaces, and (2) bioactive coatings that attract inflammatory cells induce bone resorption.

Study protocol for monitoring SARS-CoV-2 infection and its determinants in Catalonia (Spain): an observational and participatory research approach in a Sentinel Network of Schools.

INTRODUCTION: Since the SARS-CoV-2 became of concern in January 2020, many preventive measures have been adopted in educational settings to ensure the control of COVID-19 pandemic among children and staff in schools. This study aims to set up a school sentinel surveillance network with the purpose of monitoring SARS-CoV-2 infection, seroprevalence as well as to analyse the impact of preventive interventions of SARS-CoV-2 in school settings. Additionally, we will assess diverse screening strategies in a cohort of students and school staff to monitor the screening acceptance and its potential impact. Altogether, we hope this study will enable the design of more effective strategies for the prevention of COVID-19 spread. METHODS AND ANALYSIS: The sentinel schools' study is a cross-sectional, school-based project including 26 participating sentinel schools in Catalonia (Spain). Children, adolescents and staff at the schools will be invited to participate. This project will be carried out from January 2021 to June 2022 as follows: (1) twice yearly serological testing and molecular SARS-CoV-2 detection and questionnaires covering SARS-CoV-2 symptoms, tests, health, knowledge, attitudes and behaviours; (2) an environmental evaluation carried out in different classrooms; (3) SARS-CoV-2 transmission dynamics and the impact of different variants among confirmed cases and classmates; (4) a participatory process by which the participants are invited to act as coinvestigators to evaluate prevention strategies and provide recommendations to improve COVID-19 prevention in schools. Descriptive analysis will be performed for the main variables collected. The incidence and seroprevalence will be calculated and the association with sociodemographic factors and school characteristics will be determined using multivariate logistic regression. ETHICS AND DISSEMINATION: Ethical approval was obtained from the IDIAPJGol and the Hospital Universitari Vall d'Hebron ethics committees. A report will be generated quarterly. Findings will be disseminated at national and international conferences and published in peer-reviewed journals.

Biodegradable chitosan particles induce chemokine release and negligible arginase-1 activity compared to IL-4 in murine bone marrow-derived macrophages.

Alternatively activated macrophages have been implicated in the therapeutic activity of biodegradable chitosan on wound healing, however, the mechanisms of phenotypic differentiation are still unclear.In vitro, macrophages stimulated with high doses of chitosan (≥ 500 µg/mL) were reported to produce low-level markers associated with alternative activation (arginase-1) as well as classical activation (nitric oxide), and to undergo apoptosis. In this study, we tested the hypothesis that 40 kDa biodegradable chitosan (5-500 µg/mL) is sufficient to polarize mouse bone marrow-derived macrophages (BMDM) in vitro to an alternatively activated phenotype. Control cultures were stimulated with IL-4 (alternative activation), IFN-γ/LPS (classical activation), 1 µm diameter latex beads (phagocytosis), or left untreated. After 48 h of in vitro exposure, BMDM phagocytosed fluorescent chitosan particles or latex beads, and remained viable and metabolically active, although some cells detached with increasing chitosan and latex bead dosage. Arginase-1 was over 100-fold more strongly induced by IL-4 than by chitosan, which induced only sporadic and weak arginase-1 activity over untreated BMDM, and no nitric oxide. IFN-γ/LPS stimulated nitric oxide production and arginase-1 activity and high concentrations of inflammatory cytokines (IL-6, IL-1ß, TNF-α, MIP-1α/MIP-1ß), while latex beads stimulated nitric oxide and not arginase-1 activity. Chitosan or latex bead exposure, but not IL-4, tended to promote the release of several chemokines (MIP-1α/ß, GM-CSF, RANTES, IL-1ß), while all treatments promoted MCP-1 release. These data show that chitosan phagocytosis is not sufficient to polarize BMDM to the alternative or the classical pathway, suggesting that biodegradable chitosan elicits alternatively activated macrophages in vivo through indirect mechanisms.

Systemic Administration of α7-Nicotinic Acetylcholine Receptor Ligands Does Not Improve Renal Injury or Behavior in Mice With Advanced Systemic Lupus Erythematosus.

There is a critical need for safe treatment options to control inflammation in patients with systemic lupus erythematosus (SLE) since the inflammation contributes to morbidity and mortality in advanced disease. Endogenous neuroimmune mechanisms like the cholinergic anti-inflammatory pathway can be targeted to modulate inflammation, but the ability to manipulate such pathways and reduce inflammation and end organ damage has not been fully explored in SLE. Positive allosteric modulators (PAM) are pharmacological agents that inhibit desensitization of the nicotinic acetylcholine receptor (α7-nAChR), the main anti-inflammatory feature within the cholinergic anti-inflammatory pathway, and may augment α7-dependent cholinergic tone to generate therapeutic benefits in SLE. In the current study, we hypothesize that activating the cholinergic anti-inflammatory pathway at the level of the α7-nAChR with systemic administration of a partial agonist, GTS-21, and a PAM, PNU-120596, would reduce inflammation, eliminating the associated end organ damage in a mouse model of SLE with advanced disease. Further, we hypothesize that systemic α7 ligands will have central effects and improve behavioral deficits in SLE mice. Female control (NZW) and SLE mice (NZBWF1) were administered GTS-21 or PNU-120596 subcutaneously via minipumps for 2 weeks. We found that the increased plasma dsDNA autoantibodies, splenic and renal inflammation, renal injury and hypertension usually observed in SLE mice with advanced disease at 35 weeks of age were not altered by GTS-21 or PNU-120596. The anxiety-like behavior presented in SLE mice was also not improved by GTS-21 or PNU-120596. Although no significant beneficial effects of α7 ligands were observed in SLE mice at this advanced stage, we predict that targeting this receptor earlier in the pathogenesis of the disease may prove to be efficacious and should be addressed in future studies.

Increasing URiM Family Medicine Residents at University of Utah Health.

INTRODUCTION: The Department of Family and Preventive Medicine is home for the University of Utah's Family Medicine Residency program. Although Utah's diversity is steadily increasing, the race/ethnic diversity of the program's family medicine residency does not reflect the state's general population. METHODS: From 2017 to 2021, the residency instituted several adjustments to recruitment processes, including modification of an existing screening system to better highlight resiliency in overcoming challenging life experiences; promotion of commitment to diversity during interview days; incorporation of increased participation from diverse faculty and residents on interview days; and addition of outreach from the Office of Health, Equity, Diversity, and Inclusion. Underrepresented in medicine (URiM) applicants were the first to be offered interviews in an identical screening score cohort, and were ranked highest in rank lists in cohorts with identical final rank scores. RESULTS: Over the past five match cycles, Latinx residents have increased from zero to six, and underrepresented Asian residents from zero to two. In the 2021 match cycle, five of 10 incoming residents (50%) are URiM. Overall, URiM residents are now 30%, and residents of color 36%, of a total of 30 residents across all 3 training years. We found that eight URiM interviews were needed for every one URiM match. CONCLUSION: Intentional resident recruitment initiatives can transform racial/ethnic diversity in a family medicine residency program in a short amount of time.

Tolerance and effectiveness of eccentric vs. concentric muscle strengthening in rotator cuff partial tears and moderate to severe shoulder pain. A randomized pilot study.

INTRODUCTION: Rotator cuff tears are the main cause of shoulder pain and disability. First line of treatment is conservative; there is evidence regarding the advantage of using eccentric over concentric exercises in tendinopathies, but there are no evidence-based recommendations on starting strengthening exercise during painful phases nor on the effectiveness and advantages of eccentric vs. concentric exercise in rotator cuff tears. OBJECTIVE: To evaluate the tolerance of a resistance strengthening program and to compare eccentric vs. concentric programs. DESIGN: A pilot, experimental, randomized controlled study. SETTING: Outpatient Rehabilitation Center. PATIENTS: Twenty-six patients with a diagnosis of partial rotator cuff tear were randomly assigned to: the experimental group (eccentric, n = 12) and the control (concentric, n = 14). INTERVENTION: The experimental group performed muscle strengthening with eccentric technique directed to shoulder and scapular girdle muscles, while the control group performed the concentric technique. MAIN OUTCOME MEASURES: Visual Analogue Scale (VAS), Constant Scale, Strength, Structure (Ultrasound report). RESULTS: The tolerance rate was 96% in both groups. Median age (experimental vs. control) was 54.5 vs. 54 years (p = 0.69). Results at baseline, and at months 1, 3, and 12 (median) were as follows: for VAS (mm), experimental: 55, 30, 30, and 10, p < 0.001 (intra-group); control: 50, 30, 30, and 5, p = 0.01; Constant scale (points): experimental 58.5, 88, 93, and 85, p < 0.001; control 50.62, 80, and 91.5, p = 0.038; normalized strength (Kg); experimental: 0.23, 0.29, 0.73, and 0.72, p = 0.001, and control: 0.24, 0.21, 0.54, and 0.66, p = 0.01. We found inter-group differences in the Constant scale at 1 and 3 months (p < 0.05), and in strength at months 1 and 3 (p < 0.05). We observed structural differences in tendon (healing) between groups at 3 and 12 months. CONCLUSIONS: Eccentric and concentric strengthening were well tolerated; both show early improvement in pain, functionality and tendon structure. Eccentric training appears to be more effective than concentric in the early improvement of functionality, strength and tendon healing.

Biophysical characterization of iron in mitochondria isolated from respiring and fermenting yeast.

The distributions of Fe in mitochondria isolated from respiring, respiro-fermenting, and fermenting yeast cells were determined with an integrative biophysical approach involving Mossbauer and electronic absorption spectroscopies, electron paramagnetic resonance, and inductively coupled plasma emission mass spectrometry. Approximately 40% of the Fe in mitochondria from respiring cells was present in respiration-related proteins. The concentration and distribution of Fe in respiro-fermenting mitochondria, where both respiration and fermentation occur concurrently, were similar to those of respiring mitochondria. The concentration of Fe in fermenting mitochondria was also similar, but the distribution differed dramatically. Here, levels of respiration-related Fe-containing proteins were diminished approximately 3-fold, while non-heme HS Fe(II) species, non-heme mononuclear HS Fe(III), and Fe(III) nanoparticles dominated. These changes were rationalized by a model in which the pool of non-heme HS Fe(II) ions serves as feedstock for Fe-S cluster and heme biosynthesis. The integrative approach enabled us to estimate the concentration of respiration-related proteins.

A nonheme high-spin ferrous pool in mitochondria isolated from fermenting Saccharomyces cerevisiae.

Mössbauer spectroscopy was used to detect pools of Fe in mitochondria from fermenting yeast cells, including those consisting of nonheme high-spin (HS) Fe(II) species, Fe(III) nanoparticles, and mononuclear HS Fe(III) species. At issue was whether these species were located within mitochondria or on their exterior. None could be removed by washing mitochondria extensively with ethylene glycol tetraacetic acid or bathophenanthroline sulfonate (BPS), Fe(II) chelators that do not appear to penetrate mitochondrial membranes. However, when mitochondrial samples were sonicated, BPS coordinated the Fe(II) species, forming a low-spin Fe(II) complex. This treatment also diminished the levels of both Fe(III) species, suggesting that all of these Fe species are encapsulated by mitochondrial membranes and are protected from chelation until membranes are disrupted. 1,10-Phenanthroline is chemically similar to BPS but is membrane soluble; it coordinated nonheme HS Fe(II) in unsonicated mitochondria. Further, the HS Fe(III) species and nanoparticles were not reduced by dithionite until the detergent deoxycholate was added to disrupt membranes. There was no correlation between the percentage of nonheme HS Fe(II) species in mitochondrial samples and the level of contaminating proteins. These results collectively indicate that the observed Fe species are contained within mitochondria. Mossbauer spectra of whole cells were dominated by HS Fe(III) features; the remainder displayed spectral features typical of isolated mitochondria, suggesting that the Fe in fermenting yeast cells can be coarsely divided into two categories: mitochondrial Fe and (mostly) HS Fe(III) ions in one or more non-mitochondrial locations.

Association of a genetic variant in the ALOX5AP with higher risk of ischemic stroke: a case-control, meta-analysis and functional study.

BACKGROUND: Variants in the 5-lipoxygenase-activating protein (ALOX5AP) and phosphodiesterase 4D (PDE4D) genes have first been associated with ischemic stroke (IS) through whole-genome linkage screens. However, association studies obtained conflicting results. We aimed to investigate the contribution of selected single nucleotide polymorphisms (SNPs) in these genes for the first time in a large Iberian population. METHODS: A case-control design was used to analyze one SNP in ALOX5AP and five SNPs in PDE4D in a total of 1,092 IS patients and 781 healthy controls of two different subsets from Spain and Portugal. The analysis was adjusted for confounding variables and the results were integrated in a meta-analysis of all case-control studies. In addition, ALOX5AP gene expression levels were determined in controls and IS cases. RESULTS: A first meta-analysis of both subsets showed that the T allele of the SG13S114 SNP in ALOX5AP was a risk factor for IS after Bonferroni correction [OR = 1.22 (1.06-1.40); p = 0.006]. A second meta-analysis of white populations confirmed these results [OR = 1.18 (1.07-1.31); p = 0.001]. ALOX5AP gene expression analysis in a subset of controls and cases revealed that the SG13S114 genotypes modulate mRNA levels of ALOX5AP (p = 0.001) and mRNA levels were higher in IS cases (2.8 +/- 2.4%) than in controls (1.4 +/- 1.3%; p = 0.003). No association of the variants in PDE4D with IS was observed in our study. CONCLUSIONS: The ALOX5AP SG13S114 variant is an independent risk factor for IS in the Iberian population and is associated with ALOX5AP expression levels. The role of this gene in stroke merits further investigation.

Clinical studies support a role for trem-like transcript-1 during the progression of sepsis.

Sepsis is a multi-factorial disease that kills an estimated 1,400 people a day worldwide. The Triggering Receptor Expressed in Myeloid (TREM) cells Like Transcript (TLT)-1 is a platelet receptor expressed on activated platelets. Translational studies of TLT-1 suggest that TLT-1 affects hemostatic and immunological parameters that lead to the formation of disseminated intravascular coagulation (DIC). Evaluation of mice suffering from endotoxic shock shows a dramatic increase of soluble TLT-1 (sTLT-1) in their blood. Accordingly, when we evaluated the blood of septic patients we find increased levels of sTLT-1 that correlate with the presence of DIC in humans. Based on current data we hypothesize that TLT-1 plays an important role in maintaining vascular integrity during sepsis; perhaps by modulation of both the immune and hemostatic systems, and that TLT-1 makes an attractive target not only for better understanding of sepsis, but also as a point of therapeutic intervention as well.

EPR and Mössbauer spectroscopy of intact mitochondria isolated from Yah1p-depleted Saccharomyces cerevisiae.

Yah1p, an [Fe 2S 2]-containing ferredoxin located in the matrix of Saccharomyces cerevisiae mitochondria, functions in the synthesis of Fe/S clusters and heme a prosthetic groups. EPR, Mossbauer spectroscopy, and electron microscopy were used to characterize the Fe that accumulates in Yah1p-depleted isolated intact mitochondria. Gal- YAH1 cells were grown in standard rich media (YPD and YPGal) under O 2 or argon atmospheres. Mitochondria were isolated anaerobically, then prepared in the as-isolated redox state, the dithionite-treated state, and the O 2-treated state. The absence of strong EPR signals from Fe/S clusters when Yah1p was depleted confirms that Yah1p is required in Fe/S cluster assembly. Yah1p-depleted mitochondria, grown with O 2 bubbling through the media, accumulated excess Fe (up to 10 mM) that was present as 2-4 nm diameter ferric nanoparticles, similar to those observed in mitochondria from yfh1Delta cells. These particles yielded a broad isotropic EPR signal centered around g = 2, characteristic of superparamagnetic relaxation. Treatment with dithionite caused Fe (3+) ions of the nanoparticles to become reduced and largely exported from the mitochondria. Fe did not accumulate in mitochondria isolated from cells grown under Ar; a significant portion of the Fe in these organelles was in the high-spin Fe (2+) state. This suggests that the O 2 used during growth of Gal- YAH1 cells is responsible, either directly or indirectly, for Fe accumulation and for oxidizing Fe (2+) --> Fe (3+) prior to aggregation. Models are proposed in which the accumulation of ferric nanoparticles is caused either by the absence of a ligand that prevents such precipitation in wild-type mitochondria or by a more oxidizing environment within the mitochondria of Yah1p-depleted cells exposed to O 2. The efficacy of reducing accumulated Fe along with chelating it should be considered as a strategy for its removal in diseases involving such accumulations.

Novel mutation of FKBP10 in a pediatric patient with osteogenesis imperfecta type XI identified by clinical exome sequencing.

Osteogenesis imperfecta (OI) is a hereditary disease characterized by bone fragility caused by mutations in the proteins that support the formation of the extracellular matrix in the bone. The diagnosis of OI begins with clinical suspicion, from phenotypic findings at birth, low-impact fractures during childhood or family history that may lead to it. However, the variability in the semiology of the disease does not allow establishing an early diagnosis in all cases, and unfortunately, specific clinical data provided by the literature only report 28 patients with OI type XI. This information is limited and heterogeneous, and therefore, detailed information on the natural history of this disease is not yet available. This paper reports the case of a male patient who, despite undergoing multidisciplinary management, did not have a diagnosis for a long period of time, and could only be given one with the use of whole-exome sequencing. The use of the next-generation sequencing in patients with ultrarare genetic diseases, including skeletal dysplasias, should be justified when clear clinical criteria and an improvement in the quality of life of the patients and their families are intended while reducing economic and time costs. Thus, this case report corresponds to the 29th patient affected with OI type XI, and the 18th mutation in FKBP10, causative of this pathology.