De novo mutations in childhood cases of sudden unexplained death that disrupt intracellular Ca2+ regulation.

Sudden unexplained death in childhood (SUDC) is an understudied problem. Whole-exome sequence data from 124 "trios" (decedent child, living parents) was used to test for excessive de novo mutations (DNMs) in genes involved in cardiac arrhythmias, epilepsy, and other disorders. Among decedents, nonsynonymous DNMs were enriched in genes associated with cardiac and seizure disorders relative to controls (odds ratio = 9.76, P = 2.15 × 10-4). We also found evidence for overtransmission of loss-of-function (LoF) or previously reported pathogenic variants in these same genes from heterozygous carrier parents (11 of 14 transmitted, P = 0.03). We identified a total of 11 SUDC proband genotypes (7 de novo, 1 transmitted parental mosaic, 2 transmitted parental heterozygous, and 1 compound heterozygous) as pathogenic and likely contributory to death, a genetic finding in 8.9% of our cohort. Two genes had recurrent missense DNMs, RYR2 and CACNA1C Both RYR2 mutations are pathogenic (P = 1.7 × 10-7) and were previously studied in mouse models. Both CACNA1C mutations lie within a 104-nt exon (P = 1.0 × 10-7) and result in slowed L-type calcium channel inactivation and lower current density. In total, six pathogenic DNMs can alter calcium-related regulation of cardiomyocyte and neuronal excitability at a submembrane junction, suggesting a pathway conferring susceptibility to sudden death. There was a trend for excess LoF mutations in LoF intolerant genes, where ≥1 nonhealthy sample in denovo-db has a similar variant (odds ratio = 6.73, P = 0.02); additional uncharacterized genetic causes of sudden death in children might be discovered with larger cohorts.

Single-cell RNA sequencing identifies precise tolerogenic cellular and molecular pathways induced by depigmented-polymerized grass pollen allergen extract.

BACKGROUND: Immunologic mechanism of action of allergoids remains poorly understood. Previous models of allergenicity and immunogenicity have yielded suboptimal knowledge of these immunotherapeutic vaccine products. Novel single-cell RNA sequencing technology offers a bridge to this gap in knowledge. OBJECTIVE: We sought to identify the underpinning tolerogenic molecular and cellular mechanisms of depigmented-polymerized Phleum pratense (Phl p) extract. METHODS: The molecular mechanisms underlying native Phl p, depigmented Phl p (DPG-Phl p), and depigmented-polymerized (DPG-POL-Phl p) allergoid were investigated by single-cell RNA sequencing. Allergen-specific TH2A, T follicular helper (Tfh), and IL-10+ regulatory B cells were quantified by flow cytometry in peripheral blood mononuclear cells from 16 grass pollen-allergic and 8 nonatopic control subjects. The ability of Phl p, DPG-Phl p, and DPG-POL-Phl p to elicit FcεRI- and FcεRII-mediated IgE responses was measured by basophil activation test and IgE-facilitated allergen binding assay. RESULTS: Analysis revealed that DPG-POL-Phl p downregulated genes associated with TH2 signaling, induced functional regulatory T cells exhibiting immunosuppressive roles through CD52 and Siglec-10, modulated genes encoding immunoproteasome that dysregulate the processing and presentation of antigens to T cells and promoted a shift from IgE toward an IgA1 and IgG responses. In grass pollen-allergic subjects, DPG-POL-Phl p exhibited reduced capacity to elicit proliferation of TH2A, IL-4+ Tfh and IL-21+ Tfh cells while being the most prominent at inducing IL-10+CD19+CD5hi and IL-10+CD19+CD5hiCD38intCD24int regulatory B-cell subsets compared to Phl p (all P < .05). Furthermore, DPG-POL-Phl p demonstrated a hypoallergenic profile through basophil activation and histamine release compared to Phl p (31.54-fold, P < .001). CONCLUSIONS: Single-cell RNA sequencing provides an in-depth resolution of the mechanisms underlying the tolerogenic profile of DPG-POL-Phl p.

Absolute quantification of Bet v 1 in birch polymerized allergenic extracts via mass spectrometry-targeted analysis.

BACKGROUND: Quantifying major allergens is essential for evaluating the quality and efficacy of allergenic extracts. They are usually measured in non-polymerized extracts using immunoassays. However, the direct measurement of allergens in allergoids is currently not supported. This study set out to develop a method for quantifying Bet v 1 in polymerized birch extracts using mass spectrometry-based targeted analysis. METHODS: Three isotopically labelled peptide sequences of Bet v 1 were synthetized and used as internal standards for the development of a mass spectrometry-based targeted analysis. The calibration curves of the three peptides to assess the linearity and limit of detection, as well as reverse calibration curves with a constant amount of sample, were constructed. The Bet v 1 content was determined and measured in 18 batches of depigmented (native extracts purified by a mild acid treatment) and depigmented-polymerized extracts. RESULTS: Bet v 1 isoforms were identified in both type of extracts by mass spectrometry. According to mass spectrometry-targeted analysis depigmented and depigmented-polymerized extracts exhibited mean values of 70.5 and 73.5 µg Bet v 1/mg of lyophilized extract, respectively. A statistically significant correlation between the allergen content of both extracts was identified. Statistically significant differences were observed when the Bet v 1 content in non-polymerized extracts was measured via mass spectrometry (70.5 ± 11.6 µg/mg) or immunoassay (83.7 ± 19.8 µg/mg). CONCLUSIONS: These results represent the first direct quantification of Bet v 1 in allergoids using mass spectrometry-based targeted analysis. The proposed method demonstrates robustness and reliability and constitutes a promising alternative for detailed characterization of polymerized allergenic extracts.

Computational model of cardiomyocyte apoptosis identifies mechanisms of tyrosine kinase inhibitor-induced cardiotoxicity.

Despite clinical observations of cardiotoxicity among cancer patients treated with tyrosine kinase inhibitors (TKIs), the molecular mechanisms by which these drugs affect the heart remain largely unknown. Mechanistic understanding of TKI-induced cardiotoxicity has been limited in part due to the complexity of tyrosine kinase signaling pathways and the multi-targeted nature of many of these drugs. TKI treatment has been associated with reactive oxygen species generation, mitochondrial dysfunction, and apoptosis in cardiomyocytes. To gain insight into the mechanisms mediating TKI-induced cardiotoxicity, this study constructs and validates a computational model of cardiomyocyte apoptosis, integrating intrinsic apoptotic and tyrosine kinase signaling pathways. The model predicts high levels of apoptosis in response to sorafenib, sunitinib, ponatinib, trastuzumab, and gefitinib, and lower levels of apoptosis in response to nilotinib and erlotinib, with the highest level of apoptosis induced by sorafenib. Knockdown simulations identified AP1, ASK1, JNK, MEK47, p53, and ROS as positive functional regulators of sorafenib-induced apoptosis of cardiomyocytes. Overexpression simulations identified Akt, IGF1, PDK1, and PI3K among the negative functional regulators of sorafenib-induced cardiomyocyte apoptosis. A combinatorial screen of the positive and negative regulators of sorafenib-induced apoptosis revealed ROS knockdown coupled with overexpression of FLT3, FGFR, PDGFR, VEGFR, or KIT as a particularly potent combination in reducing sorafenib-induced apoptosis. Network simulations of combinatorial treatment with sorafenib and the antioxidant N-acetyl cysteine (NAC) suggest that NAC may protect cardiomyocytes from sorafenib-induced apoptosis.

Characterisation of corneas following different time and storage methods for their use as a source of stem-like limbal epithelial cells.

The transplantation of expansions of limbal epithelial stem cells (LESC) remains one of the most efficient therapies for the treatment of limbal stem cell deficiency (LSCD) to date. However, the available donor corneas are scarce, and the corneas conserved for long time, under hypothermic conditions (after 7 days) or in culture (more than 28 days), are usually discarded due to poor viability of the endothelial cells. To establish an objective criterion for the utilisation or discarding of corneas as a source of LESC, we characterized, by immunohistochemistry analysis, donor corneas conserved in different conditions and for different periods of time. We also studied the potency of LESCs isolated from these corneas and maintained in culture up to 3 cell passages. We hoped that the study of markers of LESCs present in both the corneoscleral histological sections and the cell cultures would show the adequacy of the methods used for cell isolation and how fit the LESC enrichment of the obtained cell populations to be expanded was. Thus, the expressions of markers of the cells residing in the human limbal and corneal epithelium (cytokeratin CK15 and CK12, vimentin, Collagen VII, p63α, ABCG2, Ki67, Integrin ß4, ZO1, and melan A) were analysed in sections of corneoscleral tissues conserved in hypothermic conditions for 2-9 days with post-mortem time (pmt) < 8 h or for 1 day with pmt > 16 h, and in sclerocorneal rims maintained in an organ culture medium for 29 days. Cell populations isolated from donor corneoscleral tissues were also assessed based on these markers to verify the adequacy of isolation methods and the potential of expanding LESCs from these tissues. Positivity for several putative stem cell markers such as CK15 and p63α was detected in all corneoscleral tissues, although a decrease was recorded in the ones conserved for longer times. The barrier function and the ability to adhere to the extracellular matrix were maintained in all the analysed tissues. In limbal epithelial cell cultures, a simultaneous decrease in the melan A melanocyte marker and the putative stem cell markers was detected, suggesting a close relationship between the melanocytes and the limbal stem cells of the niche. Holoclones stained with putative stem cell markers were obtained from long-term, hypothermic, stored sclerocorneal rims. The results showed that the remaining sclerocorneal rims after corneal transplantation, which were conserved under hypothermic conditions for up to 7 days and would have been discarded at a first glance, still maintained their potential as a source of LESC cultures.

Ole e 3, a Candidate for in vivo Diagnosis of Polcalcin Sensitization.

INTRODUCTION: Polcalcins belong to the family of calcium-binding proteins. They are ubiquitous in the plant kingdom and highly conserved, which leads to these panallergens showing a high degree of inter-cross-reactivity. They are responsible for allergic polysensitization, and therefore, their diagnosis is necessary for correct selection of immunotherapy. The objectives were to develop a method to purify native polcalcin with intact allergenic properties and to validate its use for diagnosis of polcalcin sensitization. METHODS: Ole e 3 was purified by immunoaffinity chromatography using anti-rChe a 3 polyclonal antibodies and identified by mass spectrometry. Calcium-binding assays were performed in immunoblot and ELISA assays. Diagnostic capacity of Ole e 3 was analyzed by ELISA and compared to ImmunoCAP with sera from a pollen-sensitized population. Cross-reactivity with other polcalcins was investigated by ImmunoCAP inhibition. RESULTS: Immunogenicity of purified Ole e 3 was not affected by the addition of calcium. However, the presence of a calcium chelator agent completely inhibited IgG binding by immunoblot and produced a 32.3% reduction in IgE binding by ELISA. Ole e 3 enabled diagnosis of polcalcin-sensitized patients, and a good correlation was revealed with ImmunoCAP. A 50% inhibition in IgE binding was obtained with 2.8 ng of Ole e 3 for rBet v 4 and 3.9 ng for rPhl p 7. DISCUSSION/CONCLUSION: Native Ole e 3 was purified by maintaining its allergenic properties. This innovative method enables obtaining this active native allergen to be used for in vivo diagnosis of polcalcin sensitization.

Specific Dermatophagoides farinae extract for canine immunotherapy.

BACKGROUND: Canine atopic dermatitis (cAD) is a pruritic allergic skin disease most often caused by Dermatophagoides farinae. Differences in the sensitization profile to D. farinae have been reported between people and dogs. However, allergic dogs traditionally have been treated with extracts intended for human immunotherapy. HYPOTHESIS/OBJECTIVES: To develop a specific allergen immunotherapy for veterinary practice enriched in canine major allergens and to demonstrate its in vitro efficacy. ANIMALS: Twenty privately owned dogs, clinically diagnosed with cAD, and three healthy dogs. METHODS AND MATERIALS: A veterinary D. farinae allergen extract was manufactured and characterized compared to D. farinae extract used for human immunotherapy. The protein profile was analysed by SDS-PAGE and size exclusion chromatography and Der f 15 and Der f 18 allergens quantified by mass spectrometry. The allergenic profile was studied by immunoblot and the biological potency by enzyme-linked immunosorbent assay-inhibition assays. The extract's capacity to induce cytokine production [interleukin (IL)-10, interferon (IFN)-Ɣ] by peripheral blood mononuclear cells also was evaluated. RESULTS: The veterinary extract showed a higher content of high molecular weight proteins, preferentially recognized by atopic dog sera. The fold-increases in Der f 15 and Der f 18 with respect to the human extract were 2.07 ± 0.32 and 1.63 ± 0.15, respectively. The veterinary extract showed higher biological potency (0.062 versus 0.132 µg required for 50% inhibition of dogs sera) compared to the human extract and induced significantly higher levels of IL-10 (1,780 pg/mL) and IFN-Ɣ (50.4 pg/mL) with respect to the negative control. CONCLUSIONS AND CLINICAL IMPORTANCE: A veterinary D. farinae extract with a higher content of dog major allergens was developed and in vitro efficacy demonstrated by immunological parameters.

Laser-facilitated epicutaneous immunotherapy with depigmented house dust mite extract alleviates allergic responses in a mouse model of allergic lung inflammation.

BACKGROUND: Skin-based immunotherapy of type 1 allergies has recently been re-investigated as an alternative for subcutaneous injections. In the current study, we employed a mouse model of house dust mite (HDM)-induced lung inflammation to explore the potential of laser-facilitated epicutaneous allergen-specific treatment. METHODS: Mice were sensitized against native Dermatophagoides pteronyssinus extract and repeatedly treated by application of depigmented D pteronyssinus extract via laser-generated skin micropores or by subcutaneous injection with or without alum. Following aerosol challenges, lung function was determined by whole-body plethysmography and bronchoalveolar lavage fluid was analyzed for cellular composition and cytokine levels. HDM-specific IgG subclass antibodies were determined by ELISA. Serum as well as cell-bound IgE was measured by ELISA, rat basophil leukemia cell assay, and ex vivo using a basophil activation test, respectively. Cultured lymphocytes were analyzed for cytokine secretion profiles and cellular polarization by flow cytometry. RESULTS: Immunization of mice by subcutaneous injection or epicutaneous laser microporation induced comparable IgG antibody levels, but the latter preferentially induced regulatory T cells and in general downregulated T cell cytokine production. This effect was found to be a result of the laser treatment itself, independent from extract application. Epicutaneous treatment of sensitized animals led to induction of blocking IgG, and improvement of lung function, superior compared to the effects of subcutaneous therapy. During the whole therapy schedule, no local or systemic side effects occurred. CONCLUSION: Allergen-specific immunotherapy with depigmented HDM extract via laser-generated skin micropores offers a safe and effective treatment option for HDM-induced allergy and lung inflammation.

Expansion of Human Limbal Epithelial Stem/Progenitor Cells Using Different Human Sera: A Multivariate Statistical Analysis.

Transplantation of human cultured limbal epithelial stem/progenitor cells (LESCs) has demonstrated to restore the integrity and functionality of the corneal surface in about 76% of patients with limbal stem cell deficiency. However, there are different protocols for the expansion of LESCs, and many of them use xenogeneic products, being a risk for the patients' health. We compared the culture of limbal explants on the denuded amniotic membrane in the culture medium-supplemental hormone epithelial medium (SHEM)-supplemented with FBS or two differently produced human sera. Cell morphology, cell size, cell growth rate, and the expression level of differentiation and putative stem cell markers were examined. Several bioactive molecules were quantified in the human sera. In a novel approach, we performed a multivariate statistical analysis of data to investigate the culture factors, such as differently expressed molecules of human sera that specifically influence the cell phenotype. Our results showed that limbal cells cultured with human sera grew faster and contained similar amounts of small-sized cells, higher expression of the protein p63α, and lower of cytokeratin K12 than FBS cultures, thus, maintaining the stem/progenitor phenotype of LESCs. Furthermore, the multivariate analysis provided much data to better understand the obtaining of different cell phenotypes as a consequence of the use of different culture methodologies or different culture components.

Hyaluronic Acid Combined with Serum Rich in Growth Factors in Corneal Epithelial Defects.

The aim of this study is to assess if an adhesive biopolymer, sodium hyaluronate (NaHA), has synergistic effects with s-PRGF (a serum derived from plasma rich in growth factors and a blood derivative that has already shown efficacy in corneal epithelial wound healing), to reduce time of healing or posology. In vitro proliferation and migration studies, both in human corneal epithelial (HCE) cells and in rabbit primary corneal epithelial (RPCE) cultures, were carried out. In addition, we performed studies of corneal wound healing in vivo in rabbits treated with s-PRGF, NaHA, or the combination of both. We performed immunohistochemistry techniques (CK3, CK15, Ki67, ß4 integrin, ZO-1, α-SMA) in rabbit corneas 7 and 30 days after a surgically induced epithelial defect. In vitro results show that the combination of NaHA and s-PRGF offers the worst proliferation rates in both HCE and RPCE cells. Addition of NaHA to s-PRGF diminishes the re-epithelializing capability of s-PRGF. In vivo, all treatments, given twice a day, showed equivalent efficacy in corneal epithelial healing. We conclude that the combined use of s-PRGF and HaNA as an adhesive biopolymer does not improve the efficacy of s-PRGF alone in the wound healing of corneal epithelial defects.

Hospital-acquired hyponatremia: Does the type of fluid therapy affect children admitted to intensive care? / Hiponatremia adquirida en el hospital: ¿influye el tipo de fluidoterapia en los niños ingresados a cuidados intensivos?

INTRODUCTION: The objective of this study was to evaluate the association between the type of initial fluid therapy used (isotonic or hypotonic solutions) and the development of hyponatremia, the plas ma chlorine values and the tolerance of venous access. PATIENTS AND METHOD: Retrospective cohort study in a Pediatric Intensive Care Unit (PICU) of a high complexity hospital. There were included children younger than 15 years old hospitalized during the first semester of 2010 and 2013 who recei ved intravenous maintenance fluid therapy, excluding patients undergoing cardiac surgery, kidney transplant and admissions that lasted less than 24 hours. Epidemiological, comorbidity and admis sion-related data were collected, including type of solution received, sodium and chlorine values in the first 72 hours of hospitalization and the incidence of extravasation of peripheral intravenous lines. RESULTS: 111 children were included; 68 children (61.3%) were treated with hypotonic solutions and 43 (38.7%) with isotonic solutions. There were no differences in pathology and severity, and also in the volume of fluid received. Among the patients who received hypotonic solutions, 28 (41.2%) de veloped hyponatremia, wich was moderate (Na <130 mEq/Kg) in 11 cases, compared with 8 children (18.6%) who received isotonic solutions, with only one case of moderate hyponatremia (p = 0.027). No cases of hypernatremia were recorded, and there were no differences in plasma chlorine values. There was also no increased frequency of venous access loss in patients treated with isotonic solutions (4.7% versus 7.4%, p = 0.704). CONCLUSION: Treatment with initial isotonic solutions in children hos pitalized in PICU is associated with a lower incidence and severity of hyponatremia, without changes in the plasma chlorine values and it is well tolerated by peripheral intravenous lines.

In vitro evidence of efficacy and safety of a polymerized cat dander extract for allergen immunotherapy.

BACKGROUND: Allergy to cat epithelia is highly prevalent, being the major recommendation for allergy sufferers its avoidance. However, this is not always feasible. Allergen specific immunotherapy is therefore recommended for these patients. The use of polymerized allergen extracts, allergoids, would allow to achieve the high allergen doses suggested to be effective while maintaining safety. RESULTS: Cat native extract and its depigmented allergoid were manufactured and biochemically and immunochemically characterized. Protein and chromatographic profiles showed significant modification of the depigmented allergoid with respect to its corresponding native extract. However, the presence of different allergens (Fel d 1, Fel d 2, Fel d 3, Fel d 4 and Fel d 7) was confirmed in the allergoid. Differences in IgE-binding capacity were observed as loss of biological potency and lower stability of the IgE-allergen complex on surface plasmon resonance. The allergoid induced production of IgG antibodies able to block IgE-binding to native extract. Finally, studies carried out with peripheral-blood mononuclear cells from cat allergic patients showed that the allergoid induced IFN-γ and IL-10 production similar to that induced by native extract. CONCLUSIONS: Cat depigmented allergoid induced production of cytokines involved in a Th1 and Treg response, was able to induce production of IgG-antibodies that blocks IgE-binding to cat native extract, and showed reduced interaction with IgE, suggesting greater safety than native extract while maintaining in vitro efficacy.

In vivo diagnosis with purified tropomyosin in mite and shellfish allergic patients.

BACKGROUND: Tropomyosin is the most studied shellfish allergen and has been involved in cross-reactivity among different invertebrates (crustacean, mollusks, mites, insects, and nematodes). OBJECTIVE: To determine the relevance of tropomyosin in mite- and shellfish-sensitized patients using tropomyosin skin testing. METHODS: Patients were divided into 3 groups: group M included mite allergic patients (ie, individuals with respiratory symptoms and a positive result on skin prick testing [SPT] to house dust mites), group S included shellfish allergic patients (ie, individuals who reported symptoms with shellfish), and group MS included mite- and shellfish allergic patients (ie, individuals who simultaneously fulfilled the inclusion criteria for groups M and S). Tropomyosin was purified from shrimp, characterized, and used in SPT for diagnosis in the patient population. RESULTS: Eight hundred fifty patients were included in the study: 790 (92.9%) in group M, 21 (2.5%) in group S, and 39 (4.6%) in group MS. Tropomyosin was purified from shrimp with a purity higher than 95%. Forty-two individuals tested positive to tropomyosin: the prevalence was 2.7% in group M, 28.6% in group S, and 38.5% in patients of group MS. Twenty-one (50%) of the tropomyosin-positive individuals had symptoms with shellfish, and 3 (14.3%) reported anaphylaxis. CONCLUSION: The prevalence of tropomyosin was low in mite-sensitized patients (2.7 %) and high in shellfish allergic patients (28.6%). The higher prevalence of tropomyosin was found in patients sensitized to both mite and shellfish (38.5%). The selection of tropomyosin-sensitized patients by SPT might help in the choice of appropriate treatments or management for these patients.

Structural characterization of a polymorphic repeat at the CACNA1C schizophrenia locus.

Genetic variation within intron 3 of the CACNA1C calcium channel gene is associated with schizophrenia and bipolar disorder, but analysis of the causal variants and their effect is complicated by a nearby variable-number tandem repeat (VNTR). Here, we used 155 long-read genome assemblies from 78 diverse individuals to delineate the structure and population variability of the CACNA1C intron 3 VNTR. We categorized VNTR sequences into 7 Types of structural alleles using sequence differences among repeat units. Only 12 repeat units at the 5' end of the VNTR were shared across most Types, but several Types were related through a series of large and small duplications. The most diverged Types were rare and present only in individuals with African ancestry, but the multiallelic structural polymorphism Variable Region 2 was present across populations at different frequencies, consistent with expansion of the VNTR preceding the emergence of early hominins. VR2 was in complete linkage disequilibrium with fine-mapped schizophrenia variants (SNPs) from genome-wide association studies (GWAS). This risk haplotype was associated with decreased CACNA1C gene expression in brain tissues profiled by the GTEx project. Our work suggests that sequence variation within a human-specific VNTR affects gene expression, and provides a detailed characterization of new alleles at a flagship neuropsychiatric locus.

Reappraisal of the outcome of healthcare-associated and community-acquired bacteramia: a prospective cohort study.

BACKGROUND: Healthcare-associated (HCA) bloodstream infections (BSI) have been associated with worse outcomes, in terms of higher frequencies of antibiotic-resistant microorganisms and inappropriate therapy than strict community-acquired (CA) BSI. Recent changes in the epidemiology of community (CO)-BSI and treatment protocols may have modified this association. The objective of this study was to analyse the etiology, therapy and outcomes for CA and HCA BSI in our area. METHODS: A prospective multicentre cohort including all CO-BSI episodes in adult patients was performed over a 3-month period in 2006-2007. Outcome variables were mortality and inappropriate empirical therapy. Adjusted analyses were performed by logistic regression. RESULTS: 341 episodes of CO-BSI were included in the study. Acquisition was HCA in 56% (192 episodes) of them. Inappropriate empirical therapy was administered in 16.7% (57 episodes). All-cause mortality was 16.4% (56 patients) at day 14 and 20% (71 patients) at day 30. After controlling for age, Charlson index, source, etiology, presentation with severe sepsis or shock and inappropriate empirical treatment, acquisition type was not associated with an increase in 14-day or 30-day mortality. Only an stratified analysis of 14th-day mortality for Gram negatives BSI showed a statically significant difference (7% in CA vs 17% in HCA, p = 0,05). Factors independently related to inadequate empirical treatment in the community were: catheter source, cancer, and previous antimicrobial use; no association with HCA acquisition was found. CONCLUSION: HCA acquisition in our cohort was not a predictor for either inappropriate empirical treatment or increased mortality. These results might reflect recent changes in therapeutic protocols and epidemiological changes in community pathogens. Further studies should focus on recognising CA BSI due to resistant organisms facilitating an early and adequate treatment in patients with CA resistant BSI.

Composite alginate-gelatin hydrogels incorporating PRGF enhance human dental pulp cell adhesion, chemotaxis and proliferation.

The increasing prevalence of tissue injuries is fueling the development of autologous biological treatments for regenerative medicine. Here, we investigated the potential of three different bioinks based on the combination of gelatin and alginate (GA), enriched in either hydroxyapatite (GAHA) or hydroxyapatite and PRGF (GAHAP), as a favorable microenvironment for human dental pulp stem cells (DPSCs). Swelling behaviour, in vitro degradation and mechanical properties of the matrices were evaluated. Morphological and elemental analysis of the scaffolds were also performed along with cytocompatibility studies. The in vitro cell response to the different scaffolds was also assessed. Results showed that all scaffolds presented high swelling capacity, and those that contained HA showed higher Young's modulus. GAHAP had the lowest degradation rate and the highest values of cytocompatibility. Cell adhesion and chemotaxis were significantly increased when PRGF was incorporated to the matrices. GAHA and GAHAP compositions promoted the highest proliferative rate as well as significantly stimulated osteogenic differentiation. In conclusion, the enrichment with PRGF improves the regenerative properties of the composites favouring the development of personalized constructs.

Proteogenomic analysis of cancer aneuploidy and normal tissues reveals divergent modes of gene regulation across cellular pathways.

How cells control gene expression is a fundamental question. The relative contribution of protein-level and RNA-level regulation to this process remains unclear. Here, we perform a proteogenomic analysis of tumors and untransformed cells containing somatic copy number alterations (SCNAs). By revealing how cells regulate RNA and protein abundances of genes with SCNAs, we provide insights into the rules of gene regulation. Protein complex genes have a strong protein-level regulation while non-complex genes have a strong RNA-level regulation. Notable exceptions are plasma membrane protein complex genes, which show a weak protein-level regulation and a stronger RNA-level regulation. Strikingly, we find a strong negative association between the degree of RNA-level and protein-level regulation across genes and cellular pathways. Moreover, genes participating in the same pathway show a similar degree of RNA- and protein-level regulation. Pathways including translation, splicing, RNA processing, and mitochondrial function show a stronger protein-level regulation while cell adhesion and migration pathways show a stronger RNA-level regulation. These results suggest that the evolution of gene regulation is shaped by functional constraints and that many cellular pathways tend to evolve one predominant mechanism of gene regulation at the protein level or at the RNA level.

Ex Vivo Maturation of 3D-Printed, Chondrocyte-Laden, Polycaprolactone-Based Scaffolds Prior to Transplantation Improves Engineered Cartilage Substitute Properties and Integration.

OBJECTIVE: The surgical management of nasal septal defects due to perforations, malformations, congenital cartilage absence, traumatic defects, or tumors would benefit from availability of optimally matured septal cartilage substitutes. Here, we aimed to improve in vitro maturation of 3-dimensional (3D)-printed, cell-laden polycaprolactone (PCL)-based scaffolds and test their in vivo performance in a rabbit auricular cartilage model. DESIGN: Rabbit auricular chondrocytes were isolated, cultured, and seeded on 3D-printed PCL scaffolds. The scaffolds were cultured for 21 days in vitro under standard culture media and normoxia or in prochondrogenic and hypoxia conditions, respectively. Cell-laden scaffolds (as well as acellular controls) were implanted into perichondrium pockets of New Zealand white rabbit ears (N = 5 per group) and followed up for 12 weeks. At study end point, the tissue-engineered scaffolds were extracted and tested by histological, immunohistochemical, mechanical, and biochemical assays. RESULTS: Scaffolds previously matured in vitro under prochondrogenic hypoxic conditions showed superior mechanical properties as well as improved patterns of cartilage matrix deposition, chondrogenic gene expression (COL1A1, COL2A1, ACAN, SOX9, COL10A1), and proteoglycan production in vivo, compared with scaffolds cultured in standard conditions. CONCLUSIONS: In vitro maturation of engineered cartilage scaffolds under prochondrogenic conditions that better mimic the in vivo environment may be beneficial to improve functional properties of the engineered grafts. The proposed maturation strategy may also be of use for other tissue-engineered constructs and may ultimately impact survival and integration of the grafts in the damaged tissue microenvironment.

Immunoinformatic epitope prediction to select monoclonal antibodies for Phl p 1 quantification.

BACKGROUND: Allergen quantification has become a relevant parameter for allergen extract characterization and to guarantee the consistency of the manufacturing process at allergen immunotherapy. The aim of this study was to develop and validate a method to quantify the major allergen Phl p 1 based on a prediction of the antigenic regions by immunoinformatic strategies. METHODS: Phl p 1 was purified from a Phleum pratense native extract by chromatographic methods. Immunoinformatic tools were used to predict B-cell epitopes. In silico predictions were verified by mapping linear epitopes with a peptide library and used to select the appropriate regions for producing the mAbs to develop an ELISA method, which was validated. Phl p 1 was quantified in 24 batches of P. pratense extracts. RESULTS: Phl p 1 was purified with 95 % purity and completely functional. Eight B-cell epitopes in each of the two Phl p 1 isoforms were predicted. Two of the predicted B-cell epitopes overlapped with the experimentally determined peptides recognized by two mAbs selected for development of the kit. The quantification method demonstrated to be specific to Phl p 1, linear, accurate and precise in the range from 7.7 to 123.3 µg/mg. Mean Phl p 1 content was 28.95 µg of allergen/mg of lyophilized native extract and 44.23 µg of allergen/mg of lyophilized depigmented extract. CONCLUSIONS: An ELISA method for measuring Phl p 1 in P. pratense extracts was developed and validated by producing the appropriate mAbs against epitopes selected by immunoinformatic tools.

Three-Dimensional Bioprinting Scaffolding for Nasal Cartilage Defects: A Systematic Review.

BACKGROUND: In recent years, three-dimensional (3D)-printing of tissue-engineered cartilaginous scaffolds is intended to close the surgical gap and provide bio-printed tissue designed to fit the specific geometric and functional requirements of each cartilage defect, avoiding donor site morbidity and offering a personalizing therapy. METHODS: To investigate the role of 3D-bioprinting scaffolding for nasal cartilage defects repair a systematic review of the electronic databases for 3D-Bioprinting articles pertaining to nasal cartilage bio-modelling was performed. The primary focus was to investigate cellular source, type of scaffold utilization, biochemical evaluation, histological analysis, in-vitro study, in-vivo study, animal model used, length of research, and placement of experimental construct and translational investigation. RESULTS: From 1011 publications, 16 studies were kept for analysis. About cellular sources described, most studies used primary chondrocyte cultures. The cartilage used for cell isolation was mostly nasal septum. The most common biomaterial used for scaffold creation was polycaprolactone alone or in combination. About mechanical evaluation, we found a high heterogeneity, making it difficult to extract any solid conclusion. Regarding biological and histological characteristics of each scaffold, we found that the expression of collagen type I, collagen Type II and other ECM components were the most common patterns evaluated through immunohistochemistry on in-vitro and in-vivo studies. Only two studies made an orthotopic placement of the scaffolds. However, in none of the studies analyzed, the scaffold was placed in a subperichondrial pocket to rigorously simulate the cartilage environment. In contrast, scaffolds were implanted in a subcutaneous plane in almost all of the studies included. CONCLUSION: The role of 3D-bioprinting scaffolding for nasal cartilage defects repair is growing field. Despite the amount of information collected in the last years and the first surgical applications described recently in humans. Further investigations are needed due to the heterogeneity on mechanical evaluation parameters, the high level of heterotopic scaffold implantation and the need for quantitative histological data.