De novo complex X chromosome rearrangement unmasking maternally inherited CSF2RA deletion in a girl with pulmonary alveolar proteinosis.

We report on a 3-year-old girl with a de novo complex X chromosome rearrangement associated with congenital pulmonary alveolar proteinosis (PAP) and short stature. Array comparative genome hybridization and FISH analyses contributed to characterize the complex rearrangement consisting of a 7.37 Mb terminal deletion of Xp22.33p22.2, a 17.3 Mb interstitial inverted duplication of Xp22.2p21.3, and a 10.14 Mb duplication of Xq27.3q28. PCR analysis of microsatellite markers supported a paternal origin of the X chromosome rearrangement. A pre-meiotic two-step mechanism may explain the occurrence of this complex X rearrangement: an inverted duplication deletion event on Xp, and duplication of the Xq27.3qter region through a telomere capture event stabilizing the broken chromosome Xp end. The girl has also inherited from her healthy mother an X chromosome with a colony stimulating factor 2 receptor, alpha (CSF2RA) gene deletion. Consistent with the recessive mode of inheritance, the de novo paternal Xp22.33p22.2 deletion combined to the maternally inherited CSF2RA gene deletion led to homozygous deletion of CSF2RA and PAP diagnosis in the girl. The Xp deletion encompasses the pseudoautosomal region 1 (PAR1) which contains genes that escape X inactivation. Short stature homeobox (SHOX) haploinsufficiency explains growth retardation. Absence of other symptoms in relation to the X deletion/amplification is most probably due to skewed X inactivation. Finally, inherited deletions may unmask rare pathogenic genomic rearrangement and contribute to clinical phenotypes by a recessive mode of gene action.

In vitro comparison of whey protein isolate and hydrolysate for their effect on glucose homeostasis markers.

Research on new strategies to regulate glucose homeostasis to prevent or manage type 2 diabetes is a critical challenge. Several studies have shown that protein-rich diets could improve glucose homeostasis. Whey protein hydrolysis allows the release of amino acids and bioactive peptides, which exert numerous well-documented bioactivities. This study evaluates and compares the hypoglycemic potential of a whey protein hydrolysate and a whey protein isolate after static in vitro simulated gastrointestinal digestion (SGID) using the INFOGEST protocol. The peptide molecular mass distributions of the digested samples were evaluated by size exclusion chromatography and show that after digestion, the whey hydrolysate is significantly more hydrolyzed. After SGID, the whey protein hydrolysate induces a significative greater secretion of GLP-1 after two hours of contact with the enteroendocrine STC-1 cell line than the whey protein after isolation. In addition, the digested whey hydrolysate increases preproglucagon (GCG) and pro-convertase-1 (PCSK1) expression. The digested hydrolysate also inhibits the DPP-IV activity after an intestinal barrier passage challenge using a Caco-2/HT29-MTX mixed-cell model. Our results highlight that the prehydrolysis of whey proteins modify the intestinal peptidome, leading to a potentially greater hypoglycemic effect. This study confirms the previously observed in vitro hypoglycemic effect of this hydrolysate and evidences the beneficial impact of the industrial hydrolysis process.

Identification, production and bioactivity of casein phosphopeptides - A review.

Milk and dairy products are significant sources of proteins and peptides impacting human health. In this way, the interest in CPPs, bioactive phosphorylated peptides resulting from the hydrolysis of caseins, has grown in the past years. CPPs were mainly studied for their capacity to chelate and increase the bioavailability of essential minerals involved in multiple physiological processes. Moreover, CPPs harbour interesting antioxidant and anti-inflammatory properties. Recent in vivo and in vitro studies demonstrated that these different roles are strongly linked to the intrinsic properties of CPPs and CPP concentrate preparations. This review first comments on the different methods of CPP analytical characterization, focusing on recent techniques. Then, the CPP release occurring during the gastrointestinal digestion was reviewed, followed by the different CPP obtention processes and their impact on their physicochemical characteristics. Finally, the different bioactive roles attributed to CPPs, including mineral chelating properties, are discussed. We show that CPPs have a promising role in treating various pathologies, notably to compensate for deficiencies in certain nutrients and an anti-oxidant and anti-inflammatory role. Nevertheless, the mechanisms by which CPPs exert their role remain to be elucidated, and this requires precise characterization of CPPs. This work highlights the key parameters to be considered to study and produce CPPs and the different ways to be investigated in the future to elucidate their roles in vivo and characterize their potential for human health.

Partial-, Double-Enzymatic Dephosphorylation and EndoGluC Hydrolysis as an Original Approach to Enhancing Identification of Casein Phosphopeptides (CPPs) by Mass Spectrometry.

The identification of phosphopeptides is currently a challenge when they are part of a complex matrix of peptides, such as a milk protein enzymatic hydrolysate. This challenge increases with both the number of phosphorylation sites on the phosphopeptides and their amino acid length. Here, this paper reports a four-phase strategy from an enzymatic casein hydrolysate before a mass spectrometry analysis in order to enhance the identification of phosphopeptides and phosphosites: (i) the control protein hydrolysate, (ii) a two-step enzymatic dephosphorylation of the latter, allowing for the almost total dephosphorylation of peptides, (iii) a one-step enzymatic dephosphorylation, allowing for the partial dephosphorylation of the peptides and (iv) an additional endoGluC enzymatic hydrolysis, allowing for the cleavage of long-size peptides into shorter ones. The reverse-phase high-pressure liquid chromatography-tandem mass spectrometry (RP-HPLC-MS/MS) analyses of hydrolysates that underwent this four-phase strategy allowed for the identification of 28 phosphorylation sites (90%) out of the 31 referenced in UniprotKB/Swiss-Prot (1 June 2021), compared to 17 sites (54%) without the latter. The alpha-S2 casein phosphosites, referenced by their similarity in the UniProt database, were experimentally identified, whereas pSer148, pThr166 and pSer187 from a multiphosphorylated long-size kappa-casein were not. Data are available via ProteomeXchange with identifier PXD027132.

Anti-CD3 monoclonal antibody in new-onset type 1 diabetes mellitus.

BACKGROUND: Type 1 diabetes mellitus is a chronic autoimmune disease caused by the pathogenic action of T lymphocytes on insulin-producing beta cells. Previous clinical studies have shown that continuous immune suppression temporarily slows the loss of insulin production. Preclinical studies suggested that a monoclonal antibody against CD3 could reverse hyperglycemia at presentation and induce tolerance to recurrent disease. METHODS: We studied the effects of a nonactivating humanized monoclonal antibody against CD3--hOKT3gamma1(Ala-Ala)--on the loss of insulin production in patients with type 1 diabetes mellitus. Within 6 weeks after diagnosis, 24 patients were randomly assigned to receive either a single 14-day course of treatment with the monoclonal antibody or no antibody and were studied during the first year of disease. RESULTS: Treatment with the monoclonal antibody maintained or improved insulin production after one year in 9 of the 12 patients in the treatment group, whereas only 2 of the 12 controls had a sustained response (P=0.01). The treatment effect on insulin responses lasted for at least 12 months after diagnosis. Glycosylated hemoglobin levels and insulin doses were also reduced in the monoclonal-antibody group. No severe side effects occurred, and the most common side effects were fever, rash, and anemia. Clinical responses were associated with a change in the ratio of CD4+ T cells to CD8+ T cells 30 and 90 days after treatment. CONCLUSIONS: Treatment with hOKT3gamma1(Ala-Ala) mitigates the deterioration in insulin production and improves metabolic control during the first year of type 1 diabetes mellitus in the majority of patients. The mechanism of action of the anti-CD3 monoclonal antibody may involve direct effects on pathogenic T cells, the induction of populations of regulatory cells, or both.

Family physicians' attitudes and practices regarding assessments of medical fitness to drive in older persons.

BACKGROUND: Higher crash rates per mile driven in older drivers have focused attention on the assessment of older drivers. OBJECTIVE: To examine the attitudes and practices of family physicians regarding fitness-to-drive issues in older persons. DESIGN: Survey questionnaire. PARTICIPANTS: The questionnaire was sent to 1,000 randomly selected Canadian family physicians. Four hundred sixty eligible physicians returned completed questionnaires. MEASUREMENTS: Self-reported attitudes and practices towards driving assessments and the reporting of medically unsafe drivers. RESULTS: Over 45% of physicians are not confident in assessing driving fitness and do not consider themselves to be the most qualified professionals to do so. The majority (88.6%) feel that they would benefit from further education in this area. About 75% feel that reporting a patient as an unsafe driver places them in a conflict of interest and negatively impacts on the patient and the physician-patient relationship. Nevertheless, most (72.4%) agree that physicians should be legally responsible for reporting unsafe drivers to the licensing authorities. Physicians from provinces with mandatory versus discretionary reporting requirements are more likely to report unsafe drivers (odds ratio [OR], 2.78; 95% confidence interval [CI], 1.58 to 4.91), but less likely to perform driving assessments (OR, 0.58; 95% CI, 0.39 to 0.85). Most driving assessments take between 10 and 30 minutes, with much variability in the components included. CONCLUSIONS: Family physicians lack confidence in performing driving assessments and note many negative consequences of reporting unsafe drivers. Education about assessing driving fitness and approaches that protect the physician-patient relationship when reporting occurs are needed.

Metrics for Success: Strategies for Enabling Core Facility Performance and Assessing Outcomes.

Core Facilities are key elements in the research portfolio of academic and private research institutions. Administrators overseeing core facilities (core administrators) require assessment tools for evaluating the need and effectiveness of these facilities at their institutions. This article discusses ways to promote best practices in core facilities as well as ways to evaluate their performance across 8 of the following categories: general management, research and technical staff, financial management, customer base and satisfaction, resource management, communications, institutional impact, and strategic planning. For each category, we provide lessons learned that we believe contribute to the effective and efficient overall management of core facilities. If done well, we believe that encouraging best practices and evaluating performance in core facilities will demonstrate and reinforce the importance of core facilities in the research and educational mission of institutions. It will also increase job satisfaction of those working in core facilities and improve the likelihood of sustainability of both facilities and personnel.

Genotype-Phenotype Relationship in Patients and Relatives with SHOX Region Anomalies in the French Population.

BACKGROUND: The aim of our study was to describe a large population with anomalies involving the SHOX region, responsible for idiopathic short stature and Léri-Weill dyschondrosteosis (LWD), and to identify a possible genotype/phenotype correlation. METHODS: We performed a retrospective multicenter study on French subjects with a SHOX region anomaly diagnosed by multiplex ligation-dependent probe amplification or Sanger sequencing. Phenotypes were collected in each of the 7 genetic laboratories practicing this technique for SHOX analysis. RESULTS: Among 205 index cases and 100 related cases, 91.3% had LWD. For index cases, median age at evaluation was 11.7 (9.0; 15.9) years and mean height standard deviation score was -2.3 ± 1.1. A deletion of either SHOX or PAR1 or both was found in 74% of patients. Duplications and point mutations/indels affected 8 and 18% of the population, respectively. Genotype-phenotype correlation showed that deletions were more frequently associated with Madelung deformity and mesomelic shortening in girls, as well as with presence of radiologic anomalies, than duplications. CONCLUSIONS: Our results highlight genotype-phenotype relationships in the French population with a SHOX defect and provide new information showing that clinical expression is milder in cases of duplication compared to deletions.

Differentiating Transient Idiopathic Hyperglycaemia and Neonatal Diabetes Mellitus in Preterm Infants.

BACKGROUND/AIMS: Transient idiopathic hyperglycaemia (TIH) is partly due to defective processing of proinsulin to insulin in preterm neonates, whereas transient neonatal diabetes mellitus (TNDM) is a rare genetic form of pancreatic ß-cell dysfunction. Distinguishing these two conditions is difficult yet essential to allow personalised management and genetic testing. Here we investigated whether metabolic or therapeutic features contributed to the diagnosis in preterm neonates. METHODS: We prospectively included 13 preterm neonates with TIH between 2008 and 2011, and we identified 2 patients with TNDM in the French neonatal diabetes cohort registry. All of them were born before 32 weeks of gestation. We compared clinical features, glycaemic profiles, insulin dosages, and nutritional intakes. RESULTS: TNDM patients had higher day-1 glycaemia levels before insulin therapy [median 23.5 (20-27) vs. 13.6 (10.7-19.8) mmol/l, p = 0.025] and higher insulin requirements [median 1.2 (0.9-1.5) vs. 0.8 (0.3-0.9) IU/kg/day, p = 0.037] compared to TIH. They also required insulin therapy earlier [median 0.75 (0.5-1) vs. 2 (0.5-7) days, p = 0.036] and for a longer time [median 85 (57-113) vs. 11 (4-15) days, p = 0.036]. CONCLUSION: TNDM and TIH are different clinical and genetic entities with specific pathophysiological mechanisms. Metabolic and therapeutic features may help to detect TNDM in preterm neonates as soon as day-1 of hyperglycaemia.

Best practices for core facilities: handling external customers.

This article addresses the growing interest among U.S. scientific organizations and federal funding agencies in strengthening research partnerships between American universities and the private sector. It outlines how core facilities at universities can contribute to this partnership by offering services and access to high-end instrumentation to both nonprofit organizations and commercial organizations. We describe institutional policies (best practices) and procedures (terms and conditions) that are essential for facilitating and enabling such partnerships. In addition, we provide an overview of the relevant federal regulations that apply to external use of academic core facilities and offer a set of guidelines for handling them. We conclude by encouraging directors and managers of core facilities to work with the relevant organizational offices to promote and nurture such partnerships. If handled appropriately, we believe such partnerships can be a win-win situation for both organizations that will support research and bolster the American economy.

CTLA-4 regulates the requirement for cytokine-induced signals in T(H)2 lineage commitment.

The relative importance of the cytokine milieu versus cytolytic T lymphocyte-associated antigen 4 (CTLA-4) and T cell receptor signal strength on T cell differentiation remains unclear. Here we have generated mice deficient for signal transducer and activator of transcription 6 (STAT6) and CTLA-4 to determine the role of CTLA-4 in cytokine-driven T cell differentiation. CTLA-4-deficient T cells bypass the need for STAT6 in the differentiation of T helper type 2 (T(H)2) cells. T(H)2 differentiation of cells deficient for both STAT6 and CTLA-4 is accompanied by induction of GATA-3 and the migration of T(H)2 cells to peripheral tissues. CTLA-4 deficiency also affects the balance of the nuclear factors NFATc1 and NFATc2, and enhances activation of NF-kappaB. These results suggest that CTLA-4 has a critical role in T cell differentiation and that STAT6-dependent T(H)2 lineage commitment and stabilization can be bypassed by increasing the strength of signaling through the T cell receptor.

Modified anti-CD3 therapy in psoriatic arthritis: a phase I/II clinical trial.

OBJECTIVE: Treatment of autoimmune diseases with therapies that tolerize pathogenic lymphocytes may obviate the need for longterm global immunosuppression. In vitro, non-Fc receptor binding derivatives of anti-murine CD3 monoclonal antibodies tolerize type 1 T cells and stimulate type 2 T cells. Recently, a humanized non-FcR binding derivative of the anti-human CD3 Mab OKT3, huOKT3gamma1(ala-ala), has been described. We hypothesized that this Mab may be safe and efficacious in the treatment of type 1 T lymphocyte mediated chronic autoimmune diseases such as psoriatic arthritis (PsA). METHODS: In a Phase I/II trial, 7 patients with PsA were treated with escalating daily doses of huOKT3gamma1(ala-ala) for 12 to 14 days. Number of tender and swollen joints and a visual analog pain scale were used to rate disease activity at entry and Day 30 and Day 90 after treatment. RESULTS: At Day 30, 6 of 7 patients had > or = 75% improvement in the number of inflamed joints and an average 63% improvement on the patient pain scale. Two of 6 responders had sustained improvement at Day 90. No patient treated with an initial dose < or = 1 mg had significant side effects, nor did they have detectable increases in serum cytokines. One patient treated with 4 mg without escalation developed mild cytokine release symptoms associated with elevation of interleukin 10. Transient T cell depletion occurred following treatment with the maximum dose of 4 mg, which resolved by Day 30. Antiidiotypic antibodies developed in 2 patients; however, there was no concurrent decrease in efficacy. CONCLUSION: These data indicate that huOKT3gamma1(ala-ala) may be useful in treating PsA.