Molecular chaperones as enzymes that catalytically unfold misfolded polypeptides.

Stress-denatured or de novo synthesized and translocated unfolded polypeptides can spontaneously reach their native state without assistance of other proteins. Yet, the pathway to native folding is complex, stress-sensitive and prone to errors. Toxic misfolded and aggregated conformers may accumulate in cells and lead to degenerative diseases. Members of the canonical conserved families of molecular chaperones, Hsp100s, Hsp70/110/40s, Hsp60/CCTs, the small Hsps and probably also Hsp90s, can recognize and bind with high affinity, abnormally exposed hydrophobic surfaces on misfolded and aggregated polypeptides. Binding to Hsp100, Hsp70, Hsp110, Hsp40, Hsp60, CCTs and Trigger factor may cause partial unfolding of the misfolded polypeptide substrates, and ATP hydrolysis can induce further unfolding and release from the chaperone, leading to spontaneous refolding into native proteins with low-affinity for the chaperones. Hence, specific chaperones act as catalytic polypeptide unfolding isomerases, rerouting cytotoxic misfolded and aggregated polypeptides back onto their physiological native refolding pathway, thus averting the onset of protein conformational diseases.

Carences en vitamine B12 et fer: du diagnostic au suivi [Vitamin B12 and iron deficiencies: from diagnostic to follow-up].

Vitamin B12 and iron deficiencies are common problems in consultations of general internal medicine. They cause different symptoms that can be non-specific. This article makes it possible, from a clinical frame of reference, to answer the following questions: What value of vitamin B12 should we consider a "deficiency", and what is the role of methylmalonate? What is the role of vitamin B12 oral supplements? How should we interpret values of ferritine? How should iron deficiency be investigated? What is the place of intravenous iron administration?

A Novel Pulse-Chase SILAC Strategy Measures Changes in Protein Decay and Synthesis Rates Induced by Perturbation of Proteostasis with an Hsp90 Inhibitor.

Standard proteomics methods allow the relative quantitation of levels of thousands of proteins in two or more samples. While such methods are invaluable for defining the variations in protein concentrations which follow the perturbation of a biological system, they do not offer information on the mechanisms underlying such changes. Expanding on previous work [1], we developed a pulse-chase (pc) variant of SILAC (stable isotope labeling by amino acids in cell culture). pcSILAC can quantitate in one experiment and for two conditions the relative levels of proteins newly synthesized in a given time as well as the relative levels of remaining preexisting proteins. We validated the method studying the drug-mediated inhibition of the Hsp90 molecular chaperone, which is known to lead to increased synthesis of stress response proteins as well as the increased decay of Hsp90 "clients". We showed that pcSILAC can give information on changes in global cellular proteostasis induced by treatment with the inhibitor, which are normally not captured by standard relative quantitation techniques. Furthermore, we have developed a mathematical model and computational framework that uses pcSILAC data to determine degradation constants kd and synthesis rates Vs for proteins in both control and drug-treated cells. The results show that Hsp90 inhibition induced a generalized slowdown of protein synthesis and an increase in protein decay. Treatment with the inhibitor also resulted in widespread protein-specific changes in relative synthesis rates, together with variations in protein decay rates. The latter were more restricted to individual proteins or protein families than the variations in synthesis. Our results establish pcSILAC as a viable workflow for the mechanistic dissection of changes in the proteome which follow perturbations. Data are available via ProteomeXchange with identifier PXD000538.

Pédiatrie. 4. Dépistage de la dénutrition chez les enfants: nouvelles pratiques alimentaires à l'Hôpital de l'Enfance de Lausanne [Pediatrics. Screening for nutritional deficiencies in hospitalized children: new practices at the Hôpital de I'Enfance in Lausanne].

Screening for undernutrition among hospitalized children requires a systematic assessment of dietary intake. The development of a new tool for quick and playful assessment of dietary intake, called "Fleur" ("Flower"), at the Hôpital de l'Enfance in Lausanne allows to identify children at risk of undernutrition and to adapt their nutrition to their specific needs.

Evaluation of the prenatal diagnosis of limb reduction deficiencies. EUROSCAN Study Group.

Ultrasound scans in the mid-trimester of pregnancy are now a routine part of antenatal care in most European countries. Using data from registries of congenital anomalies a study was undertaken in Europe. The objective of the study was to evaluate prenatal detection of limb reduction deficiencies (LRD) by routine ultrasonographic examination of the fetus. All LRDs suspected prenatally and all LRDs (including chromosome anomalies) confirmed at birth were identified from 20 Congenital Malformation Registers from the following 12 European countries: Austria, Croatia, Denmark, France, Germany, Italy, Lithuania, Spain, Switzerland, The Netherlands, UK and Ukrainia. These registries are following the same methodology. During the study period (1996-98) there were 709,030 births, and 7,758 cases with congenital malformations including LRDs. If more than one LRD was present the case was coded as complex LRD; 250 cases of LRDs with 63 (25.2%) termination of pregnancies were identified including 138 cases with isolated LRD, 112 with associated malformations, 16 with chromosomal anomalies and 38 non chromosomal recognized syndromes. The prenatal detection rate of isolated LRD was 24.6% (34 out of 138 cases) compared with 49.1% for associated malformations (55 out of 112; p<0.01). The prenatal detection of isolated terminal transverse LRD was 22.7% (22 out of 97), 50% (3 out of 6) for proximal intercalary LRD, 8.3% (1 out of 12) for longitudinal LRD and 0 for split hand/foot; for multipli-malformed children with LRD those percentages were 46.1% (30 out of 65), 66.6% (6 out of 9), 57.1% (8 out of 14) and 0 (0 out of 2), respectively. The prenatal detection rate of LRDs varied in relation with the ultrasound screening policies from 20.0% to 64.0% in countries with at least one routine fetal scan.

Nutritional deficiencies after Roux-en-Y gastric bypass for morbid obesity often cannot be prevented by standard multivitamin supplementation

BACKGROUND: Despite the increasing use of Roux-en-Y gastric bypass (RYGBP) in the treatment of morbid obesity, data about postoperative nutritional deficiencies and their treatment remain scarce. OBJECTIVE: The aim of this study was to evaluate the efficacy of a standard multivitamin preparation in the prevention and treatment of nutritional deficiencies in obese patients after RYGBP. DESIGN: This was a retrospective study of 2 y of follow-up of obese patients after RYGBP surgery. Between the first and the sixth postoperative months, a standardized multivitamin preparation was prescribed for all patients. Specific requirements for additional substitutive treatments were systematically assessed by a biologic workup at 3, 6, 9, 12, 18, and 24 mo. RESULTS: A total of 137 morbidly obese patients (110 women and 27 men) were included. The mean (+/-SD) age at the time of surgery was 39.9 +/- 10.0 y, and the body mass index (in kg/m(2)) was 46.7 +/- 6.5. Three months after RYGBP, 34% of these patients required at least one specific supplement in addition to the multivitamin preparation. At 6 and 24 mo, this proportion increased to 59% and 98%, respectively. Two years after RYGBP, a mean amount of 2.9 +/- 1.4 specific supplements had been prescribed for each patient, including vitamin B-12, iron, calcium + vitamin D, and folic acid. At that time, the mean monthly cost of the substitutive treatment was $34.83. CONCLUSION: Nutritional deficiencies are very common after RYGBP and occur despite supplementation with the standard multivitamin preparation. Therefore, careful postoperative follow-up is indicated to detect and treat those deficiencies.

Déficits dans les apports nutritionnels chez les candidats a la chirurgie bariatrique [Nutritional deficiencies in candidates for bariatric surgery]

98% of patients who have undergone a gastric bypass for treating severe obesity develop multiple micronutrient deficits. However, prior to surgery, it isn't rare to find nutrient deficiencies. Indeed, the dietary intakes of surgery candidates are often unbalanced, lacking in variety especially in high vitamin and mineral nutrients. We present the preliminary results concerning the qualitative and quantitative analysis in a group of patients waiting for a gastric bypass. The recommended daily amounts in vitamin B9, vitamin D and iron are insufficient in the majority of the patients. The correction of nutritional intakes is advisable, even before the surgery, in order to reduce the risks of developing biological deficiencies.

FGFR1 and PROKR2 rare variants found in patients with combined pituitary hormone deficiencies.

The genetic aetiology of congenital hypopituitarism (CH) is not entirely elucidated. FGFR1 and PROKR2 loss-of-function mutations are classically involved in hypogonadotrophic hypogonadism (HH), however, due to the clinical and genetic overlap of HH and CH; these genes may also be involved in the pathogenesis of CH. Using a candidate gene approach, we screened 156 Brazilian patients with combined pituitary hormone deficiencies (CPHD) for loss-of-function mutations in FGFR1 and PROKR2. We identified three FGFR1 variants (p.Arg448Trp, p.Ser107Leu and p.Pro772Ser) in four unrelated patients (two males) and two PROKR2 variants (p.Arg85Cys and p.Arg248Glu) in two unrelated female patients. Five of the six patients harbouring the variants had a first-degree relative that was an unaffected carrier of it. Results of functional studies indicated that the new FGFR1 variant p.Arg448Trp is a loss-of-function variant, while p.Ser107Leu and p.Pro772Ser present signalling activity similar to the wild-type form. Regarding PROKR2 variants, results from previous functional studies indicated that p.Arg85Cys moderately compromises receptor signalling through both MAPK and Ca(2) (+) pathways while p.Arg248Glu decreases calcium mobilization but has normal MAPK activity. The presence of loss-of-function variants of FGFR1 and PROKR2 in our patients with CPHD is indicative of an adjuvant and/or modifier effect of these rare variants on the phenotype. The presence of the same variants in unaffected relatives implies that they cannot solely cause the phenotype. Other associated genetic and/or environmental modifiers may play a role in the aetiology of this condition.

TRAIP is a regulator of the spindle assembly checkpoint.

Accurate chromosome segregation during mitosis is temporally and spatially coordinated by fidelity-monitoring checkpoint systems. Deficiencies in these checkpoint systems can lead to chromosome segregation errors and aneuploidy, and promote tumorigenesis. Here, we report that the TRAF-interacting protein (TRAIP), a ubiquitously expressed nucleolar E3 ubiquitin ligase important for cellular proliferation, is localized close to mitotic chromosomes. Its knockdown in HeLa cells by RNA interference (RNAi) decreased the time of early mitosis progression from nuclear envelope breakdown (NEB) to anaphase onset and increased the percentages of chromosome alignment defects in metaphase and lagging chromosomes in anaphase compared with those of control cells. The decrease in progression time was corrected by the expression of wild-type but not a ubiquitin-ligase-deficient form of TRAIP. TRAIP-depleted cells bypassed taxol-induced mitotic arrest and displayed significantly reduced kinetochore levels of MAD2 (also known as MAD2L1) but not of other spindle checkpoint proteins in the presence of nocodazole. These results imply that TRAIP regulates the spindle assembly checkpoint, MAD2 abundance at kinetochores and the accurate cellular distribution of chromosomes. The TRAIP ubiquitin ligase activity is functionally required for the spindle assembly checkpoint control.

Exploring Parallels Between Molecular Changes Induced in PNS by Aging and Demyelinating Neuropathies

The peripheral nervous system (PNS) is involved in many age-dependent neurological deficits, including numbness, pain, restless legs, trouble with walking and balance that are commonly found in the elderly. These symptoms generally result from demyelination and/or loss of axonal integrity. However, the precise identity of age-regulated molecular changes in either neuronal or glial compartments of the nerve is unclear. Interestingly, these deficiencies are also present in inherited neuropathies, where the expressivity of the rapid and early onset phenotypes is undeniably more severe than in normal aging. Nevertheless, especially the molecular changes underlying loss of axonal integrity in neuropathy condition are also poorly understood. To unravel molecular mechanisms affected by PNS aging, we used wildtype mice at 17 time-points from day of birth until senescence (28 months-old). For the neuropathy study, we focused on 56 day-old Schwann cell-specific neuropathy-inducing mutants, MPZCre/1/ LpinfE2-3/fE2-3 and MPZCre/1/ScapfE1/fE1 mice, that have, at this age, already developed neuropathic symptoms. Transcriptomes of dissected Schwann cell-containing endoneurium or sensory neuron-containing dorsal root ganglia have been analyzed throughout time or genotypes, using Illumina Bead Chips. Following data validation, we identified groups of differentially expressed genes in the development, aging and in the neuropathic mutants, in both glial and neuronal compartments. We detected substantial differences in the dynamics of changes in gene expression during development and aging between these two compartments. Furthermore, considering the above-mentioned phenotypic similarities, we integrated aging and mutant data. Interestingly, we observed that there are some parallels at the molecular level between processes involved in aging, which leads to less severe and more progressive PNS alterations, and in the rapid onset peripheral neuropathies. Apart from helping the understanding of molecular alterations underlying age-related PNS phenotypes, this data should also contribute to the identification of pathways that could be used as targets for therapeutical approaches to prevent complications associated with both aging and inherited forms of neuropathies.

NALP3 is not necessary for early protection against experimental tuberculosis.

In vitro, Toll-like receptors (TLR)2, 4 and 9 as well as NOD-like receptor 2 critically determine macrophage responses to Mycobacterium tuberculosis (Mtb) infection. However, in low-dose experimental murine tuberculosis, single or multiple deficiencies in TLRs 2, 4, 9 or NOD2 have little, if any, impact on early mycobacterial growth containment, granuloma formation and survival. Here, we analyzed the relevance of NALP3, one component of the danger-signaling inflammasome, for (i) Mtb-induced cytokine secretion in vitro and in vivo, (ii) restriction of Mtb replication in infected organs and (iii) granuloma formation. In the absence of functional NALP3, there was no IL-1beta and IL-18 production in Mtb-infected dendritic cells and macrophages in vitro, whereas secretion of IL-1alpha, IL-12p40 and TNF remained unaffected. After three weeks of infection, NALP3-deficient as well as IL-18-deficient mice were as capable as wildtype mice of restricting Mtb loads at a plateau level within well-differentiated granulomas. In conclusion, despite its involvement in cytokine processing, NALP3 is not essential for induction of protective immunity to Mtb.

Elevated resource availability sufficient to turn opportunistic into virulent fish pathogens.

There is mounting evidence that organic or inorganic enrichment of aquatic environments increases the risk of infectious diseases, with disease agents ranging from helminth parasites to fungal, bacterial, and viral pathogens. The causal link between microbial resource availability and disease risk is thought to be complex and, in the case of so-called "opportunistic pathogens," to involve additional stressors that weaken host resistance (e.g., temperature shifts or oxygen deficiencies). In contrast to this perception, our experiment shows that the link between resource levels and infection of fish embryos can be very direct: increased resource availability can transform benign microbial communities into virulent ones. We find that embryos can be harmed before further stresses (e.g., oxygen depletion) weaken them, and treatment with antibiotics and fungicides cancels the detrimental effects. The changed characteristics of symbiotic microbial communities could simply reflect density-dependent relationships or be due to a transition in life-history strategy. Our findings demonstrate that simple microhabitat changes can be sufficient to turn "opportunistic" into virulent pathogens.

Loss of single HLA class I allospecificities in melanoma cells due to selective genomic abbreviations.

Expression of human leucocyte antigen (HLA) Class I molecules is essential for the recognition of malignant melanoma (MM) cells by CD8(+) T lymphocytes. A complete or partial loss of HLA Class I molecules is a potent strategy for MM cells to escape from immunosurveillance. In 2 out of 55 melanoma cell cultures we identified a complete phenotypic loss of HLA allospecificities. Both patients have been treated unsuccessfully with HLA-A2 peptides. To identify the reasons underlying the loss of single HLA-A allospecificities, we searched for genomic alterations at the locus for HLA Class I alpha-chain on chromosome 6 in melanoma cell cultures established from 2 selected patients with MM in advanced stage. This deficiency was associated with alterations of HLA-A2 gene sequences as determined by polymerase chain reaction-sequence specific primers (PCR-SSP). Karyotyping revealed a chromosomal loss in Patient 1, whereas melanoma cell cultures established from Patient 2 displayed 2 copies of chromosome 6. Loss of heterozygosity (LOH) using markers located around position 6p21 was detected in both cases. By applying group-specific primer-mixes spanning the 5'-flanking region of the HLA-A2 gene locus the relevant region was amplified by PCR and subsequent sequencing allowed alignment with the known HLA Class I reference sequences. Functional assays using HLA-A2-restricted cytotoxic T-cell clones were performed in HLA-A2 deficient MM cultures and revealed a drastically reduced susceptibility to CTL lysis in HLA-A2 negative cells. We could document the occurrence of selective HLA-A2 deficiencies in cultured advanced-stage melanoma metastases and identify their molecular causes as genomic alterations within the HLA-A gene locus.