Dissecting the Nuclear Import of the Ribosomal Protein Rps2 (uS5).

The ribosome is assembled in a complex process mainly taking place in the nucleus. Consequently, newly synthesized ribosomal proteins have to travel from the cytoplasm into the nucleus, where they are incorporated into nascent ribosomal subunits. In this study, we set out to investigate the mechanism mediating nuclear import of the small subunit ribosomal protein Rps2. We demonstrate that an internal region in Rps2, ranging from amino acids 76 to 145, is sufficient to target a 3xyEGFP reporter to the nucleus. The importin-ß Pse1 interacts with this Rps2 region and is involved in its import, with Rps2 residues arginine 95, arginine 97, and lysine 99 being important determinants for both Pse1 binding and nuclear localization. Moreover, our data reveal a second import mechanism involving the N-terminal region of Rps2, which depends on the presence of basic residues within amino acids 10 to 28. This Rps2 segment overlaps with the binding site of the dedicated chaperone Tsr4; however, the nuclear import of Rps2 via the internal as well as the N-terminal nuclear-targeting element does not depend on Tsr4. Taken together, our study has unveiled hitherto undescribed nuclear import signals, showcasing the versatility of the mechanisms coordinating the nuclear import of ribosomal proteins.

Rbp95 binds to 25S rRNA helix H95 and cooperates with the Npa1 complex during early pre-60S particle maturation.

Eukaryotic ribosome synthesis involves more than 200 assembly factors, which promote ribosomal RNA (rRNA) processing, modification and folding, and assembly of ribosomal proteins. The formation and maturation of the earliest pre-60S particles requires structural remodeling by the Npa1 complex, but is otherwise still poorly understood. Here, we introduce Rbp95 (Ycr016w), a constituent of early pre-60S particles, as a novel ribosome assembly factor. We show that Rbp95 is both genetically and physically linked to most Npa1 complex members and to ribosomal protein Rpl3. We demonstrate that Rbp95 is an RNA-binding protein containing two independent RNA-interacting domains. In vivo, Rbp95 associates with helix H95 in the 3' region of the 25S rRNA, in close proximity to the binding sites of Npa1 and Rpl3. Additionally, Rbp95 interacts with several snoRNAs. The absence of Rbp95 results in alterations in the protein composition of early pre-60S particles. Moreover, combined mutation of Rbp95 and Npa1 complex members leads to a delay in the maturation of early pre-60S particles. We propose that Rbp95 acts together with the Npa1 complex during early pre-60S maturation, potentially by promoting pre-rRNA folding events within pre-60S particles.

Dedicated chaperones coordinate co-translational regulation of ribosomal protein production with ribosome assembly to preserve proteostasis.

The biogenesis of eukaryotic ribosomes involves the ordered assembly of around 80 ribosomal proteins. Supplying equimolar amounts of assembly-competent ribosomal proteins is complicated by their aggregation propensity and the spatial separation of their location of synthesis and pre-ribosome incorporation. Recent evidence has highlighted that dedicated chaperones protect individual, unassembled ribosomal proteins on their path to the pre-ribosomal assembly site. Here, we show that the co-translational recognition of Rpl3 and Rpl4 by their respective dedicated chaperone, Rrb1 or Acl4, reduces the degradation of the encoding RPL3 and RPL4 mRNAs in the yeast Saccharomyces cerevisiae. In both cases, negative regulation of mRNA levels occurs when the availability of the dedicated chaperone is limited and the nascent ribosomal protein is instead accessible to a regulatory machinery consisting of the nascent-polypeptide-associated complex and the Caf130-associated Ccr4-Not complex. Notably, deregulated expression of Rpl3 and Rpl4 leads to their massive aggregation and a perturbation of overall proteostasis in cells lacking the E3 ubiquitin ligase Tom1. Taken together, we have uncovered an unprecedented regulatory mechanism that adjusts the de novo synthesis of Rpl3 and Rpl4 to their actual consumption during ribosome assembly and, thereby, protects cells from the potentially detrimental effects of their surplus production.

Living cells are packed full of molecules known as proteins, which perform many vital tasks the cells need to survive and grow. Machines called ribosomes inside the cells use template molecules called messenger RNAs (or mRNAs for short) to produce proteins. The newly-made proteins then have to travel to a specific location in the cell to perform their tasks. Some newly-made proteins are prone to forming clumps, so cells have other proteins known as chaperones that ensure these clumps do not form. The ribosomes themselves are made up of several proteins, some of which are also prone to clumping as they are being produced. To prevent this from happening, cells control how many ribosomal proteins they make, so there are just enough to form the ribosomes the cell needs at any given time. Previous studies found that, in yeast, two ribosomal proteins called Rpl3 and Rpl4 each have their own dedicated chaperone to prevent them from clumping. However, it remained unclear whether these chaperones are also involved in regulating the levels of Rpl3 and Rpl4. To address this question, Pillet et al. studied both of these dedicated chaperones in yeast cells. The experiments showed that the chaperones bound to their target proteins (either units of Rpl3 or Rpl4) as they were being produced on the ribosomes. This protected the template mRNAs the ribosomes were using to produce these proteins from being destroyed, thus allowing further units of Rpl3 and Rpl4 to be produced. When enough Rpl3 and Rpl4 units were made, there were not enough of the chaperones to bind them all, leaving the mRNA templates unprotected. This led to the destruction of the mRNA templates, which decreased the numbers of Rpl3 and Rpl4 units being produced. The work of Pillet et al. reveals a feedback mechanism that allows yeast to tightly control the levels of Rpl3 and Rpl4. In the future, these findings may help us understand diseases caused by defects in ribosomal proteins, such as Diamond-Blackfan anemia, and possibly also neurodegenerative diseases caused by clumps of proteins forming in cells. The next step will be to find out whether the mechanism uncovered by Pillet et al. also exists in human and other mammalian cells.

Seronegative autoimmune hepatitis in children: Spectrum of disorders.

BACKGROUND: A few children with acute or chronic liver disease display histological features compatible with autoimmune hepatitis, but lack specific serological markers. AIM: To describe features, management and outcome of childhood seronegative autoimmune hepatitis. METHODS: From 1988 to 2010, 38 children were included under the following criteria: negative virological studies, no serum autoantibodies, exclusion of other causes of liver diseases, and liver histology compatible with autoimmune hepatitis. RESULTS: Four groups were identified: (1) 12 with increased serum gamma globulin concentrations; (2) 10 with normal or low serum gamma globulins and no combined blood disease; (3) 10 with combined aplastic anemia; and (4) 6 with peripheral thrombocytopenia with/without neutropenia. Immunosuppressive treatment was associated with aminotransferases normalization in all but one child who required liver transplantation. Relapses occurred in 10 children. Lymphocytopenia was found at the time of the diagnosis of hepatitis in 13 children, 12 in groups 3 or 4. All 38 children are alive after 4-17 years, 18 still under immunosuppression. CONCLUSIONS: Childhood seronegative autoimmune hepatitis includes a spectrum of disorders. Early liver histology is recommended and, if compatible with autoimmune hepatitis, immunosuppressive treatment should be started. Initial lymphocytopenia may indicate future hematological complication.

Early postpartum discharge and breastfeeding: an observational study from France.

BACKGROUND: Early postpartum discharge is a recent practice in France, but the influence of a shortened hospital stay on subsequent breastfeeding is unknown. The objective of the present study was to compare the breastfeeding mode after early discharge (ED) and conventional discharge (CD) from a hospital maternity unit. METHODS: An observational study was conducted in a French university hospital among 135 breastfeeding mothers, who delivered between 1 January and 31 July 2006. Forty-five ED mothers were matched with 90 CD mothers on 13 criteria. A structured questionnaire was used to collect data regarding feeding practices at 10 weeks postpartum, the period corresponding to paid maternity leave. RESULTS: Exclusive breast-, mixed, and bottle feedings were reported by, respectively, 35 (77.8%), three (6.7%) and seven (15.5%) ED mothers and 64 (71.1%), 12 (13.3%) and 14 (15.6%) CD mothers (no significant differences). Satisfaction with support for breastfeeding and reasons for switching to mixed or bottle feeding were comparable in the two groups. Multivariate analysis indicated that only the planned duration of breastfeeding and the mother's dissatisfaction with help significantly influenced breastfeeding prevalence. CONCLUSIONS: Early postpartum hospital discharge organized by skilled professionals is compatible with a satisfactory rate of exclusive breastfeeding up to the return to work. Formalized programs of instruction for perinatal professionals would help to reduce early abandonment.

HCV/HIV co-infection, HCV viral load and mode of delivery: risk factors for mother-to-child transmission of hepatitis C virus?

BACKGROUND: Hepatitis C virus (HCV) infection in children is mainly acquired via maternal transmission. Our aim was to identify mother-to-child-transmission (MTC) risk factors, namely those associated with maternal virological characteristics and mode of delivery. METHODS: Women were included between October 1998 and September 2002 in six hospitals in Southern France on the basis of positive HCV serological status. Recorded data included maternal characteristics, circumstances of delivery and laboratory data concerning mother and child. Paediatric follow-up lasted 1 year, and 2 years for children with circulating HCV RNA. RESULTS: A total of 214 mother-and-child pairs were analysed; 55 (26%) were HCV/HIV co-infected. The probability of HCV transmission was three-fold higher for HCV/HIV-co-infected women (P = 0.05). Twelve children were HCV RNA positive at 1 year of age (MTC = 5.6%); three became HCV RNA negative between 12 (M12) and 18 months of age (M18) and recovered normal alanine aminotransferase levels. Circulating HCV RNA was found in 137 (69%) mothers. Mothers of infected children all displayed HCV viraemia (MTC = 8.8%): six children were born of HCV/HIV-co-infected HCV RNA positive women (MTC = 13.6%) and six from HCV monoinfected women with positive HCV RNA (MTC = 6.5%). When maternal HCV RNA levels were below 6 log-IU/ml, the rate of transmission was significantly higher in the case of HCV/HIV co-infection (odds ratio = 8.3, 95% confidence interval, 1.4-47.5) P = 0.01. This association did not, however, exist for HCV RNA-positive mothers with levels of at least 6 log-IU/ml. Rate of transmission did not differ significantly between children born by vaginal delivery or caesarean section after membrane rupture and those born by elective caesarean section independently of HIV status.

Clinical effects of heliox administration for acute bronchiolitis in young infants.

OBJECTIVE: To assess the effect of heliox, a helium-oxygen mixture, on respiratory distress symptoms in young infants. DESIGN: Prospective, randomized, double-blind study. SETTING: Pediatric ICU (PICU) of a university hospital. PATIENTS: Twenty infants, all < 3 months old, admitted to the PICU with moderate-to-severe acute respiratory syncytial virus bronchiolitis. INTERVENTIONS: All infants were randomly and blindly assigned to inhale either heliox or an air-oxygen mixture (airox) for 1 h under an oxyhood. MEASUREMENTS AND RESULTS: After 1 h, the respiratory distress score was significantly lower in the heliox group compared with the airox group (3.05 vs 5.5, p < 0.01), with a significant reduction in accessory muscles use (p < 0.05) and expiratory wheezing (p < 0.01). In contrast, inspiratory breath sounds and cyanosis did not significantly differ between groups. The ex-premature infants of the heliox group had a higher respiratory distress score at baseline compared with the term infants of this group (5.8 vs 5.2, p < 0.05) and a comparable decrease in the score at 60 min. CONCLUSIONS: In young infants, even those born prematurely, heliox breathing induced a rapid reduction in accessory muscles use and expiratory wheezing. Further studies are needed to confirm the decreased respiratory muscle work of breathing during heliox inhalation in this population.

Real-time PCR for chloroquine sensitivity assay and for pfmdr1-pfcrt single nucleotide polymorphisms in Plasmodium falciparum.

Plasmodium falciparum drug resistance is a major problem in malaria endemic areas. Molecular markers and in vitro tests have been developed to study and monitor drug resistance. However, none, used alone, can provide sufficient data concerning the level of drug resistance and to issue precise guidelines for drug use policies in endemic areas. We propose real-time PCR for the simultaneous detection of pfcrt and pfmdr1 genes mutations and to determine the half-maximal inhibitory response (IC(50)) of antimalarial drug. Using hybridization probes and SybrGreen technology on LightCycler instrument, point mutations of pfcrt and pfmdr1 genes have been successfully detected in 161 human blood samples and determination of IC values was applied to chloroquine-sensitive and chloroquine-resistant strains. Moreover, mixed infections caused by P. falciparum clones with wild-type or mutant alleles could be efficiency separated. The aim of this study was not to provide definitive data concerning the rate of mutations in an endemic area, but to describe a powerful method allowing the quantification of DNA for IC(50) determination and the detection of major pfmdr1 and pfcrt mutations.