Protein N-Homocysteinylation Induces the Formation of Toxic Amyloid-Like Protofibrils

Previous works reported that a mild increase in homocysteine level is a risk factor for cardiovascular and neurodegenerative diseases in humans. Homocysteine thiolactone is a cyclic thioester, most of which is produced by an error-editing function of methionyl-tRNA synthetase, causing in vivo post-translational protein modifications by reacting with the ?-amino group of lysine residues. In cells, the rate of homocysteine thiolactone synthesis is strictly dependent on the levels of the precursor metabolite, homocysteine. In this work, using bovine serum albumin as a model, we investigated the impact of N-homocysteinylation on protein conformation as well as its cellular actions. Previous works demonstrated that protein N-homocysteinylation causes enzyme inactivation, protein aggregation, and precipitation. In addition, in the last few years, several pieces of evidence have indicated that protein unfolding and aggregation are crucial events leading to the formation of amyloid fibrils associated with a wide range of human pathologies. For the first time, our results reveal how the low level of protein N-homocysteinylation can induce mild conformational changes leading to the formation of native-like aggregates evolving over time, producing amyloid-like structures. Taking into account the fact that in humans about 70% of circulating homocysteine is N-linked to blood proteins such as serum albumin and hemoglobin, the results reported in this article could have pathophysiological relevance and could contribute to clarify the mechanisms underlying some pathological consequences described in patients affected by hyperhomocysteinemia.     

Tec kinases Itk and Rlk are required for CD8+ T cell responses to virus infection independent of their role in CD4+ T cell help

Itk and Rlk are members of the Tec kinase family of nonreceptor protein tyrosine kinases that are expressed in T cells, NK cells, and mast cells. These proteins are involved in the regulation of signaling processes downstream of the TCR in CD4(+) T cells, particularly in the phosphorylation of phospholipase C-gamma1 after TCR activation; furthermore, both Itk and Rlk are important in CD4(+) T cell development, differentiation, function, and homeostasis. However, few studies have addressed the roles of these kinases in CD8(+) T cell signaling and function. Using Itk(-/-) and Itk(-/-)Rlk(-/-) mice, we examined the roles of these Tec family kinases in CD8(+) T cells, both in vitro and in vivo. These studies demonstrate that the loss of Itk and Rlk impairs TCR-dependent signaling, causing defects in phospholipase C-gamma1, p38, and ERK activation as well as defects in calcium flux and cytokine production in vitro and expansion and effector cytokine production by CD8(+) T cells in response to viral infection. These defects cannot be rescued by providing virus-specific CD4(+) T cell help, thereby substantiating the important role of Tec kinases in CD8(+) T cell signaling.     

Complex rearrangement involving 9p deletion and duplication in a syndromic patient: genotype/phenotype correlation and review of the literature

We describe a 7-year-old boy with a complex rearrangement involving the whole short arm of chromosome 9 defined by means of molecular cytogenetic techniques. The rearrangement is characterized by a 18.3 Mb terminal deletion associated with the inverted duplication of the adjacent 21,5 Mb region. The patient shows developmental delay, psychomotor retardation, hypotonia. Other typical features of 9p deletion (genital disorders, midface hypoplasia, long philtrum) and of the 9p duplication (brachycephaly, down slanting palpebral fissures and bulbous nasal tip) are present. Interestingly, he does not show trigonocephaly that is the most prominent dysmorphism associated with the deletion of the short arm of chromosome 9. Patient's phenotype and the underlying flanking opposite 9p imbalances are compared with that of reported patients and the proposed critical regions for 9p deletion and 9p duplication syndromes.     

The natriuretic peptide time-course in end-stage heart failure patients supported by left ventricular assist device implant: focus on NT-proCNP

This study aimed to evaluate left ventricular assist device (LVAD) effects on natriuretic peptide (NP) prohormone plasma levels in end-stage heart failure (HF) patients, especially NT-proCNP, in order to better characterize the NP system during hemodynamic recovery by LVAD. HF patients (n=17, NYHA III-IV) undergoing LVAD were studied: 6 died of multi-organ failure syndrome (NS) and 11 survived (S). Total sequential organ failure assessment (t-SOFA) score and blood samples were obtained at admission (T1) and at 24, 72h and 1, 2, 4 weeks (T2-T6) after LVAD. In S, NT-proANP and NT-proCNP significantly increased at 24h after implantation, reaching a reduction to basal levels at 4 weeks following LVAD [NT-proANP: T1 vs. T2 p=0.017, NT-proCNP: T1 vs. T2 p=0.028, T1 vs. T3 p=0.043]. Elevated NT-proBNP plasma levels were observed at all times. In NS, NP plasma levels sustained higher with respect to S. No statistical variation was observed for NT-proCNP and NT-proANP in S and NS while NT-proBNP reached significant differences at T4 in NS. Considering S+NS, only NT-proCNP strongly correlated with t-SOFA score at T1 (rho=0.554, p=0.04) while subdividing patients NT-proCNP positively correlated in NS with t-SOFA score (rho=0.988, p=0.002) only at T4. In NS a correlation between NT-proCNP and NT-proBNP at T1 was observed (rho=-0.900, p=0.037). Both IL-6 and TNF-alpha sustained higher in NS patients than in S; in particular, statistical significance was observed for IL-6. The study of new peptides, such as NT-proCNP, would provide additional information for identifying patients who are more likely to recover.     

Plasma C-type natriuretic peptide levels in healthy children

C-type natriuretic peptide (CNP) is assuming increasing importance in cardiovascular disease, and in adults its plasma levels are related to clinical and functional disease severity. Data are scarce regarding the reference values for CNP in infancy. Aim of this study was to assess the reference intervals for CNP in human healthy newborns and infants. Plasma CNP was measured in 121 healthy children divided into: 41 newborns (age 0-3 days), 24 newborns (4-30 days), 22 infants (1-12 months) and 32 children (1-12 years). A group of 32 healthy adult subjects (age 64 ± 1 years) was also studied. CNP was measured by a specific radioimmunoassay. Between- and within-assay variability resulted ≤ 30 and 20%, respectively and analytical sensitivity 0.77 ± 0.05 pg/tube. Plasma CNP resulted significantly higher in children than in adult subjects (13.6 ± 1.2 pg/ml vs. 7.4 ± 1.0 pg/ml, p=0.030). When the results were analyzed as a function of the age the reference intervals for plasma CNP resulted: 11.6 ± 2.1 pg/ml for newborns (0-3 days), 16.4 ± 3.7 pg/ml for newborns (4-30 days), 15.4 ± 2.7 pg/ml for infants (1-12 months), 13.6 ± 2.3 pg/ml for children (1-12 years) [p=0.01 newborns (4-30 days) vs. adults; p=0.03 infants (1-12 months) vs. adults]. CNP showed the highest concentrations after 12h of life with a peak between 4 and 5 days of life and with a progressive decline afterwards. According to these data at least five different reference intervals for CNP determinations should be used. These observations may be helpful for future clinical application of CNP in human children.     

RACK1 is a ribosome scaffold protein for β-actin mRNA/ZBP1 complex

In neurons, specific mRNAs are transported in a translationally repressed manner along dendrites or axons by transport ribonucleic-protein complexes called RNA granules. ZBP1 is one RNA binding protein present in transport RNPs, where it transports and represses the translation of cotransported mRNAs, including β-actin mRNA. The release of β-actin mRNA from ZBP1 and its subsequent translation depends on the phosphorylation of ZBP1 by Src kinase, but little is known about how this process is regulated. Here we demonstrate that the ribosomal-associated protein RACK1, another substrate of Src, binds the β-actin mRNA/ZBP1 complex on ribosomes and contributes to the release of β-actin mRNA from ZBP1 and to its translation. We identify the Src binding and phosphorylation site Y246 on RACK1 as the critical site for the binding to the β-actin mRNA/ZBP1 complex. Based on these results we propose RACK1 as a ribosomal scaffold protein for specific mRNA-RBP complexes to tightly regulate the translation of specific mRNAs.     

Evidence for the co-circulation of dengue virus type 3 genotypes III and V in the Northern region of Brazil during the 2002-2004 epidemics

The reintroduction of dengue virus type 3 (DENV-3) in Brazil in 2000 and its subsequent spread throughout the country was associated with genotype III viruses, the only DENV-3 genotype isolated in Brazil prior to 2002. We report here the co-circulation of two different DENV-3 genotypes in patients living in the Northern region of Brazil during the 2002-2004 epidemics. Complete genomic sequences of viral RNA were determined from these epidemics, and viruses belonging to genotypes V (Southeast Asia/South Pacific) and III were identified. This recent co-circulation of different DENV-3 genotypes in South America may have implications for pathological and epidemiological dynamics.     

BioTimer Assay, a new method for counting Staphylococcus spp. in biofilm without sample manipulation applied to evaluate antibiotic susceptibility of biofilm

The medical device-related infections are frequently a consequence of Staphylococcus biofilm, a lifestyle enhancing bacterial resistance to antibiotics. Antibiotic susceptibility tests are usually performed on planktonic forms of clinical isolates. Some methods have been developed to perform antibiotic susceptibility tests on biofilm. However, none of them counts bacterial inoculum. As antibiotic susceptibility is related to bacterial inoculum, the test results could be mistaken. Here, a new method, BioTimer Assay (BTA), able to count bacteria in biofilm without any manipulation of samples, is presented. Moreover, the BTA method is applied to analyze antibiotic susceptibility of six Staphylococcus strains in biofilm and to determine the number of viable bacteria in the presence of sub-inhibitory doses of four different antibiotics. To validate BTA, the new method was compared to reference methods both for counting and antibiotic susceptibility tests. A high agreement between BTA and reference methods is found on planktonic forms. Therefore, BTA was employed to count bacteria in biofilm and to analyze biofilm antibiotic susceptibility. Results confirm the high resistance to antibiotics of Staphylococcus biofilm. Moreover, BTA counts the number of viable bacteria in the presence of sub-inhibitory doses of antibiotics. The results show that the number of viable bacteria depends on sub-inhibitory doses, age of biofilm and type of antibiotic. In particular, differently to gentamicin and ampicillin, sub-inhibitory doses of ofloxacin and azithromycin reduce the number of viable bacteria at lower extent in young than in old biofilm. In conclusion, BTA is a reliable, rapid, easy-to-perform, and versatile method, and it can be considered a useful tool to analyze antibiotic susceptibility of Staphylococcus spp. in biofilm.     

A90V TDP-43 variant results in the aberrant localization of TDP-43 in vitro

TAR DNA-binding protein-43 (TDP-43) is a highly conserved, ubiquitously expressed nuclear protein that was recently identified as the disease protein in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). Pathogenic TDP-43 gene (TARDBP) mutations have been identified in familial ALS kindreds, and here we report a TARDBP variant (A90V) in a FTLD/ALS patient with a family history of dementia. Significantly, A90V is located between the bipartite nuclear localization signal sequence of TDP-43 and the in vitro expression of TDP-43-A90V led to its sequestration with endogenous TDP-43 as insoluble cytoplasmic aggregates. Thus, A90V may be a genetic risk factor for FTLD/ALS because it predisposes nuclear TDP-43 to redistribute to the cytoplasm and form pathological aggregates.