The nanostructured secretome.

The term secretome, which traditionally strictly refers to single proteins, should be expanded to also include the great variety of nanoparticles secreted by cells (secNPs) into the extracellular space, which ranges from high-density lipoproteins of a few nanometers to extracellular vesicles and fat globules of hundreds of nanometers. Widening the definition is urged by the ever-increasing understanding of the role of secNPs as regulators/mediators of key physiological and pathological processes, which also puts them in the running as breakthrough cell-free therapeutics and diagnostics. "Made by cells for cells", secNPs are envisioned as a sweeping paradigm shift in nanomedicine, promising to overcome the limitations of synthetic nanoparticles by unsurpassed circulation and targeting abilities, precision and sustainability. From a longer/wider perspective, advanced manipulation would possibly make secNPs available as building blocks for future "biogenic" nanotechnology. However, the current knowledge is fragmented and sectorial (the majority of the studies being focused on a specific biological and/or medical aspect of a given secNP class or subclass), the understanding of the nanoscale and interfacial properties is limited and the development of bioprocesses and regulatory initiatives is in the early days. We believe that new multidisciplinary competencies and synergistic efforts need to be attracted and augmented to move forward. This review will contribute to the effort by attempting for the first time to rationally gather and elaborate secNPs and their traits into a unique concise framework - from biogenesis to colloidal properties, engineering and clinical translation - disclosing the overall view and easing comparative analysis and future exploitation.

Role of BDNF Val66Met functional polymorphism in Alzheimer's disease-related depression.

BACKGROUND: The gene encoding brain-derived neurotrophic factor (BDNF) has been suggested as a candidate for major depression, and for depression susceptibility in different neurological and psychiatric diseases. No study has investigated the role of BDNF genetic variation and depressive symptoms in Alzheimer's disease (AD). OBJECTIVE: The aim of this study was to assess the genetic contribution of BDNF Val66Met functional polymorphism to AD-related depression. METHODS: Two-hundred and sixty-four AD patients underwent clinical and neuropsychological examination as well as an evaluation of behavioral and psychiatric disturbances. They were subsequently divided into two subgroups according to the presence (AD-D) or the absence (AD-nD), based on DSM-IV criteria for depression in AD. In each subject, BDNF Val66Met functional polymorphism and apolipoprotein E (APOE) genotype were evaluated. RESULTS: In our sample, 35.2% of patients (n=93) reported AD-related depressive symptoms. Compared to patients bearing no polymorphisms (BDNF G/G), BDNF G/A carriers showed more than twofold-time risk (OR=2.38; 95%CI=1.38-4.13), and BDNF A/A carriers had a threefold-time risk (OR=3.04; 95%CI=1.15-8.00) for depression in AD. Accordingly, considering the allele frequencies, BDNF A allele was significantly over-represented in AD-D (32.8%) compared to AD-nD (19.0%) (OR=2.08; 95%CI=1.38-3.13). An association between the number of carried A allele and the severity of depressive symptoms was observed (P<0.002). No effect of APOE genotype on risk for depression was found. CONCLUSIONS: The present findings provide evidence of BDNF genetic variation role in the susceptibility to AD-related depression. This study puts emphasis on the usefulness of considering genetic background for better defining individualized risk profiles in AD.

Technical note: simultaneous identification of CSN1S2 A, B, C, and E alleles in goats by polymerase chain reaction-single strand conformation polymorphism.

Most variability in goat caseins originates from the high number of genetic polymorphisms often affecting the specific protein expression, with strong effects on milk composition traits and technological properties. At least 7 alleles have been found in the goat alpha(S2)-CN gene (CSN1S2). Five of them (CSN1S2*A, CSN1S2*B, CSN1S2*C, CSN1S2*E, and CSN1S2*F) are widespread in most breeds, whereas the other 2 (CSN1S2*D and CSN1S2*0) are rarer alleles. Four different PCR-RFLP tests are needed to detect all of these variants at the DNA level. The objective of this study was to develop and validate a rapid method for typing 4 of the 5 most-common goat CSN1S2 alleles by means of PCR-single strand conformation polymorphism (SSCP). The method was validated by analyzing 37 goat samples at the protein and DNA level, respectively, by milk isoelectrofocusing and PCR-RFLP methods already described. The genotypes obtained using the PCR-SSCP approach were in full agreement with those obtained by the validation analyses. The newly developed PCR-SSCP approach provides an accurate and inexpensive assay highly suitable for genotyping goat CSN1S2.

Tyrosine83 is essential for the activity of E. coli galactoside transacetylase.

The gene (lacA) coding for Escherichia coli galactoside transacetylase was cloned into the pTrcHisB plasmid, and the corresponding hexahistidine-tagged enzyme was over-expressed and purified. The kinetic constants of the tagged protein were determined, yielding values in excellent agreement with previous observations reported for the natural enzyme. LacA Tyrosine83 was then substituted with a Valine: by comparing the K(m) and k(cat) values observed for wild type and mutant enzymes using isopropyl-thio-beta-d-galactopyranoside or p-nitrophenyl-beta-d-galactopyranoside as substrates, Tyrosine83 was identified as an essential residue for the catalytic activity of E. coli galactoside transacetylase.

Directed evolution of beta-galactosidase from Escherichia coli by mutator strains defective in the 3'-->5' exonuclease activity of DNA polymerase III.

Directed evolution of Escherichia coli beta-galactosidase into variants featuring beta-glucosidase activity was challenged. To this end, mutagenesis of lacZ was performed by replication in E. coli CC954, a mutator strain containing a DNA polymerase III defective in 3'-->5' exonuclease activity. beta-Galactosidase variants can be isolated upon mutagenesis of lacZ hosted into the self-transmissible episome F'128. Optimal evolution of lacZ can be achieved by propagation of E. coli CC954/F'128 cultures for 15 generations; further growth of mutator cultures for 37 or 55 generations imposes a high mutational load on lacZ and hinders the selection of efficiently evolved clones.

Expression of the RNA recognition motif-containing protein SEB-4 during Xenopus embryonic development.

RNA binding proteins play key roles in the post-transcriptional regulation of gene expression. Here we present the molecular cloning and spatio-temporal expression of Xseb-4, which codes for a putative RNA binding protein containing a single RNA recognition motif (RRM). XSEB-4 shares 60-65% identity with the mammalian SEB-4 proteins. Xseb-4 is strongly expressed maternally. Zygotic transcription is initiated in the early gastrula embryo in paraxial mesoderm that is fated to give rise to somites. During the course of gastrulation and neurulation Xseb-4 expression in somitic paraxial mesoderm is centered within the XmyoD expression domain. As development proceeds Xseb-4 expression is in addition initiated in the cardiac primordium and the lens vesicle. In the heart expression is confined to the myocardium. Thus, the RRM-containing putative RNA binding protein XSEB-4 is differentially expressed during embryonic development in Xenopus.