Acidophilic character of yeast PID261/BUD32, a putative ancestor of eukaryotic protein kinases

Yeast piD261/Bud32 and its homologues are present in eukaryotes and in archaea but not in bacteria and are believed to make up a primordial branch of the eukaryotic protein kinase superfamily. Here, we show that, at variance with the majority of Ser/Thr protein kinases which recognize phosphoacceptor sites specified by basic and/or proline residues, piD261 phosphorylates in vitro a number of acidic proteins and peptides, and it recognizes seryl residues specified by carboxylic side chains. These data suggest that recognition of acidic sites might have been a primordial trait of protein kinases, which was modified during evolution to cope with the increasing complexity of protein phosphorylation in eukaryotes.     

Human Immunodeficiency Virus Type 1 Strains R5 and X4 Induce Different Pathogenic Effects in hu-PBL-SCID Mice, Depending on the State of Activation/Differentiation of Human Target Cells at the Time of Primary Infection

In a previous study, we had found that the extent of T-cell dysfunctions induced by a T-tropic strain of human immunodeficiency virus type 1 (HIV-1) in SCID mice reconstituted with human peripheral blood lymphocytes (hu-PBLs) (hu-PBL-SCID mice) was related to the in vivo state of activation of the human lymphocytes. In this article, we compared the effect of infection of hu-PBL-SCID mice with either T-tropic (X4) or M-tropic (R5) strains of HIV-1 by performing virus inoculation at either 2 h or 2 weeks after the hu-PBL transfer, when the human T cells exhibited a marked activation state or a predominant memory phenotype, respectively. A comparable level of infection was found when hu-PBL-SCID mice were challenged with either the SF162 R5 or the IIIB X4 strain of HIV at 2 h postreconstitution, while at 2 weeks, the R5 virus infection resulted in a higher level of HIV replication than the X4 virus. The R5 strain induced a marked human CD4(+) T-cell depletion along with a drop in levels of human immunoglobulin M in serum and release of soluble factors at both infection times, while the X4 virus induced severe immune dysfunctions only at 2 h. Of interest, injection of hu-PBLs into SCID mice resulted in a marked up-regulation of CCR5 on human CD4(+) T cells. The percentage of CXCR4(+) cells did not change after transplantation, even though a significant decrease in antigen expression was observed. Comparative experiments with two molecular clones of HIV-1 (X4 SF2 and R5 SF162) and two envelope recombinant viruses generated from these viruses showed that R5 viruses (SF162 and the chimeric env-SF162-SF2) caused an extensive depletion of human CD4(+) T cells in SCID mice at both 2 h and 2 weeks after reconstitution, while the X4 viruses (SF2 and the chimeric env-SF2-SF162) induced CD4 T-cell depletion only when infection was performed at the 2-h reconstitution time. These results emphasize the importance of the state of activation/differentiation of human CD4(+) T cells and gp120-coreceptor interactions at the time of primary infection in determining HIV-1 pathogenicity in the hu-PBL-SCID mouse model.     

Antibody Complementarity-Determining Regions (CDRs): A Bridge between Adaptive and Innate Immunity

Background

It has been documented that, independently from the specificity of the native antibody (Ab) for a given antigen (Ag), complementarity determining regions (CDR)-related peptides may display differential antimicrobial, antiviral and antitumor activities.

Methodology/Principal Findings

In this study we demonstrate that a synthetic peptide with sequence identical to V_HCDR3 of a mouse monoclonal Ab (mAb) specific for difucosyl human blood group A is easily taken up by macrophages with subsequent stimulation of: i) proinflammatory cytokine production; ii) PI3K-Akt pathway and iii) TLR-4 expression. Significantly, V_HCDR3 exerts therapeutic effect against systemic candidiasis without possessing direct candidacidal properties.

Conclusions/Significance

These results open a new scenario about the possibility that, beyond the half life of immunoglobulins, Ab fragments may effectively influence the antiinfective cellular immune response in a way reminiscent of regulatory peptides of innate immunity.     

The actin regulatory protein HS1 is required for antigen uptake and presentation by dendritic cells

The hematopoietic actin regulatory protein hematopoietic lineage cell-specific protein 1 (HS1) is required for cell spreading and signaling in lymphocytes, but the scope of HS1 function in Ag presentation has not been addressed. We show that dendritic cells (DCs) from HS1(-/-) mice differentiate normally and display normal LPS-induced upregulation of surface markers and cytokines. Consistent with their normal expression of MHC and costimulatory molecules, HS1(-/-) DCs present OVA peptide efficiently to CD4(+) T cells. However, presentation of OVA protein is defective. Similarly, MHC class I-dependent presentation of VSV8 peptide to CD8(+) T cells occurs normally, but cross-presentation of GRP94/VSV8 complexes is defective. Analysis of Ag uptake pathways shows that HS1 is required for receptor-mediated endocytosis, but not for phagocytosis or macropinocytosis. HS1 interacts with dynamin 2, a protein involved in scission of endocytic vesicles. However, HS1(-/-) DCs showed decreased numbers of endocytic invaginations, whereas dynamin-inhibited cells showed accumulation of these endocytic intermediates. Taken together, these studies show that HS1 promotes an early step in the endocytic pathway that is required for efficient Ag presentation of exogenous Ag by DCs.     

Search Algorithms as a Framework for the Optimization of Drug Combinations

Combination therapies are often needed for effective clinical outcomes in the management of complex diseases, but presently they are generally based on empirical clinical experience. Here we suggest a novel application of search algorithms—originally developed for digital communication—modified to optimize combinations of therapeutic interventions. In biological experiments measuring the restoration of the decline with age in heart function and exercise capacity in Drosophila melanogaster, we found that search algorithms correctly identified optimal combinations of four drugs using only one-third of the tests performed in a fully factorial search. In experiments identifying combinations of three doses of up to six drugs for selective killing of human cancer cells, search algorithms resulted in a highly significant enrichment of selective combinations compared with random searches. In simulations using a network model of cell death, we found that the search algorithms identified the optimal combinations of 6–9 interventions in 80–90% of tests, compared with 15–30% for an equivalent random search. These findings suggest that modified search algorithms from information theory have the potential to enhance the discovery of novel therapeutic drug combinations. This report also helps to frame a biomedical problem that will benefit from an interdisciplinary effort and suggests a general strategy for its solution.     

UVB-radiation-induced apoptosis in Jurkat cells: a coordinated fourier transform infrared spectroscopy-flow cytometry study

We studied the induction of apoptosis in Jurkat cells by UVB radiation (wavelength 290-320 nm) at a dose of 310 mJ/ cm2. We combined Fourier transform infrared (FTIR) spectroscopy with flow cytometry to determine whether the combination of both techniques could provide new and improved information about cell modifications. To do this, we looked for correspondences and correlations between spectroscopy and flow cytometry data and found three highly probable spectroscopic markers of apoptosis. The behavior of the wave number shift of both the Amide I beta-sheet component and the area of the 1083 cm(-1) band reproduced, with a high correlation, the behavior of the early apoptotic cell population, while the behavior of the Amide I area showed a high correlation with the early plus late apoptotic cell population.     

Direct and label‐free detection of the human growth hormone in urine by an ultrasensitive bimodal waveguide biosensor

A label‐free interferometric transducer showing a theoretical detection limit for homogeneous sensing of 5 × 10^–8 RIU, being equivalent to a protein mass coverage resolution of 2.8 fg mm^–2, is used to develop a high sensitive biosensor for protein detection. The extreme sensitivity of this transducer combined with a selective bioreceptor layer enables the direct evaluation of the human growth hormone (hGH) in undiluted urine matrix in the 10 pg mL^–1 range.

     

Mode of binding of the cytotoxic alkaloid berberine with the double helix oligonucleotide d(AAGAATTCTT)(2)

Berberine, an isoquinoline plant alkaloid, belongs to the structural class of protoberberines. Recently, the ability of these compounds to act as Topoisomerase I or II poisons, was related to the antitumor activity. The binding of protoberberins to DNA has been studied and the partial intercalation into the double helix has been considered responsible for their activity. We have studied the interaction of berberine with the double helix oligonucleotides d(AAGAATTCTT)(2), d(GCGATCGC)(2), d(CGTATACG)(2), d(CGTACG)(2), 5'-d(ACCTTTTTGATGT)-3'/5(ACATCAAAAAGGT)-3' and with the single strand 5'-d(ACATCAAAAAGGT)-3', by 1H, 31P NMR and UV spectroscopy. Phosphorus resonance experiments were performed to detect small conformational changes of the phosphoribose backbone, in the case that an intercalation process occurs. Our data reveal that berberine does not intercalate into the duplexes studied, and binds preferentially to AT rich sequences. The structure of the complex with d(AAGAATTCTT)(2) was determined by using proton 2D NOESY spectra, which allowed to obtain several NOE contacts between the drug and the nucleotide. Structural models were built up by Molecular Mechanics (MM) and Molecular Dynamics (MD) calculations, by using the inter-proton distances derived from the NOE values. Berberine results to be located in the minor groove, lying with the convex side on the helix groove and presenting the positively charged nitrogen atom close to the negative ionic surface of the oligomer. The large 1H chemical shifts variation, observed for the drug when it is added to the above duplexes, as well as to the single strand oligomer, was interpreted with non-specific ionic interactions. The binding constants were measured by UV and NMR spectroscopy. They are strongly affected by the ionic strength and by the self-association process, which commonly occurs with this type of drugs. A dimerisation constant was measured and the value was included in the calculations of the binding constants. The results obtained show that the non-specific ionic interactions represent the major contribution to the values of the binding constants. These parameters, as well as the protons chemical shift variation of the ligand, are thus not diagnostic for the identification of a drug/DNA complex.     

Concentration-dependent, size-independent toxicity of citrate capped AuNPs in Drosophila melanogaster

The expected potential benefits promised by nanotechnology in various fields have led to a rapid increase of the presence of engineered nanomaterials in a high number of commercial goods. This is generating increasing questions about possible risks for human health and environment, due to the lack of an in-depth assessment of the physical/chemical factors responsible for their toxic effects. In this work, we evaluated the toxicity of monodisperse citrate-capped gold nanoparticles (AuNPs) of different sizes (5, 15, 40, and 80 nm) in the model organism Drosophila melanogaster, upon ingestion. To properly evaluate and distinguish the possible dose- and/or size-dependent toxicity of the AuNPs, we performed a thorough assessment of their biological effects, using two different dose-metrics. In the first approach, we kept constant the total surface area of the differently sized AuNPs (Total Exposed Surface area approach, TES), while, in the second approach, we used the same number concentration of the four different sizes of AuNPs (Total Number of Nanoparticles approach, TNN). We observed a significant AuNPs-induced toxicity in vivo, namely a strong reduction of Drosophila lifespan and fertility performance, presence of DNA fragmentation, as well as a significant modification in the expression levels of genes involved in stress responses, DNA damage recognition and apoptosis pathway. Interestingly, we found that, within the investigated experimental conditions, the toxic effects in the exposed organisms were directly related to the concentration of the AuNPs administered, irrespective of their size.     

An Automated High Throughput Screening-Compatible Assay to Identify Regulators of Stem Cell Neural Differentiation

The use of Embryonic Stem Cells (ESCs) holds considerable promise both for drug discovery programs and the treatment of degenerative disorders in regenerative medicine approaches. Nevertheless, the successful use of ESCs is still limited by the lack of efficient control of ESC self-renewal and differentiation capabilities. In this context, the possibility to modulate ESC biological properties and to obtain homogenous populations of correctly specified cells will help developing physiologically relevant screens, designed for the identification of stem cell modulators. Here, we developed a high throughput screening-suitable ESC neural differentiation assay by exploiting the Cell ^maker robotic platform and demonstrated that neural progenies can be generated from ESCs in complete automation, with high standards of accuracy and reliability. Moreover, we performed a pilot screening providing proof of concept that this assay allows the identification of regulators of ESC neural differentiation in full automation.