Whole-bacterium ribosome display selection for isolation of highly specific anti-Staphyloccocus aureus Affitins for detection- and capture-based biomedical applications

Detection and capture methods using antibodies have been developed to ensure identification of pathogens in biological samples. Though antibodies have many attractive properties, they also have limitations and there are needs to expand the panel of available affinity proteins with different properties. Affitins, that we developed from the Sul7d proteins, are a solid class of affinity proteins, which can be used as substitutes to antibodies or to complement them. We report the generation and characterization of antibacterial Affitins with high specificity for Staphylococcus aureus. For the first time, ribosome display selections were carried out using whole-living-cell and naïve combinatorial libraries, which avoid production of protein targets and immunization of animals. We showed that Affitin C5 exclusively recognizes S. aureus among dozens of strains, including clinical ones. C5 binds staphylococcal Protein A (SpA) with a K _D of 108 ± 2 nM and has a high thermostability (T _m = 77.0°C). Anti-S. aureus C5 binds SpA or bacteria in various detection and capture applications, including ELISA, western blot analysis, bead-fishing, and fluorescence imaging. Thus, novel anti-bacteria Affitins which are cost-effective, stable, and small can be rapidly and fully designed in vitro with high affinity and specificity for a surface-exposed marker. This class of reagents can be useful in diagnostic and biomedical applications.     

Biochemical and immunochemical characterization of boar sperm flagellar protein with role in hyperactivation/capacitation process

Investigations on specific and functionally active sperm antigens could bring about the elucidation of the mechanisms of gamete interaction and help the search for new approaches in prognosis and regulation of fertility. Previously, we reported that the monoclonal antibody (Mab) 3G4 against capacitated boar spermatozoa was capable of inhibiting boar sperm-porcine zona pellucida binding due to its inhibitory effect on sperm hyperactivation and capacitation. The cell and tissue specificity of Mab 3G4 was demonstrated in indirect immunofluorescence (IIF) and ELISA experiments against spermatozoa from different vertebrate species, as well as against extracts of boar reproductive and somatic organs. In the present IIF experiments, it was shown that Mab 3G4 recognized an antigen determinant on the flagellar midpiece region of ejaculated and capacitated boar spermatozoa. It was speculated that the Mab 3G4-corresponding antigen participates in pyruvate/lactate metabolism because of its specific localization in the sperm structure, which is responsible for producing forward motility and its involvement in processes that require the metabolism of pyruvate and lactate. As a possible approach toward investigating the participation of Ag 3G4 in pyruvate/lactate metabolism, Mab 3G4's effect on lactate dehydrogenase (LDH) was examined. Using an electrophoretic approach we provided evidence that Mab 3G4 stimulates LDH activity in the Triton X-100 and NP40 protein fractions of capacitated boar spermatozoa. In addition, we found that LDH isoenzymes stimulated by Mab 3G4 are of gametic C type. In Western blot, under nonreducing conditions, Mab 3G4 identified a single protein band with a molecular weight of 140 kDa. The biochemical and immunochemical experiments provided evidence supporting the involvement of 3G4 antigen in the sperm pyruvate/lactate metabolism.     

DNA-PKcs, but not TLR9, is required for activation of Akt by CpG-DNA

CpG-DNA and its related synthetic CpG oligodeoxynucleotides (CpG-ODNs) play an important role in immune cell survival. It has been suggested that Akt is one of the CpG-DNA-responsive serine/threonine kinases; however, the target protein of CpG-DNA that leads to Akt activation has not been elucidated. Here, we report that ex vivo stimulation of bone marrow-derived macrophages (BMDMs) from mice lacking the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) results in defective phosphorylation and activation of Akt by CpG-DNA. Unexpectedly, loss of the Toll-like receptor 9 has a minimal effect on Akt activation in response to CpG-DNA. Further in vitro analysis using purified DNA-PK and recombinant Akt proteins reveals that DNA-PK directly induces phosphorylation and activation of Akt. In addition, in BMDMs, DNA-PKcs associates with Akt upon CpG-DNA stimulation and triggers transient nuclear translocation of Akt. Thus, our findings establish a novel role for DNA-PKcs in CpG-DNA signaling and define a CpG-DNA/DNA-PKcs/Akt pathway.     

Monoclonal antibody against a cell wall marker protein for embryogenic potential of <Emphasis Type="Italic">Dactylis glomerata</Emphasis> L. suspension cultures

We identified and isolated a monoclonal antibody (MAb 3G2) raised against extracellular proteins from microcluster cells of orchard grass (Dactylis glomerata L.) embryogenic suspension culture. MAb 3G2 recognized with high specificity an antigen ionically bound within the primary cell wall and in the culture medium of microcluster cells. Two-dimensional polyacrylamide gel analysis and blotting of proteins on PVDF membrane showed that MAb 3G2 detected a single polypeptide of apparent molecular mass of 48 kDa and an isoelectric point (pI) of 5.2, designated EP48. A transient expression during somatic embryogenesis was observed for EP48. Indirect immunofluorescence showed that this protein highly accumulated in the cell walls of some single cells, microclusters and partly in proembryogenic masses (PEMs), but not in globular embryos of the embryogenic cell line and microclusters from the non-embryogenic cell line. Signal intensity varied between individual cells of the same population and in successive stages of somatic embryo development. Screening of several D. glomerata L. embryogenic and non-embryogenic cell lines with MAb 3G2 indicated the presence of ECP48 in only embryogenic suspension cultures at early stages of embryo development long before morphological changes have taken place and thus it could serve as an early marker for embryogenic potential in D. glomerata L. suspension cultures.     

Condensin dysfunction in human cells induces nonrandom chromosomal breaks in anaphase,with distinct patterns for both unique and repeated genomic regions

Condensin complexes are essential for chromosome condensation and segregation in mitosis, while condensin dysfunction, among other pathways leading to chromosomal bridging in mitosis, may play a role in tumor genomic instability, including recently discovered chromotripsis. To characterize potential double-strand breaks specifically occurring in late anaphase, human chromosomes depleted of condensin were analyzed by γ-H2AX ChIP followed by high-throughput sequencing (ChIP-seq). In condensin-depleted cells, the nonrepeated parts of the genome were shown to contain distinct γ-H2AX enrichment zones 75% of which overlapped with known hemizygous deletions in cancers. Furthermore, some tandemly repeated DNA sequences, analyzed separately from the rest of the genome, showed significant γ-H2AX enrichment in condensin-depleted anaphases. The most commonly occurring targets of such enrichment included simple repeats, centromeric satellites, and rDNA. The two latter categories indicate that acrocentric human chromosomes are especially susceptible to breaks upon condensin deficiency. The genomic regions that are specifically destabilized upon condensin dysfunction may constitute a condensin-specific chromosome destabilization pattern.     

Rate transition and regulatory coupling in endocytosis of interferon-alpha and tumor necrosis factor-alpha in human epithelial tumor cells.

The time-dependent concentrations of interferon-alpha and tumor necrosis factor-alpha associated with the membrane and internalized by cells contain information on the kinetics of endocytosis and their cellular processing. This information can be reduced quantitatively by application of the respective compartmental models. In our studies of human epithelial tumor cells interacting with human interferon-alpha and human tumor necrosis factor-alpha, we accounted only for actual endocytosis and elimination of the tracer from cells by a novel method sensitive to changes in the rate of endocytosis, to the delay in tracer elimination, and to the nonlinear regulatory coupling between endocytosis and the internalized ligand. Data reduced by this method resulted in best-fit parameter values statistically superior to values obtained by previous methods (Bajzer et al., 1989). The results indicate a change with time in the rate of endocytosis of tumor necrosis factor-alpha and the inhibition of endocytosis by the endocytosed ligand-receptor complex. We conclude that sorting and processing of interferon-alpha and tumor necrosis factor-alpha are restricted by the type of both the receptor and the cell.     

Physical activity and exercise as an essential medical strategy for the COVID-19 pandemic and beyond

COVID-19 disease has been a problem in today’s society, which has worldwide effects on different areas, especially on the economy; also, from a health perspective, the disease affects the daily life quality. Physical activity is one major positive factor with regard to enhancing life quality, as it can improve the whole psychological, social, and physical health conditions. Current measures such as social distancing are focused on preventing the viral spread. However, the consequences on other areas are yet to be investigated. Elderly, people with chronic diseases, obese, and others benefit largely from exercise from the perspective of improved health, and preventive measures can drastically improve daily living. In this article, we elaborate the effects of exercise on the immune system and the possible strategies that can be implemented toward greater preventive potential.     

Lipidation by the Host Prenyltransferase Machinery Facilitates Membrane Localization of Legionella pneumophila Effector Proteins

The intracellular human pathogen Legionella pneumophila translocates multiple proteins in the host cytosol known as effectors, which subvert host cellular processes to create a membrane-bound organelle that supports bacterial replication. It was observed that several Legionella effectors encode a prototypical eukaryotic prenylation CAAX motif (where C represents a cysteine residue and A denotes an aliphatic amino acid). These bacterial motifs mediated posttranslational modification of effector proteins resulting in the addition of either a farnesyl or geranylgeranyl isoprenyl lipid moiety to the cysteine residue of the CAAX tetrapeptide. Lipidation enhanced membrane affinity for most Legionella CAAX motif proteins and facilitated the localization of these effector proteins to host organelles. Host farnesyltransferase and class I geranylgeranyltransferase were both involved in the lipidation of the Legionella CAAX motif proteins. Perturbation of the host prenylation machinery during infection adversely affected the remodeling of the Legionella-containing vacuole. Thus, these data indicate that Legionella utilize the host prenylation machinery to facilitate targeting of effector proteins to membrane-bound organelles during intracellular infection.