A protein which masks galactose receptor mediated phage susceptibility in Lactococcus lactis subsp. lactis MPL56

A 28.5-kb plasmid, isolated from Lactococcus lactis subsp. lactis MPL56, causes complete inhibition of four lactococcal phages. Cell wall characteristics of wild-type strain MPL56 were compared with its 28.5 kb plasmid-cured, phage-sensitive derivative MPL56-22. After proteolytic enzyme treatments, adsorption of phages occurred at high levels, an example is 94.6–98.5% in MPL56 cells. Analysis of cell wall extracts of MPL56-22 by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) indicated that the only difference between strains was the 55.4 kDa band in protein patterns of MPL56. Adsorption of the four phages was completely inhibited when MPL56-22 cells were subjected to SDS, Triton-X-100, HCl and NaOH treatments. Lectins that were specific for glucose/mannose and N-acetylglucosamine did not prevent adsorption of phages in cell wall extracts of MPL 56-22. However a lectin specific for galactose (MCA; Momordica charantia ) completely inhibited adsorption of these phages in cell wall extracts of MPL56-22. HPLC patterns of cell wall carbohydrates of MPL56-22 and its HCl treated preparations showed that the most prevalent difference was the galactose on untreated MPL56-22 cell wall chromatograms.     

A Novel Artificially Humanized Anti-Cripto-1 Antibody Suppressing Cancer Cell Growth

Cripto-1 is a member of the EGF-CFC/FRL1/Cryptic family and is involved in embryonic development and carcinogenesis. We designed a novel anti-Cripto-1 artificial antibody and assessed the recognition to the antigen and the potential to suppress the growth of cancer stem cells. First, single chain antibody clones were isolated by bio-panning with the affinity to recombinant Cripto-1 protein from our original phage-display library. Then, the variable regions of heavy chain VH and light chain VL in each clone were fused to constant regions of heavy chain CH and light chain CL regions respectively. These fused genes were expressed in ExpiCHO-S cells to produce artificial humanized antibodies against Cripto-1. After evaluation of the expression levels, one clone was selected and the anti-Cripto-1 antibody was produced and purified. The purified antibody showed affinity to recombinant Cripto-1 at 1.1 pmol and immunoreactivity to cancer tissues and cell lines. The antibody was available to detect the immunoreactivity in tissue microarrays of malignant tumors as well as in Cripto-1 overexpressing cells. Simultaneously, the antibody exhibited the potential to suppress the growth of human colon cancer derived GEO cells overexpressing Cripto-1 with IC₅₀ at approximately 110 nM. The artificially humanized antibody is proposed to be a good candidate to target cancer cells overexpressing Cripto-1.     

In Vitro Characterization of Neutralizing Hen Antibodies to Coxsackievirus A16

Coxsackievirus A16 (CA16) is one of the major causative agents of hand, foot, and mouth disease (HFMD). Children aged <5 years are the most affected by CA16 HFMD globally. Although clinical symptoms of CA16 infections are usually mild, severe complications, such as aseptic meningitis or even death, have been recorded. Currently, no vaccine or antiviral therapy for CA16 infection exists. Single-chain variable fragment (scFv) antibodies significantly inhibit viral infection and could be a potential treatment for controlling the infection. In this study, scFv phage display libraries were constructed from splenocytes of a laying hen immunized with CA16-infected lysate. The pComb3X vector containing the scFv genes was introduced into ER2738 Escherichia coli and rescued by helper phages to express scFv molecules. After screening with five cycles of bio-panning, an effective scFv antibody showing favorable binding activity to proteins in CA16-infected lysate on ELISA plates was selected. Importantly, the selected scFv clone showed a neutralizing capability against the CA16 virus and cross-reacted with viral proteins in EV71-infected lysate. Intriguingly, polyclonal IgY antibody not only showed binding specificity against proteins in CA16-infected lysate but also showed significant neutralization activities. Nevertheless, IgY-binding protein did not cross-react with proteins in EV71-infected lysate. These results suggest that the IgY- and scFv-binding protein antibodies provide protection against CA16 viral infection in in vitro assays and may be potential candidates for treating CA16 infection in vulnerable young children.     

Antibody Libraries as Tools to Discover Functional Antibodies and Receptor Pleiotropism

Most antibodies currently in use have been selected based on their binding affinity. However, nowadays, antibodies that can not only bind but can also alter the function of cell surface signaling components are increasingly sought after as therapeutic drugs. Therefore, the identification of such functional antibodies from a large antibody library is the subject of intensive research. New methods applied to combinatorial antibody libraries now allow the isolation of functional antibodies in the cellular environment. These selected agonist antibodies have provided new insights into important issues of signal transduction. Notably, when certain antibodies bind to a given receptor, the cell fate induced by them may be the same or different from that induced by natural agonists. In addition, combined with phenotypic screening, this platform allows us to discover unexpected experimental results and explore various phenomena in cell biology, such as those associated with stem cells and cancer cells.     

Chemical Modification of Phage-Displayed Helix-Loop-Helix Peptides to Construct Kinase-Focused Libraries

Conformationally constrained peptides hold promise as molecular tools in chemical biology and as a new modality in drug discovery. The construction and screening of a target-focused library could be a promising approach for the generation of de novo ligands or inhibitors against target proteins. Here, we have prepared a protein kinase-focused library by chemically modifying helix-loop-helix (HLH) peptides displayed on phage and subsequently tethered to adenosine. The library was screened against aurora kinase A (AurA). The selected HLH peptide Bip - 3 retained the α-helical structure and bound to AurA with a K_D value of 13.7 μM. Bip - 3 and the adenosine-tethered peptide Bip - 3 - Adc provided IC₅₀ values of 103 μM and 7.7 μM, respectively, suggesting that Bip - 3 - Adc bivalently inhibited AurA. In addition, the selectivity of Bip - 3 - Adc to several protein kinases was tested, and was highest against AurA. These results demonstrate that chemical modification can enable the construction of a kinase-focused library of phage-displayed HLH peptides.     

Bias in bacteriophage morphological classification by transmission electron microscopy due to breakage or loss of tail structures

Virtually every study that has used transmission electron microscopy (TEM) to estimate viral diversity has acknowledged that loss of phage tails during sample preparation may have biased the results. However, the magnitude of this potential bias has yet to be constrained. To characterize biases in virus morphological diversity due to tail loss, six phage strains representing the order Caudovirales were inoculated into sterile sediments and soils. Phage particles were then extracted using standard methods. Morphologies of extracted phage particles were compared to those of unmanipulated control samples to determine the extent of tail breakage incurred by extraction procedures. Podoviruses exhibited the smallest frequency of tail loss during extraction (1.2-14%), myoviruses were moderately susceptible to tail breakage (15-40%), and siphoviruses were highly susceptible (32-76%). Thus, TEM assessments of viral diversity in soils or sediments by distribution of tail morphologies may be biased toward podoviruses and virions lacking tails, while simultaneously underestimating the abundance of siphoviruses. However, since the majority of viral capsids observed under TEM were intact, estimates of viral diversity based on the distribution of capsid diameters may provide a more reliable basis for morphological comparisons within and across ecosystems.     

Characterization and Comparative Genomics Analysis of a New Bacteriophage BUCT610 against Klebsiella pneumoniae and Efficacy Assessment in Galleria mellonella Larvae

The spread of multidrug-resistant Klebsiella pneumoniae (MDR-KP) has become an emerging threat as a result of the overuse of antibiotics. Bacteriophage (phage) therapy is considered to be a promising alternative treatment for MDR-KP infection compared with antibiotic therapy. In this research, a lytic phage BUCT610 was isolated from hospital sewage. The assembled genome of BUCT610 was 46,774 bp in length, with a GC content of 48%. A total of 83 open reading frames (ORFs) and no virulence or antimicrobial resistance genes were annotated in the BUCT610 genome. Comparative genomics and phylogenetic analyses showed that BUCT610 was most closely linked with the Vibrio phage pYD38-A and shared 69% homology. In addition, bacteriophage BUCT610 exhibited excellent thermal stability (4–75 °C) and broad pH tolerance (pH 3–12) in the stability test. In vivo investigation results showed that BUCT610 significantly increased the survival rate of Klebsiella pneumonia-infected Galleria mellonella larvae from 13.33% to 83.33% within 72 h. In conclusion, these findings indicate that phage BUCT610 holds great promise as an alternative agent with excellent stability for the treatment of MDR-KP infection.     

Norovirus and MS2 inactivation kinetics of UV-A and UV-B with and without TiO2

Germicidal ultraviolet, such as 254-nm UV-C, is a common method of disinfection of pathogenic enteric viruses. However, the disinfection efficacies of UV-A or -B in terms of inactivating waterborne viruses such as norovirus have not been characterized. We evaluated the inactivation kinetics of MS2 bacteriophage and murine norovirus (MNV), a surrogate of human norovirus (NoV), by UV-A and -B. In addition to UV disinfection, we further investigated whether the presence of TiO₂ could enhance the virus inactivation kinetics of UV-A and -B. Both MS2 and MNV were highly resistant to UV-A. However, the addition of TiO₂ enhanced the efficacy of UV-A for inactivating these viruses. UV-A dose of 1379 mJ/cm² resulted in a 4 log₁₀ reduction. In comparison, UV-B alone effectively inactivated both MS2 and MNV, as evidenced by the 4 log₁₀ reduction by 367 mJ/cm² of UV-B. The addition of TiO₂ increased the inactivation of MS2; however, it did not significantly increase the efficacy of UV-B disinfection for inactivating MNV. When these treatments were applied to field water such as groundwater, the results were generally consistent with the laboratory findings. Our results clearly indicated that UV-B is useful for the disinfection of waterborne norovirus. However, MNV was quite resistant to UV-A, and UV-A effectively inactivated the tested viruses only when used in combination with TiO₂.