Laccase immobilization on enzymatically functionalized polyamide 6,6 fibres

Polyamide matrices, such as membranes, gels and non-wovens, have been applied as supports for enzyme immobilization, although in literature the enzyme immobilization on woven nylon matrices is rarely reported. In this work, a protocol for a Trametes hirsuta laccase immobilization using woven polyamide 6,6 (nylon) was developed. A 2⁴ full factorial design was used to study the influence of pH, spacer (1,6-hexanediamine), enzyme and crosslinker concentration on the efficiency of immobilization. The factors enzyme dosage and spacer seem to have played a critical role in the immobilization of laccase onto nylon support. Under optimized working conditions (29 U mL⁻¹ of laccase, 10% of glutaraldehyde, pH = 5.5, with the presence of the spacer), the half-life time attained was about 78 h (18% higher than that of free enzyme), the protein retention was 30% and the immobilization yield was 2%. The immobilized laccase has potential for application in the continuous decolourization of textile effluents, where it can be applied into a membrane reactor.     

Biosynthesis of vitamin B12 by Propionibacterium freudenreichii subsp. shermanii ATCC 13673 using liquid acid protein residue of soybean as culture medium

Vitamin B12 deficiency still persists, mainly caused by low intake of animal food products affecting vegetarians, vegans, and populations of underdeveloped countries. In this study, we investigate the biosynthesis of vitamin B12 by potential probiotic bacterium using an agroindustry residue, the liquid acid protein residue of soybean (LAPRS), as a low-cost, animal derivate-free alternative culture medium. Cultures of Propionibacterium freudenreichii subsp. shermanii ATCC 13673 growing in LAPRS for vitamin B12 biosynthesis were studied using the Plackett–Burman experimental approach, followed by a central composite design 2² to optimize the concentration of significant variables. We also performed a proteolytic treatment of LAPRS and evaluated the optimized–hydrolyzed medium influence on the microbial growth and metabolism in shaker flask and bioreactor experiments. In this all-plant source medium, P. freudenreichii subsp. shermanii produced high concentrations of cells and high amounts of vitamin B12 (0.6 mg/g cells) after process optimization. These results suggest the possibility of producing vitamin B12 by a potential probiotic bacterium in a very cheap, animal derivate-free medium to address the needs of specific population groups, at the same time reducing the production costs of this essential vitamin.     

Airborne pollen in Padua (NE-Italy): A comparison between two pollen samplers

During recent years a gradual decrease inallergenic airborne pollen concentration hasbeen observed in the monitoring station ofPadua (Italy). Because technical checks of thesampler were not able to explain this trend,the results obtained from two twinpollen-samplers (Lanzoni VPPS 2000), placed twometres apart, were compared.An eight-week sampling was carried out duringthe year 2000 from July to September.Subsequent analysis revealed no statisticallysignificant difference between the dataobtained with the two instruments. On the otherhand, both samplers captured high levels offungal spores. We conclude that the observednegative trend in pollen count is real and notrelated to technical biases.     

Viroporins

Viroporins are a group of proteins that participate in several viral functions, including the promotion of release of viral particles from cells. These proteins also affect cellular functions, including the cell vesicle system, glycoprotein trafficking and membrane permeability. Viroporins are not essential for the replication of viruses, but their presence enhances virus growth. Comprising some 60-120 amino acids, viroporins have a hydrophobic transmembrane domain that interacts with and expands the lipid bilayer. Some viroporins also contain other motifs, such as basic amino acid residues or a domain rich in aromatic amino acids that confers on the protein the ability to interact with the interfacial lipid bilayer. Viroporin oligomerization gives rise to hydrophilic pores at the membranes of virus-infected cells. As the list of known viroporins steadily grows, recent research efforts focus on deciphering the actions of the viroporins poliovirus 2B, alphavirus 6K, HIV-1 Vpu and influenza virus M2. All these proteins can enhance the passage of ions and small molecules through membranes depending on their concentration gradient. Future work will lengthen the list of viroporins and will provide a deeper understanding of their mechanisms of action.     

Oligomeric State and Thermal Stability of Apo- and Holo- Human Ornithine δ-Aminotransferase

Human ornithine δ-aminotransferase (hOAT) (EC 2.6.1.13) is a mitochondrial pyridoxal 5′-phosphate (PLP)-dependent aminotransferase whose deficit is associated with gyrate atrophy, a rare autosomal recessive disorder causing progressive blindness and chorioretinal degeneration. Here, both the apo- and holo-form of recombinant hOAT were characterized by means of spectroscopic, kinetic, chromatographic and computational techniques. The results indicate that apo and holo-hOAT (a) show a similar tertiary structure, even if apo displays a more pronounced exposure of hydrophobic patches, (b) exhibit a tetrameric structure with a tetramer-dimer equilibrium dissociation constant about fivefold higher for the apoform with respect to the holoform, and (c) have apparent T_m values of 46 and 67?°C, respectively. Moreover, unlike holo-hOAT, apo-hOAT is prone to unfolding and aggregation under physiological conditions. We also identified Arg217 as an important hot-spot at the dimer–dimer interface of hOAT and demonstrated that the artificial dimeric variant R217A exhibits spectroscopic properties, T_m values and catalytic features similar to those of the tetrameric species. This finding indicates that the catalytic unit of hOAT is the dimer. However, under physiological conditions the apo-tetramer is slightly less prone to unfolding and aggregation than the apo-dimer. The possible implications of the data for the intracellular stability and regulation of hOAT are discussed.     

Opsonizing properties of natural antibodies of rainbow trout, Oncorhynchus mykiss (Walbaum)

In order to confirm previous observations in which a protective effect of rainbow trout natural antibodies against furunculosis was suspected, phagocytosis studies wereconducted in vitro , using combinations of rainbow trout sera with high or low levels of natural antibodies and active or inactivated complement as opsonizing factors. Opsonization was observed in all the cases where complement was present, and to a lesser degree with sera containing only natural antibodies. The results confirm the prime importance of the complement system and provide additional evidence for a possible role of natural antibodies in antimicrobial defences.     

Adaptation of signature-tagged mutagenesis to Escherichia coli K1 and the infant-rat model of invasive disease

With the exception of the polysialic acid capsule (K1 antigen), little is known about other virulence factors needed for systemic infection by Escherichia coli K1, the leading cause of Gram-negative neonatal meningitis in humans. In this work, the functional genomics method of signature-tagged mutagenesis (STM) was adapted to E. coli K1 and the infant-rat model to identify non-capsule virulence genes. Validation of the method was demonstrated by the failure to recover a reconstructed acapsular mutant from bacterial pools used to systemically infect 5-day-old rats. Three new genes required for systemic disease were identified from a total of 192 mutants screened by STM (1.56% hit rate). Gut colonization, Southern blot hybridization, mixed-challenge infection, and DNA sequence analyses showed that the attenuating defects in the mutants were associated with transposon insertions in rfaL (O antigen ligase), dsbA (thiol:disulfide oxidoreductase), and a new gene, puvA (previously unidentified virulence gene A), with no known homologues. The results indicate the ability of STM to identify novel systemic virulence factors in E. coli K1.     

Role of PCIF1‐mediated 5′‐cap N6‐methyladeonsine mRNA methylation in colorectal cancer and anti‐PD‐1 immunotherapy

Adenosine N6‐methylation (m6A) and N6,2′‐O‐dimethylation (m6Am) are regulatory modifications of eukaryotic mRNAs. m6Am formation is catalyzed by the methyl transferase phosphorylated CTD‐interacting factor 1 (PCIF1); however, the pathophysiological functions of this RNA modification and PCIF1 in cancers are unclear. Here, we show that PCIF1 expression is upregulated in colorectal cancer (CRC) and negatively correlates with patient survival. CRISPR/Cas9‐mediated depletion of PCIF1 in human CRC cells leads to loss of cell migration, invasion, and colony formation in vitro and loss of tumor growth in athymic mice. Pcif1 knockout in murine CRC cells inhibits tumor growth in immunocompetent mice and enhances the effects of anti‐PD‐1 antibody treatment by decreasing intratumoral TGF‐β levels and increasing intratumoral IFN‐γ, TNF‐α levels, and tumor‐infiltrating natural killer cells. We further show that PCIF1 modulates CRC growth and response to anti‐PD‐1 in a context‐dependent mechanism with PCIF1 directly targeting FOS, IFITM3, and STAT1 via m6Am modifications. PCIF1 stabilizes FOS mRNA, which in turn leads to FOS‐dependent TGF‐β regulation and tumor growth. While during immunotherapy, Pcif1‐Fos‐TGF‐β, as well as Pcif1‐Stat1/Ifitm3‐IFN‐γ axes, contributes to the resistance of anti‐PD‐1 therapy. Collectively, our findings reveal a role of PCIF1 in promoting CRC tumorigenesis and resistance to anti‐PD‐1 therapy, supporting that the combination of PCIF1 inhibition with anti‐PD‐1 treatment is a potential therapeutic strategy to enhance CRC response to immunotherapy. Finally, we developed a lipid nanoparticles (LNPs) and chemically modified small interfering RNAs (CMsiRNAs)‐based strategy to silence PCIF1 in vivo and found that this treatment significantly reduced tumor growth in mice. Our results therefore provide a proof‐of‐concept for tumor growth suppression using LNP‐CMsiRNA to silence target genes in cancer.