Efficient double-stranded DNA cleavage by artificial zinc-finger nucleases composed of one zinc-finger protein and a single-chain FokI dimer

Zinc-finger–FokI nucleases (ZFNs) are useful for manipulating genomic DNA, but two ZFNs are required to cleave one site of double-stranded DNA (dsDNA), which limits the choice of targets. To refine ZFN technology, we constructed artificial zinc-finger nucleases containing an artificial zinc-finger protein (AZP) and a single-chain FokI dimer with nine different peptide linkers between two FokI molecules (designated AZP–scFokI). DNA cleavage assays revealed that the AZP–scFokI variant possessing the longest peptide linker cleaved dsDNA with equal or greater reactivity than the corresponding AZP–FokI dimer. The DNA cleavage pattern of AZP–scFokI suggests that the enhanced dsDNA cleavage was due to increased formation of FokI dimer in AZP–scFokI. Furthermore, we demonstrated that AZP–scFokI site-specifically cleaved its target DNA due to the AZP moiety discriminating one base pair difference. Thus, a single AZP–scFokI molecule is able to cleave dsDNA efficiently and site-specifically, and enhances the usefulness of the ZFN approach.     

Inhibition of DNA replication of human papillomavirus by artificial zinc finger proteins

Recently, we demonstrated that plant DNA virus replication was inhibited in planta by using an artificial zinc finger protein (AZP) and created AZP-based transgenic plants resistant to DNA virus infection. Here we apply the AZP technology to the inhibition of replication of a mammalian DNA virus, human papillomavirus type 18 (HPV-18). Two AZPs, designated AZP(HPV)-1 and AZP(HPV)-2, were designed by using our nondegenerate recognition code table and were constructed to block binding of the HPV-18 E2 replication protein to the replication origin. Both of the newly designed AZPs had much higher affinities towards the replication origin than did the E2 protein, and they efficiently blocked E2 binding in vitro. In transient replication assays, both AZPs inhibited viral DNA replication, especially AZP(HPV)-2, which reduced the replication level to approximately 10%. We also demonstrated in transient replication assays, using plasmids with mutant replication origins, that AZP(HPV)-2 could precisely recognize the replication origin in mammalian cells. Thus, it was demonstrated that the AZP technology could be applied not only to plant DNA viruses but also to mammalian DNA viruses.     

Hospital admissions for skin and soft tissue infections in a population with endemic scabies: A prospective study in Fiji, 2018–2019

Scabies is an important predisposing factor for impetigo but its role in more serious skin and soft tissue infections (SSTIs) is not well understood. Information is limited on incidence of SSTIs in the presence of endemic scabies. We conducted a prospective study of hospital admissions for SSTIs in the Northern Division of Fiji (population: 131,914). Prospective surveillance for admissions with impetigo, abscess, cellulitis, wound infection, pyomyositis, necrotizing fasciitis, infected scabies, and crusted scabies was conducted at the Division’s referral hospital between 2018 to 2019. Information was collected on demographic characteristics, clinical features, microbiology, treatment and outcomes. Over the study period, 788 SSTI admissions were recorded corresponding to a population incidence 647 per 100,000 person-years (95%CI 571–660). Incidence was highest at the extremes of age with peak incidence in children aged <5 years (908 per 100,000) and those aged ≥65 years (1127 per 100,000). Incidence was 1.7 times higher among the Indigenous Fijian population (753 per 100,000) compared to other ethnicities (442 per 100,000). Overall case fatality rate was 3.3%, and 10.8% for those aged ≥65 years. Scabies was diagnosed concurrently in 7.6% of all patients and in 24.6% of admitted children <5 years. There is a very high burden of hospital admissions for SSTIs in Fiji compared to high-income settings especially among the youngest, oldest and indigenous population which is concordant with scabies and impetigo distribution in this population. Our findings highlight the need for strategies to reduce the burden of SSTIs in Fiji and similar settings.     

Effects of Zn(II) complex with vitamins C and U, and carnitine on metabolic syndrome model rats

The insulinomimetic activity of a Zn(ii) complex is reported. The effects of the Zn(ii) complex with ascorbic acid (Vitamin C; VC), methylmethionine sulfonium chloride (Vitamin U; VU) and l-carnitine were assessed in diet-induced metabolic syndrome model rats. Zn(VU)(2)Cl(2) and Zn(VC)Cl(2) were suggested to be useful supplementary materials for preventing metabolic syndrome by reducing visceral adipose tissues or accelerating blood fluidity.     

Synergistic effects of 2A-mediated polyproteins on the production of lignocellulose degradation enzymes in tobacco plants

Cost-effective bioethanol production requires a supply of various low-cost enzymes that can hydrolyse lignocellulosic materials consisting of multiple polymers. Because plant-based enzyme expression systems offer low-cost and large-scale production, this study simultaneously expressed β-glucosidase (BglB), xylanase (XylII), exoglucanase (E3), and endoglucanase (Cel5A) in tobacco plants, which were individually fused with chloroplast-targeting transit peptides and linked via the 2A self-cleaving oligopeptideex from foot-and-mouth disease virus (FMDV) as follows: [RsBglB-2A-RaCel5A], [RsXylII-2A-RaCel5A], and [RsE3-2A-RaCel5A]. The enzymes were targeted to chloroplasts in tobacco cells and their activities were confirmed. Similarly to the results of a transient assay using Arabidopsis thaliana protoplasts, when XylII was placed upstream of the 2A sequence, the [RsXylII-2A-RaCel5A] transgenic tobacco plant had a more positive influence on expression of the protein placed downstream. The [RsBglB-2A-RaCel5A] and [RsE3-2A-RaCel5A] transgenic lines displayed higher activities towards carboxylmethylcellulose (CMC) compared to those in the [RsXylII-2A-RaCel5A] transgenic line. This higher activity was attributable to the synergistic effects of the different cellulases used. The [RsBglB-2A-RaCel5A] lines exhibited greater efficiency (35-74% increase) of CMC hydrolysis when the exoglucanase CBHII was added. Among the various exoglucanases, E3 showed higher activity with the crude extract of the [RsBglB-2A-RaCel5A] transgenic line. Transgenic expression of 2A-mediated multiple enzymes induced synergistic effects and led to more efficient hydrolysis of lignocellulosic materials for bioethanol production.     

Analysis of Trace and Major Elements in Bronchoalveolar Lavage Fluid of Mycoplasma Bronchopneumonia in Calves

The aim of this study was to evaluate the reliability and effectiveness of direct determination of trace and major element concentrations in bronchoalveolar lavage fluid samples from Holstein calves with Mycoplasma bronchopneumonia (n = 21) and healthy controls (n = 20). The samples were obtained during bronchoscopy using a standard examination method. A total of 18 elements (aluminum, bromine, calcium, chlorine, chromium, copper, iron, potassium, magnesium, manganese, molybdenum, nickel, phosphorous, sulfur, silicon, strontium, titanium, and zinc) were detected by particle-induced X-ray emission. The average bromine, iron, potassium, magnesium, and phosphorous concentrations were higher in calves with bronchopneumonia than in controls (p < 0.05). They were found to have higher amounts of calcium and zinc, and a higher zinc–copper ratio than that in healthy calves (p < 0.001). Based on the receiver operating characteristics curves, we propose a diagnostic cutoff point for zinc–copper ratio for identification of Mycoplasma pneumonia of 8.676. Our results indicate that assessment of the elemental composition of broncholaveolar lavage fluid is a promising diagnostic tool for Mycoplasma bronchopneumonia.