Plant Expression of Hydrophobin Fused K39 Antigen for Visceral Leishmaniasis Immunodiagnosis.

Visceral leishmaniasis is a Neglected Tropical Disease of high mortality caused by the protozoan Leishmania infantum. Its transmission cycle is complex, and it has in the domestic dog its main reservoir. The diagnostic tests currently used rely on prokaryotic systems' proteins, but their low sensitivity increases the disease's burden. The plant transient expression of recombinant proteins allows the production of complex antigens. However, this system has limited competitiveness against the bacterial production of purified antigens. Thus, we have shown that the L. infantum K39 antigen's fusion to a hydrophobin allows its production for diagnostic tests without the need for intensive purification. The sera of naturally infected dogs specifically detect the semi-purified rK39-HFBI protein. The test validation against a panel of 158 clinical samples demonstrates the platform's viability, resulting in sensitivity and specificity of 90.7 and 97.5%, respectively. Thus, the use of semi-purified antigens fused to hydrophobins can become the standard platform for large-scale antigens production to expand diagnostic tests for other human and veterinary diseases worldwide.

Transient Expression of Dengue Virus NS1 Antigen in Nicotiana benthamiana for Use as a Diagnostic Antigen.

Dengue is a viral disease that represents a significant threat to global public health since billions of people are now at risk of infection by this mosquito-borne virus. The implementation of extensive screening tests is indispensable to control this disease, and the Dengue virus non-structural protein 1 (NS1) is a promising antigen for the serological diagnosis of dengue fever. Plant-based systems can be a safe and cost-effective alternative for the production of dengue virus antigens. In this work, two strategies to produce the dengue NS1 protein in Nicotiana benthamiana leaves were evaluated: Targeting NS1 to five different subcellular compartments to assess the best subcellular organelle for the expression and accumulation of NS1, and the addition of elastin-like polypeptide (ELP) or hydrophobin (HFBI) fusion tags to NS1. The transiently expressed proteins in N. benthamiana were quantified by Western blot analysis. The NS1 fused to ELP and targeted to the ER (NS1 ELP-ER) showed the highest yield (445 mg/kg), approximately a forty-fold increase in accumulation levels compared to the non-fused protein (NS1-ER), representing the first example of transient expression of DENV NS1 in plant. We also demonstrated that NS1 ELP-ER was successfully recognized by a monoclonal anti-dengue virus NS1 glycoprotein antibody, and by sera from dengue virus-infected patients. Interestingly, it was found that transient production of NS1-ER and NS1 ELP-ER using vacuum infiltration of whole plants, which is easier to scale up, rather than syringe infiltration of leaves, greatly improved the accumulation of NS1 proteins. The generated plant made NS1, even without extensive purification, showed potential to be used for the development of the NS1 diagnostic tests in resource-limited areas where dengue is endemic.

Addition of L-arginine to the fertilization medium enhances subsequent bovine embryo development rates.

Although L-Arginine (ARG) has been reported as a promising bovine sperm capacitation agent, its effects on embryo development are still poorly understood. Herein, we compared the effects of ARG and/or heparin (HEP) addition to the fertilization medium for bovine oocytes on sperm capacitation and embryo development. We chose 10 mM ARG based on blastocyst development rates in a titration experiment. Addition of ARG and/or HEP to the fertilization medium resulted in similar rates of blastocyst development (P > 0.05). However, when ARG, but not HEP, was combined with a nitric oxide (NO) synthase inhibitor (N-Nitro-L-ARG-methyl ester, 10 mM) blastocyst development was decreased (P < 0.05). To assess the effects on capacitation, bovine sperm were incubated for 0, 3, and 6 hours in fertilization medium containing ARG and/or HEP and/or N-Nitro-L-ARG-methyl esterand acrosomal exocytosis rates were evaluated using fluorescein isothiocyanate conjugated Pisum sativum lectin (FITC-PSA) staining and flow cytometry. With HEP, acrosomal exocytosis rates were highest by 3 hours of incubation; however, by 6 hours, rates were similar for HEP and/or ARG (P > 0.05) and higher than those in control media (P < 0.05). Although both ARG and HEP increased sperm NO production (P < 0.05), combination with L-NAME only precluded acrosomal exocytosis when ARG added alone in the medium (P > 0.05). These results suggest that although both ARG and HEP supported sperm capacitation, only the effects of the former were driven via NO production. Moreover, ARG was also as effective as HEP at improving blastocyst development rates. Therefore, ARG may be used as a low-cost alternative sperm capacitation agent for bovine in vitro embryo production.

Novel antibiofilm chemotherapy targets exopolysaccharide synthesis and stress tolerance in Streptococcus mutans to modulate virulence expression in vivo.

Fluoride is the mainstay of dental caries prevention, and yet current applications offer incomplete protection and may not effectively address the infectious character of the disease. Therefore, we evaluated the effectiveness of a novel combination therapy (CT; 2 mM myricetin, 4 mM tt-farnesol, 250 ppm of fluoride) that supplements fluoride with naturally occurring, food-derived, antibiofilm compounds. Treatment regimens simulating those experienced clinically (twice daily for ≤60 s) were used both in vitro over a saliva-coated hydroxyapatite biofilm model and in vivo with a rodent model of dental caries. The effectiveness of CT was evaluated based on the incidence and severity of carious lesions (compared to fluoride or vehicle control). We found that CT was superior to fluoride (positive control, P < 0.05); topical applications dramatically reduced caries development in Sprague-Dawley rats, all without altering the Streptococcus mutans or total populations within the plaque. We subsequently identified the underlying mechanisms through which applications of CT modulate biofilm virulence. CT targets expression of key Streptococcus mutans genes during biofilm formation in vitro and in vivo. These are associated with exopolysaccharide matrix synthesis (gtfB) and the ability to tolerate exogenous stress (e.g., sloA), which are essential for cariogenic biofilm assembly. We also identified a unique gene (SMU.940) that was severely repressed and may represent a potentially novel target; its inactivation disrupted exopolysaccharide accumulation and matrix development. Altogether, CT may be clinically more effective than current anticaries modalities, targeting expression of bacterial virulence associated with pathogenesis of the disease. These observations may have relevance for development of enhanced therapies against other biofilm-dependent infections.

Chemical composition and botanical origin of red propolis, a new type of brazilian propolis.

Red propolis is a new type of Brazilian propolis. This material, as well as the secretions of 20 plant species that are often mentioned as its probable botanical source, have been investigated by RP-HPTLC. Phytochemical evidence based on UV-VIS spectra, RP-HPLC and GC-MS, showed Dalbergia ecastophyllum (L.) Taub. to be the main source of red propolis in Alagoas state. The propolis and plant resin showed high relative percentages of the isoflavonoids 3-Hydroxy-8,9-dimethoxypterocarpan and medicarpin. To our knowledge this is the first report of the secretion of a leguminous species being the source of propolis.