Neuromodulation of Acid-Sensitive Ion Channels (ASICs) and Anti-Inflammatory Potential by Lichenxanthone in Adult Zebrafish (Danio rerio): Experimental and Docking Studies.

The xanthone lichenxanthone did not show toxic effects (LC50>1.0 mg/mL). lichenxanthone prevented nociceptive behavior induced by acidic saline, and its analgesic effect was blocked by amiloride, highlighting the involvement of neuromodulation of acid-sensitive ion channels (ASICs). In the analysis of anti-inflammatory activity, concentrations of 0.1 and 0.5 mg/mL of lichenxanthone reduced the edema induced by k-carrageenan 3.5 %, observed from the fourth hour of analysis. This effect was similar to that observed with ibuprofen (positive control). No leukocyte infiltrates were observed in lichenxanthone, suggesting that the compound acts in the acute inflammatory response. The results of the molecular docking study revealed that lichenxanthone exhibited better affinity energy when compared to the ibuprofen control against the two targets evaluated.

A Redox-Probe-Free Immunosensor Based on Electrocatalytic Prussian Blue Nanostructured Film One-Step-Prepared for Zika Virus Diagnosis.

The Zika virus (ZIKV) is a great concern for global health due to its high transmission, including disseminating through blood, saliva, urine, semen and vertical transmission. In some cases, ZIKV has been associated with microcephaly, neurological disorders, and Guillain−Barré syndrome. There is no vaccine, and controlling the disease is a challenge, especially with the co-circulation of the Dengue virus, which causes a severe cross-reaction due to the similarity between the two arboviruses. Considering that electrochemical immunosensors are well-established, sensitive, and practical tools for diagnosis, in this study we developed a sensor platform with intrinsic redox activity that facilitates measurement readouts. Prussian blue (PB) has a great ability to form electrocatalytic surfaces, dispensing redox probe solutions in voltammetric measurements. Herein, PB was incorporated into a chitosan−carbon nanotube hybrid, forming a nanocomposite that was drop-casted on a screen-printed electrode (SPE). The immunosensor detected the envelope protein of ZIKV in a linear range of 0.25 to 1.75 µg/mL (n = 8, p < 0.01), with a 0.20 µg/mL limit of detection. The developed immunosensor represents a new method for electrochemical measurements without additional redox probe solutions, and it is feasible for application in point-of-care diagnosis.

New insights into the recombinant proteins and monoclonal antibodies employed to immunodiagnosis and control of Zika virus infection: A review.

An emergent positive-stranded RNA virus, transmitted by mosquitoes with its first case of vertical transmission confirmed in 2015 in Brazil. The Zika virus (ZIKV) fever has received particular attention, mainly related to neurological diseases such as microcephaly in newborns. However, the laboratory diagnosis for ZIKV still faces some challenges due to its cross-reactivity with other flaviviruses, requiring a correct and differential diagnosis, contributing to the good prognosis of patients, especially in pregnant women. Among these, for early diagnosis, the CDC considers the RT-PCR the gold standard, more sensitive and specific, but expensive. Serological tests for the diagnosis of ZIKV can also be found beyond the period when the viral components are detectable in the serum. Inputs to produce more sensitive and specific diagnostic kits and the possibility of viral detection in less invasive samples are among the objectives of recent research on ZIKV. This review outlines recent advances in developing recombinant antigen and antibody-based diagnostic tools for the main flaviviruses in Northeast Brazil, such as ZIKV and Dengue virus (DENV).

Non-structural protein 1 from Zika virus: Heterologous expression, purification, and potential for diagnosis of Zika infections.

Zika virus (ZIKV) infections were associated with neurological disorders only after the Brazilian outbreak in 2015. The lack of vaccines and precise diagnosis requires a precise method to detect ZIKV infection. This study aimed to evaluate three ZIKV recombinant proteins for the development of ZIKV infections. Here, it was purified stable recombinant ZIKV Capsid (r-ZIKV-c), non-structural proteins NS1 (r-ZIKV-NS1), and NS3 (r-ZIKV-NS3) for detection of the infection by ZIKV in blood sera of patients. A commercial polyclonal antibody recognized the r-ZIKV-NS1. Here, among three proteins, NS1 showed the best result for diagnostic purposes using serum samples, despite the high similarity with NS1 from DENV, and could differentiate the infections. The recombinant NS1 was used to produce a monoclonal antibody to differentiate between DENV and ZIKV NS1. As for recombinant proteins, the result for r-ZIKV-NS1 values showed 77% and 100% sensitivity and specificity, respectively, in the IgM assay. Our data showed the protein could successfully differentiate between sera of ZIKV infected patients from sera of those not infected with the virus and differentiate from sera of DENV infected patients. Thus, the generated recombinant proteins have great potential for serological diagnosis of ZIKV in Brazil, where it is indispensable.

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.

Saliva proteomics from children with caries at different severity stages.

OBJECTIVE: To perform a comparative analysis of saliva protein profile of patients with early childhood caries at different levels of severity and caries-free individuals. MATERIALS AND METHODS: Stimulated saliva samples were collected from 126 children (2-6 years old), classified according to the ICDAS II, and divided into 3 groups (n = 42): caries-free (CF), enamel caries (EC), and dentine caries (DC). Samples were digested and analyzed by nanoUPLC coupled with a mass spectrometry. Data analyses were conducted with Progenesis QI for Proteomics Software v2.0. Gene Ontology (GO) terms and protein-protein interaction analysis were obtained. RESULTS: A total of 306 proteins (≈6 peptides) were identified. Among them, 122 were differentially expressed in comparisons among children with different caries status. Out of the 122 proteins, the proteins E2AK4 and SH3L2 were exclusively present in groups CF and EC, respectively, and 8 proteins (HAUS4, CAH1, IL36A, IL36G, AIMP1, KLHL8, KLH13, and SAA1) were considered caries-related proteins when compared to caries-free children; they were up-regulated proteins in the caries groups (EC and DC). CONCLUSION: The identification of exclusive proteins for caries-free or carious-related conditions may help in understanding the mechanisms of caries and predicting risk as well as advancing in caries control or anti-caries approaches.

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.

Antinociceptive activity of ethanolic extract of Azadirachta indica A. Juss (Neem, Meliaceae) fruit through opioid, glutamatergic and acid-sensitive ion pathways in adult zebrafish (Danio rerio).

Neem fruit (Azadirachta indica A. Juss.) are popularly used to treat infections, diarrhea, fever, bronchitis, skin diseases, infected burns and hypertension. Although the antinociceptive and anti-inflammatory potential of A. indica has already been investigated in experimental models of pain and inflammation in mice, the current research is the first to report the evaluation of the capacity of A. indica fruit ethanolic extract (EtFrNeem) in acute pain attenuation using the adult zebrafish (Danio rerio) as an alternative model to the use in rodents. EtFrNeem was submitted to antioxidant action, preliminary chemical prospecting, FT-IR and determination of phenol and flavonoid content tests. Subsequently, EtFrNeem was tested for acute nociception and abdominal inflammation, locomotor activity, and acute toxicity in adult zebrafish. Possible neuromodulation mechanisms were also evaluated. EtFrNeem showed low antioxidant activity, but was shown to be rich in flavonoids. EtFrNeem showed no anti-inflammatory action, did not alter the locomotor system, and it was not toxic. However, EtFrNeem significantly reduced the nociceptive behavior induced by formalin, glutamate and acidic saline, when compared to the control group. These effects of EtFrNeem were significantly similar to those of morphine, used as a positive control. The antinociceptive effect of EtFrNeem was inhibited by naloxone, ketamine and amiloride. EtFrNeem has the pharmacological potential for acute pain treatment and this effect is modulated by the opioid system, NMDA receptors and ASICs channels. These results lead us to studies of isolation and characterization of EtFrNeem bioactive principles, using adult zebrafish as an experimental model.

Orofacial antinociceptive effect of Mimosa tenuiflora (Willd.) Poiret.

Mimosa tenuiflora (Willd.) Poiret, popularly known in Brazil as "jurema-preta" is widely used against bronchitis, fever, headache and inflammation. Its antioxidant, anti-inflammatory and antinociceptive potential has already been reported. To assess the orofacial antinociceptive effect of M. tenuiflora, ethanolic extracts of M. tenuiflora (leaves, twigs, barks and roots) were submitted to in vitro tests of antioxidant activity. The extract with the highest antioxidant potential was partitioned and subjected to preliminary chemical prospecting, GC-MS, measurement of phenolic content and cytotoxicity tests of the fraction with the highest antioxidant activity. The nontoxic fraction with the highest antioxidant activity (FATEM) was subjected to tests of acute and chronic orofacial nociception and locomotor activity. The possible mechanisms of neuromodulation were also assessed. The EtOAc fraction, obtained from the ethanolic extract of M. tenuiflora barks, was the one with the highest antioxidant potential and nontoxic (FATEM), and Benzyloxyamine was the major constituent (34.27%). FATEM did not alter the locomotor system of mice and reduced significantly the orofacial nociceptive behavior induced by formalin, glutamate, capsaicin, cinnamaldehyde or acidic saline compared to the control group. FATEM also inhibited formalin- or mustard oil-induced temporomandibular nociception. In addition, it also reduced mustard oil-induced orofacial muscle nociception. However, FATEM did not alter hypertonic saline-induced corneal nociception. Neuropathic nociception was reversed by treatment with FATEM. The antinociceptive effect of FATEM was inhibited by naloxone, L-NAME and glibenclamide. FATEM has pharmacological potential for the treatment of acute and neuropathic orofacial pain and this effect is modulated by the opioid system, nitric oxide and ATP-sensitive potassium channels. These results lead us to studies of isolation and characterization of bioactive principles.

Larvicidal activity of Tagetes erecta against Aedes aegypti.

The aim of this study was to evaluate the activity of essential oil from Tagetes erecta against 3rd instars of Aedes aegypti and to determine the amounts of larvicidal thiophenes in all plant tissues. The oil obtained by steam distillation and analyzed by gas chromatography/mass spectrometry showed 14 compounds. The main compounds were piperitone (45.72%), D-limonene (9.67%), and piperitenone (5.89%). The essential oil was active against larvae of Ae. aegypti, with LC50 of 79.78 microg/ml and LC90 of 100.84 microg/ml. The larvicidal thiophene contents were higher in the roots and flowers as demonstrated by high-performance liquid chromatography analysis. Thus, T. erecta constitutes a good source of varied compounds showing larvicidal activity against Ae. aegypti.

Practical use of immobilized lysozyme for the remediation process of Escherichia coli in aqueous solution

The lysozyme enzyme was immobilized on vitreous surface (fragments with diameters of 0.3 and 1.0 mm) for remediation of the microorganism Escherichia coli JM 109 into fresh water and saline solutions with 0.9 percent NaCl (w/v). Characterization of enzymatic film was carried out by infrared spectroscopy and atomic force microscopy techniques. Bactericide activity of the enzyme was evaluated by spectrophotometric analysis. It was verified that the enzymatic film was strongly coupled with the vitreous surface. The topographic analysis demonstrated that the deposited film was uniform and homogeneous. It was observed bactericide activity of film deposited on vitreous surface with 0.3 mm in fresh and saline solutions. This fact was not verified to vitreous fragments with 1.0 mm of diameter.