Modification of the Trizol Method for the Extraction of RNA from Prorocentrum triestinum ACIZ_LEM2.

In samples of harmful algal blooms (HABs), seawater can contain a high abundance of microorganisms and elemental ions. Along with the hardness of the walls of key HAB dinoflagellates such as Prorocentrum triestinum, this makes RNA extraction very difficult. These components interfere with RNA isolation, causing its degradation, in addition to the complex seawater properties of HABs that could hinder RNA isolation for effective RNA sequencing and transcriptome profiling. In this study, an RNA isolation technique was established through the modification of the Trizol method by applying the Micropestle System on cell pellets of P. triestinum frozen at -20 °C, obtained from 400 mL of culture with a total of 107 cells/mL. The results of the modified Trizol protocol generated quality RNA samples for transcriptomics sequencing, as determined by their measurement in Analyzer Agilent 4150.

Development of an alternative saliva test for diagnosis of SARS-CoV-2 using TRIzol: Adapting to countries with lower incomes looking for a large-scale detection program.

The use of saliva for the diagnosis of SARS-CoV-2 has shown to be a good alternative to nasopharyngeal swabs (NPS), since it permits self-collection, avoids the exposure of healthy persons to infected patients, reduces waiting times, eliminates the need of personal protective equipment and is non-invasive. Yet current saliva testing is still expensive due to the need of specialized tubes containing buffers to stabilize the RNA of SARS-CoV-2 and inactivate the virus. These tubes are expensive and not always accessible in sufficient quantities. We now developed an alternative saliva testing method, using TRIzol for extraction, viral inactivation, and storage of SARS-CoV-2 RNA, combined with RT-qPCR, which was comparable in its performance to NPS. Paired saliva samples and NPS were taken from 15 asymptomatic healthcare workers and one patient with SARS-CoV-2. Further 13 patients with SARS-CoV-2 were only saliva-tested. All the tests were performed according to CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel. Saliva (4 mL) was taken in sterile 50 mL tubes, 1.5 mL TRIzol were added and mixed. Our results show that 5 µL of saliva RNA extracted with TRIzol allow for an adequate detection of the virus in patients positive for SARS-CoV-2 and was equally sensitive to NPS in TRIzol. We conclude that saliva testing using TRIzol is a recommendable method for diagnosis of SARS-CoV-2 since it has several advantages over currently used saliva tests: it can be done with normal sterile tubes, does not need cold-chain handling, is stable at room temperature, is non-invasive and less costly, making it more accessible for low-income countries. Cheaper saliva testing using TRIzol is especially relevant for low-income countries to optimize diagnosis and help define quarantine durations for families, healthcare workers, schools, and other public workplaces, thus decreasing infections and mortality caused by SARS-CoV-2.

Efficient method for isolation of high-quality RNA from Psidium guajava L. tissues.

Acquiring high-quality RNA in sufficient amounts is crucial in plant molecular biology and genetic studies. Several methods for RNA extraction from plants are available in the literature, mainly due to the great biochemical diversity present in each species and tissue, which can complicate or prevent the extraction. Psidium guajava (Myrtaceae family) is a perennial fruit tree of medicinal and economic value; nevertheless, only a few molecular studies are available for the species. One reason is the difficulty in obtaining RNA due to the content of the samples, which are rich in polyphenols, polysaccharides, and secondary metabolites. Furthermore, there are few studies available for the isolation of RNA from guava or Psidium samples, which hampers advances in the study of the genus. Here, quality and yields of RNA isolates were compared using six extraction protocols: two protocols based on the application of cetyltrimethylammonium bromide (CTAB) lysis buffer, one protocol which uses the TRIzol reagent, one which applies guanidine thiocyanate lysis buffer followed by organic phase extraction, and two commercial kits (PureLink RNA Mini Kit and RNeasy Plant Mini Kit). The CTAB-based method provided the highest RNA yields and quality for five different tissues (flower bud, immature leaf, young leaf, mature leaf, and root), genotypes, and stress conditions. For the most efficient protocol, the average yield of RNA from guava leaves was 203.06 µg/g of tissue, and the A260/A280 and A260/A230 ratios were 2.1 and 2.2, respectively. RT-qPCR analysis demonstrated that the purity of the samples was sufficient for molecular biology experiments. CTAB-based methods for RNA isolation were found to be the most efficient, providing the highest RNA yields and quality for tissues from P. guajava. Additionally, they were compatible for downstream RNA-based applications, besides being simple and cost-effective.

PHMB-loaded PDMS and its antimicrobial properties for biomedical applications.

Given the global panorama of demands in the health area, the development of biomaterials becomes irreducible for the maintenance and/or improvement in the quality of life of the human being. Aiming to reduce the impacts related to infections in the healing processes of the dermal structure, the present work proposes the development of polydimethylsiloxane (PDMS) based membranes with the incorporated polyhexamethylenebiguanide (PHMB) antimicrobial agent. In the present study, the antimicrobial and antibiofilm properties of polydimethylsiloxane (PDMS) films incorporated with 0.1, 0.3, and 0.5% (w/w) of polyhexamethylene biguanide (PHMB) were evaluated, aiming the development of a protective biomaterial that avoids cutaneous infections from the autochthonous and allochthonous microbiota. The disk diffusion of PHMB-loaded PDMS has shown the growth inhibition of Escherichia coli (ATCC 9637), Pseudomonas aeruginosa (ATCC 27953), Acinetobacter baumannii (ATCC 19606), Staphylococcus aureus (ATCC 6538), Staphylococcus epidermidis (ATCC 12228), Streptococcus pyogenes (ATCC 19615), Bacillus subtilis (ATCC 6633) and also yeast-like fungi Candida albicans, all microorganisms found on the epidermal surface. Likewise, the present study demonstrated low cytotoxicity of the PHMB-loaded PDMS on HaCaT and L929 cells at lower concentrations (0.1% w/w), indicating the possibility of using the developed material as a dressing for wounds, burns, and post-surgical procedures.

Magnetic ß-Cyclodextrin Nanosponges for Potential Application in the Removal of the Neonicotinoid Dinotefuran from Wastewater.

This article describes the use of ß-cyclodextrin-based carbonate nanosponges (NSs) decorated with superparamagnetic Fe3O4 nanoparticles to study and investigate the potential removal of dinotefuran (DTF) from wastewater. The NS-DTF inclusion compound was characterized by transmission electron microscopy (TEM), energy-dispersive spectroscopy (EDS), UV-visible spectroscopy (UV-VIS), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), X-ray powder diffraction (XRPD) and proton nuclear magnetic resonance (1H-NMR). The adsorption efficiency of NSs was evaluated as function of different contact times. The results confirmed that the NSs have a favourable sorption capacity for the chosen guest, as the polymers exhibited a maximum adsorption of 4.53 × 10-3 mmol/g for DTF. We also found that magnetic NSs show good reusability as they maintain their efficiency after eight adsorption and desorption cycles. Our studies and characterization by means of SEM, TEM, EDS, vibrating sample magnetometer (VSM) and UV-VIS also show that NSs with magnetic properties are excellent tools for insecticide removal from aqueous environments.

Chemosensitizing Effect of Cernumidine Extracted from Solanum cernuum on Bladder Cancer Cells in Vitro.

Cernumidine (CER) is a guanidinic alkaloid isolated from Solanum cernuum leaves. In this work, we investigated the cytotoxicity, chemosensitizing effect of cernumidine to cisplatin (cDDP) and the possible mechanism of action of the combination on bladder cancer cells. Cernumidine showed cytotoxicity and could sensitize bladder cancer cells to cisplatin. The combination of CER+cDDP inhibited cell migration on T24 cells. CER+cDDP down-regulated MMP-2/9 and p-ERK1/2, while it increased EGFR activity corroborating the observed cell migration inhibition. Down-regulation of Bcl-2 and up-regulation pro-apoptotic Bax and further depletion of the mitochondrial membrane potential (ΔΨm) indicates that mitochondria play a central role in the combination treatment inducing the mitochondrial signaling pathway of apoptosis in T24 cells. Our data showed that the alkaloid cernumidine is worthy of further studies as a chemosensitizing agent to be used in complementary chemotherapy.

Antibacterial, chemical and physical properties of sealants with polyhexamethylene guanidine hydrochloride.

The aim of this study was to evaluate the influence of polyhexamethylene guanidine hydrochloride (PHMGH) in the physico-chemical properties and antibacterial activity of an experimental resin sealant. An experimental resin sealant was formulated with 60 wt.% of bisphenol A glycol dimethacrylate and 40 wt.% of triethylene glycol dimethacrylate with a photoinitiator/co-initiator system. PHMGH was added at 0.5 (G0.5%), 1 (G1%), and 2 (G2%) wt.% and one group remained without PHMGH, used as control (GCTRL). The resin sealants were analyzed for degree of conversion (DC), Knoop hardness (KHN), and softening in solvent (ΔKHN), ultimate tensile strength (UTS), contact angle (θ) with water or α-bromonaphthalene, surface free energy (SFE), and antibacterial activity against Streptococcus mutans for biofilm formation and planktonic bacteria. There was no significant difference for DC (p > 0.05). The initial Knoop hardness ranged from 17.30 (±0.50) to 19.50 (± 0.45), with lower value for GCTRL (p < 0.05). All groups presented lower KHN after immersion in solvent (p < 0.05). The ΔKHN ranged from 47.22 (± 4.30) to 57.22 (± 5.42)%, without significant difference (p > 0.05). The UTS ranged from 54.72 (± 11.05) MPa to 60.46 (± 6.50) MPa, with lower value for G2% (p < 0.05). PHMGH groups presented no significant difference compared to GCTRL in θ (p > 0.05). G2% showed no difference in SFE compared to GCTRL (p > 0.05). The groups with PHMGH presented antibacterial activity against biofilm and planktonic bacteria, with higher antibacterial activity for higher PHMGH incorporation (p < 0.05). PHMGH provided antibacterial activity for all resin sealant groups and the addition up to 1 wt.% showed reliable physico-chemical properties, maintaining the caries-protective effect of the resin sealant over time.

Antibacterial, chemical and physical properties of sealants with polyhexamethylene guanidine hydrochloride

Abstract The aim of this study was to evaluate the influence of polyhexamethylene guanidine hydrochloride (PHMGH) in the physico-chemical properties and antibacterial activity of an experimental resin sealant. An experimental resin sealant was formulated with 60 wt.% of bisphenol A glycol dimethacrylate and 40 wt.% of triethylene glycol dimethacrylate with a photoinitiator/co-initiator system. PHMGH was added at 0.5 (G0.5%), 1 (G1%), and 2 (G2%) wt.% and one group remained without PHMGH, used as control (GCTRL). The resin sealants were analyzed for degree of conversion (DC), Knoop hardness (KHN), and softening in solvent (ΔKHN), ultimate tensile strength (UTS), contact angle (θ) with water or α-bromonaphthalene, surface free energy (SFE), and antibacterial activity against Streptococcus mutans for biofilm formation and planktonic bacteria. There was no significant difference for DC (p > 0.05). The initial Knoop hardness ranged from 17.30 (±0.50) to 19.50 (± 0.45), with lower value for GCTRL (p < 0.05). All groups presented lower KHN after immersion in solvent (p < 0.05). The ΔKHN ranged from 47.22 (± 4.30) to 57.22 (± 5.42)%, without significant difference (p > 0.05). The UTS ranged from 54.72 (± 11.05) MPa to 60.46 (± 6.50) MPa, with lower value for G2% (p < 0.05). PHMGH groups presented no significant difference compared to GCTRL in θ (p > 0.05). G2% showed no difference in SFE compared to GCTRL (p > 0.05). The groups with PHMGH presented antibacterial activity against biofilm and planktonic bacteria, with higher antibacterial activity for higher PHMGH incorporation (p < 0.05). PHMGH provided antibacterial activity for all resin sealant groups and the addition up to 1 wt.% showed reliable physico-chemical properties, maintaining the caries-protective effect of the resin sealant over time.

Evidence for the involvement of TNF-α, IL-1ß and IL-10 in the antinociceptive and anti-inflammatory effects of indole-3-guanylhydrazone hydrochloride, an aromatic aminoguanidine, in rodents.

BACKGROUND: Indole-3-guanylhydrazone hydrochloride (LQM01) is a new derivative of aminoguanidine hydrochloride, an aromatic aminoguanidine. METHODS: Mice were treated with LQM01 (5, 10, 25 or 50 mg/kg, i.p.), vehicle (0.9% saline i.p.) or a standard drug. The mice were subjected to carrageenan-induced pleurisy, abdominal writhing induced by acetic acid, the formalin test and the hot-plate test. The model of non-inflammatory chronic muscle pain induced by saline acid was also used. Mice from the chronic protocol were assessed for withdrawal threshold, muscle strength and motor coordination. LQM01 or vehicle treated mice were evaluated for Fos protein. RESULTS: LQM01 inhibits TNF-α and IL-1ß production, as well as leukocyte recruitment during inflammation process. The level of IL-10 in LQM01-treated mice increased in pleural fluid. In addition, LQM01 decreased the nociceptive behavior in the acetic acid induced writhing test, the formalin test (both phases) and increased latency time on the hot-plate. LQM01 treatment also decreased mechanical hyperalgesia in mice with chronic muscle pain, with no changes in muscle strength and motor coordination. LQM01 reduced the number of Fos positive cells in the superficial dorsal horn. This compound exhibited antioxidant properties in in vitro assays. CONCLUSIONS: LQM01 has an outstanding anti-inflammatory and analgesic profile, probably mediated through a reduction in proinflammatory cytokines release, increase in IL-10 production and reduction in neuron activity in the dorsal horn of the spinal cord in mice. GENERAL SIGNIFICANCE: Beneficial effects of LQM01 suggest that it has some important clinical features and can play a role in the management of 'dysfunctional pain' and inflammatory diseases.

Aminoguanidine hydrazones (AGH's) as modulators of norfloxacin resistance in Staphylococcus aureus that overexpress NorA efflux pump.

One of the promising fields for improving the effectiveness of antimicrobial agents is their combination with efflux pump inhibitors (EPIs), which besides expanding the use of existing antibiotics. The goal of this research was to evaluate a series of aminoguanidine hydrazones (AGH's, 1-19) as antibacterial agents and NorA efflux pump inhibitors in Staphylococcus aureus strain SA-1199B. Molecular modeling and docking studies were also performed in order to explain at the molecular level the interactions of the compounds with the generated NorA efflux pump model. The MICs of the antibiotic and ethidium bromide were determined by microdilution assay in absence or presence of a subinhibitory concentration of aminoguanidine hydrazones and macrophages viability was determined through MTT assay. Bioinformatic software Swiss-Model and AutoDock 4.2 were used to perform modeling and docking studies, respectively. As results, all AGH's were able to potentiate the action for the antibiotic norfloxacin, causing MIC's reduction of 16-fold and 32-fold to ethidium bromide. In the cell viability test, the concentration of 10 µg/mL showed better results than 90% and the concentration of 1000 µg/mL showed the lowest viability, reaching a maximum of 50% for the analyzed aminoguanidine hydrazones. Molecular docking studies showed that both norfloxacin and derivative 13 were recognized by the same binding site of NorA pump, suggesting a competitive mechanism. The present work demonstrated for the first time that AGH derivatives have potential to be putative inhibitors of NorA efflux pump, showing a promising activity as an antibacterial drug development.

The chemistry and biology of guanidine natural products.

Covering: 2015 and 2016The chemistry and biology of natural guanidines isolated from microbial culture media, from marine invertebrates, as well as from terrestrial plants and animals, are reviewed. Emphasis is directed to the biosynthesis, total synthesis, ecological roles as well as on the evolution of guanidines isolated from natural sources.

Analogues of Marine Guanidine Alkaloids Are in Vitro Effective against Trypanosoma cruzi and Selectively Eliminate Leishmania (L.) infantum Intracellular Amastigotes.

Synthetic analogues of marine sponge guanidine alkaloids showed in vitro antiparasitic activity against Leishmania (L.) infantum and Trypanosoma cruzi. Guanidines 10 and 11 presented the highest selectivity index when tested against Leishmania. The antiparasitic activity of 10 and 11 was investigated in host cells and in parasites. Both compounds induced depolarization of mitochondrial membrane potential, upregulation of reactive oxygen species levels, and increased plasma membrane permeability in Leishmania parasites. Immunomodulatory assays suggested an NO-independent effect of guanidines 10 and 11 on macrophages. The same compounds also promoted anti-inflammatory activity in L. (L.) infantum-infected macrophages cocultived with splenocytes, reducing the production of cytokines MCP-1 and IFN-γ. Guanidines 10 and 11 affect the bioenergetic metabolism of Leishmania, with selective elimination of parasites via a host-independent mechanism.

Synthesis and Anticancer Activities of Novel Guanylhydrazone and Aminoguanidine Tetrahydropyran Derivatives.

In this paper we present the convenient syntheses of six new guanylhydrazone and aminoguanidine tetrahydropyran derivatives 2-7. The guanylhydrazone 2, 3 and 4 were prepared in 100% yield, starting from corresponding aromatic ketones 8a-c and aminoguanidine hydrochloride accessed by microwave irradiation. The aminoguanidine 5, 6 and 7 were prepared by reduction of guanylhydrazone 2-4 with sodium cyanoborohydride (94% yield of 5, and 100% yield of 6 and 7). The aromatic ketones 8a-c were prepared from the Barbier reaction followed by the Prins cyclization reaction (two steps, 63%-65% and 95%-98%). Cytotoxicity studies have demonstrated the effects of compounds 2-7 in various cancer and normal cell lines. That way, we showed that these compounds decreased cell viabilities in a micromolar range, and from all the compounds tested we can state that, at least, compound 3 can be considered a promising molecule for target-directed drug design.

5-Carba-pterocarpens: A new scaffold with anti-HCV activity.

The synthesis of a series of 5-carba-pterocarpens derivatives involving the cyclization of α-aryl-α-tetralones is described. Several compounds demonstrated potent activity and selectivity in vitro against HCV replicon reporter cells. The best profile in Huh7/Rep-Feo1b replicon reporter cells was observed with 2h (EC50 = 5.5 µM/SI = 20), while 2e was the most active in Huh7.5-FGR-JC1-Rluc2A replicon reporter cells (EC50 = 1.5 µM/SI = 70). Hydroxy groups at A- and D-rings are essential for anti-HCV activity, and substitutions in the A-ring at positions 3 and 4 resulted in enhanced activity of the compounds.

The chemistry and biology of guanidine natural products.

The present review discusses the isolation, structure determination, synthesis, biosynthesis and biological activities of secondary metabolites bearing a guanidine group. Topics include non-ribosomal peptides, alkaloids, guanidine-bearing terpenes, polyketides and shikimic acid derivatives from natural sources. A critical analysis of some yet underdeveloped aspects of guanidine metabolites is also presented.

Anti-parasitic Guanidine and Pyrimidine Alkaloids from the Marine Sponge Monanchora arbuscula.

HPLC-UV-ELSD-MS-guided fractionation of the anti-parasitic extract obtained from the marine sponge Monanchora arbuscula, collected off the southeastern coast of Brazil, led to the isolation of a series of guanidine and pyrimidine alkaloids. The pyrimidines monalidine A (1) and arbusculidine A (7), as well as the guanidine alkaloids batzellamide A (8) and hemibatzelladines 9-11, represent new minor constituents that were identified by analysis of spectroscopic data. The total synthesis of monalidine A confirmed its structure. Arbusculidine A (7), related to the ptilocaulin/mirabilin/netamine family of tricyclic guanidine alkaloids, is the first in this family to possess a benzene ring. Batzellamide A (8) and hemibatzelladines 9-11 represent new carbon skeletons that are related to the batzelladines. Evaluation of the anti-parasitic activity of the major known metabolites, batzelladines D (12), F (13), L (14), and nor-L (15), as well as of synthetic monalidine A (1), against Trypanosoma cruzi and Leishmania infantum is also reported, along with a detailed investigation of parasite cell-death pathways promoted by batzelladine L (14) and norbatzelladine L (15).

Antipodal crambescin A2 homologues from the marine sponge Pseudaxinella reticulata. Antifungal structure-activity relationships.

Investigation of antifungal natural products from the marine sponge Pseudaxinella reticulata from the Bahamas led to the discovery of new crambescin homologues (1, 2) and enantiomers (3, 4) of known natural products. The cyclic-guanidine structures were solved through analysis of 2D NMR, MS-MS, and CD data. The absolute configurations of 1-4 were established as 13R-opposite of known homologues reported from Crambe crambe obtained from the Mediterranean Sea-by comparison of their CD spectra with predicted Cotton effects obtained from DFT calculations. Antifungal activities of 1-4 against the pathogenic strains Candida albicans and Cryptococcus sp. were observed to correlate potency (MIC50 and MIC90) with the length of the alkyl side chain.

Alchornedine, a new anti-trypanosomal guanidine alkaloid from Alchornea glandulosa.

Bioactivity-guided fractionation of the MeOH extract from the leaves of Alchornea glandulosa afforded a new guanidine alkaloid named alchornedine, as well as two other inactive derivatives (pteroginine and pteroginidine). The structure of alchornedine, which shows a very rare ring system, was elucidated based on NMR, IR, and MS spectral analyses. This compound displayed antiprotozoal activity against Trypanosoma cruzi (Y strain). By using the MTT assay, the trypomastigotes showed an IC50 value of 93 µg/mL (443 µM), a similar effectiveness to the standard drug benznidazole. Alchornedine also showed activity against the intracellular amastigotes, with an IC50 value of 27 µg/mL (129 µM). Using benznidazole as a standard drug, this guanidine alkaloid was approximately 3-fold more effective against the intracellular form of T. cruzi. The mammalian cytotoxicity of alchornedine was verified against NCTC cells and demonstrated an IC50 of 50 µg/mL (237 µM), but this compound demonstrated a selective elimination of parasites inside macrophages without affecting the morphology of the host cells. Alchornedine was effective against both clinical forms of T. cruzi and could be used as a scaffold for future drug design studies against American trypanosomiasis.

In Vitro Antibacterial Activity of Prenylated Guanidine Alkaloids from Pterogyne nitens and Synthetic Analogues.

The present investigation deals with the antibiotic activity of eight natural guanidine alkaloids and two synthetic analogues against a variety of clinically relevant methicillin-resistant Staphylococcus aureus strains. Galegine (1) and pterogynidine (2) were the most potent compounds, with a minimum inhibitory concentration of 4 mg/L, to all tested strains. The preliminary chemical features correlating to anti-MRSA activity showed that the size of the side chain and the substitution pattern in the guanidine core played a key role in the antibacterial activity of the imino group. Guanidine alkaloids 1 and 2 are promising molecular models for further synthetic derivatives and, thus, for medicinal chemistry studies.

Chemometric quality inspection control of pyrantel pamoate, febantel and praziquantel in veterinary tablets by mid infrared spectroscopy.

This paper describes the development and validation of a new multivariate calibration method based on diffuse reflectance mid infrared spectroscopy for direct and simultaneous determination of three veterinary pharmaceutical drugs, pyrantel pamoate, praziquantel and febantel, in commercial tablets. The best synergy interval partial least squares (siPLS) model was obtained by selecting three spectral regions, 3715-3150, 2865-2583, and 2298-1733 cm(-1), preprocessed by first derivative and Savitzky-Golay smoothing followed by mean centering. This model was built with five latent variables and provided root mean square errors of prediction (RMSEP) equal or lower than 0.69 mg per 100 mg of powder for the three analytes. The method was validated according the appropriate regulations through the estimate of figures of merit, such as trueness, precision, linearity, analytical sensitivity, bias and residual prediction deviation (RPD). Then, it was applied to three different veterinary pharmaceutical formulations found in the Brazilian market, in a situation of multi-product calibration, since the excipient composition of these commercial products, which was not known a priori, was modeled by an experimental design that scanned the likely content range of the possible constituents. The results were verified with high performance liquid chromatography with diode array detection (HPLC-DAD) and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and were in agreement with the predicted values at 95% confidence level. The developed method presented the advantages of being simple, rapid, solvent free, and about ten times faster than the HPLC ones.