Comparative performances of seven quantitative Reverse-Transcription Polymerase Chain Reaction assays (RT-qPCR) for detecting SARS-CoV-2 infection in samples from individuals suspected of COVID-19 in São Paulo, Brazil.

Introduction: Brazil is the second largest country with COVID-19 positive cases worldwide. Due to the potent spread of the virus and the scarcity of kits and supplies, the Brazilian Ministry of Health has granted authorization for the use of kits available during this emergency, without an accurate evaluation of their performance. This study compared the performance and cost-effectiveness of seven molecular assays/kits available in São Paulo, Brazil, for SARS-CoV-2 diagnosis. Materials and methods: A total of 205 nasopharyngeal/oropharyngeal samples from suspected cases of COVID-19, were tested using the following assays: (i) GeneFinder COVID-19 plus RealAmp kit; (ii) 2019-nCoV RNA PCR-Fluorescence Probing, Da An Gene Co.; (iii) in-house RT-qPCR SARS-CoV-2 IAL; (iv) 2019-nCoV kit, IDT; (v) molecular SARS-CoV-2 (E) kit, Bio-Manguinhos; (vi) Allplex 2019-nCoV modified Assay, Seegene Inc, and (vii) Biomol one-step COVID-19 kit, IBMP. The criteria for determining a SARS-CoV-2 true positive result included the cycle threshold cut-off values, the characteristics of exponential/linear curves, the gene target diversity, and a positive result in at least two assays. Results: The overall sensitivity of the assays listed were GeneFinder 83.6%, Da An Gene 100.0%, IAL 90.4%, IDT 94.6%, Bio-Manguinhos 87.7%, Allplex 97.3%, and IBMP 87.7%. The minor sensitive gene target was RdRP. Although all assays had a Cohen's Kappa index ≥0.893, the best tests used multiplex assays identifying N-gene and/or E-gene targets. Conclusion: All assays tested accurate for diagnosis, but considering cost-effectiveness (cost, time consumption, number of samples tested, and performance), the in-house IAL assay was ideal for COVID-19 diagnosis in São Paulo, Brazil.

A Highly Sensitive Fluorogenic Probe for Imaging Glycoproteins and Mucine Activity in Live Cells in the Near-Infrared Region.

A novel fluorescent molecular probe is reported, which is able to detect glycoproteins, especially mucins, with high sensitivity and with a turn-on response along with a large Stokes shift (>130 nm), within the biologically active window. The probe contains an aminotricarbocyanine as the fluorescent reporter with a linked benzoboroxole as the recognition unit, which operates through a dynamic covalent reaction between the boronic hemiester residue of the receptor and cis-diols of the analyte. The superior selectivity of the probe is displayed by the labeling of mucins present in Calu-3 cells. The new benzoboroxole fluorescent derivative gathers together key properties to make it a highly rated molecular probe: specificity, excellent solubility in water, and off-on near infrared emission. This probe is expected to be an excellent tool for imaging intracellular mucin to evaluate mucus-related diseases as well as a sensing strategy towards glycosylated structures with a high potential for theranostics approaches in biological samples.

Mesoscopic modelling of Cy3 and Cy5 dyes attached to DNA duplexes.

Cy3 and Cy5 dyes linked to the 5' end of a double stranded DNA molecule are known to attach to both strands in a way that is very similar to an additional base pair and has a stabilizing effect on the oligonucleotide. Here we adapt the Peyrard-Bishop mesoscopic model to incorporate cyanine dyes and use the technique of thermal equivalence to obtain the appropriate parameters from existing melting temperatures. We have found that the stacking parameters are in the same range of ordinary AT and CG base pairs, in particular Cy3-A was found to be most rigidly stacked. While the cyanines stabilize the AT hydrogen bonds quite strongly the CG bonds are mostly unaffected.

Photocytotoxicity of a cyanine dye with two chromophores toward melanoma and normal cells.

BACKGROUND: Due to high optical absorption, triplet quantum yield and affinity to biological structures bichromophoric cyanine dyes (BCDs) can be considered promising sensitizers for application in photodynamic therapy (PDT). In this work, we report on the study of the BCD photocytotoxicity toward melanoma and normal cells in comparison with that of commercial photosensitizer Photogem®. METHODS: The cytotoxic and phototoxic effects were measured by standard tests of cell viability. The drug uptake was obtained by the flow cytometry and optical absorption techniques. The BCD intracellular distribution was obtained by the fluorescence image microscopy using specific organelle markers. RESULTS: Both drugs demonstrated increased cytotoxicity under irradiation, while in darkness their cytotoxic effect at concentrations lower than 20 µM after 24 h of incubation did not exceed 20%. For 5 h of incubation, BCD photocytotoxicity in relation to melanoma cells reached 100% already at concentrations below 5 µM, while for normal cells the effect did not exceed 70% even for the 20 µM concentration. It is shown that BCD penetrates into the cells and is located predominantly in perinuclear cytoplasmic structures. CONCLUSIONS: The BCD photosensitizing characteristics appear more adequate for application in PDT than that of the actually applied commercial photosensitizer Photogem®. Higher light absorption by BCD in the near IR region and its preferential localization in mitochondria can explain its high photocytotoxicity. GENERAL SIGNIFICANCE: BCD can be considered as a new promising photosensitizer class for cancer PDT.

The Edinger-Westphal nucleus II: Hypothalamic afferents in the rat.

Numerous functions have been attributed to the Edinger-Westphal nucleus (EW), including those related to feeding behavior, pain control, alcohol consumption and the stress response. The EW is thought to consist of two parts: one controls accommodation, choroidal blood flow and pupillary constriction, primarily comprising cholinergic cells and projecting to the ciliary ganglion; and the other would be involved in the non-ocular functions mentioned above, comprising peptide-producing neurons and projecting to the brainstem, spinal cord and prosencephalic regions. Despite the fact that the EW is well known, its connections have yet to be described in detail. The aim of this work was to produce a map of the hypothalamic sources of afferents to the EW in the rat. We injected the retrograde tracer Fluoro-Gold into the EW, and using biotinylated dextran amine, injected into afferent sources as the anterograde control. We found retrogradely labeled cells in the following regions: subfornical organ, paraventricular hypothalamic nucleus, arcuate nucleus, lateral hypothalamic area, zona incerta, posterior hypothalamic nucleus, medial vestibular nucleus and cerebellar interpositus nucleus. After injecting BDA into the paraventricular hypothalamic nucleus, lateral hypothalamic area and posterior hypothalamic nucleus, we found anterogradely labeled fibers in close apposition to and potential synaptic contact with urocortin 1-immunoreactive cells in the EW. On the basis of our findings, we can suggest that the connections between the EW and the hypothalamic nuclei are involved in controlling stress responses and feeding behavior.

Exchangeable aluminum evaluation in acid soils

One of the main factors limiting agricultural production in tropical climate regions is mainly related to the presence of exchangeable aluminum (Al3+) in highly weathered acid soils. Four methods of Al3+ determination extracted with neutral 1 mol L¹ KCl solution were evaluated: three colorimetric methods (aluminon plus ascorbic acid, and eriochrome cyanine R by FIA) and the usual titrimetric method with back-titration. Surface samples from 20 soils of different Brazilian regions, with active acidity (0.01 mol L¹ CaCl2 pH) ranging from very high to medium (3.82 to 5.52), were used. The variance analysis revealed significant interaction among Al3+ determination methods and soil. Mean methods comparisons within each soil (Tukey, P 0.05) indicated that, for most of the soils, the methods differed among each other, although there were high correlations between the obtained values. Al3+ values determined for soil samples by titration varied between 0.15 and 14.71 mmol c dm³. The colorimetric methods showed higher values than the titration method, mainly for those with aluminon (up to 18.75 mmol c dm-3). The Al3+ contents of colorimetric methods correlated quadraticaly with the titration values, for the soil samples with Al3+ > 10 mmol c dm³. Among colorimetric methods, in operational terms, the eriochrome with FIA method presented analytical performance up to 50 samples per hour, easiness and sensibility for routine Al analysis in soil samples. However, due to the specificity, the titration/back-titration method should be used, despite the moroseness, when the Al3+ ions are the objective.

Um dos principais fatores que afetam a produção agrícola em regiões de clima tropical, é a presença de alumínio trocável (Al3+) em solos ácidos altamente intemperizados. Foram avaliados quatro métodos de determinação de alumínio trocável em solução neutra de KCl 1 mol L¹: três métodos colorimétricos (aluminon, aluminon + ácido ascórbico e eriocromo cianina R por FIA) e o método titulométrico usual com retrotitulação ácida. Para obtenção dos extratos de KCl, foram usadas amostras superficiais de 20 solos de diferentes regiões brasileiras, com acidez ativa (pH em CaCl2 0,01 mol L¹) variando de muito alta a média (3,82 a 5,52). A análise de variância revelou interação significativa entre os métodos de determinação e amostras de solos. O teste de médias (Tukey, P 0,05) de métodos dentro de amostras evidenciou, para a maioria das amostras de solos, que os métodos analíticos diferenciaram entre si, embora tenha havido forte correlação entre os valores obtidos. Os valores de Al3+ determinados por titulação variaram de 0,15 a 14,71 mmol c dm³. Os métodos colorimétricos apresentaram valores superiores aos da titulação, notadamente aqueles com aluminon (até 18,75 mmol c dm³). Os teores de Al3+ dos métodos colorimétricos se relacionaram quadraticamente com os teores obtidos por titulação, em amostras de solos com Al3+ > 10 mmol c dm³. Dentre os métodos colorimétricos, o reagente eriocromo cianina R, em sistema FIA, mostrou-se operacionalmente confiável e eficiente para a análise de Al, de amostras de solos, em rotina, com freqüência analítica de 50 determinações por hora. Todavia, devido à especificidade, o método titulométrico com retrotitulação deve ser usado, não obstante a morosidade, quando o objetivo é a determinação do íon Al3+.

Exchangeable aluminum evaluation in acid soils

One of the main factors limiting agricultural production in tropical climate regions is mainly related to the presence of exchangeable aluminum (Al3+) in highly weathered acid soils. Four methods of Al3+ determination extracted with neutral 1 mol L¹ KCl solution were evaluated: three colorimetric methods (aluminon plus ascorbic acid, and eriochrome cyanine R by FIA) and the usual titrimetric method with back-titration. Surface samples from 20 soils of different Brazilian regions, with active acidity (0.01 mol L¹ CaCl2 pH) ranging from very high to medium (3.82 to 5.52), were used. The variance analysis revealed significant interaction among Al3+ determination methods and soil. Mean methods comparisons within each soil (Tukey, P 0.05) indicated that, for most of the soils, the methods differed among each other, although there were high correlations between the obtained values. Al3+ values determined for soil samples by titration varied between 0.15 and 14.71 mmol c dm³. The colorimetric methods showed higher values than the titration method, mainly for those with aluminon (up to 18.75 mmol c dm-3). The Al3+ contents of colorimetric methods correlated quadraticaly with the titration values, for the soil samples with Al3+ > 10 mmol c dm³. Among colorimetric methods, in operational terms, the eriochrome with FIA method presented analytical performance up to 50 samples per hour, easiness and sensibility for routine Al analysis in soil samples. However, due to the specificity, the titration/back-titration method should be used, despite the moroseness, when the Al3+ ions are the objective.

Um dos principais fatores que afetam a produção agrícola em regiões de clima tropical, é a presença de alumínio trocável (Al3+) em solos ácidos altamente intemperizados. Foram avaliados quatro métodos de determinação de alumínio trocável em solução neutra de KCl 1 mol L¹: três métodos colorimétricos (aluminon, aluminon + ácido ascórbico e eriocromo cianina R por FIA) e o método titulométrico usual com retrotitulação ácida. Para obtenção dos extratos de KCl, foram usadas amostras superficiais de 20 solos de diferentes regiões brasileiras, com acidez ativa (pH em CaCl2 0,01 mol L¹) variando de muito alta a média (3,82 a 5,52). A análise de variância revelou interação significativa entre os métodos de determinação e amostras de solos. O teste de médias (Tukey, P 0,05) de métodos dentro de amostras evidenciou, para a maioria das amostras de solos, que os métodos analíticos diferenciaram entre si, embora tenha havido forte correlação entre os valores obtidos. Os valores de Al3+ determinados por titulação variaram de 0,15 a 14,71 mmol c dm³. Os métodos colorimétricos apresentaram valores superiores aos da titulação, notadamente aqueles com aluminon (até 18,75 mmol c dm³). Os teores de Al3+ dos métodos colorimétricos se relacionaram quadraticamente com os teores obtidos por titulação, em amostras de solos com Al3+ > 10 mmol c dm³. Dentre os métodos colorimétricos, o reagente eriocromo cianina R, em sistema FIA, mostrou-se operacionalmente confiável e eficiente para a análise de Al, de amostras de solos, em rotina, com freqüência analítica de 50 determinações por hora. Todavia, devido à especificidade, o método titulométrico com retrotitulação deve ser usado, não obstante a morosidade, quando o objetivo é a determinação do íon Al3+.