Spatially resolved qualified sewage spot sampling to track SARS-CoV-2 dynamics in Munich - One year of experience.

Wastewater-based epidemiology (WBE) is a tool now increasingly proposed to monitor the SARS-CoV-2 burden in populations without the need for individual mass testing. It is especially interesting in metropolitan areas where spread can be very fast, and proper sewage systems are available for sampling with short flow times and thus little decay of the virus. We started in March 2020 to set up a once-a-week qualified spot sampling protocol in six different locations in Munich carefully chosen to contain primarily wastewater of permanent residential areas, rather than industry or hospitals. We used RT-PCR and sequencing to track the spread of SARS-CoV-2 in the Munich population with temporo-spatial resolution. The study became fully operational in mid-April 2020 and has been tracking SARS-CoV-2 RNA load weekly for one year. Sequencing of the isolated viral RNA was performed to obtain information about the presence and abundance of variants of concern in the Munich area over time. We demonstrate that the evolution of SARS-CoV-2 RNA loads (between <7.5 and 3874/ml) in these different areas within Munich correlates well with official seven day incidence notification data (between 0.0 and 327 per 100,000) obtained from the authorities within the respective region. Wastewater viral loads predicted the dynamic of SARS-CoV-2 local incidence about 3 weeks in advance of data based on respiratory swab analyses. Aligning with multiple different point-mutations characteristic for certain variants of concern, we could demonstrate the gradual increase of variant of concern B.1.1.7 in the Munich population beginning in January 2021, weeks before it became apparent in sequencing results of swabs samples taken from patients living in Munich. Overall, the study highlights the potential of WBE to monitor the SARS-CoV-2 pandemic, including the introduction of variants of concern in a local population.

Effects of Qualea grandiflora Extract on the Expression of MMP-14 and HIF-1α in Cultured Fibroblasts and Preosteoblasts

Abstract Background: Qualea grandiflora (QG) (Vochysiaceae), also known as "pau-ferro", "pau-terra" or "pau-de-tucano", is a very common deciduous tree in the Brazilian Cerrado used in traditional medicine to treat inflammations, ulcers, diarrhea, and infections. There are reports in the scientific literature that demonstrate the medicinal effects of the bark and leaf of the QG. However, studies involving this plant are rather imited. Aim of the study: To perform the phytochemical analysis of the QG hydroalcoholic extract (HAE) of leaves, and to investigate it effects on fibroblast and preosteoblasts. Methods: Phytochemical analysis was done by HPLC-DAD. Murine NIH/3T3 fibroblasts and MC3T3-E1 preosteoblasts cell lines (ATCC) were used for the experiments. Cell viability was assessed by the MTT colorimetric assay and the expression of MMP-14 and HIF-1α by immunofluorescence. Results and conclusion: The following compounds were identified by HPLC-DAD, such as quinic acid, ethyl galate, ellagic acid derivatives as O-methylellagic acid O-galloyl, O-methylellagic acid O-deoxyhexoside, galloyl derivatives, flavonol glycoside as kaempferol-O-deoxyhexoside, quercetin-O-deoxyhexoside, myricetin-O-deoxyhexoside and the pentacyclic triterpene arjunglucoside. Cell viability results demonstrated no cytotoxic effects in the studied concentrations. We found in QG HAE some compounds with therapeutic properties that can increase the expression of MMP-14 and HIF-1α, in fibroblasts and preosteoblasts. These data suggest that QG HAE has an action on these two molecules widely involved in physiological conditions, such as collagen remodeling, bone development and growth and pathological processes as HIF signaling in cancer metastasis.

Liver Proteome of Mice with Distinct Genetic Susceptibilities to Fluorosis Treated with Different Concentrations of F in the Drinking Water.

Appropriate doses of fluoride (F) have therapeutic action against dental caries, but higher levels can cause disturbances in soft and mineralized tissues. Interestingly, the susceptibility to the toxic effects of F is genetically determined. This study evaluated the effects of F on the liver proteome of mice susceptible (A/J) or resistant (129P3/J) to the effects of F. Weanling male A/J (n = 12) and 129P3/J (n = 12) mice were housed in pairs and assigned to two groups given low-F food and drinking water containing 15 or 50 ppm F for 6 weeks. Liver proteome profiles were examined using nano-LC-ESI-MS/MS. Difference in expression among the groups was determined using the PLGS software. Treatment with the lower F concentration provoked more pronounced alterations in fold change in liver proteins in comparison to the treatment with the higher F concentration. Interestingly, most of the proteins with fold change upon treatment with 15 ppm F were increased in the A/J mice compared with their 129P3/J counterparts, suggesting an attempt of the former to fight the deleterious effects of F. However, upon treatment with 50 ppm F, most proteins with fold change were decreased in the A/J mice compared with their 129P3/J counterparts, especially proteins related to oxidative stress and protein folding, which might be related to the higher susceptibility of the A/J animals to the deleterious effects of F. Our findings add light into the mechanisms underlying genetic susceptibility to fluorosis.

Proteomic analysis of liver in mice with different susceptibilities to fluorosis / Análise proteômica do fígado de camundongos com diferentes suscetibilidades à fluorose

Fluoride (F) is a potent anti-cariogenic element, but only an appropriate dose is effective to have therapeutic action, else systemic toxicity may be observed. Additionally, two factors, amount of F and time of exposure, drive its action. Surprisingly, the susceptibility to toxic effects of F is genetically determined. The present study identified the effects of F on the liver proteome of mice susceptible (A/J) or resistant (129P3/J) to the effects of F. Weanling male A/J (n=6) and 129P3/J mice (n=6) were housed in pairs and assigned to three groups given low-F diet and drinking water containing 0, 15 or 50 ppm F for 7 weeks. Liver proteome profiles were examined using nano-LC-ESI-MS/MS. Protein function was classified by GO biological process (Cluego v2.0.7 + Clupedia v1.0.8). Difference in expression among the groups was determined using the PLGS software. In the control group (0 ppm F), most proteins with fold change were increased in A/J mice. Precisely the proteins related to energy flux and oxidative stress were quite significant in this context, suggesting the high susceptibility of these mice to the effects of F, since the exposure also induces oxidative stress. Treatment with the lower F concentration provoked more pronounced alterations in fold change in liver proteins in comparison to the treatment with the higher F concentration. Strikingly, most of the proteins with fold change upon following 15 ppm F treatment, were increased in the A/J mice compared with their 129P3/J counterparts, thus suggesting attempt of the former to fight against the toxic effects of F. With respect to 50 ppm F, most proteins with fold change were decreased in the A/J mice compared with their 129P3/J counterparts, especially proteins related to oxidative stress and protein folding, which might be related to the higher susceptibility of the A/J animals to the deleterious effects of F. Our findings can provide new insights into the molecular mechanisms underlying genetic susceptibility to fluorosis by indicating key protein players which need to be better addressed in future experimental studies.(AU)

O Fluoreto (F) é um potente elemento anti-cariogênico, mas é somente efetivo terapeuticamente em uma dose apropriada. Por outro lado, doses acima das recomendadas levam a toxicidade sistêmica. Em adição, dois fatores decidem sua efetividade de ação: quantidade de F e tempo de exposição. A suscetibilidade aos efeitos tóxicos do F é determinada geneticamente. O presente estudo avaliou os efeitos do F no proteoma do fígado de camundongos suscetíveis (A/J) ou resistentes (129P3/J) aos efeitos do F. Camundongos machos desmamados A/J (n=6) e 129P3/J (n=6) foram alojados em pares e divididos em três grupos tratados com ração com baixo teor de F e água contendo 0, 15, ou 50 ppm de F por 7 semanas. Perfis proteômicos do fígado foram examinados usando nano-LC-ESI-MS/MS. A função de proteínas foi classificada pelo processamento biológico GO (Cluego v2.0.7 + Clupedia v1.0.8). A diferença de expressão entre os grupos foi determinada usando o software PLGS. No grupo controle (0 ppm F), a expressão da maioria das proteínas foi aumentada nos camundongos A/J e precisamente as proteínas relacionadas ao fluxo de energia e estresse oxidativo foram significativas neste contexto, sugerindo portanto, a alta sucetibilidade destes camundongos aos efeitos do F, já que a exposição também induz o estresse oxidativo. O tratamento com baixa concentração de F provocou alterações mais pronunciadas em proteínas do fígado comparado ao tratamento com alta concentração de F. Notadamente, a maioria das proteínas encontradas no fígado dos animais tratados com 15 ppm de F foi aumentada em camundongos A/J comparados aos camundongos 129P3/J, demonstrando portanto, uma tentativa dos A/J de neutralizar os efeitos tóxicos do F. Já nos animais tratados com 50 ppm de F, a maioria das proteínas foi diminuída nos camundongos comparados aos seus pares 129P3/J, especialmente proteínas relacionadas ao estresse oxidativo e enovelamento de proteínas, o que pode estar relacionado à alta suscetibilidade dos animais A/J aos efeitos deletérios do F. Nossos achados podem fornecer novos insights que podem contribuir para a interpretação os mecanismos moleculares relacionados à suscetibilidade genética à fluorose, indicando proteínas chaves que precisam ser melhor estudadas em estudos futuros.(AU)

Liver proteome of mice with different genetic susceptibilities to the effects of fluoride.

OBJECTIVE: In this study, we investigated the differential pattern of protein expression in the liver of these mice to provide insights on why they have different responses to F. MATERIAL AND METHODS: Weanling male A/J and 129P3/J mice (n=10 from each strain) were pared and housed in metabolic cages with ad libitum access to low-F food and deionized water for 42 days. Liver proteome profiles were examined using nLC-MS/MS. Protein function was classified by GO biological process (Cluego v2.0.7 + Clupedia v1.0.8) and protein-protein interaction network was constructed (PSICQUIC, Cytoscape). RESULTS: Most proteins with fold change were increased in A/J mice. The functional category with the highest percentage of altered genes was oxidation-reduction process (20%). Subnetwork analysis revealed that proteins with fold change interacted with Disks large homolog 4 and Calcium-activated potassium channel subunit alpha-1. A/J mice had an increase in proteins related to energy flux and oxidative stress. CONCLUSION: This could be a possible explanation for the high susceptibility of these mice to the effects of F, since the exposure also induces oxidative stress.

Liver proteome of mice with different genetic susceptibilities to the effects of fluoride

ABSTRACT A/J and 129P3/J mice strains have been widely studied over the last few years because they respond quite differently to fluoride (F) exposure. 129P3/J mice are remarkably resistant to the development of dental fluorosis, despite excreting less F in urine and having higher circulating F levels. These two strains also present different characteristics regardless of F exposure. Objective In this study, we investigated the differential pattern of protein expression in the liver of these mice to provide insights on why they have different responses to F. Material and Methods Weanling male A/J and 129P3/J mice (n=10 from each strain) were pared and housed in metabolic cages with ad libitum access to low-F food and deionized water for 42 days. Liver proteome profiles were examined using nLC-MS/MS. Protein function was classified by GO biological process (Cluego v2.0.7 + Clupedia v1.0.8) and protein-protein interaction network was constructed (PSICQUIC, Cytoscape). Results Most proteins with fold change were increased in A/J mice. The functional category with the highest percentage of altered genes was oxidation-reduction process (20%). Subnetwork analysis revealed that proteins with fold change interacted with Disks large homolog 4 and Calcium-activated potassium channel subunit alpha-1. A/J mice had an increase in proteins related to energy flux and oxidative stress. Conclusion This could be a possible explanation for the high susceptibility of these mice to the effects of F, since the exposure also induces oxidative stress.