Sustained Hyperglycemia and Its Relationship with the Outcome of Hospitalized Patients with Severe COVID-19: Potential Role of ACE2 Upregulation.

Chronic hyperglycemia increases the risk of developing severe COVID-19 symptoms, but the related mechanisms are unclear. A mean glucose level upon hospital admission >166 mg/dl correlates positively with acute respiratory distress syndrome in patients with hyperglycemia. The objective of this study was to evaluate the relationship between sustained hyperglycemia and the outcome of hospitalized patients with severe COVID-19. We also evaluated the effect of high glucose concentrations on the expression of angiotensin-converting enzyme 2 (ACE2). We carried out a case-control study with hospitalized patients with severe COVID-19 with and without sustained hyperglycemia. In a second stage, we performed in vitro assays evaluating the effects of high glucose concentrations on ACE2 gene expression. Fifty hospitalized patients with severe COVID-19 were included, of which 28 (56%) died and 22 (44%) recovered. Patients who died due to COVID-19 and COVID-19 survivors had a high prevalence of hyperglycemia (96.4% versus 90.9%), with elevated central glucose upon admission (197.7 mg/dl versus 155.9 mg/dl, p = 0.089) and at discharge (185.2 mg/dl versus 134 mg/dl, p = 0.038). The mean hypoxemia level upon hospital admission was 81% in patients who died due to COVID-19 complications and 88% in patients who survived (p = 0.026); at the time of discharge, hypoxemia levels were also different between the groups (68% versus 92%, p ≤ 0.001). In vitro assays showed that the viability of A549 cells decreased (76.41%) as the glucose concentration increased, and the ACE2 gene was overexpressed 9.91-fold after 72 h (p ≤ 0.001). The relationship between hyperglycemia and COVID-19 in hospitalized patients with COVID-19 plays an important role in COVID-19-related complications and the outcome for these patients. In patients with chronic and/or sustained hyperglycemia, the upregulation of ACE2, and its potential glycation and malfunction, could be related to complications observed in patients with COVID-19.

The aflatoxin inhibitors capsaicin and piperine from Capsicum chinense and Piper nigrum fruits modulate the antioxidant system in Aspergillus parasiticus.

Several aflatoxin inhibitors can modulate the antioxidant system in fungi. In this work, the effect of the ethanolic extract of Capsicum chinense and Piper nigrum fruits, capsaicin, and piperine on the expression of the aflE, aflG, aflH, aflI, aflK, aflL, aflO, aflP, and aflQ genes involved in the aflatoxin biosynthetic pathway in Aspergillus parasiticus were studied by qRT-PCR analysis. As well as, the effect on the expression of fungal antioxidant genes (sod1, catA, and cat2) and enzymatic activity of catalase (CAT) and superoxide dismutase (SOD). Results reveal that the highest (p < 0.05) radial growth inhibition (68 and 86%) and aflatoxins production inhibition (73 and 80%) was observed with capsaicin and piperine respectively, at 300 µg/mL, instead of the ethanolic extract at the same concentration. The qRT-PCR analysis showed that compounds and extracts at 300 µg/mL induced a down-regulation of aflatoxin genes and an up-regulation on the fungal antioxidant genes. CAT activity increased by 23.15, 36.65, 51.40, and 65.50%, in the presence of C. chinense and P. nigrum extract, capsaicin, and piperine exposure, respectively. While SOD activity was not significantly impacted (p > 0.05). In conclusion, the capsaicin and piperine, two antifungal and anti-aflatoxigenic compounds produce an up-regulation of antioxidant defense genes accompanied by an enhancement of catalase enzymatic activity in A. parasiticus.

Down-regulation of aflatoxin biosynthetic genes in Aspergillus parasiticus by Heliopsis longipes roots and affinin for reduction of aflatoxin production.

Affinin present in Heliopsis longipes roots has been identified as an anti-aflatoxin molecule. However, its mechanism of action has yet to be clarified. Aflatoxins biosynthesis involves not less than 27 enzymatic reactions. In this work, the genes aflG, aflH, aflI, aflK, aflL, aflM, aflO, aflP, and aflQ of the aflatoxins cluster and the aflS gene encoding an internal regulatory factor involved in aflatoxins biosynthesis in Aspergillus parasiticus, were studied by qRT-PCR. Results demonstrated that ethanolic extract of H. longipes roots and affinin inhibit aflatoxin biosynthesis and fungal growth in a dose-dependent manner. At 300 µg/mL, ethanolic extract and affinin presented the highest inhibition of radial growth (86% and 94%) and aflatoxin production (68% and 80%). The qRT-PCR analysis demonstrated that nine tested genes were down-regulated by affinin and ethanolic extract. The most down-regulated was the aflK, a gene that encodes an enzyme cyclase with double function during the aflatoxin biosynthesis. While no significant down-regulation was obtaining for aflH gene. Exposure to affinin also resulted in decreased transcript levels of the internal regulator factor aflS. Based on our results, a model showing the regulatory mechanism in aflatoxin biosynthesis and its role in gene expression was proposed. In conclusion, affinin modulates the expression of several aflatoxin biosynthetic genes, leading to mycotoxin biosynthesis inhibition. Therefore, H. longipes roots is a suitable candidate to developed control strategies via lowering gene expressions as a future perspective in reducing aflatoxin contamination.

Antifungal and anti-aflatoxigenic activity of Heliopsis longipes roots and affinin/spilanthol against Aspergillus parasiticus by downregulating the expression of alfD and aflR genes of the aflatoxins biosynthetic pathway.

In the present study, ethanolic extract from Heliopsis longipes roots and affinin/spilanthol against Aspergillus parasiticus growth and aflatoxins production were studied in relation to the expression of aflD and aflR, two key genes of aflatoxins biosynthetic pathway. Phytochemical analysis of the ethanolic extract by GC-EIMS identified affinin/spilanthol (7.84 ± 0.27 mg g-1) as the most abundant compounds in H. longipes roots. The antifungal and anti-aflatoxigenic assays showed that affinin/spilanthol at 300 µg mL-1 produced the higher inhibition of radial growth (95%), as well as, the higher aflatoxins production inhibition (61%) in comparison to H. longipes roots (87% and 48%, respectively). qRT-PCR revealed that the expression of aflD and aflR genes showed a higher downregulation in affinin/spilanthol at 300 µg mL-1. The expression ratio of alfD was suppressed by affinin/spilanthol in 79% and aflR in 84%, while, a lower expression ratio suppressed by H. longipes was obtained, alfD (55%) and aflR (59%). Affinin/spilanthol possesses higher antifungal and anti-aflatoxigenic activity against A. parasiticus rather than H. longipes roots, and this anti-aflaxotigenic activity occurring via downregulation of the aflD and aflR genes. Thus, H. longipes roots and affinin/spilanthol can be considered potent antifungal agents against aflatoxigenic fungus, especially, affinin/spilanthol.

Continuous flow micro-bioreactors for the production of biopharmaceuticals: the effect of geometry, surface texture, and flow rate.

We used continuous flow micro-devices as bioreactors for the production of a glycosylated pharmaceutical product (a monoclonal antibody). We cultured CHO cells on the surface of PMMA/PDMS micro-channels that had been textured by micromachining and coated with fibronectin. Three different micro-channel geometries (a wavy channel, a zigzag channel, and a series of donut-shape reservoirs) were tested in a continuous flow regime in the range of 3 to 6 µL min(-1). Both the geometry of the micro-device and the flow rate had a significant effect on cell adhesion, cell proliferation, and monoclonal antibody production. The most efficient configuration was a series of donut-shaped reservoirs, which yielded mAb concentrations of 7.2 mg L(-1) at residence times lower than one minute and steady-state productivities above 9 mg mL(-1) min(-1). These rates are at about 3 orders of magnitude higher than those observed in suspended-cell stirred tank fed-batch bioreactors.