Strategies for Increasing the Throughput of Genetic Screening: Lessons Learned from the COVID-19 Pandemic within a University Community.

Amidst the COVID-19 pandemic, the Polytechnic University of Setúbal (IPS) used its expertise in molecular genetics to establish a COVID-19 laboratory, addressing the demand for community-wide testing. Following standard protocols, the IPS COVID Lab received national accreditation in October 2020 and was registered in February 2021. With the emergence of new SARS-CoV-2 variants and safety concerns for students and staff, the lab was further challenged to develop rapid and sensitive diagnostic technologies. Methodologies such as sample-pooling extraction and multiplex protocols were developed to enhance testing efficiency without compromising accuracy. Through Real-Time Reverse Transcription Polymerase Chain Reaction (RT-qPCR) analysis, the effectiveness of sample pooling was validated, proving to be a clear success in COVID-19 screening. Regarding multiplex analysis, the IPS COVID Lab developed an in-house protocol, achieving a sensitivity comparable to that of standard methods while reducing operational time and reagent consumption. This approach, requiring only two wells of a PCR plate (instead of three for samples), presents a more efficient alternative for future testing scenarios, increasing its throughput and testing capacity while upholding accuracy standards. The lessons learned during the SARS-CoV-2 pandemic provide added value for future pandemic situations.

Kinetic Changes in B7 Costimulatory Molecules and IRF4 Expression in Human Dendritic Cells during LPS Exposure.

A key aspect of the inflammatory phenomenon is the involvement of costimulatory molecules expressed by antigen-presenting cells (APCs) and their ability to secrete cytokines to set instructions for an adaptive immune response and to generate tolerance or inflammation. In a novel integrative approach, we aimed to evaluate the kinetic expression of the membrane and soluble B7 costimulatory molecules CD86, ICOS-L, PDL1, PDL2, the transcription factor Interferon Regulatory Factor 4 (IRF4), and the cytokines produced by monocyte-derived dendritic cells (Mo-DCs) after challenging them with different concentrations of stimulation with E. coli lipopolysaccharide (LPS) for different lengths of time. Our results showed that the stimuli concentration and time of exposure to an antigen are key factors in modulating the dynamic expression pattern of membrane and soluble B7 molecules and cytokines.

The donating capacity of the elderly: A case report of vascular dementia.

Seniors may be more vulnerable to the influence of others, being less able to make judicious decisions. The legal legitimacy of donating capacity is based on the clinical assumption that, at the time of the event, the donor is able to understand the nature of the transaction, weigh its consequences and determine his/her actions according to it. From the point of view of forensic psychiatry, the evaluation of such premises is a challenge, reflecting the difficulty in assessing, retrospectively, the subject's cognitive functions at the time of the event. In cases where the cross-sectional evaluation favours the diagnosis of dementia it is crucial to objectify the onset and course of the illness. The authors report and discuss the case of a donation from father to sons, involving a great deal of money, made by an 85-year-old man, who had suffered an ischaemic cerebrovascular accident 5 years before.

Effect of acute vs chronic H2O2-induced oxidative stress on antioxidant enzyme activities.

H2O2 can freely crosses membranes and in the presence of Fe2+ (or Cu+) it is prone to participate in Fenton reaction. This study evaluated the concentration and time-dependent effects of H2O2-induced oxidative stress on MnSOD, Se:GPx and catalase and on aconitase. Acute and chronic H2O2 treatments were able to induce oxidative stress in HeLa cells as they significantly decreased aconitase activity and also caused a very significant decrease on antioxidant enzyme activities. The inhibition of enzyme activities was time- and concentration-dependent. Chronic treatment with 5 microM H2O2/h after 24 h was able to decrease all enzyme activities almost at the same level as the acute treatment. Acute and chronic treatments on antioxidant enzyme activities were prevented by cell treatment with ascorbic acid or N-acetylcysteine. These results indicate that antioxidant enzymes can also be affected by the same ROS they produce or neutralize if the time of exposure is long enough.

Oxidative stress expression status associated to Helicobacter pylori virulence in gastric diseases.

OBJECTIVES: To analyze the status of expression of inflammation markers, antioxidant and oxidant enzymes in biopsies from patients diagnosed with gastritis, gastric ulcer (GU) and gastric cancer (GC) and the Helicobacter pylori virulence from these isolated biopsies in order to evaluate a possible association among these factors. METHODS: H. pylori genotype from isolated biopsies was performed by PCR. The pattern of expression of inflammation (TNF-alpha, IL-1beta, IL-8, IL-10 and IL-12), oxidant (iNOS and Nox1) and antioxidant markers (MnSOD, GPX and CAT) of biopsies from gastritis, GU, GC and control groups was performed by RT-PCR. RESULTS: Different from other gastric diseases studied here, gastritis is characterized by an oxidative stress with significant expression of TNF-alpha, IL-8, IL-12, iNOS and Nox and significant absence of MnSOD and GPX expression. Gastritis was the only condition where there was an association between TNF-alpha or IL-8 expression and H. pylori cagA+/vacAs1 genotype. In this case, TNF-alpha expression was about 3 times higher when compared to control subjects. CONCLUSION: In this study, only gastritis was found to be associated with significant oxidative stress marker expression of TNF-alpha and IL-8 that was also related to H. pylori virulence, suggesting that they are the main oxidant stress markers responsible to trigger an increase in ROS level that contributes to decrease the expression of the MnSOD and GPX.