Essential dynamics of ubiquitin in water and in a natural deep eutectic solvent.

Natural deep eutectic solvents (NADESs) comprised of osmolytes are of interest as potential biomolecular (cryo)protectants. However, the way these solvents influence the structure and dynamics of biomolecules as well as the role of water remains poorly understood. We carried out principal component analysis of various secondary structure elements of ubiquitin in water and a betaine : glycerol : water (1 : 2 : ζ; ζ = 0, 1, 2, 5, 10, 20, 45) NADES, from molecular dynamics trajectories, to gain insight into the protein dynamics as it undergoes a transition from a highly viscous anhydrous to an aqueous environment. A crossover of the protein's essential dynamics at ζ ∼ 5, induced by solvent-shell coupled fluctuations, is observed, indicating that ubiquitin might (re)fold in the NADES upon water addition at ζ > ∼5. Further, in contrast to water, the anhydrous NADES preserves ubiquitin's essential modes at high temperatures explaining the protein's seemingly enhanced thermal stability.

Protein stability in a natural deep eutectic solvent: Preferential hydration or solvent slaving?

Deep eutectic solvents (DESs) emerged as potential alternative solvent media in multiple areas, including biomolecular (cryo)preservation. Herein, we studied the stability of a small protein (ubiquitin) in water and a betaine-glycerol-water (B:G:W) (1:2:ζ; ζ = 0, 1, 2, 5, 10) DES, through molecular dynamics. An AMBER-based model that accurately describes the density and shear viscosity of the DES is proposed. We find that water molecules are largely trapped in the solvent, precluding the formation of a full hydration layer, seemingly opposite to osmolytes' preferential exclusion/preferential hydration mechanism. Although the protein is stable in the DES, structural fluctuations are largely suppressed and only recovered upon sufficient hydration. This is explained by a solvent-slaving mechanism where ß-fluctuations are key, with the non-monotonic hydration of some amino acids with the water content providing an explanation to the non-monotonic folding of some proteins in aqueous DESs. A major thermal stability enhancement in the DES is also observed, caused by a similar slowdown of the backbone torsional dynamics. Our results support a kinetic stabilization of the protein in the DES, whereas a possible thermodynamic stabilization does not follow a preferential hydration or water entrapment mechanism.

Can a new noninvasive method for assessment of intracranial pressure predict intracranial hypertension and prognosis?

PURPOSE: Individuals with TBI are at risk of intracranial hypertension (ICH), and monitoring of intracranial pressure (ICP) is usually indicated. However, despite many new noninvasive devices, none is sufficiently accurate and effective for application in clinical practice, particularly in the management of TBIs. This study aimed to compare the noninvasive Brain4Care system (nICP) with invasive ICP (iICP) curve parameters in their ability to predict ICH and functional prognosis in severe TBI. METHODS: Observational, descriptive-analytical, and prospective study of 22 patients between 2018 and 2021, simultaneously monitored with nICP and iICP. The independent variables evaluated were the presence of ICH and functional prognoses. The dependent variables were the P2/P1 pressure ratio metrics, time to peak (TTP), and TTP × P2/P1. RESULTS: We found a good nonlinear correlation between iICP and nICP waveforms, despite a moderate Pearson's linear correlation. The noninvasive parameters of P2/P1, P2/P1 × TTP, and TTP were not associated with outcomes or ICH. The nICP P2/P1 ratio showed sensitivity/specificity/accuracy (%) of 100/0/56.3, respectively for 1-month outcomes and 77.8/22.2/50 for 6-month outcomes. The nICP TTP ratio had values of 100/0/56.3 for 1-month and 99.9/42.9/72.2 for 6-month outcomes. The nICP P2/P1 × TTP values were 100/0/56.3 for 1-month outcomes and 81.8/28.6/61.1 for 6-month outcomes. CONCLUSION: Brain4Care's noninvasive method showed low specificity and accuracy and cannot be used as the sole means of monitoring ICP in patients with severe TBI. Future studies with a larger sample of patients with P2 > P1 and new nICP curve parameters are warranted.

Impact of parabens on drinking water bacteria and their biofilms: The role of exposure time and substrate materials.

Parabens have been detected in drinking water (DW) worldwide, however, their impact on DW microbial communities remains to be explored. Microorganisms can easily adapt to environmental changes. Therefore, their exposure to contaminants of emerging concern, particularly parabens, in DW distribution systems (DWDS) may affect the microbiological quality and safety of the DW reaching the consumers tap. This work provides a pioneer evaluation of the effects of methylparaben (MP), propylparaben (PP), butylparaben (BP), and their combination (MIX), in bacterial biofilms formed on different surfaces, representative of DWDS materials - high-density polyethylene (HDPE), polypropylene (PPL) and polyvinyl chloride (PVC). Acinetobacter calcoaceticus and Stenotrophomonas maltophilia, isolated from DW, were used to form single and dual-species biofilms on the surface materials selected. The exposure to MP for 7 days caused the most significant effects on biofilms, by increasing their cellular culturability, density, and thickness up to 233%, 150%, and 224%, respectively, in comparison to non-exposed biofilms. Overall, more pronounced alterations were detected for single biofilms than for dual-species biofilms when HDPE and PPL, demonstrating that the surface material used affected the action of parabens on biofilms. Swimming motility and the production of virulence factors (protease and gelatinase) by S. maltophilia were increased up to 141%, 41%, and 73%, respectively, when exposed to MP for 7 days. The overall results highlight the potential of parabens to interfere with DW bacteria in planktonic state and biofilms, and compromise the DW microbiological quality and safety.

Acceptance of SARS-CoV-2 vaccination among cancer patients in Portugal: attitudes and associated factors.

OBJECTIVE: COVID-19 vaccines have shown efficacy and safety in healthy people. However, cancer patients under active immunosuppressive treatment were not included in the clinical trials conducted to test vaccines' efficacy and safety. This study aimed to evaluate the COVID-19 vaccine acceptance in cancer patients undergoing immunosuppressive therapy. METHODS: A total of 200 adult cancer patients received a questionnaire between March 8 and April 2, 2021, before the beginning of cancer patients' vaccination in Portugal. The questionnaire adapted from previously conducted studies included 11 close-ended items, evaluating variables such as patient sociodemographic and clinical characteristics, and the acceptance and underlying reasons to be or not to be vaccinated. The primary outcome was the intended acceptance of the COVID-19 vaccine in cancer patients. Multiple logistic regression was performed to identify factors associated with intended acceptance. RESULTS: Among the 200 delivered questionnaires, only 169 were included in this study. From those, 142 (84%) patients intended to be vaccinated against COVID-19. Only 27 participants (16%) had not yet decided or were reluctant to COVID-19 vaccination. High school degree (odds ratio (OR) 0.133, 95% confidence interval (C.I.) 0.031-0.579, p = 0.007], rural residence (OR 0.282, 95% C.I. 0.081-0.984, p = 0.047), and reluctance in believing in the vaccine efficacy (OR 0.058, 95% C.I. 0.016-0.204, p < 0.001] were identified predictors factor for COVID-19 vaccine hesitancy. CONCLUSION: Most patients intended to be vaccinated against COVID-19, and specific factors such as education level, rural residence and the belief in vaccine efficacy were related to vaccine acceptance.

Sporadic Creutzfeldt-Jakob disease presenting as epilepsia partialis continua and non-ictal nystagmus.

BACKGROUND AND PURPOSE: Creutzfeldt-Jakob disease (CJD) is a rare form of rapidly progressive neurodegenerative disorder. Seizures are uncommon in the early stage of CJD, increasing diagnostic difficulty. METHODS: An autopsy-proven case of CJD presenting initially as an epilepsia partialis continua is reported, in which the initial workup was unremarkable. Retrospectively, the presence of nystagmus, which proved to be non-epileptic, pointed to a cerebellar lesion before a diagnosis of clinically probable CJD was made. RESULTS: A 70-year-old man presented with a 3-week history of intermittent rhythmic jerking tremors in his left limbs, interfering with his gait. Examination showed left body clonic movements. Electroencephalography revealed an ictal right centroparietal pattern of focal status epilepticus. Video-oculography revealed right-beating nystagmus (mean slow phase velocity [SPV] 3.4º/s) in the dark and left-beating nystagmus (SPV 2.6º/s) in the light, left-beating nystagmus after head shaking (SPV 4º/s) and during mastoid vibration (SPV 11º/s) and mildly hypoactive horizontal head impulses. Search for occult malignancy, serologies, cerebrospinal fluid analyses, anti-onconeural antigen, auto-immune panel and brain magnetic resonance imaging were unrevealing. Rapid neurological decline was observed. Three weeks later, cerebrospinal fluid was positive for 14.3.3 protein, electroencephalography showed generalized periodic sharp wave complexes and brain magnetic resonance imaging revealed diffusion restriction and T2/fluid-attenuated inversion recovery hyperintensities in the cerebellum, basal ganglia, thalamus and cortex. He died 1 month later. Neuropathological study confirmed the diagnosis of CJD. CONCLUSION: This case highlights that CJD should be considered in the differential diagnosis of new onset epilepsia partialis continua and that neuro-ophthalmological examination can be helpful in pointing to early asymmetric cerebellar involvement.

Comparison of Waveforms Between Noninvasive and Invasive Monitoring of Intracranial Pressure.

Intracranial pressure (ICP) is an important invasive monitoring parameter in management of patients with acute brain injury and compromised compliance. This study aimed to compare waveforms obtained from standard ICP monitoring and noninvasive ICP monitoring (nICP) methods.We analyzed continuous arterial blood pressure (ABP) waves, ICP (with standard monitoring), and nICP recorded simultaneously. All signal recordings were sliced into data chunks, each 1 min in duration, and from the mean pulse, we determined the time to peak (Tp) and the ratio between tidal and percussion waves (P2/P1). We also calculated the Isomap projection of the pulses into a bidimensional space-K1 and K2. The defined nICP and ICP parameters were compared using a unilateral Wilcoxon-Mann-Whitney test. The Pearson correlation coefficient and normalized mutual information were used to verify the association between parameters.In total, 1504 min of monitoring from ten patients were studied. Nine of the patients were male. The mean age of the patients was 58.4 ± 10.4 years, and they had an initial Glasgow Coma Scale of 9 ± 4, a mean Simplified Acute Physiology Score (SAPS II) of 45.6, and an intensive care unit stay of 44 ± 45 days. With the exception of Tp, all parameters showed a weak linear association but presented a strong nonlinear association.Mutual information analysis and a bigger sample size would be helpful to build more refined models and to improve understanding of the waveform relationships.

Standardized reactors for the study of medical biofilms: a review of the principles and latest modifications.

Biofilms can cause severe problems to human health due to the high tolerance to antimicrobials; consequently, biofilm science and technology constitutes an important research field. Growing a relevant biofilm in the laboratory provides insights into the basic understanding of the biofilm life cycle including responses to antibiotic therapies. Therefore, the selection of an appropriate biofilm reactor is a critical decision, necessary to obtain reproducible and reliable in vitro results. A reactor should be chosen based upon the study goals and a balance between the pros and cons associated with its use and operational conditions that are as similar as possible to the clinical setting. However, standardization in biofilm studies is rare. This review will focus on the four reactors (Calgary biofilm device, Center for Disease Control biofilm reactor, drip flow biofilm reactor, and rotating disk reactor) approved by a standard setting organization (ASTM International) for biofilm experiments and how researchers have modified these standardized reactors and associated protocols to improve the study and understanding of medical biofilms.

Measurement of adsorption constants of laccase on gold nanoparticles to evaluate the enhancement in enzyme activity of adsorbed laccase.

Adsorption of enzymes to nanoparticles and the mechanisms responsible for enzyme activity modulation of adsorbed enzymes are not well understood. In this work, gold nanoparticles were used for electrostatic adsorption of a plant-derived laccase. Adsorption constants were determined by four independent techniques: dynamic light scattering, electrophoretic light scattering, agarose gel electrophoresis and fluorescence quenching. Stable bionanoconjugates were formed with log K in the range 6.8-8.9. An increase in enzyme activity was detected, in particular at acidic and close to neutral pH values, a feature that expands the useful pH range of the enzyme. A model for the adsorption was developed, based on geometrical considerations and volume increase data from dynamic light scattering. This indicates that enzymes adsorbed to gold nanoparticles are ca. 9 times more active than the free enzyme.

Long-term results of percutaneous balloon valvuloplasty in neonatal critical pulmonary valve stenosis: a 20-year, single-centre experience.

Introduction Percutaneous balloon valvuloplasty is the primary treatment for critical pulmonary valve stenosis in neonates. Thus far, a few studies have reported long-term results of this technique in neonatal critical pulmonary valve stenosis. METHODS: We carried out a retrospective study of all consecutive newborns with critical pulmonary valve stenosis subjected to percutaneous balloon valvuloplasty at a single centre, between 1994 and 2014, to assess its immediate and long-term safety and efficacy. RESULTS: A total of 24 neonates presented with critical pulmonary valve stenosis. The mean diameter of the pulmonary annulus was 7 mm (±1.19); 33.3% had a dysplastic pulmonary valve, and 92% were started on prostaglandin E1 treatment. Percutaneous balloon valvuloplasty was performed at a mean age of 4.0±4.3 days using, on average, a balloon-to-pulmonary annulus ratio of 1.18 mm (with a range from 0.9 to 1.43). Immediate success was achieved in 22/24 patients (92%) with a reduction in the pulmonary transvalvular peak gradient (p<0.05) and in the right ventricle/systemic pressure ratio (p<0.05). There was one death (4%) 6 days after the procedure, and 29.2% of them had transient rhythm complications. For a mean follow-up time of 8.4 years, the re-intervention rate was 42.9%. In total, 14 re-interventions were performed in nine neonates, including surgery in six. Freedom from re-intervention was 50% at 8 years and 43% at 10 and 15 years. CONCLUSION: This series, to the best of our knowledge, has had the longest follow-up of neonates with critical pulmonary valve stenosis. Percutaneous balloon valvuloplasty is a safe and effective treatment, and in our study 75% of the patients were exclusively treated using this technique.

Vascular function long term after Kawasaki disease: another piece of the puzzle?

BACKGROUND: Kawasaki disease is an acute systemic vasculitis. Cardiac complications are frequent and include endothelial dysfunction in patients with coronary anomalies. Thus far, endothelial dysfunction in patients with no coronary lesions is poorly understood. Our aim was to access the vascular function in adolescents and young adults long term after Kawasaki disease, but without coronary aneurysms or any other cardiac risk factors. METHODS: We carried out a single-centre prospective study in a Portuguese population. We evaluated two groups of subjects: (1) Kawasaki disease patients over 11 years of age, diagnosed >5 years ago, with no coronary lesions or any other risk factors for cardiovascular disease; (2) control group of individuals without cardiovascular risk factors. Patients and controls were clinically assessed. Endo-PAT and carotid intima-media thickness assessment were performed to determine vascular function. RESULTS: In total, 43 Kawasaki disease patients were assessed and compared with 43 controls. Kawasaki disease patients presented a decreased reactive hyperaemia index compared with controls (1.59±0.45 versus 1.98±0.41; p<0.001). Augmentation index was similar in both groups (-4.5±7 versus -5±9%; p 0.6). The mean carotid intima-media thickness was not significantly increased in the Kawasaki disease group. There were no statistically significant changes with regard to laboratory data. CONCLUSIONS: Children with Kawasaki disease may have long-term sequelae, even when there is no discernible coronary artery involvement in the acute stage of the disease. Further research is needed to assess whether known strategies to improve endothelial function would bring potential benefits to Kawasaki disease patients.

Conservation planning for biodiversity and wilderness: a real-world example.

Several of the most important conservation prioritization approaches select markedly different areas at global and regional scales. They are designed to maximize a certain biodiversity dimension such as coverage of species in the case of hotspots and complementarity, or composite properties of ecosystems in the case of wilderness. Most comparisons between approaches have ignored the multidimensionality of biodiversity. We analyze here the results of two species-based methodologies-hotspots and complementarity-and an ecosystem-based methodology-wilderness-at local scale. As zoning of protected areas can increase the effectiveness of conservation, we use the data employed for the management plan of the Peneda-Gerês National Park in Portugal. We compare the approaches against four criteria: species representativeness, wilderness coverage, coverage of important areas for megafauna, and for regulating ecosystem services. Our results suggest that species- and ecosystem-based approaches select significantly different areas at local scale. Our results also show that no approach covers well all biodiversity dimensions. Species-based approaches cover species distribution better, while the ecosystem-based approach favors wilderness, areas important for megafauna, and for ecosystem services. Management actions addressing different dimensions of biodiversity have a potential for contradictory effects, social conflict, and ecosystem services trade-offs, especially in the context of current European biodiversity policies. However, biodiversity is multidimensional, and management and zoning at local level should reflect this aspect. The consideration of both species- and ecosystem-based approaches at local scale is necessary to achieve a wider range of conservation goals.

Regucalcin is an androgen-target gene in the rat prostate modulating cell-cycle and apoptotic pathways.

BACKGROUND: Regucalcin (RGN) is a calcium (Ca(2+) )-binding protein underexpressed in prostate adenocarcinoma comparatively to non-neoplastic prostate or benign prostate hyperplasia cases. Moreover, RGN expression is negatively associated with the cellular differentiation of prostate adenocarcinoma, suggesting that loss of RGN may be associated with tumor onset and progression. However, the RGN actions over the control of prostate cell growth have not been investigated. METHODS: Androgens are implicated in the promotion of prostate cell proliferation, thus we studied the in vivo effect of androgens on RGN expression in rat prostate. The role of RGN modulating cell proliferation and apoptotic pathways in rat prostate was investigated using transgenic animals (Tg-RGN) overexpressing the protein. RESULTS: In vivo stimulation with 5α-dihydrotestosterone (DHT) down-regulated RGN expression in rat prostate. Cell proliferation index and prostate weight were reduced in Tg-RGN, which was concomitant with altered expression of cell-cycle regulators. Tg-RGN presented diminished expression of the oncogene H-ras and increased expression of cell-cycle inhibitor p21. Levels of anti-apoptotic Bcl-2, as well as the Bcl-2/Bax protein ratio were increased in prostates overexpressing RGN. Both caspase-3 expression and enzyme activity were decreased in the prostates of Tg-RGN. CONCLUSIONS: Overexpression of RGN resulted in inhibition of cell proliferation and apoptotic pathways, which demonstrated its role maintaining prostate growth balance. Thus, deregulation of RGN expression may be an important event favoring the development of prostate cancer. Moreover, the DHT effect down-regulating RGN expression in rat prostate highlighted for the importance of this protein in prostatic physiology.

The effects of methylparaben exposure on biofilm tolerance to chlorine disinfection.

Parabens are emerging contaminants that have been detected in drinking water. Their presence in DW distribution systems (DWDS) can alter bacterial behaviour, characteristics, and structure, which may compromise DW disinfection. This work provides insights into the impact of methylparaben (MP) on the tolerance to chlorine disinfection and antibiotics from dual-species biofilms formed by Acinetobacter calcoaceticus and Stenotrophomonas maltophilia isolated from DW and grown on high-density polyethylene (HDPE) and polypropylene (PPL). Results showed that dual-species biofilms grown on PPL were more tolerant to chlorine disinfection, expressing a decrease of over 50 % in logarithmic reduction values of culturable cells in relation to non-exposed biofilms. However, bacterial tolerance to antibiotics was not affected by MP presence. Although MP-exposed dual-species biofilms grown on HDPE and PPL were metabolically more active than non-exposed counterparts, HDPE seems to be the material with lower impact on DW risk management and disinfection, if MP is present. Overall, results suggest that MP presence in DW may compromise chlorine disinfection, and consequently affect DW quality and stability, raising potential public health issues.

Rare Presentation and Diagnostic Challenge of a Germ Cell Tumor of the Yolk Sac Type.

Opsoclonus-ataxia paraneoplastic syndrome (OAPS) is a rare neurological disorder often associated with malignancies. This case report highlights an unusual instance of OAPS linked to a yolk sac (germ cell) tumor, a correlation underrepresented in the medical literature. The patient presented with distinct neurological symptoms alongside mediastinal lymphadenopathies. The subsequent diagnostic journey revealed a yolk sac germ cell tumor. Following incisional biopsies and treatment, the patient experienced fluctuations in mental status, leading to challenges in initiating chemotherapy. Despite these complications, a multidisciplinary approach involving neurologists, oncologists, and hematologists was pivotal. The case emphasizes the complexities of managing OAPS in tandem with a germ cell tumor, underscoring the need for further research and highlighting the significance of specialized neurological evaluation in similar cases.

A review of research advances on disinfection strategies for biofilm control in drinking water distribution systems.

The presence of biofilms in drinking water distribution systems (DWDS) is responsible for water quality deterioration and a possible source of public health risks. Different factors impact the biological stability of drinking water (DW) in the distribution networks, such as the presence and concentration of nutrients, water temperature, pipe material composition, hydrodynamic conditions, and levels of disinfectant residual. This review aimed to evaluate the current state of knowledge on strategies for DW biofilm disinfection through a qualitative and quantitative analysis of the literature published over the last decade. A systematic review method was performed on the 562 journal articles identified through database searching on Web of Science and Scopus, with 85 studies selected for detailed analysis. A variety of disinfectants were identified for DW biofilm control such as chlorine, chloramine, UV irradiation, hydrogen peroxide, chlorine dioxide, ozone, and others at a lower frequency, namely, electrolyzed water, bacteriophages, silver ions, and nanoparticles. The disinfectants can impact the microbial communities within biofilms, reduce the number of culturable cells and biofilm biomass, as well as interfere with the biofilm matrix components. The maintenance of an effective residual concentration in the water guarantees long-term prevention of biofilm formation and improves the inactivation of detached biofilm-associated opportunistic pathogens. Additionally, strategies based on multi-barrier processes by optimization of primary and secondary disinfection combined with other water treatment methods improve the control of opportunistic pathogens, reduce the chlorine-tolerance of biofilm-embedded cells, as well as decrease the corrosion rate in metal-based pipelines. Most of the studies used benchtop laboratory devices for biofilm research. Even though these devices mimic the conditions found in real DWDS, future investigations on strategies for DW biofilm control should include the validity of the promising strategies against biofilms formed in real DW networks.

Efficacy, Complications and Effects of Alcides Pereira's Sutures in the Management of Uterine Atony: A Retrospective Study.

INTRODUCTION: Uterine compressive sutures are conservative measures applied in cases of severe postpartum hemorrhage unresponsive to uterotonics. Pereira's suture was introduced in 2005 and consists of two longitudinal and three transverse non-transfixes sutures. Previous studies reported favorable results, highlighting its benefits and value. The aim of this study was to assess the efficacy, complications, and impact on fertility and future pregnancies of Pereira's suture applied in cases of uterine atony and postpartum hemorrhage. METHODS: An observational retrospective study was performed by consulting the medical records of women treated with Pereira's compressive sutures in a tertiary center between January 2013 and December 2022. We registered demographic data, pregnancy outcomes, short-term complications, and outcomes of subsequent pregnancies. RESULTS: A total of 50 women were treated with Pereira's suture. The overall success rate was 96% and no hysterectomies were performed. Women who had sutures performed during an intra-cesarean section had better outcomes than those who had an after-cesarean section. Complications were reported in 12% (n = 6) of women, with the most frequent being pelvic infection (n = 3) and abdominal pain (n = 3). Regarding fertility, all women desiring a future pregnancy (n = 5) were able to conceive, resulting in three live births. CONCLUSION: Pereira's suture is a type of suture that provides numerous advantages and should be considered when first-line medical treatment fails. When applied at an early stage, the sutures may prevent maternal morbidity. The Alcides Pereira's suture is a safe technique and appears to preserve fertility.

Spontaneous Hemoperitoneum Due to Ruptured Uterine Artery: A Rare Complication During Pregnancy.

Spontaneous rupture of the uterine artery is a rare and life-threatening cause of hemoperitoneum in pregnancy, associated with high maternal and perinatal morbidity and mortality. We present a case of a 29-year-old woman, in the 36th week of gestation, with acute abdomen due to hemoperitoneum. Ultrasound revealed free fluid in the abdominal cavity, with no signs of fetal distress, and the patient was mildly hypotensive. Exploratory laparotomy and cesarean section were performed, and extensive blood clots on the upper abdominal quadrants were discovered, as well as a bleeding left uterine artery. We expect that this case raises awareness of the ruptured uterine artery as a possible etiology of hemoperitoneum during pregnancy.

A one-step low-cost molecular test for SARS-CoV-2 detection suitable for community testing using minimally processed saliva.

The gold standard for coronavirus disease 2019 diagnostic testing relies on RNA extraction from naso/oropharyngeal swab followed by amplification through reverse transcription-polymerase chain reaction (RT-PCR) with fluorogenic probes. While the test is extremely sensitive and specific, its high cost and the potential discomfort associated with specimen collection made it suboptimal for public health screening purposes. In this study, we developed an equally reliable, but cheaper and less invasive alternative test based on a one-step RT-PCR with the DNA-intercalating dye SYBR Green, which enables the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directly from saliva samples or RNA isolated from nasopharyngeal (NP) swabs. Importantly, we found that this type of testing can be fine-tuned to discriminate SARS-CoV-2 variants of concern. The saliva RT-PCR SYBR Green test was successfully used in a mass-screening initiative targeting nearly 4500 asymptomatic children under the age of 12. Testing was performed at a reasonable cost, and in some cases, the saliva test outperformed NP rapid antigen tests in identifying infected children. Whole genome sequencing revealed that the antigen testing failure could not be attributed to a specific lineage of SARS-CoV-2. Overall, this work strongly supports the view that RT-PCR saliva tests based on DNA-intercalating dyes represent a powerful strategy for community screening of SARS-CoV-2. The tests can be easily applied to other infectious agents and, therefore, constitute a powerful resource for an effective response to future pandemics.

Six transmembrane epithelial antigen of the prostate 1 is down-regulated by sex hormones in prostate cells.

BACKGROUND: STEAP1 is over-expressed in several types of tumors, especially prostate cancer, where it is localized in the plasma membrane of epithelial cells, at cell-cell junctions. Its role in prostate carcinogenesis and its regulation in prostate cells remain unknown. Therefore, we propose to study the effect of sex hormones in the regulation of STEAP1 expression in prostate cells in vitro and in vivo. METHODS: LNCaP prostate cells were incubated with fetal bovine serum (FBS), charcoal-stripped FBS (CS-FBS), 5α-dihydrotestosterone (DHT), and 17ß-estradiol (E2 ) for different periods of stimulation. In addition, adult male Wistar rats were castrated and treated with DHT and E2 . The levels of STEAP1 in response to treatments were analyzed by real-time PCR, Western blot, and immunohistochemistry. RESULTS: The treatment of LNCaP cells with DHT or E2 induces a down-regulation of STEAP1 expression, while incubation with CS-FBS has the opposite effect. Experiments using inhibitors of androgen and estrogen receptor (AR and ER) showed that down-regulation of STEAP1 is AR-dependent, but ER-independent. However, the mediation of six transmembrane epithelial antigen of the prostate 1 (STEAP1) expression by AR seems to be dependent of de novo protein synthesis. In vivo studies showed that castrated rats express higher levels of STEAP1 protein when compared to intact rats, an effect reversed by DHT or E2 replacement. CONCLUSIONS: STEAP1 is down-regulated by DHT and E2 in LNCaP cells and in rat prostate.