Study protocol: a pragmatic, cluster-randomized controlled trial to evaluate the effect of implementation of the Truenat platform/MTB assays at primary health care clinics in Mozambique and Tanzania (TB-CAPT CORE).

BACKGROUND: In 2020, the WHO-approved Molbio Truenat platform and MTB assays to detect Mycobacterium tuberculosis complex (MTB) and resistance to rifampicin directly on sputum specimens. This primary health care center-based trial in Mozambique and Tanzania investigates the effect of Truenat platform/MTB assays (intervention arm) combined with rapid communication of results compared to standard of care on TB diagnosis and treatment initiation for microbiologically confirmed TB at 7 days from enrolment. METHODS: The Tuberculosis Close the Gap, Increase Access, and Provide Adequate Therapy (TB-CAPT) CORE trial employs a pragmatic cluster randomized controlled design to evaluate the impact of a streamlined strategy for delivery of Truenat platform/MTB assays testing at primary health centers. Twenty-nine centers equipped with TB microscopy units were selected to participate in the trial. Among them, fifteen health centers were randomized to the intervention arm (which involves onsite molecular testing using Truenat platform/MTB assays, process process optimization to enable same-day TB diagnosis and treatment initiation, and feedback on Molbio platform performance) or the control arm (which follows routine care, including on-site sputum smear microscopy and the referral of sputum samples to off-site Xpert testing sites). The primary outcome of the study is the absolute number and proportion of participants with TB microbiological confirmation starting TB treatment within 7 days of their first visit. Secondary outcomes include time to bacteriological confirmation, health outcomes up to 60 days from first visit, as well as user preferences, direct cost, and productivity analyses. ETHICS AND DISSEMINATION: TB-CAPT CORE trial has been approved by regulatory and ethical committees in Mozambique and Tanzania, as well as by each partner organization. Consent is informed and voluntary, and confidentiality of participants is maintained throughout. Study findings will be presented at scientific conferences and published in peer-reviewed international journals. TRIAL REGISTRATION: US National Institutes of Health's ClinicalTrials.gov, NCT04568954. Registered 23 September 2020.

Investigator Initiated Clinical Trials (IICTs): A Systematic Search in Registries to Compare the Czech Republic and Portugal in Terms of Funding Policies and Scientific Outcomes.

OBJECTIVES: Clinical trials provide one of the highest levels of evidence to support medical practice. Investigator initiated clinical trials (IICTs) answer relevant questions in clinical practice that may not be addressed by industry. For the first time, two European Countries are compared in terms of IICTs, respective funders and publications, envisaging to inspire others to use similar indicators to assess clinical research outcomes. METHODS: A retrospective systematic search of registered IICTs from 2004 to 2017, using four clinical trials registries was carried out in two European countries with similar population, GDP, HDI and medical schools but with different governmental models to fund clinical research. Each IICT was screened for sponsors, funders, type of intervention and associated publications, once completed. RESULTS: IICTs involving the Czech Republic and Portugal were n = 439 (42% with hospitals as sponsors) and n = 328 (47% with universities as sponsors), respectively. The Czech Republic and Portuguese funding agencies supported respectively 61 and 27 IICTs. Among these, trials with medicinal products represent 52% in Czech Republic and 4% in Portugal. In the first, a higher percentage of IICTs' publications in high impact factor journals with national investigators as authors was observed, when compared to Portugal (75% vs 15%). CONCLUSION: The better performance in clinical research by Czech Republic might be related to the existence of specific and periodic funding for clinical research, although further data are still needed to confirm this relationship. In upcoming years, the indicators used herein might be useful to tracking clinical research outcomes in these and other European countries.

Efectividad de los regímenes tratar y extender vs. pro re nata para el tratamiento de degeneración macular neovascular vinculada a la edad: una comparación en un entorno real / Effectiveness of treat-and-extend versus pro re nata regimens in the treatment of Neovascular Age Macular Degeneration: A Real World Comparison

OBJETIVO: Evaluar y comparar los resultados visuales y morfológicos de regímenes de tratamiento pro re nata (PRN) y tratar-y-extender (T&E) a tres años en la práctica clínica real. MÉTODOS: Un estudio retrospectivo de pacientes con degeneración macular vinculada a la edad neovascular (DMEN) tratadas con anti-VEGF con tres años de seguimiento continuo y sin tratamientos anti-VEGF anteriores. Se midieron la mejor agudeza visual corregida (MAVC), el espesor foveal central (EFC) y el número de inyecciones intravítreas para determinar diferencias estadísticas entre ambos grupos al inicio y a lo largo del seguimiento. RESULTADOS: Se incluyeron en el estudio un total de 240 ojos, 170 en el grupo PRN y 70 en el grupo T&E. A los 12 meses la ganancia media con respecto al inicio de MAVC (en letras ETDRS) llegó a su punto más alto en el grupo T&E (+ 6,38 ± 13,32; p = 0,25). En el grupo PRN, MAVC llegó al máximo a los tres meses y disminuyó lentamente hasta el final del seguimiento. Con ambos regímenes, desde el inicio el EFC continuó disminuyendo hasta el segundo año (PRN -138,81 [-846,7 a +162,77] y T&E -81 [-604 a +100] μm, p = 0,06). Posteriormente, el grupo T&E mantuvo esta tendencia, llegando al nivel más bajo de EFC a los 36 meses, mientras que el grupo PRN mostró un aumento en los valores de EFC (PRN -104 [-807,7 a +297] μm y T&E -103 [-575 a +244], μm p = 0,63). Los pacientes tratados con el régimen T&E recibieron un número significativamente mayor de inyecciones (PRN 16,3 ± 7,6 vs. T&E 23,9 ± 9,4, p <0,01). CONCLUSIÓN: Los resultados demostraron una tendencia de T&E a conseguir valores más altos de MAVC, llegando al máximo a los 12 meses, y grosores menores de EFC al final de tres años. A pesar del mayor número de inyecciones en el grupo T&E, la media de MAVC revirtió a los valores de base a los tres años

PURPOSE: Evaluate and compare the visual and morphological results of Pro re nata (PRN) and treat-and-extend (T&E) treatment regimens at 3 years in real world clinical practice. METHODS: Retrospective study of patients with neovascular age macular: degeneration (AMD) treated with anti-VEGF with 3 years of continuous follow-up and no previous anti-VEGF treatment. Best corrected visual acuity (BCVA), central foveal thickness (CFT) and number of intravitreal injections outcomes were tested for statistical differences between the two groups at baseline and during follow-up. RESULTS: A total of 240 eyes were included in the study, 170 in the PRN group and 70 in the T&E group. At 12 months, mean BCVA (ETDRS letters) gain from baseline was at its highest point in the T&E group (+6.38 ± 13.32; p = 0.25). In the PRN group, BCVA peaked at 3 months and slowly decreased until end of follow-up. With both regimens, from baseline, CFT continued to decrease until the second year (PRN -138.81 [-846.7 to +162.77] and T&E -81 [-604 to +100] μm, p = 0.06). After that, T&E group maintained this tendency, reaching the lowest CFT value at 36 months, whereas PRN group showed an increased in CFT values (PRN -104 [-807.7 to +297] μm and T&E -103 [-575 to +244], μm p = 0.63). Patients treated with T&E regimen received a significantly higher number of injections (PRN 16.3 ± 7.6 vs T&E 23.9 ± 9.4, p < 0.01). CONCLUSION: Our results demonstrated a trend towards for T&E to achieve higher marks in BCVA, peaking at 12 months, and lower CFT thickness at the end of three years. Despite the higher number of injections performed in the T&E group the mean BCVA reverts to baseline values at 3 years

Effectiveness of treat-and-extend versus pro re nata regimens in the treatment of Neovascular Age Macular Degeneration: A Real World Comparison. / Efectividad de los regímenes tratar y extender vs. pro re nata para el tratamiento de degeneración macular neovascular vinculada a la edad: una comparación en un entorno real.

PURPOSE: Evaluate and compare the visual and morphological results of Pro re nata (PRN) and treat-and-extend (T&E) treatment regimens at 3 years in real world clinical practice. METHODS: Retrospective study of patients with neovascular age macular degeneration (AMD) treated with anti-VEGF with 3 years of continuous follow-up and no previous anti-VEGF treatment. Best corrected visual acuity (BCVA), central foveal thickness (CFT) and number of intravitreal injections outcomes were tested for statistical differences between the two groups at baseline and during follow-up. RESULTS: A total of 240 eyes were included in the study, 170 in the PRN group and 70 in the T&E group. At 12 months, mean BCVA (ETDRS letters) gain from baseline was at its highest point in the T&E group (+6.38±13.32; p=0.25). In the PRN group, BCVA peaked at 3 months and slowly decreased until end of follow-up. With both regimens, from baseline, CFT continued to decrease until the second year (PRN -138.81 [-846.7 to +162.77] and T&E -81 [-604 to +100] µm, p=0.06). After that, T&E group maintained this tendency, reaching the lowest CFT value at 36 months, whereas PRN group showed an increased in CFT values (PRN -104 [-807.7 to +297] µm and T&E -103 [-575 to +244], µm p=0.63). Patients treated with T&E regimen received a significantly higher number of injections (PRN 16.3±7.6 vs T&E 23.9 ±9.4, p<0.01). CONCLUSION: Our results demonstrated a trend towards for T&E to achieve higher marks in BCVA, peaking at 12 months, and lower CFT thickness at the end of three years. Despite the higher number of injections performed in the T&E group the mean BCVA reverts to baseline values at 3 years.

Seasonal proteome variation in intertidal shrimps under a natural setting: Connecting molecular networks with environmental fluctuations.

The ability of intertidal organisms to maintain their performance via molecular and physiological adjustments under low tide, seasonal fluctuations and extreme events ultimately determines population viability. Analyzing this capacity in the wild is extremely relevant since intertidal communities are under increased climate variability owing to global changes. We addressed the seasonal proteome signatures of a key intertidal species, the shrimp Palaemon elegans, in a natural setting. Shrimps were collected during spring and summer seasons at low tides and were euthanized in situ. Environmental variability was also assessed using hand-held devices and data loggers. Muscle samples were taken for 2D gel electrophoresis and protein identification through mass spectrometry. Proteome data revealed that 55 proteins (10.6% of the proteome) significantly changed between spring and summer collected shrimps, 24 of which were identified. These proteins were mostly involved in cytoskeleton remodelling, energy metabolism and transcription regulation. Overall, shrimps modulate gene expression leading to metabolic and structural adjustments related to seasonal differences in the wild (i.e. abiotic variation and possibly intrinsic cycles of reproduction and growth). This potentially promotes performance and fitness as suggested by the higher condition index in summer-collected shrimps. However, inter-individual variation (% coefficient of variation) in protein levels was quite low (min-max ranges were 0.6-8.3% in spring and 1.2-4.8% in summer), possibly suggesting reduced genetic diversity or physiological canalization. Protein plasticity is relevant to cope with present and upcoming environmental variation related to anthropogenic forcing (e.g. global change, pollution) but low inter-individual variation may limit evolutionary potential of shrimp populations.

Investigator-initiated clinical trials conducted by the Portuguese Clinical Research Infrastructure Network (PtCRIN).

Interventional clinical studies can provide the highest levels of evidence and generate significant results on specific investigational medicinal products or medical devices. In order to have powerful studies, attain unquestionable results and make significant discoveries, the number of patients enrolled must be high. Therefore, multinational, randomised clinical trials are necessary. The multicentre, multinational recruitment of subjects in investigator-initiated clinical trials (IICTs) increases their logistical burden, justifying the need for specific infrastructures to ease implementation. Herein, we provide for the first time an overview of the facts and figures concerning IICTs, existing infrastructures' capacity for interventional clinical research, and scientific performance of investigators in a European country, Portugal. We aim to highlight the relevance and need for investing in European infrastructures such as the European Clinical Research Infrastructure Network (ECRIN) for multinational IICTs. A public, non-profit organisation, ECRIN facilitates the conduct of multinational clinical trials in Europe by coordinating scientific partners and their networks, and providing advice, management services and tools to enhance collaboration. Currently in Portugal, few multinational randomised IICTs are coordinated by national investigators. This is most likely due to the lack of human resources dedicated to clinical trials in clinical research centres (CRCs) as well as the scarcity of professional academic clinical trial units (CTUs) providing logistics and management services at non-profit rates. With the data shown, we expect to trigger the development of similar studies in other European countries and stress the impact of government support for IICTs.

d-serine levels in Alzheimer's disease: implications for novel biomarker development.

Alzheimer's disease (AD) is a severe neurodegenerative disorder still in search of effective methods of diagnosis. Altered levels of the NMDA receptor co-agonist, d-serine, have been associated with neurological disorders, including schizophrenia and epilepsy. However, whether d-serine levels are deregulated in AD remains elusive. Here, we first measured D-serine levels in post-mortem hippocampal and cortical samples from nondemented subjects (n=8) and AD patients (n=14). We next determined d-serine levels in experimental models of AD, including wild-type rats and mice that received intracerebroventricular injections of amyloid-ß oligomers, and APP/PS1 transgenic mice. Finally, we assessed d-serine levels in the cerebrospinal fluid (CSF) of 21 patients with a diagnosis of probable AD, as compared with patients with normal pressure hydrocephalus (n=9), major depression (n=9) and healthy controls (n=10), and results were contrasted with CSF amyloid-ß/tau AD biomarkers. d-serine levels were higher in the hippocampus and parietal cortex of AD patients than in control subjects. Levels of both d-serine and serine racemase, the enzyme responsible for d-serine production, were elevated in experimental models of AD. Significantly, d-serine levels were higher in the CSF of probable AD patients than in non-cognitively impaired subject groups. Combining d-serine levels to the amyloid/tau index remarkably increased the sensitivity and specificity of diagnosis of probable AD in our cohort. Our results show that increased brain and CSF d-serine levels are associated with AD. CSF d-serine levels discriminated between nondemented and AD patients in our cohort and might constitute a novel candidate biomarker for early AD diagnosis.

D-serine prevents cognitive deficits induced by acute stress.

Increasing evidence indicates that acute stress disrupts cognitive functions mediated by glutamate-NMDA receptors, although the mechanisms are not fully understood. Here we investigated whether d-serine and glycine, the endogenous co-agonists of the NMDA receptor, are regulated by acute stress. We studied the biochemical and behavioral effects of acute restraint stress in C57BL/6 mice. Acute restraint stress decreased d-serine levels in the prefrontal cortex and glycine levels in the hippocampus. Behaviorally, acute stress impaired memory consolidation in the object recognition task and prepulse inhibition of the startle response. Importantly, d-serine administration (1 g/kg, i.p.) prevented both stress-induced impairments. Taken together, our results show for the first time an interplay between stress and d-serine and warrant further research on the role of d-serine in stress-related disorders.

Time-resolved microfocused small-angle X-ray scattering investigation of the microfluidic concentration of charged nanoparticles.

We describe the concentration process of a dispersion of silica nanoparticles undergoing evaporation in a dedicated microfluidic device. Using microfocused small-angle X-ray scattering, we measure in time and space both the concentration field of the dispersion and its structure factor. We show that the electrostatic interactions affect the concentration rate by strongly enhancing the collective diffusion coefficient of the nanoparticle dispersion. En route towards high concentrations, the nanoparticles eventually undergo a liquid-solid phase transition in which we evidence crystallites of micron size.

Nonviral gene delivery to mesenchymal stem cells using cationic liposomes for gene and cell therapy.

Mesenchymal stem cells (MSCs) hold a great promise for application in several therapies due to their unique biological characteristics. In order to harness their full potential in cell-or gene-based therapies it might be advantageous to enhance some of their features through gene delivery strategies. Accordingly, we are interested in developing an efficient and safe methodology to genetically engineer human bone marrow MSC (BM MSC), enhancing their therapeutic efficacy in Regenerative Medicine. The plasmid DNA delivery was optimized using a cationic liposome-based reagent. Transfection efficiencies ranged from approximately 2% to approximately 35%, resulting from using a Lipid/DNA ratio of 1.25 with a transgene expression of 7 days. Importantly, the number of plasmid copies in different cell passages was quantified for the first time and approximately 20,000 plasmid copies/cell were obtained independently of cell passage. As transfected MSC have shown high viabilities (>90%) and recoveries (>52%) while maintaining their multipotency, this might be an advantageous transfection strategy when the goal is to express a therapeutic gene in a safe and transient way.

A quantitative method to evaluate mesenchymal stem cell lipofection using real-time PCR.

Genetic modification of human mesenchymal stem cells (MSC) is a powerful tool to improve the therapeutic utility of these cells and to increase the knowledge on their regulation mechanisms. In this context, strong efforts have been made recently to develop efficient nonviral gene delivery systems. Although several studies addressed this question most of them use the end product of a reporter gene instead of the DNA uptake quantification to test the transfection efficiency. In this study, we established a method based on quantitative real-time PCR (RT-PCR) to determine the intracellular plasmid DNA copy number in human MSC after lipofection. The procedure requires neither specific cell lysis nor DNA purification. The influence of cell number on the RT-PCR sensitivity was evaluated. The method showed good reproducibility, high sensitivity, and a wide linear range of 75-2.5 x 106 plasmid DNA copies per cell. RT-PCR results were then compared with the percentage of transfected cells assessed by flow cytometry analysis, which showed that flow cytometry-based results are not always proportional to plasmid cellular uptake determined by RT-PCR. This work contributed for the establishment of a rapid quantitative assay to determine intracellular plasmid DNA in stem cells, which will be extremely beneficial for the optimization of gene delivery strategies.

Optimization of gene delivery to HEK293T cells by microporation using a central composite design methodology.

Microporation is an efficient method for delivering plasmid DNA molecules into cultured cells. Herein, we present the optimization of gene delivery by microporation using a Central Composite Design methodology. It was given relevance not only to the transfection efficiency but also to the cell recovery. Different amounts of DNA (1 and 3 µg) mainly affected cell viabilities and cell recoveries, which decrease from 93 to 76% and from 47 to 25% respectively, when higher DNA quantity is used. With this work we suggest an easy methodology to improve transfection of mammalian cells underlining the feasibility to achieve 60% of gene delivery efficiencies whilst recovering 50% of cells, with 90% of viability.

Effect of cationic liposomes/DNA charge ratio on gene expression and antibody response of a candidate DNA vaccine against Maedi Visna virus.

Maedi Visna virus (MVV) is an ovine lentivirus with high prevalence all over the world. Since conventional vaccines had failed in protecting animals against the infection, the development of a DNA vaccine can be an alternative. The candidate vaccine was constructed by cloning the sequence encoding MVV p25 protein and was tested both in vitro and in vivo experiments associated with cationic liposomes. The lipoplexes (plasmid DNA-liposome complexes) with charge ratios ranging from 0 to 18 were prepared in physiological saline solution and characterized at a physical-chemistry level. Agarose gel electrophoresis was used as a first approach to evaluate qualitatively the amount of unbounded DNA by the liposomes. Dynamic light scattering measurements revealed that under the studied conditions lipoplexes with theoretical charge ratios (+/-) from 3 to 6 are unstable and prone to aggregation displaying sizes higher than 1 microm. At lower and higher charge ratios lipoplex size range from 200 to 500 nm. Using a Foster Resonance Energy Transfer methodology previously reported by us, complexation efficiency of the same complexes was related to in vitro and in vivo results. Higher transfection efficiencies were obtained in vitro with lipoplexes with charge ratio (+/-)=10, where 97% of the DNA were protected by the liposomes. However, the subcutaneous immunization of mice induced higher antibody titers with lipoplexes at charge ratio (+/-)=1, in which only 23% DNA is protected by the liposomes. Moreover, use of cationic liposomes has shown an increased antibody response when compared with a naked DNA immunization.

Nail abrasion.

Certain diseases of the nail complex cause hyperkeratosis or alterations of the shape of the nail plate. These conditions may be painful, may decrease the penetration of topical medicaments and may be ugly. The nail plate abrasion, performed with dermabrader device or sandpaper, has application in patients suffering from onychomycosis, psoriasis, subnail infections and haematomas. The technique facilitates the collection of scales for mycological examination, decreases treatment time (of topical monotherapy) for onychomycosis and provides greater comfort for the patient by reducing nail plate thickness. It can also be useful for the partial removal of the nail plate in cases of haematomas and subnail infections. Nail abrasion is an effective and inexpensive method, easily applied in either nail pathologies with hyperkeratosis of the nail plate or in those requiring partial removal of the plate.

Tetrapotassium disodium decavanadate(V) decahydrate.

The title compound, K(4)Na(2)[V(10)O(28)].10H(2)O, is isostructural with the known disodium tetraammonium salt of the centrosymmetric [V(10)O(18)](6-) anion.

Lipid profile in haemodialysis patients treated with recombinant human erythropoietin.

BACKGROUND: The long-term effect of recombinant human erythropoietin (rhEPO) on the blood-lipid profile has not been well documented. The aim of this study was to evaluate whether rhEPO therapy affects the lipid pattern. METHODS: A group of 102 maintenance haemodialysis patients were treated for 2 years with rhEPO given intravenously at the end of the dialysis session. Attempts were made to keep the haemoglobin (Hb) at 10-11 g/dl and/or the haematocrit (Hct) at 30-35%. Twenty maintenance haemodialysis patients not treated with rhEPO were examined as controls. Total cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides, apolipoproteins A1 and B, and lipoprotein (a)[Lp (a)] were assessed at baseline (without rhEPO), and 1 and 2 years after the beginning of treatment. Hb, Hct and ferritin were measured monthly, and Kt/v was evaluated monthly and kept above 1.1. RESULTS: During follow-up, in both groups, there was a significant increase in Apo A1 and no significant changes in the other lipid parameters. In the treated group, Hb and Hct increased significantly after the fourth month of treatment. CONCLUSIONS: Erythropoietin therapy does not affect significantly the levels of total cholesterol, LDL- and HDL-cholesterol, triglycerides, Apo B and Lp (a) in maintenance hemodialysis patients.

[Health and education: alternative courses for the development of nursing personnel]. / Saúde e educação: cursos alternativos para desenvolvimento do pessoal de enfermagem.

To promote therapeutic educational activities for nursing personnel in order to decrease stress, to improve interpersonal relations and the search for self-knowledge are the objectives of the courses promoted by the Departament of Nursing of FCM and by The Continued Education Nursing Service of the University Hospital of UNICAMP. Respiration, relaxation, body sensibilization and awareness, and theater interpretation techniques were taught in the following courses: The Hospital and Human Relations, Dance and Creativity, Yoga and Mental Relaxation. The estrategy employed was "group experience", with the participation of nurse's aides, nurses technicians, practical nurses, and registered nurses during working hours in 15 to 20 meetings per course. "Individual statements" written by the participants were used as a research tool, and the method employed was content analysis. The evaluation demonstrated that the "experience" facilitated relations among the members of the work team, opened the space for effective communication, favored self-knowledge, and helped with the problem solving. The analysis demonstrated the importance of the continuity of alternative courses to help employes improve their relations with themselves, with others and with their work, and to properly value their health/learning.