Biological Activity and Chemical Composition of Propolis Extracts with Potential Use in Vulvovaginal Candidiasis Management.

Environmental sustainability is an increasing challenge in the pharmaceutical field, leading to the search for eco-friendly active ingredients. Among natural ingredients, propolis arises as an excellent alternative, being a complex substance with pharmacological properties. This work aims to explore the potential of propolis as a new pharmaceutical ingredient for the replacement of conventional vulvovaginal antifungals. Propolis extracts were obtained by Ultrasound-Assisted Extraction using different solvents (water, water/ethanol (50:50, v/v), and ethanol). Afterwards, the extracts were characterized regarding total phenolic content (TPC), antioxidant/antiradical activities, radical scavenging capacity, antifungal activity against strains of Candida species, and viability effect on two female genital cell lines. The aqueous extract achieved the best TPC result as well as the highest antioxidant/antiradical activities and ability to capture reactive oxygen species. A total of 38 phenolic compounds were identified and quantified by HPLC, among which ferulic acid, phloridzin and myricetin predominated. Regarding the anti-Candida spp. activity, the aqueous and the hydroalcoholic extracts achieved the best outcomes (with MIC values ranging between 128 and 512 µg/mL). The cell viability assays confirmed that the aqueous extract presented mild selectivity, while the hydroalcoholic and alcoholic extracts showed higher toxicities. These results attest that propolis has a deep potential for vulvovaginal candidiasis management, supporting its economic valorization.

Tissue-resident glial cells associate with tumoral vasculature and promote cancer progression.

Cancer cells are embedded within the tissue and interact dynamically with its components during cancer progression. Understanding the contribution of cellular components within the tumor microenvironment is crucial for the success of therapeutic applications. Here, we reveal the presence of perivascular GFAP+/Plp1+ cells within the tumor microenvironment. Using in vivo inducible Cre/loxP mediated systems, we demonstrated that these cells derive from tissue-resident Schwann cells. Genetic ablation of endogenous Schwann cells slowed down tumor growth and angiogenesis. Schwann cell-specific depletion also induced a boost in the immune surveillance by increasing tumor-infiltrating anti-tumor lymphocytes, while reducing immune-suppressor cells. In humans, a retrospective in silico analysis of tumor biopsies revealed that increased expression of Schwann cell-related genes within melanoma was associated with improved survival. Collectively, our study suggests that Schwann cells regulate tumor progression, indicating that manipulation of Schwann cells may provide a valuable tool to improve cancer patients' outcomes.

Study of the Antioxidant, Antimicrobial, and Wound Healing Properties of Raw Hydrolyzed Extract from Nile Tilapia Skin (Oreochromis niloticus).

Oreochromis niloticus (Nile tilapia) skin is a by-product of Brazilian fish farming, rich in collagen. The present study aims to evaluate the wound healing, antioxidant, and antimicrobial potential of the raw hydrolyzed extract of Nile tilapia skin, as well as the identification of the main compounds. The in vitro activity was performed using antioxidant, antimicrobial and scratch wound healing assays. An in vivo experiment was performed to evaluate the wound healing potential. On days 1, 7, 14 and 21, the lesions were photographed to assess wound retraction and on the 7th , 14th and 21st  days the skins were removed for histological evaluation and the blood of the animals was collected for glutamic oxaloacetic transaminase and glutamic pyruvic transaminase determination. The chemical study was carried out through liquid chromatography-tandem mass spectrometry and de novo sequencing of peptides. The in vitro assays showed a reduction of the gap area in 24 h, dose-dependent antimicrobial activity for both bacteria, and antioxidant activity. The chemical analysis highlighted the presence of active biopeptides. The histological evaluation showed that the raw hydrolyzed extract of Nile tilapia skin has a healing potential, and does not present toxicological effects; therefore, is promising for the treatment of wounds.

Integrative Metabolic Pathway Analysis Reveals Novel Therapeutic Targets in Osteoarthritis.

In osteoarthritis (OA), impairment of cartilage regeneration can be related to a defective chondrogenic differentiation of mesenchymal stromal cells (MSCs). Therefore, understanding the proteomic- and metabolomic-associated molecular events during the chondrogenesis of MSCs could provide alternative targets for therapeutic intervention. Here, a SILAC-based proteomic analysis identified 43 proteins related with metabolic pathways whose abundance was significantly altered during the chondrogenesis of OA human bone marrow MSCs (hBMSCs). Then, the level and distribution of metabolites was analyzed in these cells and healthy controls by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), leading to the recognition of characteristic metabolomic profiles at the early stages of differentiation. Finally, integrative pathway analysis showed that UDP-glucuronic acid synthesis and amino sugar metabolism were downregulated in OA hBMSCs during chondrogenesis compared with healthy cells. Alterations in these metabolic pathways may disturb the production of hyaluronic acid (HA) and other relevant cartilage extracellular matrix (ECM) components. This work provides a novel integrative insight into the molecular alterations of osteoarthritic MSCs and potential therapeutic targets for OA drug development through the enhancement of chondrogenesis.

C(3)1-TAg in C57BL/6 J background as a model to study mammary tumor development.

Diagnosis and prognosis of breast cancer is based on disease staging identified through histopathological and molecular biology techniques. Animal models are used to gain mechanistic insights into the development of breast cancer. C(3)1-TAg is a genetically engineered mouse model that develops mammary cancer. However, carcinogenesis caused by this transgene was characterized in the Friend Virus B (FVB) background. As most genetic studies are done in mice with C57BL/6 J background, we aimed to define the histological alterations in C3(1)-TAg C57BL/6 J animals. Our results showed that C3(1)-TAg animals with C57BL/6 J background develop solid-basaloid adenoid cystic carcinomas with increased fibrosis, decreased area of adipocytes, and a high proliferative index, which are triple-negative for progesterone, estrogen, and human epidermal growth factor receptor 2 (HER2) receptors. Our results also revealed that tumor development is slower in the C57BL/6 J background when compared with the FVB strain, providing a better model to study the different stages in breast cancer progression.

Pegylated catalase as a potential alternative to treat vitiligo and UV induced skin damage.

The metabolic function of catalase (CAT) is to prevent oxidative damage to tissues through the hydrolysis of hydrogen peroxide, which is a strong oxidizing agent. It has been suggested as an alternative to treat skin diseases related to oxidative stress, such as vitiligo. Owing to the instability associated to the protein nature, topical use of CAT is challenging and, in this sense, PEGylation can be an interesting alternative. Here, we conjugated CAT to methoxy-poly(ethylene oxide) (mPEG) of 10, 20 and 40 kDa, by means of a nucleophilic attack of ε-amino groups to an electron-deficient carbonyl group of the reactive PEG, resulting in site specifically PEGylated bioconjugates. PEGylation yields ranged from 31% ± 2% for CAT-PEG40 to 59% ± 4% for CAT-PEG20 and were strongly affected by the reaction pH owing to the protonation/deprotonation state of primary amines of lysine and N-terminal residues. PEGylated conjugates were purified by size-exclusion chromatography (purity > 95%) and characterized by circular dichroism. Irrespectively of MW, PEG did not affected CAT secondary and tertiary structure, but a decrease in specific activity was observed, more pronounced when PEGs of higher MWs were used. However, this loss of activity is compensated by the increased long-term stability, with a gain of >5 times in t1/2. In vitro antioxidant activity of CAT-PEG20 showed complete elimination of lipid peroxidation at the skin upper layer (stratum corneum) suitable for a topical use to treat vitiligo, as well as other skin conditions related to oxidative stress.

Analysis of Endogenous Peptides Released from Osteoarthritic Cartilage Unravels Novel Pathogenic Markers.

Osteoarthritis (OA) is a pathology characterized by the loss of articular cartilage. In this study, we performed a peptidomic strategy to identify endogenous peptides (neopeptides) that are released from human osteoarthritic tissue, which may serve as disease markers. With this aim, secretomes of osteoarthritic and healthy articular cartilages obtained from knee and hip were analyzed by shotgun peptidomics. This discovery step led to the identification of 1175 different peptides, corresponding to 101 proteins, as products of the physiological or pathological turnover of cartilage extracellular matrix. Then, a targeted multiple reaction monitoring-mass spectrometry method was developed to quantify the panel of best marker candidates on a larger set of samples (n = 62). Statistical analyses were performed to evaluate the significance of the observed differences and the ability of the neopeptides to classify the tissue. Eight of them were differentially abundant in the media from wounded zones of OA cartilage compared with the healthy tissue (p < 0.05). Three neopeptides belonging to Clusterin and one from Cartilage Oligomeric Matrix Protein showed a disease-dependent decrease specifically in hip OA, whereas two from Prolargin (PRELP) and one from Cartilage Intermediate Layer Protein 1 were significantly increased in samples from knee OA. The release of one peptide from PRELP showed the best metrics for tissue classification (AUC = 0.834). The present study reveals specific neopeptides that are differentially released from knee or hip human osteoarthritic cartilage compared with healthy tissue. This evidences the intervention of characteristic pathogenic pathways in OA and provides a novel panel of peptidic candidates for biomarker development.

Ablation of sensory nerves favours melanoma progression.

The tumour mass is composed not only of heterogeneous neoplastic cells, but also a variety of other components that may affect cancer cells behaviour. The lack of detailed knowledge about all the constituents of the tumour microenvironment restricts the design of effective treatments. Nerves have been reported to contribute to the growth and maintenance of numerous tissues. The effects of sensory innervations on tumour growth remain unclear. Here, by using state-of-the-art techniques, including Cre/loxP technologies, confocal microscopy, in vivo-tracing and chemical denervation, we revealed the presence of sensory nerves infiltrating within the melanoma microenvironment, and affecting cancer progression. Strikingly, melanoma growth in vivo was accelerated following genetic ablation or chemical denervation of sensory nerves. In humans, a retrospective analysis of melanoma patients revealed that increased expression of genes related to sensory nerves in tumours was associated with better clinical outcomes. These findings suggest that sensory innervations counteract melanoma progression. The emerging knowledge from this research provides a novel target in the tumour microenvironment for therapeutic benefit in cancer patients.

MALDI mass spectrometry imaging in rheumatic diseases.

Mass spectrometry imaging (MSI) is a technique used to visualize the spatial distribution of biomolecules such as peptides, proteins, lipids or other organic compounds by their molecular masses. Among the different MSI strategies, MALDI-MSI provides a sensitive and label-free approach for imaging of a wide variety of protein or peptide biomarkers from the surface of tissue sections, being currently used in an increasing number of biomedical applications such as biomarker discovery and tissue classification. In the field of rheumatology, MALDI-MSI has been applied to date for the analysis of joint tissues such as synovial membrane or cartilage. This review summarizes the studies and key achievements obtained using MALDI-MSI to increase understanding on rheumatic pathologies and to describe potential diagnostic or prognostic biomarkers of these diseases. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann.

Characterization of lipidic markers of chondrogenic differentiation using mass spectrometry imaging.

Mesenchymal stem cells (MSC) are an interesting alternative for cell-based therapy of cartilage defects attributable to their capacity to differentiate toward chondrocytes in the process termed chondrogenesis. The metabolism of lipids has recently been associated with the modulation of chondrogenesis and also with the development of pathologies related to cartilage degeneration. Information about the distribution and modulation of lipids during chondrogenesis could provide a panel of putative chondrogenic markers. Thus, the discovery of new lipid chondrogenic markers could be highly valuable for improving MSC-based cartilage therapies. In this work, MS imaging was used to characterize the spatial distribution of lipids in human bone marrow MSCs during the first steps of chondrogenic differentiation. The analysis of MSC micromasses at days 2 and 14 of chondrogenesis by MALDI-MSI led to the identification of 20 different lipid species, including fatty acids, sphingolipids, and phospholipids. Phosphocholine, several sphingomyelins, and phosphatidylcholines were found to increase during the undifferentiated chondrogenic stage. A particularly detected lipid profile was verified by TOF secondary ion MS. Using this technology, a higher intensity of phosphocholine-related ions was observed in the peripheral region of the micromasses collected at day 14.

Secretome analysis of human mesenchymal stem cells undergoing chondrogenic differentiation.

Human mesenchymal stem cells (hMSCs) can be triggered to differentiate toward chondrocytes and thus harbor great therapeutic potential for the repair of cartilage defects in osteoarthritis (OA) and other articular diseases. However, the molecular mechanisms underlying the chondrogenesis process are still in part unknown. In this work, we followed a double-stable isotope labeling by amino acids in cell culture (SILAC) strategy to evaluate the quantitative modulation of the secretome of stem cells isolated from bone marrow (hBMSCs) during the first steps of their chondrogenic differentiation. Analysis by LC-ESI-MS/MS led to the identification of 221 proteins with a reported extracellular localization. Most of them were characteristic of cartilage extracellular matrix, and 34 showed statistically significant quantitative alterations during chondrogenesis. These include, among others, cartilage markers such as Proteoglycan 4 or COMP, anticatabolic markers (TIMP1), reported markers of cartilage development (Versican), and a suggested marker of chondrogenesis, CRAC1. Altogether, our work demonstrates the usefulness of secretome analysis for understanding the mechanisms responsible for cartilage matrix formation, and it reports a panel of extracellular markers potentially useful for the evaluation of tissue development in cell therapy- or tissue engineering-based approaches for cartilage repair.

Pharmacoproteomic study of three different chondroitin sulfate compounds on intracellular and extracellular human chondrocyte proteomes.

Chondroitin sulfate (CS) is a symptomatic slow acting drug for osteoarthritis (OA) widely used for the treatment of this highly prevalent disease, characterized by articular cartilage degradation. However, little is known about its mechanism of action, and recent large scale clinical trials have reported variable results on OA symptoms. Herein, we aimed to study the modulations in the intracellular proteome and the secretome of human articular cartilage cells (chondrocytes) treated with three different CS compounds, with different origin or purity, by two complementary proteomic approaches. Osteoarthritic cells were treated with 200 µg/ml of each brand of CS. Quantitative proteomics experiments were carried out by the DIGE and stable isotope labeling with amino acids in cell culture (SILAC) techniques, followed by LC-MALDI-MS/MS analysis. The DIGE study, carried out on chondrocyte whole cell extracts, led to the detection of 46 spots that were differential between conditions in our study: 27 were modulated by CS1, 4 were modulated by CS2, and 15 were modulated by CS3. The SILAC experiment, carried out on the subset of chondrocyte-secreted proteins, allowed us to identify 104 different proteins. Most of them were extracellular matrix components, and 21 were modulated by CS1, 13 were modulated by CS2, and 9 were modulated by CS3. Each of the studied compounds induces a characteristic protein profile in OA chondrocytes. CS1 displayed the widest effect but increased the mitochondrial superoxide dismutase, the cartilage oligomeric matrix protein, and some catabolic or inflammatory factors like interstitial collagenase, stromelysin-1, and pentraxin-related protein. CS2 and CS3, on the other hand, increased a number of structural proteins, growth factors, and extracellular matrix proteins. Our study shows how, from the three CS compounds tested, CS1 induces the activation of inflammatory and catabolic pathways, whereas CS2 and CS3 induce an anti-inflammatory and anabolic response. The data presented emphasize the importance of employing high quality CS compounds, supported by controlled clinical trials, in the therapy of OA. Finally, the present work exemplifies the usefulness of proteomic approaches in pharmacological studies.

Psychopathological characteristics in patients with arginine vasopressin deficiency (central diabetes insipidus) and primary polydipsia compared to healthy controls.

OBJECTIVE: Distinguishing arginine vasopressin deficiency (AVP-D; central diabetes insipidus) from primary polydipsia (PP), commonly referred to as psychogenic polydipsia, is challenging. Psychopathologic findings, commonly used for PP diagnosis in clinical practice, are rarely evaluated in AVP-D patients, and no comparative data between the two conditions currently exist. DESIGN: Data from two studies involving 82 participants [39 AVP-D, 28 PP, and 15 healthy controls (HC)]. METHODS: Psychological evaluations were conducted using standardized questionnaires measuring anxiety [State-Trait Anxiety Inventory (STAI)], alexithymia [Toronto Alexithymia Scale (TAS-20)], depressive symptoms (Beck's Depression Inventory-II (BDI-II), and overall mental health [Short Form-36 Health Survey (SF-36)]. Higher STAI, TAS-20, and BDI-II scores suggest elevated anxiety, alexithymia, and depression, while higher SF-36 scores signify better overall mental health. RESULTS: Compared to HC, patients with AVP-D and PP showed higher levels of anxiety (HC 28 points [24-31] vs AVP-D 36 points [31-45]; vs PP 38 points [33-46], P < .01), alexithymia (HC 30 points [29-37] vs AVP-D 43 points [35-54]; vs PP 46 points [37-55], P < .01), and depression (HC 1 point [0-2] vs AVP-D 7 points [4-14]; vs PP 7 points [3-13], P < .01). Levels of anxiety, alexithymia, and depression showed no difference between both patient groups (P = .58, P = .90, P = .50, respectively). Compared to HC, patients with AVP-D and PP reported similarly reduced self-reported overall mental health scores (HC 84 [68-88] vs AVP-D 60 [52-80], P = .05; vs PP 60 [47-74], P < .01). CONCLUSION: This study reveals heightened anxiety, alexithymia, depression, and diminished overall mental health in patients with AVP-D and PP. The results emphasize the need for careful interpretation of psychopathological characteristics to differentiate between AVP-D and PP.

Utilization of ripe coconut water in the development of probiotic gelatin.

Background: Desserts with vegetable ingredients are a constantly expanding global market due to the search for alternatives to cow's milk. Fermentation of these matrices by lactic acid bacteria can add greater functionality to the product, improving its nutritional, sensory, and food safety characteristics, as well as creating bioactive components with beneficial effects on health. Concern for health and well-being has aroused interest in byproducts of the industry that have functional properties for the body, such as mature coconut water, a normally discarded residue that is rich in nutrients. This study aimed to develop a probiotic gelatin based on pulp and water from mature coconuts and evaluate the physicochemical characteristics, viability of the Lacticaseibacillus rhamnosus LR32 strain in the medium, as well as the texture properties of the product. Methods: After collection and cleaning, the physicochemical characterization, mineral analysis, analysis of the total phenolic content and antioxidant activity of mature coconut water were carried out, as well as the centesimal composition of its pulp. Afterwards, the gelling was developed with the addition of modified corn starch, gelatin, sucrose, and probiotic culture, being subjected to acidity analysis, texture profile and cell count, on the first day and every 7 days during 21 days of storage, under refrigeration at 5 °C. An analysis of the centesimal composition was also carried out. Results: The main minerals in coconut water were potassium (1,932.57 mg L-1), sodium (19.57 mg L-1), magnesium (85.13 mg L-1) calcium (279.93 mg L-1) and phosphorus (11.17 mg L- 1), while the pulp had potassium (35.96 g kg-1), sodium (0.97 g kg-1), magnesium (2.18 g kg-1), 37 calcium (1.64 g kg-1), and phosphorus (3.32 g kg-1). The phenolic content of the water and pulp was 5.72 and 9.77 mg gallic acid equivalent (GAE) 100 g-1, respectively, and the antioxidant capacity was 1.67 and 0.98 39 g of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) mg-1, respectively. The coconut pulp had 2.81 g 100 g-1of protein, 1.11 g 100 g-1 of 40 ash, 53% moisture, and 5.81 g 100 g-1 of carbohydrates. The gelatin produced during the storage period presented firmness parameters ranging from 145.82 to 206.81 grams-force (gf), adhesiveness from 692.85 to 1,028.63 gf sec, cohesiveness from 0.604 to 0.473, elasticity from 0.901 to 0.881, gumminess from 86.27 to 97.87 gf, and chewiness from 77.72 to 91.98 gf. Regarding the viability of the probiotic microorganism, the dessert had 7.49 log CFU g-1 that remained viable during the 21-day storage, reaching 8.51 CFU g-1. Acidity ranged from 0.15 to 0.64 g of lactic acid 100 g-1. The centesimal composition of the product showed 4.88 g 100 g-1 of protein, 0.54 g 100 g-1 of ash, 85.21% moisture, and 5.37g 100 g-1 of carbohydrates. The development of the gelatin made it possible to obtain a differentiated product, contributing to diversification in the food sector, providing a viable alternative for maintaining consumer health and reducing costs compared to desserts already available on the market.

Patients' prognosis with congenital heart disease followed by ten years: survival and associated factors.

OBJECTIVE: To evaluate the prognosis and influence of associated factors in patients with congenital heart disease admitted for the first time to the Intensive Care Unit of the Hospital da Criança Santo Antônio/Irmandade da Santa Casa de Misericórdia de Porto Alegre, especially those factors associated with death. METHODS: Patients were prospectively and consecutively allocated over a period of one year (August 2005 to July 2006). Now, 15 years after the initial selection, we collected data from these patients in the database of the Cytogenetics Laboratory of the Universidade Federal de Ciências da Saúde de Porto Alegre and in the medical records of the hospital. RESULTS: Of the 96 patients, 11 died and 85 were alive until 20 years old. Four patients died in the Intensive Care Unit. The survival probability up to 365 days of life was 95.8%. The survival assessment identified that the deaths occurred mainly before the patients completed one thousand days of life. We found that complex heart disease was independently associated with an odds ratio of 5.19 (95% confidence interval - CI:1.09-24.71; p=0.038) for death. CONCLUSIONS: Knowledge about the factors that interfere with the prognosis can be crucial in care practice planning, especially considering that congenital heart disease is an important cause of mortality in the first year of life.

Comparing electronic health record portals to obtain patient-entered family health history in primary care.

BACKGROUND: There is growing interest in developing systems to overcome barriers for acquiring and interpreting family health histories in primary care. OBJECTIVE: To examine the capacity of three different electronic portals to collect family history from patients and deposit valid data in an electronic health record (EHR). DESIGN: Pilot trial. PARTICIPANTS, INTERVENTION: Patients were enrolled from four primary care practices and were asked to collect family health history before a physical exam using either telephone-based interactive voice response (IVR) technology, a secure Internet portal, or a waiting room laptop computer, with portal assigned by practice. Intervention practices were compared to a "usual care" practice, where there was no standard workflow to document family history (663 participants in the three intervention arms were compared to 296 participants from the control practice). MAIN MEASURES: New documentation of any family history in a coded EHR field within 30 days of the visit. Secondary outcomes included participation rates and validity. KEY RESULTS: Demographics varied by clinic. Documentation of new family history data was significantly higher, but modest, in each of the three intervention clinics (7.5 % for IVR clinic, 20.3 % for laptop clinic, and 23.1 % for patient portal clinic) versus the control clinic (1.7 %). Patient-entered data on common conditions in first degree relatives was confirmed as valid by a genetic counselor for the majority of cases (ranging from 64 to 82 % in the different arms). CONCLUSIONS: Within primary care practices, valid patient entered family health history data can be obtained electronically at higher rates than a standard of care that depends on provider-entered data. Further research is needed to determine how best to match different portals to individual patient preference, how the tools can best be integrated with provider workflow, and to assess how they impact the use of screening and prevention.

Principles of assessment of abuse liability: US legal framework and regulatory environment.

Identifying the abuse potential of drug products in the premarketing and postmarketing environment has been a critical component in the implementation of drug abuse control laws worldwide. In the US, the Controlled Substances Act of 1970 (CSA) is a comprehensive federal law enacted to prevent the abuse or diversion of substances with abuse liability or addiction potential (for present purposes, these terms are used interchangeably). Under the jurisdiction of the Drug Enforcement Administration, the law applies to the manufacture and distribution of narcotics and other drug substances with potential of abuse. The CSA classifies substances with abuse potential into schedules I-V based on the substance's risk of diversion or abuse, and thus provides a legal framework for the assessment of abuse liability of New Molecular Entities. When the Food and Drug Administration reviews the safety and efficacy of a New Drug Application it also determines whether the drug has potential for abuse, and if so, will begin the process to schedule the drug under the CSA. As the assessment of abuse potential is a critical component of a marketing application, pharmaceutical companies (sponsors) bear the responsibility of generating a comprehensive preclinical and clinical data package for regulators to review and make decisions on labeling and the corresponding postmarketing surveillance. Recent regulatory guidelines adopted in the European Union (EU) (2006), Canada (2007), and USA (2010) provide recommendations to sponsors on preclinical and clinical methodologies for the assessment of abuse potential. This paper reviews the legal framework of the assessment of abuse liability and scheduling of controlled substances in the USA and describes the current global regulatory environment and the challenges that sponsors and regulators face when assessing abuse liability of New Molecular Entities, from the early stages of development through the late stages, review, and approval.

Schistosomiasis mansoni and hydrographical conditions in São Carlos, São Paulo, Brazil.

BACKGROUND: In Brazil, schistosomiasis mansoni cases still occur, even in non-endemic areas. This study aimed to evaluate schistosomiasis mansoni cases and to delimit water collections investigated for infested planorbidae in São Carlos, São Paulo, Brazil. METHODS: A cross-sectional descriptive study and spatial analysis of schistosomiasis mansoni cases notified in the city from January 2005 to December 2017 was conducted. The study used geographical information system software to map residential and leisure exposures to water courses and bodies and related them to planorbidae surveys of São Paulo state. RESULTS: During the study period, 32 cases were notified. The main forms were intestinal and hepatosplenic. Twenty-eight cases were allochthonous, two autochthonous and two indeterminate. Eleven patients (33.3%) had contact with water collections in São Carlos, mainly the 29 and Broa reservoirs. Three of them had contact only with water collections in the region. A third of cases lived in the Água Fria and Água Quente microbasins, highly impacted by the presence of domestic sewage, and the whole region seems to be colonized by Biomphalaria tenagophila. CONCLUSIONS: The resolution of anthropogenic contamination of water bodies is crucial for controlling schistosomiasis mansoni autochthony in São Carlos.

Metabolic labeling of human bone marrow mesenchymal stem cells for the quantitative analysis of their chondrogenic differentiation.

Human mesenchymal stem cells (hMSCs), residing in bone marrow as well as in the synovial lining of joints, can be triggered to differentiate toward chondrocytes. Thus, hMSCs harbor great therapeutic potential for the repair of cartilage defects in osteoarthritis (OA) and other articular diseases. However, the molecular mechanisms underlying the chondrogenesis process are still in part unknown. In this work, we applied for the first time the stable isotope labeling by amino acids in cell culture (SILAC) technique for the quantitative analysis of protein modulation during the chondrogenic differentiation process of hMSCs. First, we have standardized the metabolic labeling procedure on MSCs isolated from bone marrow (hBMSCs), and we have assessed the quality of chondrogenesis taking place in these conditions. Then, chondrogenic differentiation was induced on these labeled cells, and a quantitative proteomics approach has been followed to evaluate protein changes between two differentiation days. With this strategy, we could identify 622 different proteins by LC-MALDI-TOF/TOF analysis and find 65 proteins whose abundance was significantly modulated between day 2 and day 14 of chondrogenesis. Immunohistochemistry analyses were performed to verify the changes on a panel of six proteins that play different biological roles in the cell: fibronectin, gelsolin, vimentin, alpha-ATPase, mitochondrial superoxide dismutase, and cyclophilin A. All of these proteins were increased at day 14 compared to day 2 of chondrogenic induction, thus being markers of the enhanced extracellular matrix synthesis, cell adhesion, metabolism, and response to stress processes that take place in the early steps of chondrogenesis. Our strategy has allowed an additional insight into both specific protein function and the mechanisms of chondrogenesis and has provided a panel of protein markers of this differentiation process in hBMSCs.

Effect of point-of-care CD4 cell count tests on retention of patients and rates of antiretroviral therapy initiation in primary health clinics: an observational cohort study.

BACKGROUND: Loss to follow-up of HIV-positive patients before initiation of antiretroviral therapy can exceed 50% in low-income settings and is a challenge to the scale-up of treatment. We implemented point-of-care counting of CD4 cells in Mozambique and assessed the effect on loss to follow-up before immunological staging and treatment initiation. METHODS: In this observational cohort study, data for enrolment into HIV management and initiation of antiretroviral therapy were extracted retrospectively from patients' records at four primary health clinics providing HIV treatment and point-of-care CD4 services. Loss to follow-up and the duration of each preparatory step before treatment initiation were measured and compared with baseline data from before the introduction of point-of-care CD4 testing. FINDINGS: After the introduction of point-of-care CD4 the proportion of patients lost to follow-up before completion of CD4 staging dropped from 57% (278 of 492) to 21% (92 of 437) (adjusted odds ratio [OR] 0·2, 95% CI 0·15-0·27). Total loss to follow-up before initiation of antiretroviral treatment fell from 64% (314 of 492) to 33% (142 of 437) (OR 0·27, 95% CI 0·21-0·36) and the proportion of enrolled patients initiating antiretroviral therapy increased from 12% (57 of 492) to 22% (94 of 437) (OR 2·05, 95% CI 1·42-2·96). The median time from enrolment to antiretroviral therapy initiation reduced from 48 days to 20 days (p<0·0001), primarily because of a reduction in the median time taken to complete CD4 staging, which decreased from 32 days to 3 days (p<0·0001). Loss to follow-up between staging and antiretroviral therapy initiation did not change significantly (OR 0·84, 95% CI 0·49-1·45). INTERPRETATION: Point-of-care CD4 testing enabled clinics to stage patients rapidly on-site after enrolment, which reduced opportunities for pretreatment loss to follow-up. As a result, more patients were identified as eligible for and initiated antiretroviral treatment. Point-of-care testing might therefore be an effective intervention to reduce pretreatment loss to follow-up. FUNDING: Absolute Return for Kids and UNITAID.