Green Foaming of Biologically Extracted Chitin Hydrogels Using Supercritical Carbon Dioxide for Scaffolding of Human Osteoblasts.

Chitin is a structural polysaccharide abundant in the biosphere. Chitin possesses a highly ordered crystalline structure that makes its processing a challenge. In this study, chitin hydrogels and methanogels, prepared by dissolution in calcium chloride/methanol, were subjected to supercritical carbon dioxide (scCO2) to produce porous materials for use as scaffolds for osteoblasts. The control of the morphology, porosity, and physicochemical properties of the produced materials was performed according to the operational conditions, as well as the co-solvent addition. The dissolution of CO2 in methanol co-solvent improved the sorption of the compressed fluid into the hydrogel, rendering highly porous chitin scaffolds. The chitin crystallinity index significantly decreased after processing the hydrogel in supercritical conditions, with a significant effect on its swelling capacity. The use of scCO2 with methanol co-solvent resulted in chitin scaffolds with characteristics adequate to the adhesion and proliferation of osteoblasts.

Synovial membrane mesenchymal stem cells for cartilaginous tissues repair.

BACKGROUND: The present review is focused on general aspects of the synovial membrane as well as specialized aspects of its cellular constituents, particularly the composition and location of synovial membrane mesenchymal stem cells (S-MSCs). S-MSC multipotency properties are currently at the center of translational medicine for the repair of multiple joint tissues, such as articular cartilage and meniscus lesions. METHODS AND RESULTS: We reviewed the results of in vitro and in vivo research on the current clinical applications of S-MSCs, surface markers, cell culture techniques, regenerative properties, and immunomodulatory mechanisms of S-MSCs as well as the practical limitations of the last twenty-five years (1996 to 2021). CONCLUSIONS: Despite the poor interest in the development of new clinical trials for the application of S-MSCs in joint tissue repair, we found evidence to support the clinical use of S-MSCs for cartilage repair. S-MSCs can be considered a valuable therapy for the treatment of repairing joint lesions.

Design of a home-based intervention for Houston-area African-American adults with asthma: Methods and lessons learned from a pragmatic randomized trial.

A growing body of evidence demonstrates that home-based, multicomponent interventions can effectively reduce exposures to asthma triggers and decrease asthma symptoms. However, few of these studies have targeted adults. To address this and other research gaps, we designed and implemented a pragmatic randomized clinical trial, the Houston Home-based Integrated Intervention Targeting Better Asthma Control (HIITBAC) for African Americans, to assess the effectiveness of a home-based intervention to improve asthma control and quality of life in African-American adults-a population disproportionately affected by asthma. The primary goals were to help participants reduce allergens and irritants in their homes and better manage their disease through knowledge, improved medication use, and behavior change. HIITBAC had two groups: clinic-only and home-visit groups. Both groups received enhanced clinical care, but the home-visit group also received a detailed home assessment and four additional home visits spaced over roughly one year. We recruited 263 participants. Of these, 152 (57.8%) were recruited through electronic health record data, 51 (19.4%) through Emergency Medical Services data, and 60 (22.8%) through other efforts (e.g., emergency departments, community events, outreach). Seventy participants (26.6%) were lost to follow up, substantially more in the home-visit than in the clinic-only group. We describe the HIITBAC methodology and cohort, discuss lessons learned about recruitment and retention, and highlight adaptations we implemented to address these lessons.

Exosome Biogenesis in the Protozoa Parasite Giardia lamblia: A Model of Reduced Interorganellar Crosstalk.

: Extracellular vesicles (EVs) facilitate intercellular communication and are considered a promising therapeutic tool for the treatment of infectious diseases. These vesicles involve microvesicles (MVs) and exosomes and selectively transfer proteins, lipids, mRNAs, and microRNAs from one cell to another. While MVs are formed by extrusion of the plasma membrane, exosomes are a population of vesicles of endosomal origin that are stored inside the multivesicular bodies (MVBs) as intraluminal vesicles (ILVs) and are released when the MVBs fuse with the plasma membrane. Biogenesis of exosomes may be driven by the endosomal sorting complex required for transport (ESCRT) machinery or may be ESCRT independent, and it is still debated whether these are entirely separate pathways. In this manuscript, we report that the protozoan parasite, Giardia lamblia, although lacking a classical endo-lysosomal pathway, is able to produce and release exosome-like vesicles (ElV). By using a combination of biochemical and cell biology analyses, we found that the ElVs have the same size, shape, and protein and lipid composition as exosomes described for other eukaryotic cells. Moreover, we established that some endosome/lysosome peripheral vacuoles (PVs) contain ILV during the stationary phase. Our results indicate that ILV formation and ElV release depend on the ESCRT-associated AAA+-ATPase Vps4a, Rab11, and ceramide in this parasite. Interestingly, EIV biogenesis and release seems to occur in Giardia despite the fact that this parasite has lost most of the ESCRT machinery components during evolution and is unable to produce ceramide de novo. The differences in protozoa parasite EV composition, origin, and release may reveal functional and structural properties of EVs and, thus, may provide information on cell-to-cell communication and on survival mechanisms.

Identificação do Nível de Conhecimento em Cuidados Paliativos na Formação Médica em uma Escola de Medicina de Goiás / Identifying the Level of Knowledge in Palliative Care among Medical Students in Goiás

RESUMO Introdução O atual panorama mundial e, mais recentemente, brasileiro, nos permite afirmar que os cursos de Medicina carecem de disciplinas que abordem os temas de morte, luto e comunicação de más notícias, geralmente inseridos no contexto dos Cuidados Paliativos, embora não somente em disciplinas que conduzam os futuros profissionais médicos para além do conhecimento técnico-científico, tendo em vista a necessidade dos pacientes e familiares de serem cuidados também nos âmbitos psicossocial e espiritual1. Sabemos que existem barreiras para a aplicação de técnicas de cuidados paliativos no Brasil. Entre elas, podemos destacar a falta de formação em Cuidados Paliativos da equipe de saúde, especificamente, neste estudo, dos profissionais médicos. Objetivos Identificar o nível de conhecimento em Cuidados Paliativos (CP) na formação médica dos acadêmicos de Medicina do sexto ano de uma escola de Medicina do Estado de Goiás, compreender a percepção dos acadêmicos quanto à aprendizagem referente aos CP durante a sua formação médica e discutir a inclusão de CP na formação médica da instituição do estudo e no Brasil. Método Um questionário com nove questões foi aplicado para avaliar o nível de conhecimento em Cuidados Paliativos dos acadêmicos do sexto ano de Medicina de uma escola médica do Estado de Goiás. A população do estudo foi constituída de 81 indivíduos, de ambos os sexos, maiores de 18 anos. Resultados Foi observado que 73,84% dos entrevistados souberam definir ortotanásia, 43,07% acertaram a definição de distanásia e 58,73% assinalaram corretamente sobre eutanásia. Temos ainda que 36,92% dos alunos não se consideram preparados para lidar com a terminalidade, e a maioria dos acadêmicos (74,3%) referiu déficit quanto à abordagem do tema na graduação. Conclusão Embora os acadêmicos avaliados conheçam alguns princípios dos CP, estes não são suficientes. Os alunos alegam carência na abordagem do tema, enfatizando a necessidade de implementação dos Cuidados Paliativos como disciplina obrigatória na grade curricular brasileira.

ABSTRACT Introduction The current global, and more recently Brazilian, state-of-affairs, indicate that Medicine courses are lacking in disciplines that address the issues of death, grief and communicating bad news. These are themes generally inserted in the context of palliative care, although not exclusively in subjects that instruct future medical professionals beyond technical-scientific knowledge, bearing in mind the need of patients and relatives to be cared for in psychosocial and spiritual terms1. There are known barriers for the application of palliative care methods in Brazil. These include a lack of training in palliative care among health care staff, and specifically in this study, medical professionals. Objectives To identify the level of knowledge in Palliative Care among sixth-year medical students at a Medicine school in the state of Goiás, Brazil, understand their view of training in Palliative Care during the undergraduate medical course and, finally, discuss the inclusion of Palliative Care in the medical training offered at their school and throughout Brazil Method A questionnaire containing 9 questions was applied to assess the level of knowledge about Palliative Care among sixth-year medical students from a Medicine school in the state of Goiás, Brazil. The study population was formed by 81 individuals, of both sexes, all of whom were at least 18 years of age. Results It was found that 73.84% of the interviewees were able to define orthothanasia, 43.07% gave a correct definition of dysthanasia and 58.73% correctly described euthanasia. Nonetheless, 36.92% of the students did not deem themselves fit to deal with terminality, and 74.3% cited a lack of attention given to the theme during undergraduate training. Conclusion Although the assessed students knew some principles of palliative are, these are insufficient. The students allege a lack of attention given to the theme, emphasising the need to implement Palliative Care as a mandatory discipline in the Brazilian curriculum.

Lactoferrin and lactoferricin endocytosis halt Giardia cell growth and prevent infective cyst production.

Lactoferrin (LF) is an 80 KDa iron-binding glycoprotein that plays a significant role in the innate immune system and is considered to be an important microbicide molecule. It has been suggested to be effective in the treatment of giardiasis, an intestinal disease caused by the protozoan parasite G. lamblia. However, the molecular mechanisms by which LF exerts its effect on this parasite are unknown. Most of the microbicidal activity of human or bovine LF (hLF or bLF) has been associated with the N-terminal region of the mature LF - lactoferricin (LFcin). LFcin is produced by pepsin cleavage of the native protein in vitro and likely in vivo. In this work, we analyse the participation of the endocytic machinery of G. lamblia in the internalization of bLF and bLFcin and their effects on cell homeostasis. Our results show that, when bLF or bLFcin are internalized by receptor-mediated endocytosis, cell growth stops, and morphological changes are produced in the trophozoites, which ultimately will produce immature cysts. Our findings contribute to disclose the fine mechanism by which bLF and bLFcin may function as an antigiardial molecule and why they have therapeutic potential to eradicate giardiasis.

Behavior of multipotent stem cells isolated in mobilized peripheral blood from sheep after culture with human chondrogenic medium.

Today, regenerative medicine requires new sources of multipotent stem cells for their differentiation to chondrocytes using the mediums of differentiation available in the market. This study aimed to determine whether the Mesenchymal Stem Cells (MSCs) isolated from Mobilized Peripheral Blood (MPB) in sheep using the Granulocyte Colony-Stimulating Factor (G-CSF), have the ability of first acquire a fibroblast-like morphology after being forced out of the bone marrow niche by G-CSF and second, if the cells have the capacity to express collagen type-II α I in primary culture using a human commercial media of differentiation. Six Suffolk male sheep with age of 2 years were mobilized using G-CSF. One subcutaneous injection of 10 mcg per kilogram of bodyweight were administered every 24 h during three consecutive days. At day four, a sample of 20 mL of peripheral blood was harvested, afterwards, monocytes cells were separated by ficoll gradient. The mobilized MSCs were expanded in primary culture in DMEM medium supplemented with 10% adult sheep serum for three weeks and characterized by an antibody panel for surface markers: CD105, CD90, CD73, CD34, and CD45, before and after primary culture. Subsequently, an aliquot of cells in the first pass were cultured in a commercial human chondrogenic medium for three weeks. As a result, the percentage of surface markers for MSCs (CD105, CD90, CD73) in expanded cells in primary culture significantly increased, at the same time a decrease in the markers for hematopoietic cells (CD34 and CD45) was observed and the cells morphology was fibroblast-like. After three weeks of differentiation culture, the immunofluorescence analysis evidenced the expression of collagen-type-II. It was concluded that Mesenchymal Stem Cells isolated from mobilized peripheral blood in sheep have the ability to pre-differentiate into chondral like cells and express collagen type-II when are stimulated with a human commercial chondrogenic medium in monolayer culture.

Endoplasmic reticulum is the sorting core facility in the Golgi-lacking protozoan Giardia lamblia.

Our understanding of protein and lipid trafficking in eukaryotic cells has been challenged by the finding of different forms of compartmentalization and cargo processing in protozoan parasites. Here, we show that, in the absence of a Golgi compartment in Giardia, proteins destined for secretion are directly sorted and packaged at specialized ER regions enriched in COPII coatomer complexes and ceramide. We also demonstrated that ER-resident proteins are retained at the ER by the action of a KDEL receptor, which, in contrast to other eukaryotic KDEL receptors, showed no interorganellar dynamic but instead acts specifically at the limit of the ER membrane. Our study suggests that the ER-exit sites and the perinuclear ER-membranes are capable of performing protein-sorting functions. In our view, the description presented here suggests that Giardia adaptation represents an extreme example of reductive evolution without loss of function.

Antiparasitic evaluation of betulinic acid derivatives reveals effective and selective anti-Trypanosoma cruzi inhibitors.

Betulinic acid is a pentacyclic triterpenoid with several biological properties already described, including antiparasitic activity. Here, the anti-Trypanosoma cruzi activity of betulinic acid and its semi-synthetic amide derivatives (BA1-BA8) was investigated. The anti-Trypanosoma cruzi activity and selectivity were enhanced in semi-synthetic derivatives, specially on derivatives BA5, BA6 and BA8. To understand the mechanism of action underlying betulinic acid anti-T. cruzi activity, we investigated ultrastructural changes by electron microscopy. Ultrastructural studies showed that trypomastigotes incubated with BA5 had membrane blebling, flagella retraction, atypical cytoplasmic vacuoles and Golgi cisternae dilatation. Flow cytometry analysis showed that parasite death is mainly caused by necrosis. Treatment with derivatives BA5, BA6 or BA8 reduced the invasion process, as well as intracellular parasite development in host cells, with a potency and selectivity similar to that observed in benznidazole-treated cells. More importantly, the combination of BA5 and benznidazole revealed synergistic effects on trypomastigote and amastigote forms of T. cruzi. In conclusion, we demonstrated that BA5 compound is an effective and selective anti-T. cruzi agent.

Faculty Perceptions, Knowledge, and Attitudes Toward Interprofessional Education and Practice.

As interprofessional education (IPE) is incorporated into health professions programs, it is essential to understand faculty perceptions, knowledge, and attitudes about IPE and interprofessional practice (IPP). A descriptive, cross-sectional design was used. Seventy-one faculty from three campuses of two rural public universities representing seven different programs participated. Despite limited IPE experience, faculty appreciated IPE and IPP. Notably, many felt undervalued by other professions. Participants acknowledged the importance of working with other professions (mean 2.69±0.53), participating on IP teams (2.61±0.52), and integrating IPP in patient care (2.60±0.52). Faculty reported low IPE knowledge (1.74±0.66) and confidence in IPE teaching ability (1.74±0.67). These findings demonstrate a need for faculty development in both IPE and IPP across all health disciplines.

The giardial ENTH protein participates in lysosomal protein trafficking and endocytosis.

In the protozoa parasite Giardia lamblia, endocytosis and lysosomal protein trafficking are vital parasite-specific processes that involve the action of the adaptor complexes AP-1 and AP-2 and clathrin. In this work, we have identified a single gene in Giardia encoding a protein containing an ENTH domain that defines monomeric adaptor proteins of the epsin family. This domain is present in the epsin or epsin-related (epsinR) adaptor proteins, which are implicated in endocytosis and Golgi-to-endosome protein trafficking, respectively, in other eukaryotic cells. We found that GlENTHp (for G. lamblia ENTH protein) localized in the cytosol, strongly interacted with PI3,4,5P3, was associated with the alpha subunit of AP-2, clathrin and ubiquitin and was involved in receptor-mediated endocytosis. It also bonded PI4P, the gamma subunit of AP-1 and was implicated in ER-to-PV trafficking. Alteration of the GlENTHp function severely affected trophozoite growth showing an unusual accumulation of dense material in the lysosome-like peripheral vacuoles (PVs), indicating that GlENTHp might be implicated in the maintenance of PV homeostasis. In this study, we showed evidence suggesting that GlENTHp might function as a monomeric adaptor protein supporting the findings of other group indicating that GlENTHp might be placed at the beginning of the ENTH family.

Receptor-mediated endocytosis and trafficking between endosomal-lysosomal vacuoles in Giardia lamblia.

The early branching Giardia lamblia has highly polarized vacuoles, located underneath the plasma membrane, which have at least some of the characteristics of endosomes and of lysosomes. These peripheral vacuoles (PVs) are necessary for nutrient uptake and the maintenance of plasma membrane composition, but whether they carry out sorting and segregation of receptors and ligands is a matter of debate. Here, we showed that the internalization of low-density lipoprotein (LDL) to the PVs is highly dynamic in trophozoites with a rate similar to the internalization of the low-density lipoprotein receptor-related protein 1. Moreover, by analyzing receptor-mediated and fluid-phase endocytosis in living cells, we showed that after endocytosis LDL but not dextran moved laterally between the PVs. We speculate on PV functional heterogeneity and maturation in this parasite.

Giardia lamblia: Characterization of ecto-phosphatase activities.

Ecto-phosphatase activities of Giardia lamblia were characterized in intact cells, which are able to hydrolyze the artificial substrate p-nitrophenylphosphate (p-NPP) to p-nitrophenol (p-NP) at a rate of 8.4+/-0.8 nmol p-NP/h/10(7) cells. The ecto-phosphatase activities were inhibited at high pH as well as by classical inhibitors of acid phosphatases, such as sodium fluoride and sodium molybdate and by inorganic phosphate, the final product of the reaction. Experiments using a classical inhibitor of phosphotyrosine phosphatase, sodium orthovanadate, also showed that the ecto-phosphatase activity was inhibited in a dose-dependent manner. Different phosphorylated amino acids were used as substrates for the G. lamblia ecto-phosphatase activities the highest rate of phosphate release was achieved using phosphotyrosine. Not only p-NPP hydrolysis but also phosphotyrosine hydrolysis was inhibited by sodium orthovanadate. Phosphotyrosine but not phospho-serine or phospho-threonine inhibited the p-nitrophenylphosphatase activity. We also observed a positive correlation between the ecto-phosphatase activity and the capacity to encystation of G. lamblia trophozoites.

Giardia lamblia: biochemical characterization of an ecto-ATPase activity.

In this work, we describe the ability of living trophozoites of Giardia lamblia to hydrolyze extracellular ATP. In the absence of any divalent cations, a low level of ATP hydrolysis was observed (0.78+/-0.08 nmol Pi x h(-1)x10(-6) cells). The ATP hydrolysis was stimulated by MgCl(2) in a dose-dependent manner. Half maximum stimulation of ATP hydrolysis was obtained with 0.53+/-0.07 mM. ATP was the best substrate for this enzyme. The apparent K(m) for ATP was 0.21+/-0.04 mM. In the pH range from 5.6 to 8.4, in which cells were viable, this activity was not modified. The Mg(2+)-stimulated ATPase activity was insensitive to inhibitors of intracellular ATPases such as vanadate (P-ATPases), bafilomycin A(1) (V-ATPases), and oligomycin (F-ATPases). Inhibitors of acid phosphatases (molybdate, vanadate and fluoride) or alkaline phosphatases (levamizole) had no effect on the ecto-ATPase activity. The impermeant agent DIDS and suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases, decreased the enzymatic activity in a dose-dependent manner, confirming the external localization of this enzyme. Besides ATP, trophozoites were also able to hydrolyse ADP and 5 AMP, but the hydrolysis of these nucleotides was not stimulated by MgCl(2). Our results are indicative of the occurrence of a G. lamblia ecto-ATPase activity that may have a role in parasite physiology.

Design, synthesis, SAR, and biological evaluation of new 4-(phenylamino)thieno[2,3-b]pyridine derivatives.

In this work, we performed the design, synthesis, and the structure-activity relationship studies of 13 new derivatives of thieno[2,3-b]pyridine. These derivatives were prepared in high yields (96-70%) and their structures were elucidated by IR, (1)H, (13)C NMR, and MS. The biological results showed some derivatives as antiparasitic agents against Giardia lamblia. Computational analysis of HOMO and LUMO energy, HOMO orbital coefficient distribution, electrostatic potential map, dipole moment, and density HOMO was performed to gain insight into the SAR aspects. This study pointed the p-methoxy substituted derivative as a leading compound for the development of new microbicidal medicines based on thieno[2,3-b]pyridine analogs.

Contribution of microscopy to a better knowledge of the biology of Giardia lamblia.

Giardia lamblia is a flagellated protozoan of great medical and biological importance. It is the causative agent of giardiasis, one of the most prevalent diarrheal disease both in developed and third-world countries. Morphological studies have shown that G. lamblia does not present structures such as peroxisomes, mitochondria, and a well-elaborated Golgi complex. In this review, special emphasis is given to the contribution made by various microscopic techniques to a better knowledge of the biology of the protozoan. The application of video microscopy, immunofluorescence confocal laser scanning microscopy, and several techniques associated with transmission electron microscopy (thin section, enzyme cytochemistry, freeze-fracture, deep-etching, fracture-flip) to the study of the cell surface, peripheral vesicles, endoplasmic reticulum-Golgi complex system, and of the encystation vesicles found in trophozoites and during the process of trophozoite-cyst transformation are discussed.

Implicaçöes éticas do uso de animais no processo de ensino-aprendizagem nas Faculdades de Medicina do Rio de Janeiro e Niterói / Ethical issues in the use of animals in the teaching-learning process at Schools of Medicine in Rio de Janeiro and Niterói, Brazil

O principal objetivo deste trabalho foi identificar e analisar o uso de animais e dos chamados "métodos alternativos" a este uso no ensino médico, assim como caracterizar a penetraçäo do atual debate acerca da ética no uso de animais entre os professores envolvidos em disciplinas que utilizam animais. Foram identificadas sete instituiçöes que possuem disciplinas cujos professores utilizam animais em aulas práticas e/ou demonstrativas. Foram entrevistados os docentes que ministram aulas nessas disciplinas através de um questionário semi-aberto. Os resultados indicaram que quatro das sete instituiçöes utilizam animais em aulas, näo havendo, necessariamente, concidência em relaçäo às disciplinas que fazem esse uso nas diferentes escolas. O animal mais utilizado em aulas é o camundongo e dentre os métodos alternativos, os recursos audiovisuais. A maioria dos entrevistados näo conhece leis brasileiras sobre o uso de animais e considera impossível abolir o uso de animais no ensino. Este estudo demonstra a necessidade de ampliar a discussäo da ética aplicada à sua saúde, incluindo a discussäo sobre o uso de animais no âmbito da formaçäo profissional na área biomédica/biológica em nosso país.

Papilomatose traqueal do adulto / Adult tracheal papillomatosis

Homem de 56 anos apresentou dois episódios de hemoptise, nos últimos três meses. Sua radiografia do toráx demonstrou discreto espessamento peribrônquico em ambas as bases pulmonares e na sua tomografia computadorizada do toráx, observamos uma massa na parede lateral esquerda da traquéia no seu terço médio. A lesão foi ressecada utilizando-se o eletrocautério do broncofibroscópio com bom resultado.

A 56 year-old man presented with two episodes of hemoptyses in the last 3 months. His chest X-ray disclosed peribronchial thickening at both lung bases and his chets computed tomography showed a mass in the tracheal lefth side wall in its medial third. The patient underwent to a bronchofiberscopy and resection of the lesion with electrocautery with good result and he recovered completely from his symptoms.

Procedimento de desenhos de Familia com Estorias (DF-E) Avaliacoes de um Caso Clinico

O objetivo deste trabalho e investigar a eficacia do PROCEDIMENTO DE DESENHOS DE FAMILIA COM ESTORIAS (DF-E), introduzido por TRINCA como um instrumento de investigacao da personalidade. Para isto, realizou-se o confronto de varias avaliacoes independentes do extrato de um mesmo caso clinico contendo material eliciado pelo DF-E, realizadas por psicologos e um grupo de profissionais. A comparacao foi feita a partir do levantamento de topicos, procurando identificar-se os pontos de avaliacao comuns, bem como aqueles que sao especificos a determinada avaliacao. Os resultados indicam razoavel nivel de concordancia entre as avaliacoes, que se revelaram eficazes na compreensao do processo psiquico do cliente.