Effectiveness of problem-based learning methodology in undergraduate medical education: a scoping review.

BACKGROUND: Problem-based learning (PBL) is a pedagogical approach that shifts the role of the teacher to the student (student-centered) and is based on self-directed learning. Although PBL has been adopted in undergraduate and postgraduate medical education, the effectiveness of the method is still under discussion. The author's purpose was to appraise available international evidence concerning to the effectiveness and usefulness of PBL methodology in undergraduate medical teaching programs. METHODS: The authors applied the Arksey and O'Malley framework to undertake a scoping review. The search was carried out in February 2021 in PubMed and Web of Science including all publications in English and Spanish with no limits on publication date, study design or country of origin. RESULTS: The literature search identified one hundred and twenty-four publications eligible for this review. Despite the fact that this review included many studies, their design was heterogeneous and only a few provided a high scientific evidence methodology (randomized design and/or systematic reviews with meta-analysis). Furthermore, most were single-center experiences with small sample size and there were no large multi-center studies. PBL methodology obtained a high level of satisfaction, especially among students. It was more effective than other more traditional (or lecture-based methods) at improving social and communication skills, problem-solving and self-learning skills. Knowledge retention and academic performance weren't worse (and in many studies were better) than with traditional methods. PBL was not universally widespread, probably because requires greater human resources and continuous training for its implementation. CONCLUSION: PBL is an effective and satisfactory methodology for medical education. It is likely that through PBL medical students will not only acquire knowledge but also other competencies that are needed in medical professionalism.

Early Impairment of Lung Mechanics in a Murine Model of Marfan Syndrome.

Early morbidity and mortality in patients with Marfan syndrome (MFS) -a connective tissue disease caused by mutations in fibrillin-1 gene- are mainly caused by aorta aneurysm and rupture. However, the increase in the life expectancy of MFS patients recently achieved by reparatory surgery promotes clinical manifestations in other organs. Although some studies have reported respiratory alterations in MFS, our knowledge of how this connective tissue disease modifies lung mechanics is scarce. Hence, we assessed whether the stiffness of the whole lung and of its extracellular matrix (ECM) is affected in a well-characterized MFS mouse model (FBN1C1039G/+). The stiffness of the whole lung and of its ECM were measured by conventional mechanical ventilation and atomic force microscopy, respectively. We studied 5-week and 9-month old mice, whose ages are representative of early and late stages of the disease. At both ages, the lungs of MFS mice were significantly more compliant than in wild type (WT) mice. By contrast, no significant differences were found in local lung ECM stiffness. Moreover, histopathological lung evaluation showed a clear emphysematous-like pattern in MFS mice since alveolar space enlargement was significantly increased compared with WT mice. These data suggest that the mechanism explaining the increased lung compliance in MFS is not a direct consequence of reduced ECM stiffness, but an emphysema-like alteration in the 3D structural organization of the lung. Since lung alterations in MFS are almost fully manifested at an early age, it is suggested that respiratory monitoring could provide early biomarkers for diagnosis and/or follow-up of patients with the Marfan syndrome.

Vascular smooth muscle cell phenotypic changes in patients with Marfan syndrome.

OBJECTIVE: Marfan's syndrome is characterized by the formation of ascending aortic aneurysms resulting from altered assembly of extracellular matrix microfibrils and chronic tissue growth factor (TGF)-ß signaling. TGF-ß is a potent regulator of the vascular smooth muscle cell (VSMC) phenotype. We hypothesized that as a result of the chronic TGF-ß signaling, VSMC would alter their basal differentiation phenotype, which could facilitate the formation of aneurysms. This study explores whether Marfan's syndrome entails phenotypic alterations of VSMC and possible mechanisms at the subcellular level. APPROACH AND RESULTS: Immunohistochemical and Western blotting analyses of dilated aortas from Marfan patients showed overexpression of contractile protein markers (α-smooth muscle actin, smoothelin, smooth muscle protein 22 alpha, and calponin-1) and collagen I in comparison with healthy aortas. VSMC explanted from Marfan aortic aneurysms showed increased in vitro expression of these phenotypic markers and also of myocardin, a transcription factor essential for VSMC-specific differentiation. These alterations were generally reduced after pharmacological inhibition of the TGF-ß pathway. Marfan VSMC in culture showed more robust actin stress fibers and enhanced RhoA-GTP levels, which was accompanied by increased focal adhesion components and higher nuclear localization of myosin-related transcription factor A. Marfan VSMC and extracellular matrix measured by atomic force microscopy were both stiffer than their respective controls. CONCLUSIONS: In Marfan VSMC, both in tissue and in culture, there are variable TGF-ß-dependent phenotypic changes affecting contractile proteins and collagen I, leading to greater cellular and extracellular matrix stiffness. Altogether, these alterations may contribute to the known aortic rigidity that precedes or accompanies Marfan's syndrome aneurysm formation.

PLCγ1 participates in protein transport and diacylglycerol production triggered by cargo arrival at the Golgi.

Diacylglycerol (DAG) is required for membrane traffic and structural organization at the Golgi. DAG is a lipid metabolite of several enzymatic reactions present at this organelle, but the mechanisms by which they are regulated are still unknown. Here, we show that cargo arrival at the Golgi increases the recruitment of the DAG-sensing constructs C1-PKCθ-GFP and the PKD-wt-GFP. The recruitment of both constructs was reduced by PLCγ1 silencing. Post-Golgi trafficking of transmembrane and soluble proteins was impaired in PLCγ1-silenced cells. Under basal conditions, PLCγ1 contributed to the maintenance of the pool of DAG associated with the Golgi and to the structural organization of the organelle. Finally, we show that cytosolic phospholipase C (PLC) can hydrolyse phosphatidylinositol 4-phosphate in isolated Golgi membranes. Our results indicate that PLCγ1 is part of the molecular mechanism that couples cargo arrival at the Golgi with DAG production to co-ordinate the formation of transport carriers for post-Golgi traffic.

Lipid phosphate phosphatase 3 participates in transport carrier formation and protein trafficking in the early secretory pathway.

The inhibition of phosphatidic acid phosphatase (PAP) activity by propanolol indicates that diacylglycerol (DAG) is required for the formation of transport carriers at the Golgi and for retrograde trafficking to the ER. Here we report that the PAP2 family member lipid phosphate phosphatase 3 (LPP3, also known as PAP2b) localizes in compartments of the secretory pathway from ER export sites to the Golgi complex. The depletion of human LPP3: (i) reduces the number of tubules generated from the ER-Golgi intermediate compartment and the Golgi, with those formed from the Golgi being longer in LPP3-silenced cells than in control cells; (ii) impairs the Rab6-dependent retrograde transport of Shiga toxin subunit B from the Golgi to the ER, but not the anterograde transport of VSV-G or ssDsRed; and (iii) induces a high accumulation of Golgi-associated membrane buds. LPP3 depletion also reduces levels of de novo synthesized DAG and the Golgi-associated DAG contents. Remarkably, overexpression of a catalytically inactive form of LPP3 mimics the effects of LPP3 knockdown on Rab6-dependent retrograde transport. We conclude that LPP3 participates in the formation of retrograde transport carriers at the ER-Golgi interface, where it transitorily cycles, and during its route to the plasma membrane.

Phospholipid synthesis participates in the regulation of diacylglycerol required for membrane trafficking at the Golgi complex.

The lipid metabolite diacylglycerol (DAG) is required for transport carrier biogenesis at the Golgi, although how cells regulate its levels is not well understood. Phospholipid synthesis involves highly regulated pathways that consume DAG and can contribute to its regulation. Here we altered phosphatidylcholine (PC) and phosphatidylinositol synthesis for a short period of time in CHO cells to evaluate the changes in DAG and its effects in membrane trafficking at the Golgi. We found that cellular DAG rapidly increased when PC synthesis was inhibited at the non-permissive temperature for the rate-limiting step of PC synthesis in CHO-MT58 cells. DAG also increased when choline and inositol were not supplied. The major phospholipid classes and triacylglycerol remained unaltered for both experimental approaches. The analysis of Golgi ultrastructure and membrane trafficking showed that 1) the accumulation of the budding vesicular profiles induced by propanolol was prevented by inhibition of PC synthesis, 2) the density of KDEL receptor-containing punctated structures at the endoplasmic reticulum-Golgi interface correlated with the amount of DAG, and 3) the post-Golgi transport of the yellow fluorescent temperature-sensitive G protein of stomatitis virus and the secretion of a secretory form of HRP were both reduced when DAG was lowered. We confirmed that DAG-consuming reactions of lipid synthesis were present in Golgi-enriched fractions. We conclude that phospholipid synthesis pathways play a significant role to regulate the DAG required in Golgi-dependent membrane trafficking.

Diacylglycerol is required for the formation of COPI vesicles in the Golgi-to-ER transport pathway.

Diacylglycerol is necessary for trans-Golgi network (TGN) to cell surface transport, but its functional relevance in the early secretory pathway is unclear. Although depletion of diacylglycerol did not affect ER-to-Golgi transport, it led to a redistribution of the KDEL receptor to the Golgi, indicating that Golgi-to-ER transport was perturbed. Electron microscopy revealed an accumulation of COPI-coated membrane profiles close to the Golgi cisternae. Electron tomography showed that the majority of these membrane profiles originate from coated buds, indicating a block in membrane fission. Under these conditions the Golgi-associated pool of ARFGAP1 was reduced, but there was no effect on the binding of coatomer or the membrane fission protein CtBP3/BARS to the Golgi. The addition of 1,2-dioctanoyl-sn-glycerol or the diacylglycerol analogue phorbol 12,13-dibutyrate reversed the effects of endogenous diacylglycerol depletion. Our findings implicate diacylglycerol in the retrograde transport of proteins from Golgi to the ER and suggest that it plays a critical role at a late stage of COPI vesicle formation.

Effects of hypoxia, glucose deprivation and acidosis on phosphatidylcholine synthesis in HL-1 cardiomyocytes. CTP:phosphocholine cytidylyltransferase activity correlates with sarcolemmal disruption.

A decrease in [3H]Cho (choline) incorporation in to PtdCho (phos-phatidylcholine) preceded the onset of LDH (lactate dehydrogenase) release in HL-1 cardiomyocytes submitted to simulated ischaemia. This observation led us to examine the role of PtdCho synthesis in sarcolemmal disruption in HL-1 cardiomyocytes. To address this objective we analysed the individual effects of hypoxia, glucose deprivation and acidosis, three prominent components of ischaemia, on the different steps of the Kennedy pathway for the synthesis of PtdCho. Pulse and pulse-chase experiments with [3H]Cho, performed in whole HL-1 cells submitted to hypoxia or normoxia, in the presence or absence of glucose at different pHs indicated first, that CK (choline kinase) was inhibited by hypoxia and acidosis, whereas glucose deprivation exacerbated the inhibition caused by hypoxia. Second, the rate-limiting reaction in PtdCho synthesis, catalysed by CCT (CTP:phosphocholine cytidylyltransferase), was inhibited by hypoxia and glucose deprivation, but unexpectedly activated by acidosis. In cellfree system assays, acidosis inhibited both CK and CCT. In experiments performed in whole cells, the effect of acidosis was likely to be direct on CK, but indirect or intact-cell-dependent on CCT. Since hypoxia and glucose deprivation favoured membrane disruption, but acidosis prevented it, we hypothesized that the modulation of CCT could be an important determinant of cell survival. Supporting this hypothesis, we show that CCT activity in whole-cell experiments clearly correlated with LDH release, but not with ATP concentration. Altogether our results suggest a significant role for CCT activity in sarcolemmal disruption during ischaemia.

NMDA receptor overactivation inhibits phospholipid synthesis by decreasing choline-ethanolamine phosphotransferase activity.

Overactivation of NMDA receptors is believed to induce neuronal death by increasing phospholipid hydrolysis and subsequent degradation. We showed previously that NMDA releases choline and inhibits incorporation of [3H]choline into phosphatidylcholine before excitotoxic neuronal death. On the basis of these results, we hypothesized that excitotoxicity results from inhibition of synthesis rather than from increased degradation of phospholipids. We now investigated the effect of NMDA receptor overactivation on synthesis and degradation of major membrane phospholipids in the early stages of the excitotoxic process. Exposure of cortical neurons to neurotoxic concentrations of NMDA increased extracellular choline and activated hydrolysis of phosphatidylcholine and phosphatidylinositol by phospholipase A2 but did not induce significant degradation of phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, or phosphatidylserine. In contrast, NMDA strongly reduced the incorporation of [3H]choline and [3H]ethanolamine into their respective phospholipids. Metabolic labeling experiments in whole cells showed that NMDA receptor overactivation does not modify the activity of phosphocholine or phosphoethanolamine cytidylyltransferases but strongly inhibits choline-ethanolamine phosphotransferase activity. This effect was observed well before any significant membrane damage and cell death. Moreover, cholinephosphotransferase activity was lower in microsomes from NMDA-treated cells. These results show that membrane damage by NMDA is preceded by inhibition of phospholipid synthesis and not by phospholipid degradation in the early stages of the excitotoxic process, and that NMDA receptor overactivation decreases phosphatidylcholine and phosphatidylethanolamine synthesis by inhibiting choline-ethanolaminophosphotransferase activity.

Metabotropic glutamate receptors activate phospholipase D in astrocytes through a protein kinase C-dependent and Rho-independent pathway.

Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors that mediate phospholipase D (PLD) activation in brain, but the mechanism underlying this response remains unclear. Here we used primary cultures of astrocytes as a cell model to explore the mechanism that links mGluRs to PLD. Glutamate activated both phospholipase C (PLC) and PLD with equal potency and this effect was mimicked by L-cysteinesulfinic acid, a putative neurotransmitter previously shown to activate mGluRs coupled to PLD, but not PLC, in adult brain. PLD activation by glutamate was dependent on Ca(2+) mobilization and fully blocked by both protein kinase C (PKC) inhibitors and PKC down-regulation, suggesting that PLD activation is secondary to PLC stimulation. Furthermore, brefeldin A, an inhibitor of ADP-ribosylation factor (ARF) activation, partially inhibited the activation of PLD by glutamate. By contrast, pretreatment of astrocytes with Clostridium difficile toxin B, which inactivates small G proteins of the Rho family (Rho, Rac, and Cdc42), had no effect on PLD stimulation by glutamate. Taken together, these results indicate that PLD activation by mGluRs in astrocytes is dependent on PKC and small G proteins of the ARF family, but does not require Rho proteins.