Depolymerisation and rearrangement of actin filaments during exocytosis in rat peritoneal mast cells: involvement of ryanodine-sensitive calcium stores

Cytoskeletal F-actin associated with synaptic vesicles and granules plays an important role during Ca²⁺-mediated exocytosis. In the present work, we have used amperometry and confocal fluorescence to study the role of internal Ca²⁺ in the rearrangement of F-actin (visualised with phalloidin-Alexa 546) during exocytosis in rat mast cells. The F-actin-depolymerising drug, latrunculin A, and the ryanodine receptor agonists ryanodine and caffeine that, per se did not induce exocytosis, enhanced the exocytotic responses elicited by compound 48/80 (C48/80). They also induced cortical actin depolymerisation in the presence or absence of external Ca²⁺. Degranulation induced by C48/80 was accompanied by the formation of a cytoplasmic F-actin network. Depletion of internal Ca²⁺ with cyclopiazonic acid inhibited latrunculin potentiation of C48/80-stimulated exocytosis and completely blocked the formation of the cytoplasmic F-actin network. This indicates that the mobilisation of Ca²⁺ from ryanodine-sensitive intracellular stores plays an important role in the depolymerisation of the cortical F-actin barrier and possibly in the formation of the internal F-actin network during exocytotic activation of peritoneal mast cells.     

Unravelling residents’ and supervisors’ workplace interactions: an intersubjectivity study

Context

Successful engagement between residents and supervisors lies at the core of workplace learning, a process that is not exempt from challenge. Clinical encounters have unique learning potential as they offer opportunities to achieve a shared understanding between the resident and supervisor of how to accomplish a common goal. How residents and supervisors develop such a mutual understanding is an issue that has received limited attention in the literature. We used the ‘intersubjectivity’ concept as a novel conceptual framework to analyse this issue.

Methods

We conducted a constructivist grounded theory study in an anaesthesiology department in Bogota, Colombia, using focus groups and field observations. Eleven residents of different training levels and 18 supervisors with varying years of teaching experience participated in the study. Through iterative data analysis, collection and constant comparison, we constructed the final results.

Results

We found that residents and supervisors achieved a shared understanding by adapting to one another in the process of providing patient care. Continuous changes in the composition of resident–supervisor dyads exposed them to many procedural variations, to which they responded by engaging in various adaptation patterns that included compliance by residents with supervisors’ directions, negotiation by residents of supervisors’ preferences, and the sharing of decision making. In the process, the resident played an increasingly key role as a member of the supervisory dyad. Additionally, experiencing these adaptation patterns repeatedly resulted in the creation of a working repertoire: an attuned working code used by the members of each supervisory dyad to work together as a team.

Conclusions

The development of shared understanding between residents and supervisors entailed experiencing diverse adaptation patterns which resulted in the creation of working repertoires. Seeing supervisory interactions as adaptation processes has essential theoretical and practical implications regarding workplace learning in postgraduate settings. Our findings call for further exploration to understand learning in postgraduate education as a social process.

     

Prognosis of Patients With Severe Aortic Stenosis After the Decision to Perform an Intervention

Introduction and objectives

Current therapeutic options for severe aortic stenosis (AS) include transcatheter aortic valve implantation (TAVI) and surgical aortic valve replacement (SAVR). Our aim was to describe the prognosis of patients with severe AS after the decision to perform an intervention, to study the variables influencing their prognosis, and to describe the determinants of waiting time > 2 months.

Management of initial carious lesions: Iowa survey

BackgroundThe authors explore Iowa dentists’ agreement with the International Caries Classification and Management System (ICCMS) in the nonsurgical management of initial carious lesions in patients at low, moderate, and high caries risk and identify factors related to their agreement.MethodsElectronic surveys were mailed to 916 actively practicing dentists who are alumni of the College of Dentistry at The University of Iowa. Questions included clinical scenarios that used text, clinical photographs, and radiographic images of initial carious lesions. Dentists were asked what type of treatment they would recommend. Treatment options included no treatment, nonsurgical treatment, or surgical treatment. Logistic regression analyses were used to assess associations among agreement with ICCMS, characteristics of the dentist’s practice, and patients’ caries risk level.ResultsA total of 138 Iowa dentists responded to the survey. Agreement with ICCMS regarding nonsurgical management of initial carious lesions for patients at low, moderate, and high risk levels were 73%, 59%, and 51% respectively. Compared with their counterparts, dentists who agreed with the recommendations for nonsurgical treatment were more likely to dry the teeth during caries detection (95% confidence interval [CI], 1.02 to 12.67, P = .0468), use magnification (95% CI, 1.16 to 7.17, P = .0225) for caries detection, have graduated less than 20 years ago (P = .0024), practice in public health settings (P = .0089), and perform a caries risk assessment (95% CI, 1.10 to 4.29, P = .0262).ConclusionsDentists who dry teeth, use magnification for caries detection, graduated in the past 20 years, practice in a public health setting, and perform a caries risk assessment were significantly more likely to make decisions that were consistent with the guidelines of the ICCMS.Practical ImplicationsKnowledge of evidence-based options personalized for a patient’s risk status is essential for applying the best management of initial caries lesions.     

Cerebral blood flow is an earlier indicator of perfusion abnormalities than cerebral blood volume in Alzheimer's disease

The purpose of this study was to elucidate whether cerebral blood flow (CBF) can better characterize perfusion abnormalities in predementia stages of Alzheimer''s disease (AD) than cerebral blood volume (CBV) and whether cortical atrophy is more associated with decreased CBV or with decreased CBF. We compared measurements of CBV, CBF, and mean cortical thickness obtained from magnetic resonance images in a group of healthy controls, patients with mild cognitive impairment (MCI) who converted to AD after 2 years of clinical follow-up (MCI-c), and patients with mild AD. A significant decrease in perfusion was detected in the parietal lobes of the MCI-c patients with CBF parametric maps but not with CBV maps. In the MCI-c group, a negative correlation between CBF values and cortical thickness in the right parahippocampal gyrus suggests an increase in CBF that depends on cortical atrophy in predementia stages of AD. Our study also suggests that CBF deficits appear before CBV deficits in the progression of AD, as CBV abnormalities were only detected at the AD stage, whereas CBF changes were already detected in the MCI stage. These results confirm the hypothesis that CBF is a more sensitive parameter than CBV for perfusion abnormalities in MCI-c patients.     

Influence of Two Depuration Periods on the Activity and Transcription of Antioxidant Enzymes in Tilapia Exposed to Repeated Doses of Cylindrospermopsin under Laboratory Conditions

The cyanobacterial toxin Cylindrospermopsin (CYN), a potent protein synthesis inhibitor, is increasingly being found in freshwater bodies infested by cyanobacterial blooms worldwide. Moreover, it has been reported to be implicated in human intoxications and animal mortality. Recently, the alteration of the activity and gene expression of some glutathione related enzymes in tilapias (Oreochromis niloticus) exposed to a single dose of CYN has been reported. However, little is known about the effects induced by repeated doses of this toxin in tilapias exposed by immersion and the potential reversion of these biochemical alterations after two different depuration periods (3 or 7 days). In the present study, tilapias were exposed by immersion to repeated doses of a CYN-containing culture of Aphanizomenon ovalisporum during 14 days, and then were subjected to depuration periods (3 or 7 days) in clean water in order to examine the potential reversion of the effects observed. The activity and relative mRNA expression by real-time polymerase chain reaction (PCR) of the antioxidant enzymes glutathione peroxidase (GPx) and soluble glutathione-S-transferases (sGST), and also the sGST protein abundance by Western blot analysis were evaluated in liver and kidney of fish. Results showed significant alterations in most of the parameters evaluated and their recovery after 3 days (GPx activity, sGST relative abundance) or 7 days (GPx gene expression, sGST activity). These findings not only confirm the oxidative stress effects produced in fish by cyanobacterial cells containing CYN, but also show the effectiveness of depuration processes in mitigating the CYN-containing culture toxic effects.     

Simple methods to remove microbes from leaf surfaces

Endophytes have been defined as microorganisms living inside plant tissues without causing negative effects on their hosts. Endophytic microbes have been extensively studied for their plant growth-promoting traits. However, analyses of endophytes require complete removal of epiphytic microorganisms. We found that the established tests to evaluate surface sterility, polymerase chain reaction, and leaf imprints, are unreliable. Therefore, we used scanning electron microscopy (SEM) as an additional assessment of epiphyte removal. We used a diverse suite of sterilization protocols to remove epiphytic microorganisms from the leaves of a gymnosperm and an angiosperm tree to test the influence of leaf morphology on the efficacy of these methods. Additionally, leaf tissue damage was also evaluated by SEM, as damaging the leaves might have an impact on endophytes and could lead to inaccurate assessment of endophytic communities. Our study indicates, that complete removal of the leaf cuticle by the sterilization technique assures loss of epiphytic microbes, and that leaves of different tree species may require different sterilization protocols. Furthermore, our study demonstrates the importance of choosing the appropriate sterilization protocol to prevent erroneous interpretation of host-endophyte interactions. Moreover, it shows the utility of SEM for evaluating the effectiveness of surface sterilization methods and their impact on leaf tissue integrity.     

Generation and characterization of potential dengue vaccine candidates based on domain III of the envelope protein and the capsid protein of the four serotypes of dengue virus

Dengue is currently one of the most important arthropod-borne diseases, causing up to 25,000 deaths annually. There is currently no vaccine to prevent dengue virus infection, which needs a tetravalent vaccine approach. In this work, we describe the cloning and expression in Escherichia coli of envelope domain III-capsid chimeric proteins (DIIIC) of the four dengue serotypes as a tetravalent dengue vaccine candidate that is potentially able to generate humoral and cellular immunity. The recombinant proteins were purified to more than 85 % purity and were recognized by anti-dengue mouse and human sera. Mass spectrometry analysis verified the identity of the proteins and the correct formation of the intracatenary disulfide bond in the domain III region. The chimeric DIIIC proteins were also serotype-specific, and in the presence of oligonucleotides, they formed aggregates that were visible by electron microscopy. These results support the future use of DIIIC recombinant chimeric proteins in preclinical studies in mice for assessing their immunogenicity and efficacy.