SNAP23 is essential for platelet and mast cell development and required in connective tissue mast cells for anaphylaxis.

Degranulation, a fundamental effector response from mast cells (MCs) and platelets, is an example of regulated exocytosis. This process is mediated by SNARE proteins and their regulators. We have previously shown that several of these proteins are essential for exocytosis in MCs and platelets. Here, we assessed the role of the SNARE protein SNAP23 using conditional knockout mice, in which SNAP23 was selectively deleted from either the megakaryocyte/platelet or connective tissue MC lineages. We found that removal of SNAP23 in platelets results in severe defects in degranulation of all three platelet secretory granule types, i.e., alpha, dense, and lysosomal granules. The mutation also induces thrombocytopenia, abnormal platelet morphology and activation, and reduction in the number of alpha granules. Therefore, the degranulation defect might not be secondary to an intrinsic failure of the machinery mediating regulated exocytosis in platelets. When we removed SNAP23 expression in MCs, there was a complete developmental failure in vitro and in vivo. The developmental defects in platelets and MCs and the abnormal translocation of membrane proteins to the surface of platelets indicate that SNAP23 is also involved in constitutive exocytosis in these cells. The MC conditional deletant animals lacked connective tissue MCs, but their mucosal MCs were normal and expanded in response to an antigenic stimulus. We used this mouse to show that connective tissue MCs are required and mucosal MCs are not sufficient for an anaphylactic response.

Disruption of PCDH10 and TNRC18 Genes due to a Balanced Translocation.

Balanced chromosomal rearrangements are usually associated with a normal phenotype, although in some individuals, phenotypic alterations are observed. In these patients, molecular characterization of the breakpoints can reveal the pathogenic mechanism, providing the annotation of disease-associated loci and a better genotype-phenotype correlation. In this study, we describe a patient with a balanced reciprocal translocation between 4q27 and 7p22 associated with neurodevelopmental delay. We performed cytogenetic evaluation, next-generation sequencing of microdissected derivative chromosomes, and Sanger sequencing of the junction points to define the translocation's breakpoints at base pair resolution. We found that the PCDH10 and TNRC18 genes were disrupted by the breakpoints at chromosomes 4 and 7, respectively, with the formation of chimeric genes at the junction points. Gene expression studies in the patient's peripheral blood showed reduced expression of TNRC18, a gene with unknown function and clinical significance. PCDH10 plays a role in the development of the nervous system and might be involved with the patient's neurodevelopmental delay. In this study, the full molecular characterization of the junction points was shown as an efficient tool for fine breakpoint mapping in balanced translocations in order to unmask gene disruptions and investigate the potential pathogenic role of the disrupted genes.

Munc18-2, but not Munc18-1 or Munc18-3, regulates platelet exocytosis, hemostasis, and thrombosis.

Platelet degranulation, a form of regulated exocytosis, is crucial for hemostasis and thrombosis. Exocytosis in platelets is mediated by SNARE proteins, and in most mammalian cells this process is controlled by Munc18 (mammalian homolog of Caenorhabditis elegans uncoordinated gene 18) proteins. Platelets express all Munc18 paralogs (Munc18-1, -2, and -3), but their roles in platelet secretion and function have not been fully characterized. Using Munc18-1, -2, and -3 conditional knockout mice, here we deleted expression of these proteins in platelets and assessed granule exocytosis. We measured products secreted by each type of platelet granule and analyzed EM platelet profiles by design-based stereology. We observed that the removal of Munc18-2 ablates the release of alpha, dense, and lysosomal granules from platelets, but we found no exocytic role for Munc18-1 or -3 in platelets. In vitro, Munc18-2-deficient platelets exhibited defective aggregation at low doses of collagen and impaired thrombus formation under shear stress. In vivo, megakaryocyte-specific Munc18-2 conditional knockout mice had a severe hemostatic defect and prolonged arterial and venous bleeding times. They were also protected against arterial thrombosis in a chemically induced model of arterial injury. Taken together, our results indicate that Munc18-2, but not Munc18-1 or Munc18-3, is essential for regulated exocytosis in platelets and platelet participation in thrombosis and hemostasis.

Syntaxin 3, but not syntaxin 4, is required for mast cell-regulated exocytosis, where it plays a primary role mediating compound exocytosis.

Mast cells (MCs) participate in allergy, inflammation, and defense against pathogens. They release multiple immune mediators via exocytosis, a process that requires SNARE proteins, including syntaxins (Stxs). The identity of the Stxs involved in MC exocytosis remains controversial. Here, we studied the roles of Stx3 and -4 in fully developed MCs from conditional knockout mice by electrophysiology and EM, and found that Stx3, and not Stx4, is crucial for MC exocytosis. The main defect seen in Stx3-deficient MCs was their inability to engage multigranular compound exocytosis, while leaving most single-vesicle fusion events intact. We used this defect to show that this form of exocytosis is not only required to accelerate MC degranulation but also essential to achieve full degranulation. The exocytic defect was severe but not absolute, indicating that an Stx other than Stx3 and -4 is also required for exocytosis in MCs. The removal of Stx3 affected only regulated exocytosis, leaving other MC effector responses intact, including the secretion of cytokines via constitutive exocytosis. Our in vivo model of passive systemic anaphylaxis showed that the residual exocytic function of Stx3-deficient MCs was sufficient to drive a full anaphylactic response in mice.

Biological Activities of Extracts from Aerial Parts of Salvia pachyphylla Epling Ex Munz.

The antioxidant, antimicrobial, antiproliferative, and enzyme inhibitory properties of five extracts from aerial parts of Salvia pachyphylla Epling ex Munz were examined to assess the prospective of this plant as a source of natural products with therapeutic potential. These properties were analyzed by performing a set of standard assays. The extract obtained with dichloromethane showed the most variety of components, as they yielded promising results in all completed assays. Furthermore, the extract obtained with ethyl acetate exhibited the greatest antioxidant activity, as well as the best xanthine oxidase inhibitory activity. Remarkably, both extracts obtained with n-hexane or dichloromethane revealed significant antimicrobial activity against the Gram-positive bacteria; additionally, they showed greater antiproliferative activity against three representative cell lines of the most common types of cancers in women worldwide, and against a cell line that exemplifies cancers that typically develop drug resistance. Despite that, other extracts were less active, such as the methanolic or aqueous; their results are promising for the isolation and identification of novel bioactive molecules.

Prevalence of Burnout Among Physicians: A Systematic Review.

Importance: Burnout is a self-reported job-related syndrome increasingly recognized as a critical factor affecting physicians and their patients. An accurate estimate of burnout prevalence among physicians would have important health policy implications, but the overall prevalence is unknown. Objective: To characterize the methods used to assess burnout and provide an estimate of the prevalence of physician burnout. Data Sources and Study Selection: Systematic search of EMBASE, ERIC, MEDLINE/PubMed, psycARTICLES, and psycINFO for studies on the prevalence of burnout in practicing physicians (ie, excluding physicians in training) published before June 1, 2018. Data Extraction and Synthesis: Burnout prevalence and study characteristics were extracted independently by 3 investigators. Although meta-analytic pooling was planned, variation in study designs and burnout ascertainment methods, as well as statistical heterogeneity, made quantitative pooling inappropriate. Therefore, studies were summarized descriptively and assessed qualitatively. Main Outcomes and Measures: Point or period prevalence of burnout assessed by questionnaire. Results: Burnout prevalence data were extracted from 182 studies involving 109 628 individuals in 45 countries published between 1991 and 2018. In all, 85.7% (156/182) of studies used a version of the Maslach Burnout Inventory (MBI) to assess burnout. Studies variably reported prevalence estimates of overall burnout or burnout subcomponents: 67.0% (122/182) on overall burnout, 72.0% (131/182) on emotional exhaustion, 68.1% (124/182) on depersonalization, and 63.2% (115/182) on low personal accomplishment. Studies used at least 142 unique definitions for meeting overall burnout or burnout subscale criteria, indicating substantial disagreement in the literature on what constituted burnout. Studies variably defined burnout based on predefined cutoff scores or sample quantiles and used markedly different cutoff definitions. Among studies using instruments based on the MBI, there were at least 47 distinct definitions of overall burnout prevalence and 29, 26, and 26 definitions of emotional exhaustion, depersonalization, and low personal accomplishment prevalence, respectively. Overall burnout prevalence ranged from 0% to 80.5%. Emotional exhaustion, depersonalization, and low personal accomplishment prevalence ranged from 0% to 86.2%, 0% to 89.9%, and 0% to 87.1%, respectively. Because of inconsistencies in definitions of and assessment methods for burnout across studies, associations between burnout and sex, age, geography, time, specialty, and depressive symptoms could not be reliably determined. Conclusions and Relevance: In this systematic review, there was substantial variability in prevalence estimates of burnout among practicing physicians and marked variation in burnout definitions, assessment methods, and study quality. These findings preclude definitive conclusions about the prevalence of burnout and highlight the importance of developing a consensus definition of burnout and of standardizing measurement tools to assess the effects of chronic occupational stress on physicians.

In silico-designed mutations increase variable new-antigen receptor single-domain antibodies for VEGF165 neutralization.

The stability, binding, and tissue penetration of variable new-antigen receptor (VNAR) single-domain antibodies have been tested as part of an investigation into their ability to serve as novel therapeutics. V13 is a VNAR that recognizes vascular endothelial growth factor 165 (VEGF165). In the present study V13 was used as a parental molecule into which we introduced mutations designed in silico. Two of the designed VNAR mutants were expressed, and their ability to recognize VEGF165 was assessed in vitro and in vivo. One mutation (Pro98Tyr) was designed to increase VEGF165 recognition, while the other (Arg97Ala) was designed to inhibit VEGF165 binding. Compared to parental V13, the Pro98Tyr mutant showed enhanced VEGF165 recognition and neutralization, as indicated by inhibition of angiogenesis and tumor growth. This molecule thus appears to have therapeutic potential for neutralizing VEGF165 in cancer treatment.

Mistrust in Medicine: The Rise and Fall of America's First Vaccine Institute.

In 1813, the American government passed An Act to Encourage Vaccination, the first federal endorsement of a medical practice in American history. The law tasked a federal agent with maintaining a supply of the smallpox vaccine and distributing it nationwide. James Smith, a well-respected physician and proponent of vaccination, was appointed as vaccine agent. Smith was skeptical of claims that only well-trained physicians should be allowed to perform vaccination; he felt it was a simple procedure that should be available to all American citizens. In 1822, he made a tragic error that caused several deaths and left him vulnerable to criticism from political opponents and his medical peers. This ended Smith's professional career and led to the repeal of the act itself. In this article, we use the rise and fall of James Smith to provide a historical perspective on contemporary debates surrounding delayed vaccination schedules. We explain how physicians-in the 19th century and today-have worked to build public trust in vaccination in an American culture suspicious of medical expertise.

Soluble expression of an amebic cysteine protease in the cytoplasm of Escherichia coli SHuffle Express cells and purification of active enzyme.

BACKGROUND: Recombinant production of amebic cysteine proteases using Escherichia coli cells as the bacterial system has become a challenging effort, with protein insolubility being the most common issue. Since many of these enzymes need a native conformation stabilized by disulfide bonds, an elaborate process of oxidative folding is usually demanded to get a functional protein. The cytoplasm of E. coli SHuffle Express cells owns an enhanced ability to properly fold proteins with disulfide bonds. Because of this cellular feature, it was possible to assume that this strain represents a reliable expression system and worthwhile been considered as an efficient bacterial host for the recombinant production of amebic cysteine proteases. RESULTS: Using E. coli SHuffle Express cells as the bacterial system, we efficiently produce soluble recombinant EhCP1protein. Enzymatic and inhibition analyses revealed that it exhibits proper catalytic abilities, proceeds effectively over the substrate (following an apparent Michaelis-Menten kinetics), and displays a typical inhibition profile. CONCLUSIONS: We report the first feasibility study of the recombinant production of amebic cysteine proteases using E. coli SHuffle Express as the bacterial host. We present a simple protocol for the recombinant expression and purification of fully soluble and active EhCP1 enzyme. We confirm the suitability of recombinant EhCP1 as a therapeutic target. We propose an approachable bacterial system for the recombinant production of amebic proteins, particularly for those with a need for proper oxidative folding.

Platelet Munc13-4 regulates hemostasis, thrombosis and airway inflammation.

Platelet degranulation is crucial for hemostasis and may participate in inflammation. Exocytosis in platelets is mediated by SNARE proteins and should be controlled by Munc13 proteins. We found that platelets express Munc13-2 and -4. We assessed platelet granule exocytosis in Munc13-2 and -4 global and conditional knockout (KO) mice, and observed that deletion of Munc13-4 ablates dense granule release and indirectly impairs alpha granule exocytosis. We found no exocytic role for Munc13-2 in platelets, not even in the absence of Munc13-4. In vitro, Munc13-4-deficient platelets exhibited defective aggregation at low doses of collagen. In a flow chamber assay, we observed that Munc13-4 acted as a rate-limiting factor in the formation of thrombi. In vivo, we observed a dose-dependency between Munc13-4 expression in platelets and both venous bleeding time and time to arterial thrombosis. Finally, in a model of allergic airway inflammation, we found that platelet-specific Munc13-4 KO mice had a reduction in airway hyper-responsiveness and eosinophilic inflammation. Taken together, our results indicate that Munc13-4-dependent platelet dense granule release plays essential roles in hemostasis, thrombosis and allergic inflammation.

Munc18-2, but not Munc18-1 or Munc18-3, controls compound and single-vesicle-regulated exocytosis in mast cells.

Mast cells (MCs) play pivotal roles in many inflammatory conditions including infections, anaphylaxis, and asthma. MCs store immunoregulatory compounds in their large cytoplasmic granules and, upon stimulation, secrete them via regulated exocytosis. Exocytosis in many cells requires the participation of Munc18 proteins (also known as syntaxin-binding proteins), and we found that mature MCs express all three mammalian isoforms: Munc18-1, -2, and -3. To study their functions in MC effector responses and test the role of MC degranulation in anaphylaxis, we used conditional knockout (cKO) mice in which each Munc18 protein was deleted exclusively in MCs. Using recordings of plasma membrane capacitance for high-resolution analysis of exocytosis in individual MCs, we observed an almost complete absence of exocytosis in Munc18-2-deficient MCs but intact exocytosis in MCs lacking Munc18-1 or Munc18-3. Stereological analysis of EM images of stimulated MCs revealed that the deletion of Munc18-2 also abolishes the homotypic membrane fusion required for compound exocytosis. We confirmed the severe defect in regulated exocytosis in the absence of Munc18-2 by measuring the secretion of mediators stored in MC granules. Munc18-2 cKO mice had normal morphology, development, and distribution of their MCs, indicating that Munc18-2 is not essential for the migration, retention, and maturation of MC-committed progenitors. Despite that, we found that Munc18-2 cKO mice were significantly protected from anaphylaxis. In conclusion, MC-regulated exocytosis is required for the anaphylactic response, and Munc18-2 is the sole Munc18 isoform that mediates membrane fusion during MC degranulation.

An amebic protein disulfide isomerase (PDI) complements the yeast PDI1 mutation but is unable to support cell viability under ER or thermal stress.

In eukaryotic cells, protein disulfide isomerases (PDI) are oxidoreductases that catalyze the proper disulfide bond formation during protein folding. The pathobiology of the protozoan parasite Entamoeba histolytica, the causative agent of human amebiasis, depends on secretion of several virulence factors, such as pore-forming peptides and cysteine proteinases. Although the native conformation of these factors is stabilized by disulfide bonds, there is little information regarding the molecular machinery involved in the oxidative folding of amebic proteins. Whereas testing gene function in their physiological background would be the most suitable approach, we have taken advantage of the cellular benefits offered by the yeast Saccharomyces cerevisiae (as a model of eukaryotic cell) to examine the functional role of an amebic PDI (EhPDI). As the yeast PDI homolog is essential for cell viability, a functional complementation assay was carried out to test the ability of EhPDI to circumvent the lethal phenotype of a yeast PDI1 mutant. Also, its proficiency under stressful conditions was explored by examining the survival outcome following endoplasmic reticulum (ER) stress induced by a reductant agent (DTT) or thermal stress promoted by a nonpermissive temperature (37 °C). Our results indicate that EhPDI is functionally active when physiological conditions are stable. Nonetheless, when conditions are stressful (e.g., by the accumulation of misfolded proteins in the ER compartment), its functionality is exceeded, suggesting an inability to prevent unfolding, suppress aggregation, or assist refolding of proteins. Despite the latter, our findings constitute the initial step toward determining the participation of EhPDI in cellular mechanisms related to protein homeostasis.

Munc13 proteins control regulated exocytosis in mast cells.

Mast cells (MCs) are involved in host defenses against pathogens and inflammation. Stimulated MCs release substances stored in their granules via regulated exocytosis. In other cell types, Munc13 (mammalian homolog of Caenorhabditis elegans uncoordinated gene 13) proteins play essential roles in regulated exocytosis. Here, we found that MCs express Munc13-2 and -4, and we studied their roles using global and conditional knock-out (KO) mice. In a model of systemic anaphylaxis, we found no difference between WT and Munc13-2 KO mice, but global and MC-specific Munc13-4 KO mice developed less hypothermia. This protection correlated with lower plasma histamine levels and with histological evidence of defective MC degranulation but not with changes in MC development, distribution, numbers, or morphology. In vitro assays revealed that the defective response in Munc13-4-deficient MCs was limited to regulated exocytosis, leaving other MC secretory effector responses intact. Single cell capacitance measurements in MCs from mouse mutants differing in Munc13-4 expression levels in their MCs revealed that as levels of Munc13-4 decrease, the rate of exocytosis declines first, and then the total amount of exocytosis decreases. A requirement for Munc13-2 in MC exocytosis was revealed only in the absence of Munc13-4. Electrophysiology and EM studies uncovered that the number of multigranular compound events (i.e. granule-to-granule homotypic fusion) was severely reduced in the absence of Munc13-4. We conclude that although Munc13-2 plays a minor role, Munc13-4 is essential for regulated exocytosis in MCs, and that this MC effector response is required for a full anaphylactic response.

DNA Extraction with DNAzol and LAMP, Performed in a Heating Block as a Simple Procedure for Detection of Mycobacterium tuberculosis in Sputum Specimens.

Tuberculosis (TB) remains as a major public health issue in developing countries. Accurate detection is essential for the proper management of patients with active disease. Here, we present a simple DNAzol-LAMP (loop-mediated isothermal amplification) procedure for the detection of Mycobacterium tuberculosis in sputum specimens. Twenty smear-positive sputum samples were analyzed as follows: (i) Genetic material was extracted by a standard DNAzol protocol, and (ii) mycobacterial DNA was detected by a typical TB-specific loop-mediated isothermal amplification method. Results and diagnostic test performance attests to the suitability of the proposed procedure.

Frequency of Alu insertions within the ACE and PR loci in Northwestern Mexicans.

OBJECTIVE: Presently, non-LTR retrotransposons are the most active mobile elements in the human genome. Among these, Alu elements are highly represented in the modern population. Worldwide, distribution of Alu polymorphisms (insertion/deletion; I/D) shows variability between different populations. Two Alu insertion loci, ACE and PR, are significant biomarkers that have served in several genotype-phenotype association studies. In Mexico, studies concerning the frequency of these biomarkers have been conducted mainly in subpopulations from central and southern regions. Here, we screened a population sample of the northwestern region to gain further knowledge regarding the prevalence of Alu polymorphisms within ACE and PR loci. RESULTS: For ACE locus, the observed genotype frequencies were 26.5, 51.0 and 22.5% for II, ID, and DD, respectively; and allelic frequencies for I and D were 52 and 48%. Whereas respective genotype frequencies for PR locus were 2.7, 26.5 and 70.8%, and the corresponding allele frequencies were 16 and 84%. Furthermore, the insertion frequency within ACE locus was similar between central, western and northwestern subpopulations, and rather higher in southeastern subpopulation (p < 0.05). Although the occurrence of Alu polymorphisms within PR locus has not been widely examined, the insertion frequency was higher in northwestern subpopulation, as compared with western and southeastern subpopulations (p < 0.05). Based on the frequency of Alu insertions found in ACE and PR loci, subpopulations from the northwestern, western and central regions share a common genetic origin, but apparently not with the subpopulation from the southeastern region, in accordance with the notion that assumes the existence of a broad genomic diversity in the Mexican population. In addition, the high prevalence of Alu insertions reveals their potential application as biomarkers with prognostic value for the associated diseases; e.g., as part of the standard protocols for clinical diagnosis.

Prevalence of Depression, Depressive Symptoms, and Suicidal Ideation Among Medical Students: A Systematic Review and Meta-Analysis.

Importance: Medical students are at high risk for depression and suicidal ideation. However, the prevalence estimates of these disorders vary between studies. Objective: To estimate the prevalence of depression, depressive symptoms, and suicidal ideation in medical students. Data Sources and Study Selection: Systematic search of EMBASE, ERIC, MEDLINE, psycARTICLES, and psycINFO without language restriction for studies on the prevalence of depression, depressive symptoms, or suicidal ideation in medical students published before September 17, 2016. Studies that were published in the peer-reviewed literature and used validated assessment methods were included. Data Extraction and Synthesis: Information on study characteristics; prevalence of depression or depressive symptoms and suicidal ideation; and whether students who screened positive for depression sought treatment was extracted independently by 3 investigators. Estimates were pooled using random-effects meta-analysis. Differences by study-level characteristics were estimated using stratified meta-analysis and meta-regression. Main Outcomes and Measures: Point or period prevalence of depression, depressive symptoms, or suicidal ideation as assessed by validated questionnaire or structured interview. Results: Depression or depressive symptom prevalence data were extracted from 167 cross-sectional studies (n = 116 628) and 16 longitudinal studies (n = 5728) from 43 countries. All but 1 study used self-report instruments. The overall pooled crude prevalence of depression or depressive symptoms was 27.2% (37 933/122 356 individuals; 95% CI, 24.7% to 29.9%, I2 = 98.9%). Summary prevalence estimates ranged across assessment modalities from 9.3% to 55.9%. Depressive symptom prevalence remained relatively constant over the period studied (baseline survey year range of 1982-2015; slope, 0.2% increase per year [95% CI, -0.2% to 0.7%]). In the 9 longitudinal studies that assessed depressive symptoms before and during medical school (n = 2432), the median absolute increase in symptoms was 13.5% (range, 0.6% to 35.3%). Prevalence estimates did not significantly differ between studies of only preclinical students and studies of only clinical students (23.7% [95% CI, 19.5% to 28.5%] vs 22.4% [95% CI, 17.6% to 28.2%]; P = .72). The percentage of medical students screening positive for depression who sought psychiatric treatment was 15.7% (110/954 individuals; 95% CI, 10.2% to 23.4%, I2 = 70.1%). Suicidal ideation prevalence data were extracted from 24 cross-sectional studies (n = 21 002) from 15 countries. All but 1 study used self-report instruments. The overall pooled crude prevalence of suicidal ideation was 11.1% (2043/21 002 individuals; 95% CI, 9.0% to 13.7%, I2 = 95.8%). Summary prevalence estimates ranged across assessment modalities from 7.4% to 24.2%. Conclusions and Relevance: In this systematic review, the summary estimate of the prevalence of depression or depressive symptoms among medical students was 27.2% and that of suicidal ideation was 11.1%. Further research is needed to identify strategies for preventing and treating these disorders in this population.

In Their Own Words: An Analysis of the Experiences of Medical Interns Participating in a Prospective Cohort Study of Depression.

PURPOSE: To compare the subjective experiences of interns with and without symptoms of depression using a mixed-methods approach. METHOD: In 2007-2008, interns from six institutions were screened for depression before and during internship using an online survey that included the Patient Health Questionnaire (PHQ-9). At the end of internship, participants were asked what made the year difficult, easy, and memorable, and how they had changed. Computerized lexical and qualitative thematic analyses were performed to analyze their free-text responses. RESULTS: Sixty-three percent (244/388) of invited interns participated in the original cohort study. Of those, 42% (103/244) answered the open-ended questions for this analysis. Thirty-five percent (36/103) screened positive for clinically significant depression (i.e., PHQ-9 score ≥ 10) during their intern year. Respondents with symptoms of depression were more likely to report problems with cynicism, exhaustion, and stress, while those without them were more likely to mention positive patient care and educational experiences. Respondents with symptoms of depression preferentially described experiences that "broke" their confidence, sense of well-being, and belief in the medical profession, while those who did not described profoundly positive, life-changing experiences regarding interactions with patients and supportive colleagues, through which they grew personally and professionally. CONCLUSIONS: Depression during internship affects not only objective outcomes like medical errors but also how interns value the profession and themselves, with potentially profound consequences for their future career decisions. Residency programs should implement reactive interventions targeting depression and proactive interventions promoting resilience and well-being to address the issues that lead to depression.