Growing Medical Educators from Medical Students by Supporting Self-directing Learning Opportunities Born Out of Cadaveric Dissection.

This study focuses on a subset of medical students who participated in an anatomy dissection program and undertook an additional self-directed learning (SDL) project investigating incidental findings of cadaveric pathology. The value of SDL activity is explored as a means of enhancing medical student education, particularly its student perceived value in preparing and developing them as future medical educators. It was assessed whether the project advanced student interest in medical education by analyzing their motivations for participation. The results of the study highlight the potential of SDL as an experiential learning opportunity for medical students and the role of anatomic pathology in connecting multiple domains of medical education.

One-step site-specific S-alkylation of full-length caveolin-1: Lipidation modulates the topology of its C-terminal domain.

Caveolin-1 is an integral membrane protein that is known to acquire a number of posttranslational modifications upon trafficking to the plasma membrane. In particular, caveolin-1 is palmitoylated at three cysteine residues (C133, C143, and C156) located within the C-terminal domain of the protein which could have structural and topological implications. Herein, a reliable preparation of full-length S-alkylated caveolin-1, which closely mimics the palmitoylation observed in vivo, is described. HPLC and ESI-LC-MS analyses verified the addition of the C16 alkyl groups to caveolin-1 constructs containing one (C133), two (C133 and C143), and three (C133, C143, and C156) cysteine residues. Circular dichroism spectroscopy analysis of the constructs revealed that S-alkylation does not significantly affect the global helicity of the protein; however, molecular dynamics simulations revealed that there were local regions where the helicity was altered positively or negatively by S-alkylation. In addition, the simulations showed that lipidation tames the topological promiscuity of the C-terminal domain, resulting in a disposition within the bilayer characterized by increased depth.

Marking Pen Bacterial Contamination During Shoulder Surgery.

BACKGROUND: Equipment used to guide surgical incisions has been shown to be a source of bacterial contamination during surgery. PURPOSE/HYPOTHESIS: To compare the culture-positive rates of sterile marking pens used before and after skin preparation for shoulder surgery. It was hypothesized that there will be no difference in culture-positive rates from marking pens used after skin preparation compared with before skin preparation. STUDY DESIGN: Controlled laboratory study. METHODS: Overall, 43 consecutive patients undergoing elective shoulder surgery were enrolled prospectively into this study. Each patient provided 2 samples: study pens (from marking the surgical site incision after skin preparation) and positive control pens (from marking the surgical site incision before skin preparation). In addition, there were 43 negative control pens evaluated (straight from the packaging without any patient contact). Cultures were evaluated at 4 and 21 days, and all positive cultures were further evaluated for speciation, if able. Standard descriptive summaries and Fisher exact tests were used to compare the study samples. RESULTS: The average age of the 43 patients was 54 years (range, 18-76 years). There were 29 (67%) female patients, and 30 (70%) procedures were on the right shoulder. Of the 43 procedures performed, 29 (67.4%) were arthroscopic, 12 (27.9%) were open, and 2 (4.7%) were closed. Of the 43 study pens, 1 culture was positive for Propionibacterium acnes (2.3%). Of the 43 positive control pens, 2 cultures were positive for bacterial growth (4.7%): P. acnes and Gram-positive bacilli (no speciation could be obtained). Of the 43 negative control pens, none of the cultures were positive for bacterial growth (0%). There was no statistical difference in the culture-positive rate between the study pens and the positive or negative control pens (P ≥ .999). CONCLUSION: Study results indicated that sterile surgical marking pens used to plan incisions and to outline anatomic landmarks did not have a higher culture-positive rate compared with pens used on unprepared skin or pens straight from the packaging. CLINICAL RELEVANCE: As a precaution, sterile surgical marking pens should be discarded after use on the skin surface and not placed on the sterile field.

Purification and refolding protocol for cold-active recombinant esterase AaSGNH1 from Aphanizomenon flos-aquae expressed as insoluble inclusion bodies.

Microbial esterases are a highly desirable tool for numerous biosynthetic and biotechnological applications requiring ester bond cleavage. Once identified, microbial esterases are often produced recombinantly in Escherichia coli to enhance yield and ease of purification. In this study a polyhistidine-tagged SGNH esterase gene (AaSGNH1), originating from the cyanobacterium Aphanizomenon flos-aquae, was cloned into an over-expression plasmid and expressed in BL21(DE3) cells. The recombinant esterase enzyme was produced as inactive inclusion bodies which were insoluble in 8 M urea but readily solubilized by the detergent Empigen BB®. Crucially, the procurement of active enzyme required controlled removal of detergent during column chromatography and dialysis steps. The refolded esterase was characterized with respect to its ability to catalyze the cleavage of p-nitrophenol esters of different chain lengths (C2, C8, C16). In addition, the temperature and pH optima were determined and it was found that the enzyme was most active at low temperatures (5-15 °C) and under alkaline conditions (pH 8-10). It was found that the kinetic properties of AaSGNH1 were remarkably similar to other SGNH esterases described thereby validating that the protein was effectively refolded. Overall, this study provides a simple strategy for isolating cold-active recombinant esterase enzyme when expressed as inclusion bodies.

Application of the Environmental Relative Moldiness Index in Indoor Marijuana Grow Operations.

OBJECTIVES: Indoor marijuana grow operations (IMGOs) are increasing due to legalization of recreational and medicinal cannabis at the state level. However, the potential exposures of IMGO workers have not been well studied. Mold exposure has been identified as a major occupational health concern. Mold-specific quantitative polymerase chain reaction (MSQPCR) can provide quantitative exposure data for fungi at the species level. The purpose of this study was to characterize the airborne fungal burden using MSQPCR and to evaluate the applicability of an airborne Environmental Relative Moldiness Index (ERMI) in IMGOs. METHODS: Air and dust samples were collected inside and outside the IMGOs and then analyzed via MSQPCR. These data were then used to calculate IMGO-specific ERMI scores. Culturable air samples were collected on agar plates and analyzed via microscopy. Differences were evaluated between indoor and outdoor concentrations, as well as between air and dust samples. The agreement between MSQPCR and culture-based methods was also evaluated. RESULTS: Based on the geometric means for non-zero values of each fungal species across all IMGOs, the total airborne concentration was approximately 9100 spore equivalent (SE) m-3 with an interquartile range (IQR) of 222 SE m-3. The indoor/outdoor ratio of geometric means across all 36 species per IMGO ranged from 0.4 to 6.2. Significantly higher indoor concentrations of fungal species, including Aspergillus spp., were observed. An average airborne ERMI score of 7 (IQR = 7.6) indicated a relatively high burden of mold across a majority of operations. The ERMI scores were driven by the high concentrations of Group 1 species with a mean of 15.8 and an IQR of 13. There were 63 additional species identified in the culturable air samples not included in the ERMI. CONCLUSIONS: High concentrations of airborne fungi were identified in IMGOs. Our evaluation of the ERMI based on MSQPCR as a rapid diagnostic and risk assessment tool for industrial hygienists in the IMGO setting is equivocal. ERMI did not identify all relevant fungal species associated with this specific occupational environment. We identified several issues with using the ERMI calculation. At this time, the catalog of fungal species needs to optimized for the occupational setting to ensure adequate coverage, especially for those species expected to be found in this burgeoning industry. Further research is necessary to elucidate the link between the ERMI score of airborne samples, worker exposure and health effects in grows to generate an acceptable index score for use in occupational exposure assessments.

Reconstitution of full-length human caveolin-1 into phospholipid bicelles: Validation by analytical ultracentrifugation.

A significant hurdle in obtaining biophysical information on membrane proteins is developing a successful strategy for their reconstitution into a suitable membrane mimic. In particular, utilization of the more 'native-like' membrane mimics such as bicelles is generally more challenging than simple micellar solubilization. Caveolin-1, an integral membrane protein involved in membrane curvature, endocytosis, mechano-protection, and signal transduction, has been shown to be particularly recalcitrant to standard reconstitution protocols due to its highly hydrophobic characteristics. Herein we describe a robust method to incorporate recombinantly produced full-length caveolin-1 into bicelles at levels needed for biophysical experimentation. The benchmark of successful reconstitution is the obtainment of protein in a homogeneous state; therefore, we developed a validation procedure to monitor the success of the reconstitution using analytical ultracentrifugation of density-matched bicelles. Our findings indicated that our protocol produces a very homogeneous preparation of caveolin-1 associated with bicelles, and that caveolin-1 is highly α-helical (by circular dichroism spectroscopy). We believe that this methodology will serve as a general strategy to facilitate biophysical studies on membrane proteins.

Secondary structure of caveolins: a mini review.

Caveolae are 50-100 nm invaginations found within the plasma membrane of cells. Caveolae are involved in many processes that are essential for homeostasis, most notably endocytosis, mechano-protection, and signal transduction. Within these invaginations, the most important proteins are caveolins, which in addition to participating in the aforementioned processes are structural proteins responsible for caveolae biogenesis. When caveolin is misregulated or mutated, many disease states can arise which include muscular dystrophy, cancers, and heart disease. Unlike most integral membrane proteins, caveolin does not have a transmembrane orientation; instead, it is postulated to adopt an unusual topography where both the N- and C-termini lie on the cytoplasmic side of the membrane, and the hydrophobic span adopts an intramembrane loop conformation. While knowledge concerning the biology of caveolin has progressed apace, fundamental structural information has proven more difficult to obtain. In this mini-review, we curate as well as critically assess the structural data that have been obtained on caveolins to date in order to build a robust and compelling model of the caveolin secondary structure.

Low- q Bicelles Are Mixed Micelles.

Bicelles are used in many membrane protein studies because they are thought to be more bilayer-like than micelles. We investigated the properties of "isotropic" bicelles by small-angle neutron scattering, small-angle X-ray scattering, fluorescence anisotropy, and molecular dynamics. All data suggest that bicelles with a q value below 1 deviate from the classic bicelle that contains lipids in the core and detergent in the rim. Thus not all isotropic bicelles are bilayer-like.

Efficient solubilization and purification of highly insoluble membrane proteins expressed as inclusion bodies using perfluorooctanoic acid.

The purification of membrane proteins can be challenging due to their low solubility in conventional detergents and/or chaotropic solutions. The introduction of fusion systems that promote the formation of inclusion bodies has facilitated the over-expression of membrane proteins. In this protocol, we describe the use of perfluorooctanoic acid (PFOA) as an aid in the purification of highly hydrophobic membrane proteins expressed as inclusion bodies. The advantage of utilizing PFOA is threefold: first, PFOA is able to reliably solubilize inclusion bodies, second, PFOA is compatible with nickel affinity chromatography, and third, PFOA can be efficiently dialyzed away to produce a detergent free sample. To demonstrate the utility of employing PFOA, we expressed and purified a segment of the extremely hydrophobic membrane protein caveolin-1.

Reconstitution and spectroscopic analysis of caveolin-1 residues 62-178 reveals that proline 110 governs its structure and solvent exposure.

Caveolin-1 is a membrane protein that possesses an unusual topology where both N- and C-termini are cytoplasmic as a result of a membrane-embedded turn. In particular, proline 110 has been postulated to be the linchpin of this unusual motif. Using a caveolin-1 construct (residues 62-178) reconstituted into dodecylphosphocholine micelles with and without a cholesterol mimic, the changes that occurred upon P110A mutation were probed. Using far UV circular dichroism spectroscopy it was shown that cholesterol attenuated the helicity of caveolin-1, and that mutation of P110 to alanine caused a significant increase in the α-helicity of the protein. Near UV circular dichroism spectroscopy showed significant changes in structure and/or environment upon mutation that again were modulated by the presence of cholesterol. Stern-Volmer quenching and λ(max) analysis of tryptophan residues showed that the proline mutation caused W85 to become more exposed, W98 and W115 to become less exposed, and W128 showed no change. This finding provided evidence that regions proximal and far away from the proline are buried differentially upon its mutation and therefore this residue is strongly tied to maintaining the hydrophobic coverage along the caveolin-1 sequence. In the presence of cholesterol, the accessibilities of the two tryptophan residues that proceeded position 110 were altered much more significantly upon P110A mutation than the two tryptophans aft P110. Overall, this work provides strong evidence that proline 110 is critical for maintaining both the structure and hydrophobic coverage of caveolin-1 and that cholesterol also plays a significant role in modulating these parameters.

Recent progress in the topology, structure, and oligomerization of caveolin: a building block of caveolae.

Caveolae are cholesterol-rich plasma membrane invaginations that are found in a plethora of cell types. They play many roles including signal transduction, endocytosis, and mechanoprotection. The most critical protein in caveolae is the integral membrane protein, caveolin, which has been shown to be necessary for caveolae formation, and governs the major functions attributed to caveolae. Caveolin is postulated to act as a scaffold in the high molecular weight striated coat that surrounds the caveolar bulb, stabilizing it. Caveolin interacts, both directly and indirectly, with a large number of signaling molecules, and presides over the endocytosis of molecular cargo by caveolae. However, many of the key biophysical aspects of the caveolin protein, its structure, topology, and oligomeric behavior, are just beginning to come to light. Herein is an up-to-date summary and critique of the progress that has been made in understanding caveolin on a molecular and atomic level.

Probing the U-shaped conformation of caveolin-1 in a bilayer.

Caveolin induces membrane curvature and drives the formation of caveolae that participate in many crucial cell functions such as endocytosis. The central portion of caveolin-1 contains two helices (H1 and H2) connected by a three-residue break with both N- and C-termini exposed to the cytoplasm. Although a U-shaped configuration is assumed based on its inaccessibility by extracellular matrix probes, caveolin structure in a bilayer remains elusive. This work aims to characterize the structure and dynamics of caveolin-1 (D82-S136; Cav182-136) in a DMPC bilayer using NMR, fluorescence emission measurements, and molecular dynamics simulations. The secondary structure of Cav182-136 from NMR chemical shift indexing analysis serves as a guideline for generating initial structural models. Fifty independent molecular dynamics simulations (100 ns each) are performed to identify its favorable conformation and orientation in the bilayer. A representative configuration was chosen from these multiple simulations and simulated for 1 µs to further explore its stability and dynamics. The results of these simulations mirror those from the tryptophan fluorescence measurements (i.e., Cav182-136 insertion depth in the bilayer), corroborate that Cav182-136 inserts in the membrane with U-shaped conformations, and show that the angle between H1 and H2 ranges from 35 to 69°, and the tilt angle of Cav182-136 is 27 ± 6°. The simulations also reveal that specific faces of H1 and H2 prefer to interact with each other and with lipid molecules, and these interactions stabilize the U-shaped conformation.

Firefighter noise exposure during training activities and general equipment use.

Multiple noise measurements were taken on 6 types of fire station equipment and 15 types of emergency response vehicle-related equipment used by firefighters during routine and emergency operations at 10 fire stations. Five of the six types of fire station equipment, when measured at a distance of one meter and ear level, emitted noise equal to or greater than 85 dBA, including lawn maintenance equipment, snow blowers, compressors, and emergency alarms. Thirteen of 15 types of equipment located on the fire engines emitted noise levels equal to or greater than 85 dBA, including fans, saws, alarms, and extrication equipment. In addition, noise measurements were taken during fire engine operations, including the idling vehicle, vehicle sirens, and water pumps. Results indicated that idling fire-engine noise levels were below 85 dBA; however, during water pump and siren use, noise levels exceeded 85 dBA, in some instances, at different locations around the trucks where firefighters would be stationed during emergency operations. To determine if the duration and use of fire fighting equipment was sufficient to result in overexposures to noise during routine training activities, 93 firefighter personal noise dosimetry samples were taken during 10 firefighter training activities. Two training activities per sampling day were monitored during each sampling event, for a mean exposure time of 70 min per day. The noise dosimetry samples were grouped based on job description to compare noise exposures between the different categories of job tasks commonly associated with fire fighting. The three job categories were interior, exterior, and engineering. Mean personal dosimetry results indicated that the average noise exposure was 78 dBA during the training activities that lasted 70 min on average. There was no significant difference in noise exposure between each of the three job categories. Although firefighters routinely use equipment and emergency response vehicles that can produce hazardous levels of noise, this study showed that the average noise levels experienced by firefighters was below generally accepted guidelines.