A Survey of Clinical Immersion Experiences in Biomedical Engineering.

Immersion in clinical environments is generally believed to be a valuable experiential learning opportunity for students in biomedical engineering, both at the undergraduate and the graduate level. Immersion is believed to foster an understanding of medical culture, clinical operations, interprofessional collaboration, and oftentimes allows students to either identify unmet clinical needs. The National Institutes of Health supports efforts through grants to incorporate these clinical immersion programs into biomedical engineering curricula, and this has potentially facilitated an expansion of these programs across the United States. Unknown is how common clinical immersion experiences are in biomedical engineering programs, in general how these are organized and executed, and their goals. We conducted a survey of biomedical engineering programs to learn how many programs offer clinical immersion experiences, over what timeframe and in what formats, and what is known about their goals and learning outcomes. We present here the results of that survey which includes 52 clinical immersion courses and programs, 14 of which either are or were previously funded by the NIH. Each of these courses or programs engages, on average, about 27 students per year, but range in size from 2 to 160. The duration of the immersion experience likewise varies greatly from 3 to 400 h. The objectives of these programs are mostly to identify problems, develop engineering solutions to problems, or to learn clinical procedures. Despite the impressive breadth of experiences revealed by this survey, we still know relatively little about their impact on student learning, motivation, identity, or career path. Desired outcomes and assessment strategies must be better aligned with the structure of the clinical immersion experiences themselves if we are to determine if they are effective in meeting those outcomes, including those of professional preparation.

Nutrients influence the dynamics of Klebsiella pneumoniae carbapenemase producing enterobacterales in transplanted hospital sinks.

Antimicrobial resistance has been recognized as a threat to human health. The role of hospital sinks acting as a reservoir for some of the most concerning antibiotic resistant organisms, carbapenemase producing Enterobacterales (CPE) is evident but not well understood. Strategies to prevent establishment, interventions to eliminate these reservoirs and factors which drive persistence of CPE are not well established. We use a uniquely designed sink lab to transplant CPE colonized hospital sink plumbing with an aim to understand CPE dynamics in a controlled setting, notably exploiting both molecular and culture techniques. After ex situ installation the CPE population in the sink plumbing drop from previously detectable to undetectable levels. The addition of nutrients is followed by a quick rebound in CPE detection in the sinks after as many as 37 days. We did not however detect a significant shift in microbial community structure or the overall resistance gene carriage in longitudinal samples from a subset of these transplanted sinks using whole shotgun metagenomic sequencing. Comparing nutrient types in a benchtop culture study model, protein rich nutrients appear to be the most supportive for CPE growth and biofilm formation ability. The role of nutrients exposure is determining factor for maintaining a high bioburden of CPE in the sink drains and P-traps. Therefore, limiting nutrient disposal into sinks has reasonable potential with regard to decreasing the CPE wastewater burden, especially in hospitals seeking to control an environmental reservoir.

S-nitrosylation of cytoskeletal proteins.

Nitric oxide has pronounced effects on cellular functions normally associated with the cytoskeleton, including cell motility, shape, contraction, and mitosis. Protein S-nitrosylation, the covalent addition of a NO group to a cysteine sulfur, is a signaling pathway for nitric oxide that acts in parallel to cyclic guanosine monophosphate (cGMP), but is poorly studied compared to the latter. There is growing evidence that S-nitrosylation of cytoskeletal proteins selectively alters their function. We review that evidence, and find that S-nitrosylation of cytoskeletal targets has complementary but distinct effects to cyclic-GMP in motile and contractile cells-promoting cell migration, and biasing muscle contraction toward relaxation. However, the effects of S-nitrosylation on a host of cytoskeletal proteins and functions remains to be explored.

Design and implementation of a student-taught course on research in regenerative medicine.

In the Undergraduate School of Engineering and Applied Sciences (SEAS) at the University of Virginia (UVa), there are few opportunities for undergraduate students to teach, let alone develop, an introductory course for their major. As two undergraduate engineering students (D. N. Tavakol and C. J. Broshkevitch), we were among the first students to take advantage of a new initiative at UVa SEAS to offer student-led courses. As part of this new program, we designed a 1000-level, 1-credit, pass-fail course entitled Introduction to Research in Regenerative Medicine. During a student's first year at the University, opportunities to build research skills and gain exposure to topics within the field of the biomedical sciences are relatively rare, so, to fill this gap, we focused our course on teaching primarily freshman undergraduate students how to synthesize and contextualize scientific literature, covering both basic science and clinical applications. At the end of the course, students self-reported increased confidence in reading and discussing scientific papers and review articles. The critical impact of this course lies not only in an early introduction to the popularized field of regenerative medicine, but also encouragement for younger students to participate in research early on and to appreciate the value of interdisciplinary interactions. The teaching model can be extended for implementation of student-taught introductory courses across diverse undergraduate major tracks at an institution.

Spread from the Sink to the Patient: In Situ Study Using Green Fluorescent Protein (GFP)-Expressing Escherichia coli To Model Bacterial Dispersion from Hand-Washing Sink-Trap Reservoirs.

There have been an increasing number of reports implicating Gammaproteobacteria as often carrying genes of drug resistance from colonized sink traps to vulnerable hospitalized patients. However, the mechanism of transmission from the wastewater of the sink P-trap to patients remains poorly understood. Herein we report the use of a designated hand-washing sink lab gallery to model dispersion of green fluorescent protein (GFP)-expressing Escherichia coli from sink wastewater to the surrounding environment. We found no dispersion of GFP-expressing E. coli directly from the P-trap to the sink basin or surrounding countertop with coincident water flow from a faucet. However, when the GFP-expressing E. coli cells were allowed to mature in the P-trap under conditions similar to those in a hospital environment, a GFP-expressing E. coli-containing putative biofilm extended upward over 7 days to reach the strainer. This subsequently resulted in droplet dispersion to the surrounding areas (<30 in.) during faucet operation. We also demonstrated that P-trap colonization could occur by retrograde transmission along a common pipe. We postulate that the organisms mobilize up to the strainer from the P-trap, resulting in droplet dispersion rather than dispersion directly from the P-trap. This work helps to further define the mode of transmission of bacteria from a P-trap reservoir to a vulnerable hospitalized patient.IMPORTANCE Many recent reports demonstrate that sink drain pipes become colonized with highly consequential multidrug-resistant bacteria, which then results in hospital-acquired infections. However, the mechanism of dispersal of bacteria from the sink to patients has not been fully elucidated. Through establishment of a unique sink gallery, this work found that a staged mode of transmission involving biofilm growth from the lower pipe to the sink strainer and subsequent splatter to the bowl and surrounding area occurs rather than splatter directly from the water in the lower pipe. We have also demonstrated that bacterial transmission can occur via connections in wastewater plumbing to neighboring sinks. This work helps to more clearly define the mechanism and risk of transmission from a wastewater source to hospitalized patients in a world with increasingly antibiotic-resistant bacteria that can thrive in wastewater environments and cause infections in vulnerable patients.

Direct measurement of cortical force generation and polarization in a living parasite.

Apicomplexa is a large phylum of intracellular parasites that are notable for the diseases they cause, including toxoplasmosis, malaria, and cryptosporidiosis. A conserved motile system is critical to their life cycles and drives directional gliding motility between cells, as well as invasion of and egress from host cells. However, our understanding of this system is limited by a lack of measurements of the forces driving parasite motion. We used a laser trap to measure the function of the motility apparatus of living Toxoplasma gondii by adhering a microsphere to the surface of an immobilized parasite. Motion of the microsphere reflected underlying forces exerted by the motile apparatus. We found that force generated at the parasite surface begins with no preferential directionality but becomes directed toward the rear of the cell after a period of time. The transition from nondirectional to directional force generation occurs on spatial intervals consistent with the lateral periodicity of structures associated with the membrane pellicle and is influenced by the kinetics of actin filament polymerization and cytoplasmic calcium. A lysine methyltransferase regulates both the magnitude and polarization of the force. Our work provides a novel means to dissect the motile mechanisms of these pathogens.

Loop 2 of myosin is a force-dependent inhibitor of the rigor bond.

Myosin's actin-binding loop (loop 2) carries a charge opposite to that of its binding site on actin and is thought to play an important role in ionic interactions between the two molecules during the initial binding step. However, no subsequent role has been identified for loop 2 in actin-myosin binding. We used an optical trap to measure bond formation and bond rupture between actin and rigor heavy meromyosin when loaded perpendicular to the filament axis. We studied HMM with intact or proteolytically cleaved loop 2 at low and physiologic ionic strength. Here we show that the presence of intact loop 2 allows actomyosin bonds to form quickly and that they do so in a short-lived bound state. Increasing tensile load causes the transition to a long-lived state-the distinguishing behavior of a catch bond. When loop 2 was cleaved catch bond behavior was abrogated leaving only a long-lived state. These data suggest that in addition to its role in locating binding sites on actin, loop 2 is also a force-dependent inhibitor of the long-lived actomyosin complex. This may be important for reducing the duty ratio and increasing the shortening velocity of actomyosin at low forces.

Design, development, and evaluation of a novel retraction device for gallbladder extraction during laparoscopic cholecystectomy.

BACKGROUND: A source of frustration during laparoscopic cholecystectomy involves extraction of the gallbladder through port sites smaller than the gallbladder itself. We describe the development and testing of a novel device for the safe, minimal enlargement of laparoscopic port sites to extract large, stone-filled gallbladders from the abdomen. METHODS: The study device consists of a handle with a retraction tongue to shield the specimen and a guide for a scalpel to incise the fascia within the incision. Patients enrolled underwent laparoscopic cholecystectomy. Gallbladder extraction was attempted. If standard measures failed, the device was implemented. Extraction time and device utility scores were recorded for each patient. Patients returned 3-4 weeks postoperatively for assessment of pain level, cosmetic effect, and presence of infectious complications. RESULTS: Twenty (51 %) of 39 patients required the device. Average extraction time for the first eight patients was 120 s. After interim analysis, an improved device was used in 12 patients and average extraction time was 24 s. There were no adverse events. Postoperative pain ratings and incision cosmesis were comparable between patients with and without use of the device. CONCLUSION: The study device enables safe and rapid extraction of impacted gallbladders through the abdominal wall.

Laser trap measurements of flagellar membrane motility.

In addition to swimming motility, which is driven by propagation of bends along the flagellum, the unicellular green alga Chlamydomonas exhibits an unusual and alternative form of whole cell locomotion, called gliding motility. In gliding motility, a large flagellar membrane glycoprotein mediates flagellar membrane adhesion to solid substrates. This in turn activates a transmembrane signaling system that initiates the movement of a cross-linked cluster of glycoproteins within the plane of the flagellar membrane by activating and/or recruiting isoforms of the motor proteins kinesin and dynein. Flagellar membrane motility can be visualized through the bidirectional movement of microspheres adherent to the flagellar surface. This microsphere motility offers a unique, noninvasive experimental system for measuring the in vivo dynamics and regulation of microtubule-dependent molecular motors by using a laser trap transducer to capture and manipulate microspheres as they move along the flagellar surface. Detailed procedures for conducting such analyses are provided.

A High-Throughput Technique Reveals the Load- and Site Density-Dependent Kinetics of E-Selectin.

The kinetics of bond rupture between receptors and ligand are critically dependent on applied mechanical force. Force spectroscopy of single receptor-ligand pairs to measure kinetics is a laborious and time-consuming process that is generally performed using individual force probes and making one measurement at a time when typically hundreds of measurements are needed. A high-throughput approach is thus desirable. We report here a magnetic bond puller that provides high-throughput measurements of single receptor-ligand bond kinetics. Electromagnets are used to apply pN tensile and compressive forces to receptor-coated magnetic microspheres while monitoring their contact with a ligand-coated surface. Bond lifetimes and the probability of forming a bond are measured via videomicroscopy, and the data are used to determine the load dependent rates of bond rupture and bond formation. The approach is simple, customizable, relatively inexpensive, and can make dozens of kinetic measurements simultaneously. We used the device to investigate how compressive and tensile forces affect the rates of formation and rupture, respectively, of bonds between E-selectin and sialyl Lewisa (sLea), a sugar on P-selectin glycoprotein ligand-1 to which selectins bind. We confirmed earlier findings of a load-dependent rate of bond formation between these two molecules, and that they form a catch-slip bond like other selectin family members. We also make the novel observation of an "ideal" bond in a highly multivalent system of this receptor-ligand pair.

Force spectroscopy reveals multiple "closed states" of the muscle thin filament.

Tropomyosin (Tm) plays a critical role in regulating the contraction of striated muscle. The three-state model of activation posits that Tm exists in three positions on the thin filament: "blocked" in the absence of calcium when myosin cannot bind, "closed" when calcium binds troponin and Tm partially covers the myosin binding site, and "open" after myosin binding forces Tm completely off neighboring sites. However, we recently showed that actin filaments decorated with phosphorylated Tm are driven by myosin with greater force than bare actin filaments. This result cannot be explained by simple steric hindrance and suggests that Tm may have additional effects on actin-myosin interactions. We therefore tested the hypothesis that Tm and its phosphorylation state affect the rate at which single actin-myosin bonds form and rupture. Using a laser trap, we measured the time necessary for the first bond to form between actin and rigor heavy meromyosin and the load-dependent durations of those bonds. Measurements were repeated in the presence of subsaturating myosin-S1 to force Tm from the closed to the open state. Maximum bond lifetimes increased in the open state, but only when Tm was phosphorylated. While the frequency with which bonds formed was extremely low in the closed state, when a bond did form it took significantly less time to do so than with bare actin. These data suggest there are at least two closed states of the thin filament, and that Tm provides additional points of contact for myosin.

4-Azetidinyl-1-heteroatom linked cyclohexane antagonists of CCR2: patent evaluation.

This application discloses a series of di- and tri-substituted cyclohexanes as CCR2 receptor antagonists which are stated to be useful in treating inflammation and autoimmune diseases, such as type 2 diabetes and asthma. Although receptor binding of the compounds to CCR2 is demonstrated, there are no data to support the idea that these molecules are functional antagonists.

Thermal denaturation of tilapia myosin and its subunits as affected by constantly increasing temperature.

Purified tilapia myosin was digested with α-chymotrypsin and purified to obtain heavy meromyosin (HMM) and light meromyosin (LMM). Biochemical properties of tilapia myosin, HMM, and LMM were characterized. Surface hydrophobicity (S(o) ) showed an increase for myosin and HMM between 30 and 40 °C and reached a plateau at 70 °C. LMM, in a small magnitude, also showed a continuous increase to 70 °C. Total sulfhydryl content (TSH) demonstrated that the SH residue content of HMM was nearly double that of LMM. Surface reactive sulfhydryl groups (SRSH) for myosin and HMM were relatively unchanged from 10 to 30 °C but increased from 30 to 50 °C. The exposure of buried hydrophobic and sulfhydryl groups of myosin and HMM increased as the myosin and HMM were constantly heated. However, the TSH and SRSH results indicated that the stability of LMM was likely due to its α-helix conformation. Reducing and nonreducing sodium dodecylsulfate-polyacryamide gel electrophoresis helped to understand the role of disulfide bonds in thermal aggregation of tilapia myosin, HMM, and LMM.

The Effects of Load on E-Selectin Bond Rupture and Bond Formation.

Molecular dissociation rates have long been known to be sensitive to applied force. We use a laser trap to provide evidence that rates of association may also be force-dependent. We use the thermal fluctuation assay to study single bonds between E-selectin and sialyl Lewis(a) (sLe(a)), the sugar on PSGL-1 to which the three selectins bind. Briefly, an E-selectin-coated bead is held in a laser trap and pressed with various compressive loads against the vertical surface of a bead coated with sLe(a). The time it takes for a bond to form is used to calculate a specific two-dimensional on-rate, kono. We observe an increase in kono with increasing compressive force, providing single molecule evidence that on-rate, in addition to off-rate, is influenced by load. By measuring bond lifetimes at known tensile loads, we show that E-selectin, like its family members L- and P-selectin, is capable of forming catch bonds. Our data support a reverse Bell model, in which compressive forces lower the activation energy for binding. Load-dependent on-rates may be a general feature of all intermolecular bonds.

The viscoelastic properties of microvilli are dependent upon the cell-surface molecule.

We studied at nanometer resolution the viscoelastic properties of microvilli and tethers pulled from myelogenous cells via P-selectin glycoprotein ligand 1 (PSGL-1) and found that in contrast to pure membrane tethers, the viscoelastic properties of microvillus deformations are dependent upon the cell-surface molecule through which load is applied. A laser trap and polymer bead coated with anti-PSGL-1 (KPL-1) were used to apply step loads to microvilli. The lengthening of the microvillus in response to the induced step loads was fitted with a viscoelastic model. The quasi-steady state force on the microvillus at any given length was approximately fourfold lower in cells treated with cytochalasin D or when pulled with concanavalin A-coated rather than KPL-1-coated beads. These data suggest that associations between PSGL-1 and the underlying actin cytoskeleton significantly affect the early stages of leukocyte deformation under flow.

Direct regulation of striated muscle myosins by nitric oxide and endogenous nitrosothiols.

BACKGROUND: Nitric oxide (NO) has long been recognized to affect muscle contraction, both through activation of guanylyl cyclase and through modification of cysteines in proteins to yield S-nitrosothiols. While NO affects the contractile apparatus directly, the identities of the target myofibrillar proteins remain unknown. Here we report that nitrogen oxides directly regulate striated muscle myosins. PRINCIPAL FINDINGS: Exposure of skeletal and cardiac myosins to physiological concentrations of nitrogen oxides, including the endogenous nitrosothiol S-nitroso-L-cysteine, reduced the velocity of actin filaments over myosin in a dose-dependent and oxygen-dependent manner, caused a doubling of force as measured in a laser trap transducer, and caused S-nitrosylation of cysteines in the myosin heavy chain. These biomechanical effects were not observed in response to S-nitroso-D-cysteine, demonstrating specificity for the naturally occurring isomer. Both myosin heavy chain isoforms in rats and cardiac myosin heavy chain from human were S-nitrosylated in vivo. SIGNIFICANCE: These data show that nitrosylation signaling acts as a molecular "gear shift" for myosin--an altogether novel mechanism by which striated muscle and cellular biomechanics may be regulated.