In early summer, seals foraged at deeper depths resulting in lower feeding rates and mass gain. As sea ice extent decreased throughout the summer, seals foraged at shallower depths and benefited from more efficient energy intake. Changes in diving depth were not due to seasonal shifts in seal diets or horizontal space use and instead may reflect a change in the vertical distribution of prey. Correspondence between the timing of seal shallowing and the resource pulse was variable from year to year and could not be readily explained by our existing understanding of the ocean and ice dynamics. Phytoplankton advection occurred faster than ice break-out, and seal dive shallowing occurred substantially earlier than local break-out. While there remains much to be learned about the marine ecosystem, it appears that an increase in prey abundance and accessibility via shallower distributions during the resource pulse could synchronize life-history phenology across trophic levels in this high-latitude ecosystem.Seasonal resource pulses can have enormous impacts on species interactions. In marine ecosystems, air-breathing predators often drive their prey to deeper waters. However, it is unclear how ephemeral resource pulses such as near-surface phytoplankton blooms alter the vertical trade-off between predation avoidance and resource availability in consumers, and how these changes cascade to the diving behaviour of top predators. We integrated data on Weddell seal diving behaviour, diet stable isotopes, feeding success and mass gain to examine shifts in vertical foraging throughout ice break-out and the resulting phytoplankton bloom each year. We also tested hypotheses about the likely location of phytoplankton bloom origination (advected or produced  The A fundamental characteristic of ocean ecosystems is that they are vertically stratified, with depth gradients of temperature, light, nutrients and oxygen that constrain biological processes . PrimaryLeptonychotes weddellii) to infer temporal variation in the vertical distribution of lower trophic levels throughout the austral summer. We hypothesized that during spring, when resource stratification was weak, intermediate trophic levels (zooplankton and fish) would have deeper distributions as compared to summer, when sea ice break-out triggers a strong resource pulse of phytoplankton at the ocean surface. This resource pulse could increase the efficiency of energy transfer to air-breathing top predators, which must return to the surface for oxygen after foraging underwater.Our aim was to understand how sea ice break-out and resource pulses influence top predators' use of vertical space at a study site in the southern Ross Sea, which is in the most productive sector of the Southern Ocean in Antarctica ,38. We i. 2(a)We used archival biologgers to record the diving and foraging behaviour of 59 adult female Weddell seals in Erebus Bay, Antarctica in austral summers 2013–2016 . All study animals were between the ages of 10 and 20 years and had given birth at least once prior to inclusion in this study . Each in(b)in situ (i.e. autochthonous) production [−1 [−1 [−1 [Direct measurements of phytoplankton bloom timing were not available throughout our study site and the timing of ice break-out and the resulting phytoplankton bloom are generally not well understood . Becauseoduction , we consoduction  .(c)n = 2941 seal-days; For each dive, we calculated the maximum depth, total duration, bottom phase duration (defined as 80% of maximum dive depth) and number of bottom wiggles (inflection points in the depth profile of the bottom phase) using the IKNOS toolbox in MATLAB  ,48. We i(d)13C and nitrogen δ15N stable isotope values in sequential segments of whiskers grown during the biologger deployment period. We incorporated average whisker δ13C and δ15N values for each seal with stable isotope values of five published prey groups  in whiskers with the individual as a random effect. Full methodological details are provided in the electronic supplementary material.In austral summers 2013 and 2014, we plucked one whisker from each of nine seals during tag attachment and plucked the regrown whisker during tag removal. To infer seal diet across the summer period, we analysed carbon δable S1, ) into a age SIAR . We thenn, Sadou . Using l. 3(a)shallowestdives = 1.64 (± SE = 0.18) × Dateshallowestdives + 97.25, n = 4 years, R2 = 0.95, one-tailed p= 0.015).We analysed 138 506 dives from 59 Weddell seals to characterize the diving depth and foraging effort of each seal across the austral summer over 4 years The depth of benthic dives suggested that seals foraged in the same area throughout the austral summer, which aligns with findings from a prior mark–recapture study . Benthic(c)15N and δ13C isotope values showed no consistent trends over time, suggesting that seals did not shift their diet across summer (Pleurogramma antarcticum and Trematomus newnesi (diet proportion 72% (95% CI: 34%–86%) with smaller amounts of Pagothenia borchgrevinki and other spp.Trematomus   WiggleRate = −0.0043 (± SE = 0.0002) × MeanDiveDepth + 3.2589, R2 = 0.83, p < 0.0001, n = 76 days). Feeding rates increased by 16% from the first 10 deep days to the shallow period and then decreased by 11% in the last 10 deep days  and depth wiggles   (electroeep days . In addieep days . The cum(e)As expected, sea ice break-out progressed anticlockwise around Ross Island from the Northeast to the Southwest over approximately 40 days, although ice break-out progression showed marked inter-annual variation . We noteb). One possible explanation is that the Cape Royds current around Ross Island (a) may be notably faster than the currents near the seal colony that we used in our estimates [figure 5b). Additionally, seal foraging depth was better explained by the date of advected phytoplankton arrival than calendar date  for all but one data location, Northeast (figure 5b), that showed extremely limited inter-annual variability in ice break-out dates . However, the seal shallowing preceded phytoplankton advection by up to 20 days across the four study years, suggesting that phytoplankton advection arrives earlier than estimated from current velocities and ice break-out dates.We did not find consistent support for the hypothesis that seal shallowing is triggered by ice break-out or arrival of advected phytoplankton from a particular source. The period of shallowest diving in each year estimated by the GAMMs was tightly correlated with the temporal arrival of the resource pulse for some seasons (e.g. AS13) but not others (e.g. AS15) and from some data locations (e.g. West) but not others (e.g. Northeast) , bottom.figure 5s Island a may be stimates . Interes. 4We show that in Antarctic summer, ice break-out and the resulting resource pulse are associated with increased energy transfer to top predators. Foraging depth of Weddell seals halved and remained shallow for three weeks in mid-summer, suggesting that zooplankton and fishes may have shallowed , despitein situ? How does chlorophyll concentration change when ice cover drops below 50% then re-freezes ? Do advection rates around Ross Island exceed those directly measured in Erebus Bay [in situ phytoplankton production, and from which advective source(s)? How long does the resource pulse on the west side of Ross Island last? Is the spatial resolution of satellite-derived ice data (25 × 25 km) sufficient to describe this highly dynamic ecosystem? Alternatively, the mismatch could be due to ecological processes, such as seal prey avoiding peak resource aggregations to optimize risk-reward trade-offs, which in turn might contribute to the observed mismatch between seal shallowing and the arrival of advected phytoplankton [We found substantial variation in timing between seal shallowing and the resource pulse that cannot be explained by our existing understanding of the complex marine ecosystem . Severalebus Bay ? What prplankton .in situ by instrumenting seals with satellite-linked conductivity–temperature–depth–fluorescence tags [Regardless of the phytoplankton source and precise arrival timing, summer is clearly a season of opportunity for these top predators with important implications for energetics and behaviour that cascades up the food chain. In the future, these complex dynamics could be studied nce tags .Pagodroma nivea, and Antarctic petrels, Thalassoica antarctica, consume mesopelagic fishes [Pygoscelis adeliae, which rarely dive below 70 m [P. antarcticum in their diet eightfold during the phytoplankton bloom [Alle alle, also consume zooplankton which normally exist deeper than their diving abilities [Previous studies have shown that zooplankton and silverfish exhibit seasonal differences in depth distributions ,63 and tc fishes ,68 that c fishes . Similarlow 70 m , increasbilities .The short-lived absence of resource limitation and shallowing of intermediate trophic level species also appears to synchronize critical life-history events across multiple trophic levels. Krill, copepods and amphipods all spawn synchronously in surface waters to exploit the brief resource pulse ,72–74 de. 5In summary, ice break-out and resource pulses in the highly seasonal Southern Ocean appear to shift vertical prey distributions from deeper to shallower waters. Vertical redistributions of food chains are analogous to the horizontal migrations documented in other ecosystems ,80 and aClick here for additional data file.


Level of Difficulty: Beginner.)A pacemaker generator change for a frail woman was performed with a direct oral anticoagulant. After the procedure, a hematoma was exposed. Stopping direct oral anticoagulants in frail patients should be considered before the procedure. (  Her prescriptions included direct oral anticoagulants (DOACs) because of atrial fibrillation. Surgery for a pacemaker generator change was performed without interruption of DOACs. Three days after the operation, a hematoma at the surgical site increased. The surgical site was tightly packed with sterilized gauze to compress the hematoma. After several hours, the hematoma was exposed from the surgical site (An 83-year-old frail woman (body mass index: 18.7 kg/mcal site . Re-opercal site .Figure 1A recent trial reported that bleeding events after a pacemaker implantation or a pacemaker generator change were not different with or without DOACs . Accordi


Mycobacterium microti, a member of the M. tuberculosis complex, have been increasingly reported in humans and in domestic and free-ranging wild animals. At postmortem examination, infected animals may display histopathologic lesions indistinguishable from those caused by M. bovis or M. caprae, potentially leading to misidentification of bovine tuberculosis. We report 3 cases of M. microti infections in free-ranging red deer (Cervus elaphus) from western Austria and southern Germany. One diseased animal displayed severe pyogranulomatous pleuropneumonia and multifocal granulomas on the surface of the pericardium. Two other animals showed alterations of the lungs and associated lymph nodes compatible with parasitic infestation. Results of the phylogenetic analysis including multiple animal strains from the study area showed independent infection events, but no host-adapted genotype. Personnel involved in bovine tuberculosis–monitoring programs should be aware of the fastidious nature of M. microti, its pathogenicity in wildlife, and zoonotic potential.Infections with  Mycobacterium tuberculosis complex (MTBC). One animal-adapted sublineage within the complex, M. microti, was first isolated from field voles (Microtus agrestis) that had granulomatous tuberculosis-like lesions (Myodes glareolus), wood mice (Apodemus sylvaticus), and shrews (Sorex araneus), are considered to be primary reservoirs for M. microti, several other hosts have been identified, including domestic and wild animals  is one of the most prevalent zoonotic diseases worldwide and remains the leading cause of death from a single infectious agent  that have been deleted from certain species and that may confer differences in phenotype, host range, and virulence  6, locus tag Rv3875, and CFP-10, a culture filtrate protein encoded by the neighboring gene Rv3874 . The suspension was decontaminated by using 1% N-acetyl-L-cystein-NaOH solution and neutralized with 20 mL phosphate buffer (pH 6.8). We centrifuged the solution for 20 min at 3300 × g and plated the obtained pellet on 2 growth media: Löwenstein-Jensen medium with glycerin and PACT  and Stonebrink medium with pyruvate and PACT . Cultures of lung and lymph node specimens on solid Stonebrink medium yielded growth of suspicious mycobacterial colonies after 4–6 wk of incubation at 37°C. The isolates were identified by using GenoType MTBC reversed line blotting . For histologic examination, we fixed tissue samples in 10% nonbuffered formalin for ≈48 h, then trimmed and routinely embedded them in paraffin wax. Sections of 3–4 μm were prepared and stained with hematoxylin and eosin (HE) and Ziehl Neelsen (ZN) or modified ZN (We isolated mycobacteria following a standardized protocol as described elsewhere (M. tuberculosis typing (plus (https://www.miru-vntrplus.org/MIRU) server and exported it using MEGAX version 10.11 (DVR spoligotyping (direct variable repeat spacer oligonucleotide typing) was performed using a commercial microarray system  with integrated data analysis as described elsewhere . The same signature is also registered in the international spoligotyping database SpolDB4 as ST 539 and is characteristic of M. microti , integrated for further comparative genotyping, exhibited the same spoligotype signature, SB0118, characterized by the presence of spacers 37–38 ( microti . M. microti–positive stag killed in the alpine region in western Austria manifesting clinical signs of tuberculosis. Tuberculosis caused by M. caprae has been described several times in domestic animals and wildlife in this area (M. caprae for cattle in Austria, Germany, Italy, and Switzerland (M. microti has been isolated only once in Austria, from a red fox without visible lesions (M. microti–positive stag in case 1 was found. Clear evidence proving transmission of M. microti between individual animals of the same species or between species is missing. Human-to-human transmission regarding this pathogen has previously been investigated and the possibility cannot be dismissed (M. microti infections might often occur through oral ingestion and that direct transmission between animals is less likely (Case 1 in this study reported an  lesions . This foM. bovis prevalence decreased substantially after control of TB-infected possums, suggesting that wild deer may be spillover hosts that can be regularly reinfected by possums (M. microti seems to vary greatly both between host species and within the same species. Most visible lesions compatible with TB diagnosed in M. bovis– and M. caprae–infected red deer are located in the lymph nodes, particularly the medial retropharyngeal and mesenteric lymph nodes, suggesting oral rather than aerosol transmission. The respiratory tract, including the lungs and associated lymph nodes, seems to be affected by MTBC in a secondary phase of the infection, which is also likely for M. microti infections in red deer (The presence of MTBC in wild red deer seems to depend on multiple factors, such as population density, TB prevalence in nearby cattle or other wildlife species, and the morphologic structure of the habitat. Observations made from infected wild deer in New Zealand showed that M. microti lineages. The molecular background of M. microti seems highly conserved, and traceback analyses are delicate. The 11 isolates included in this study were collected over an 8-year period from regions bordering Austria, Germany, and Switzerland (M. microti genotypes was observed (M. microti genetic lineages is unlikely. MLVA has been successfully used worldwide as an ancillary tool for animal TB epidemiologic surveillance and outbreak investigations in multihost scenarios (M. microti as well (DVR spoligotyping analysis is a popular technique worldwide for molecular characterization of MTBC of animal origin with excellent resolution and cost-benefit ratio (tzerland . These sobserved , 5, whicM. microti and suggest that it might be an opportunistic pathogen rather than a host-adapted MTBC member, such as M. tuberculosis. In the past, similar to Mycobacterium bovis BCG strains, certain vole strains of M. microti have been used to develop live attenuated human TB vaccines in the United Kingdom and the former Czechoslovakia (M. microti strains are likely to exist (These findings highlight the wide host range of M. microti suggest that the natural foci and reservoirs of this animal-adapted lineage are small rodents and that the pathogen subsequently infects predators, such as cats or foxes, through ingestion; the mode of infection for herbivores, such as red deer or alpacas, remains ill defined. The lesions we observed in the lungs of the deer in case 1, however, provide strong evidence of bacterial shedding, which might occur either as a consequence of inflammatory processes that break into the airways or by infection of alveolar macrophages that are part of the exudate (M. microti, an MTBC agent.Some seemingly feasible theories about the natural transmission route of M. bovis or M. caprae is present is of primary importance for veterinary and public health authorities. To date, molecular testing of cultured bacteria remains the preferred method for differentiating MTBC species. Because of the fastidious nature of M. microti and the extremely slow growth rate of specific animal strains, this differentiation can take several months or remain incomplete in cases where the mycobacterium cannot be cultured. On the basis of published data, it can be assumed that a large proportion of M. microti infections remain culture negative, even if the incubation time is prolonged to 18 weeks (Because of the potential zoonotic risk related to MTBC members, rapidly and accurately identifying the mycobacterial species causing disease in animals hunted for human consumption is crucial. Once MTBC is detected, determining whether M. microti in red deer. Given the absence of typical pulmonary lesions in some cases, such as in the red deer in cases 2 and 3, diagnostic pathologists must remain highly alert. Incidence of this pathogen should be monitored within the framework of bTB surveillance programs and suspicious cases differentiated from classical bTB caused by M. bovis and M. caprae. The actual occurrence of M. microti in wildlife may be underestimated, and personnel involved in bTB monitoring programs should be aware of its pathogenicity and zoonotic potential. Therefore, molecular methods to differentiate this member of the MTBC should be included in the diagnostic workflow of bTB reference laboratories.These findings show the morphologic versatility of lesions induced by Mycobacterium microti identified in Germany, Austria, and Switzerland.Additional information on strains of


Introduction: The successful surgical treatment of paediatric spinal deformity relies on robust anchors to achieve correction. Uniaxial pedicle screws are designed with articulation between the screw head and screw shaft, thus reducing the risk of anchor failure whilst permitting corrective manoeuvres. The purpose of this study was to describe the incidence, nature, and chronology of implant-related complications in pediatric spinal deformity treated with uniaxial pedicle screws.Methods: A retrospective radiographic analysis was carried out on paediatric patients treated for spinal deformity with more than two years of follow-up. Each was treated with posterior instrumented spinal fusion (PISF) using a uniaxial pedicle screw system by a single surgeon at a single institution. Surgical records, post-operative radiographs, and follow-up documentation were scrutinised for details of the implants used, implant failure, and revision procedures.Results: Three hundred and eighty-nine eligible patients with a mean follow-up of 3.3 years were identified. The mean anchor density was 1.7. Seven implant complications were observed. Early complications (<12 months) occurred in four cases and late (>12 months) in three cases. None of the early complications were associated with non-union. Two early and two late complications required revision surgery to manage implant failure and non-union. Patients who underwent fusion across the lumbosacral junction showed a higher than expected rate of implant-related complication (P=0.02).Conclusion: This study shows that there is a rate of implant-related failure of 1.8% after PISF with uniaxial implants in pediatric spinal deformities. There is a distinction between early and late implant-related complications, with early failure being due to loss of construct integrity, whereas late failure is due to pseudarthrosis and construct fatigue. The surgical treatment of paediatric spinal deformity continues to evolve as the understanding of the precise shape and biomechanics of the deformity improves and implants become more versatile -3. NonetThe most fragile part of the spine-implant construct is the anchor points to the vertebrae. Anchor design has evolved to minimise the risk of failure, beginning with wires, progressing through hooks, and culminating in pedicle screws ,7. EarlyThe polyaxial screw was created to resolve these drawbacks. These devices are designed with articulation between the screw head and screw shaft, permitting the screw head to approach the rod (in addition to bringing the rod to the screw), facilitating rod seating, and reducing the strain on the screw-bone interface ,9. UnforA compromise between fixed angle and polyaxial designs is the uniplanar screw. This design accommodates variations of the pedicle screws relative to the rod only in the sagittal plane. This degree of freedom allows for adjustment in the sagittal plane thus preventing the destruction of the bone-implant interface through screw toggling ,10.The implant system used in this study  takes advantage of this technology and is one that is unique in the design of its rod-screw interface. The screw head is shaped with a convex saddle at the bottom of the implant in common with other systems, however, rather than a set screw being used to compress the rod into the head, the rod is compressed into the screw with a handle then an inverted U-shaped clip (the ‘clip’) is placed and seated under a lip on either side of the head. This configuration permits the rod to slide in a cranial-caudal direction with up to 20° of sagittal rotation during any correction manoeuvres. Once these movements are complete, a grub screw in the centre of the clip is tightened to prevent any additional cranial-caudal migration of the rod and secure the correction. The constructs are then reinforced with crosslinks as required.To date, no reports describe an investigation of implant-related complications for such a system. The purpose of this study was to describe the incidence, nature, and chronology of implant-related complications in paediatric spinal deformity treated with uniaxial pedicle screws.Local research ethics board approval was obtained. A retrospective radiographic analysis of the implants used, timing, and nature of any implant failures  in paediatric patients treated for spinal deformity were identified using a local hospital database which was carried out by a fellowship-trained spinal orthopaedic surgeon. Other complications were also recorded. Each patient was treated with a posterior instrumented spinal fusion (PSIF) using a uniaxial pedicle screw system  by a single surgeon at a single institution with additional hooks used when pedicle morphology prevented screw placement. Inclusion criteria comprised age less than 18 years at the time of PSIF with a minimum of two years completed follow-up. Demographics, body weight (BW), body mass index (BMI), American Society of Anaesthesiologists (ASA) grade, activities of daily living (ADL), and medical histories were recorded. Surgical records, post-operative radiographs, and follow-up documentation were scrutinised for details of any revision procedures.The data were analysed using STATA . The Mann-Whitney U test and Fisher’s exact tests were used to analyse numerical and categorical data. P-values of less than 0.05 were considered to indicate a statistically significant difference.Three hundred and eighty-nine eligible patients with a mean follow-up of 3.3 years (range 2-9.4 years) were identified, each undergoing instrumentation with the SpineVision PLUS  system. Of the 389, 75.5% were idiopathic, 4.6% congenital, 5.6% neuromuscular, and 14.1% syndromic deformities , two hooks (0.09%), one crosslink fracture 0.12%), and one-rod breakage (0.13%). This represents 1.8% of cases; three patients had an idiopathic deformity, two congenital deformities, and two neuromuscular deformities  and late (>one post-operative year), early complications occurred in four cases and late in three cases. The broken iliac screw, two slipped hooks and iliac screw pullout all occurred within 12 months of surgery, and the broken rod, broken crosslink, and pedicle screw pullout all occurred more than 12 months following surgery. The late broken rod and pedicle screw pullout were associated with non-union. None of the early complications were associated with non-union, and there were no cases of postoperative clip detachment.Two early complications required revision surgery , and two late complications required revision surgery to manage implant failure and non-union . Patients who underwent fusion across the lumbosacral junction showed a higher than expected implant-related complication rate (P=0.02).In this series of 389 cases of paediatric spinal deformity treated with the SpineVision PLUS system, we observed an implant-related complication in 1.8% of cases or seven out of 9,578 implants (0.07%). Of these, three were late complications associated with pseudarthrosis. The remainder were early complications associated with failure of the implant mechanisms or interfaces. Patients fused to the ilium were at a greater relative risk of encountering implant-related complications than those in whom the lowest instrumented level was more proximal . Four of the seven implant failures required revision surgery, two for non-union in addition to the revision of the failed implants.Previous reports that have included patients treated with a range of alternative implant systems have found that the rate of implant-related complications after PSIF for spinal deformity surgery in paediatric patients is rare, ranging from 0.6% to 1.1% in idiopathic cases, 1.5% in congenital scoliosis and 2.1-12.5% in neuromuscular deformity, though they are likely underreporting of these due to the methodology employed ,12. In gNo previous reports have detailed the failure of the implant mechanisms as a cause for failure. There is an important distinction to be made between early (<one year) and late (>one year) complications, a distinction made because 12 months is a reasonable time for fusion to occur in the lumbar spine . Early cThese data confirm that there is a higher risk of implant-related complications when spinal fusions are extended across the lumbosacral junction. Moreover, the rate of complication of the distal-most anchors is lower than that seen in previous studies, which have shown that the rate of loosening of the iliac screw in neuromuscular spine deformity surgery is between 1.8% and 39.2% and the incidence of iliac screw breakage between 21.4% and 23.6% ,16.There are limitations in this study in addition to its retrospective design. This study investigates a single implant system with a unique rod-anchor interface, used by a single surgeon, which may introduce a bias to the conclusions. Furthermore, although the system is uniaxial, it has a unique rod-screw interface that is not directly comparable to other systems. Lastly, one of the early failures was due to a patient failing to follow post-operative instructions which may influence conclusions. Further investigations would benefit from including alternative surgical implant systems with different rod-screw interface mechanisms, used by multiple surgeons to increase the generalisability of the conclusions.This study shows that there is a rate of implant-related failure of 1.8% after PISF with uniaxial implants in paediatric spinal deformities. It identifies that there is a distinction between early and late implant-related complications, with early failure being due to loss of construct integrity, whereas late failure is due to pseudarthrosis and construct fatigue. Similar studies should be undertaken with other implant systems to evaluate more precisely their respective mechanical characteristics.


Among the few techniques described for the treatment of coronoid fractures, osteosynthesis techniques include screw osteosynthesis from anterior to posterior (AP) or from posterior to anterior (PA) and plate osteosynthesis. The aim of this study was to test the biomechanical stability of screw osteosynthesis and plate osteosynthesis using anatomical plates in coronoid process fractures.On a total of 25 biomechanical synthetical ulnae, a coronoid shear fracture including 70% of the coronoid height was simulated. Osteosynthesis was then performed using two 2.7 mm screws from anterior, posterior and with use of three different anatomical plates of the coronoid process. For the biomechanical testing, axial load was applied to the fragment with 1000 cycles from 5 to 250 N, load to failure and load at 100 µm displacement. Displacements were measured using a point-based three-dimensional motion analysis system.Osteosynthesis using the PA-screw showed significant more displacement during cyclic loading compared with all other osteosyntheses (0.99 mm), whereas AP-screw showed the smallest displacement (0.10 mm) during cyclic loading. The PA-screw technique showed a significant lower load to failure compared to all other osteosynthesis with the highest load in AP-screw osteosynthesis. The load for 100 µm displacement was the smallest in PA-screw with a significant difference to the AP-screw and one plate osteosynthesis.Osteosynthesis of large coronoid shear fractures with two small-fragment screws from anterior allows stable fixation that is not inferior to anterior plate osteosynthesis and might be an alternative in specific fracture types. Posterior screw fixation was found as the weakest fixation method.Basic science study Injuries to the coronoid process can destabilize the elbow as it resists axial loading with varus and valgus stress . HistoriWe hypothesized, that (1) an osteosynthesis of the coronoid process by the use of either buttress plating or screw osteosynthesis can result in axial stability to allow for immediate mobilization and (2) this stability in a screw osteosynthesis is highly dependent on the direction of the inserted screw.We used 25 biomechanical synthetic ulnae  for this investigation. A custom-made plaster form was created for standardized osteotomies producing Regan-Morrey type III fractures (70% of the coronoid height). For the osteotomies, a 1 mm saw blade was used. To create a realistic fracture pattern, the distal ¼ of the fracture was broken after osteotomy of the proximal ¾.The fractures produced were divided into five groups for the different osteosynthesis: 1) screw osteosynthesis using full threaded screws from anterior to posterior (AP-screw)  in anterio-posterior and posterior-anterior orientation. Screws were inserted in a standardized fashion bicortically, perpendicular to the articular surface. Drill-holes were previously applied along defined anatomical structures on the proximal ulna but resulted in slightly different positioning of the screws in the fracture fragment. Since compression of the fracture fragments was applied using a reduction clamp, full-threaded screws were used for the screw-osteosynthesis instead of partially threaded screws.For the analysis of plate osteosynthesis, three different anatomical plate-types with specific features were used to test common biomechanical plate-constructs. Differences in plate-designs that could alter biomechanical results are the used screw-diameters (2.7 mm (Acumed), 2.5 mm (Medartis), or 2.0 mm (Zimmer-Biomet)), the use of locking screws at the proximal coronoid fracture fragment (Medartis and Zimmer-Biomet), or the positioning and variability of screw-holes. All plate osteosyntheses were fixated according to the manufactures’ instructions. The Acumed plate was used with non-locking 2.7 mm screws at the coronoid tip and monoaxial locking 2.7 mm screws for the distal fixation. The Medartis coronoid plate is used with 2.0 mm polyaxial locking screws except for the gliding holes. Since the gliding holes were not used for the osteosynthesis, we only used the locking screws in this plate. The Zimmer-Biomet plate can be used with either monoaxial locking or non-locking screws. We only used the 2.5 mm monoaxial locking screws with this plate. To standardize the number of screws used in each osteosynthesis, six screws were used for the fixation in all plate osteosyntheses. All anatomical plates used in this study allow for fixation at the coronoid base with a bracket crossing the fracture. This bracket was used in all osteosyntheses so that the plate crossed the osteotomy. To allow for ideal fit, plates were bend to guarantee bony contact. Distal screws at the shaft of the ulna were fixated bicortically in all plate osteosyntheses, whereas screws below the articular surface were fixated monocortically. Screw length was measured for every osteosynthesis and screws were used accordingly to reach the subcortical bone in monocortical screws.All ulnae were embedded with Methyl Methacrylate in a custom rig. For the biomechanical testing, we used a biaxial torsion-tension machine (Instron 8675) with a custom testing protocol. After osteosynthesis, the coronoid was placed on a metallic cylindrical tube. Although axial pressure and frictional contact on the tube restricts movement of the coronoid, sliding on the metallic cylinder during axial compression was possible so that the ulna could move towards the maximal contact area of the fracture fragment, simulating axial force of the distal humerus Fig. . DislocaTo detect differences between osteosyntheses, we performed a Welch-test between samples in load to failure and for displacements under cyclic loading. The level of significance was defined as p < 0.05. To summarize results of each biomechanical setup, means and standard deviations were used.p = 0.1946). The smallest axial displacement could be found in AP-screw. Displacements during cyclic loading are depicted in Fig. None of the osteosynthesis failed during cyclic loading. During cyclic loading, axial displacement of all plate osteosyntheses ranged between a mean of 0.11 mm and 0.20 mm (Table p = 0.046) and to the Zimmer Biomet plate . No significant difference was found between different plate osteosynthesis.The load for a dislocation of the fragment of 100 µm is depicted in Fig. p = 0.013).The mean load to failure of each osteosynthesis is depicted in Fig. In the present study, we could find a higher biomechanical stability in all available plate osteosyntheses and screw osteosynthesis from anterior for the treatment of large coronoid shear fractures compared to screw osteosynthesis from posterior. Screw osteosynthesis from posterior was significant less stable resulting in lower load to failure and a larger displacement in cyclic loading compared to anatomical plating or screw osteosynthesis from anterior.Indication for operative treatment of coronoid fractures is based on morphology, orientation of the fragment and accompanying injuries . When trOnly few studies address biomechanical properties of different osteosynthetic techniques for fixation of the fragment , 18–20. Budoff et al. tested the stability of a single compression screw, a plate osteosynthesis and the combination of both during axial loading . The comMorellato et al. performed a study investigating the stability of screw osteosynthesis from anterior with locking and non-locking non-anatomical plates in simulated anteromedial facet fractures on artificial bones . All excMorellato et al. did not reveal any significant differences between different plates (locking and non-locking) indicating the general high stability of those constructs, regardless of the use of locking or non-locking screws . The majAll plates tested in this study have the advantage of a bracket that reaches to the sublime tubercle. These implants are particular useful when fixating smaller fragments of the anteromedial facet using locking- or non-locking screws. They also allow for a stable fixation if a fracture fragment is too small to be fixated using screw osteosynthesis. In these cases, the anatomical plate works as a buttress to stabilize the coronoid process fragment. Besides considerations of the stability of the osteosynthesis, fixation of anteromedial fragments requires an additional medial approach if treated with anatomical plate osteosynthesis. Fractures of the coronoid tip on the other hand regularly occur in combination with radial head injuries and dislocation of the elbow. In these cases, a lateral approach offers the possibility of osteosynthesis of the lateral aspect of the coronoid and the radial head or the lateral ligament complex.Another technique that is used for fixation of coronoid process fractures are sutures in combination with anchors or as a lasso technique . IannuzzFor injuries to the coronoid process, the amount of compressive forces an osteosynthesis needs to withstand are difficult to predict. Compressive forces at the fracture are highly dependent on the rotation of the forearm, degree of flexion of the elbow, the contraction of muscles across the elbow and whether a varus or valgus stress is applied to the elbow , 23. WheThere are several limitations in the study. First, use of artificial bones in general does not necessarily reflect the biomechanical properties and differences between individuals with limitations in drawn conclusions, while, however, allowing for better comparability in our study. Secondly, osteotomies produced with the use of an oscillating saw are not directly comparable with realistic fracture patterns. The high standard deviations in our study reflects the high variability of each individual osteosynthesis. However, this variability will also be present in clinical practice and might therefore give a realistic impression of the variance in stability. Therefore, we believe the overall biomechanical properties in relation to the screw orientation and plate versus screw osteosynthesis are applicable to the clinical setting. Although biomechanical analysis in our study using cyclic testing, load-to-failure and load for displacement of 100 µm can be overlapping especially in cases with a displacement > 100 µm during cyclic testing, we believe the different testing scenarios can broaden the picture of the biomechanical stability of each osteosynthesis. In general, the direction of a screw osteosynthesis and whether a screw or plate osteosynthesis is used should not only be made according the present fracture type, but also the individual surgeons’ experience and accompanying injuries. Also, forces that are transmitted through the coronoid process highly depend on the position of the arm and cannot simply be reduced to axial force alone. Especially, varus forces are regularly applied during activities of daily living when lifting the elbow. Biomechanical properties of osteosynthesis could alter when applying varus or valgus forces to the coronoid process.In conclusion, screw and plate osteosynthesis can provide comparable stability during axial loading of large coronoid shear fractures dependent on the orientation of the screw osteosynthesis. Screw osteosynthesis from posterior showed larger displacements compared to the other fixation methods tested.


Organic extracts were assessed for mutagenicity using Ames test (TA98 and TA100 strains), cell viability (WST-1 and LDH assays) and genotoxicity (Comet assay) with human bronchial cells (BEAS-2B) and estrogenic activity using a gene reporter assay (MELN cells). In all sites, high levels of PM10 and PM2.5 were measured during the fire suggesting that near and distant sites were influenced by fire pollutants. The PM10 and PM2.5 extracts induced a significant mutagenicity in all sites and the mutagenic effect was increased with respect to historical data. All extracts induced a slight increase of the estrogenic activity but a possible antagonistic activity of PM samples collected near fire was observed. No cytotoxicity or DNA damage was detected. Results confirm that fires could be relevant for human health, since they can worsen the air quality increasing PM concentrations, mutagenic and estrogenic effects.Wildland fires, increasing in recent decades in the Mediterranean region due to climate change, can contribute to PM levels and composition. This study aimed to investigate biological effects of PM Wildland fires are complex phenomena influenced by numerous factors such as land management, human activities, and weather conditions .All over the world climate change has been causing an increase in the frequency and intensity of extreme weather events  affectinWildland fires induce both environmental and human health effects on firefighters as well as on the general population . It was Wildland fires can produce PM, aromatic hydrocarbons, aldehydes, metals, dioxins, furans, benzene, formaldehyde and other airborne pollutants ,10,11. H2.5 (Ø < 2.5 µm) from wildland fires is 0.9–16 g for each kg of burned dry biomass ; (iii) and total mutagenicity factor per 20 m3, TMF (TMF = MR TA98 + MR TA98 (+S9) + MR TA100 + MR TA100 (+S9)). For each strain, with or without S9, the mutagenic effect was considered significant when mean of total revertants were at least twice than mean of spontaneous revertants (MR ≥ 1), while an extract was considered mutagen when at least one strain, with or without S9, showed an MR ≥ 1. Blank filter extract was also tested to verify that it did not induce any mutagenic effect.The results were expressed as: (i) total revertants; (ii) mutagenicity ratio per 20 mv/v), L-glutamine (4 mM), penicillin-streptomycin (100 U/mL–100 µg/mL)). MELN (70% confluent) were seeded in 96-well plates with a flat clear bottom (4 × 104 cells/well) and cultured overnight. The day after, test medium was replaced with organic extracts  were tested as a standard positive curve. The extracts (1 m3/mL) were also tested in combination with tamoxifen (10−6 M) in order to confirm whether the effects observed were due to activation of estrogen-receptors (ERs) and in combination with 17β-estradiol (10−10 M) to test the interaction between each extract and 17β-estradiol. Blank filter extract was also tested to verify that it did not induce any estrogenic activity.Estrogenic activity was assessed with the luciferase gene reporter assay using the One-Glo Luciferase Assay System (Promega). The assay was carried out as described by Balaguer et al.  with somThe luciferase activity was expressed as fold induction with respect to negative control (fold induction = 1). All experiments were performed in quadruplicate .t-test and the one-way ANOVA test followed by post-hoc Dunnett test were used to assess significant differences between effects induced by extracts and effects induced by negative controls. Moreover, the t-test was used to assess significant differences among the mutagenic effects induced by the extracts collected in different sites. The differences were considered significant for p < 0.05.Statistical analysis was performed using SPSS 26.0 . The In 10 were analysed from January 2016 to December 2018. In order to establish whether the high values of PM in the different sites were influenced by the wildland fire, for each site the monthly means of PM10 was registered during the wildland fire month (October 2017). This evidence suggests that not only the sites near the wildland fire but also distant sites, such as N, were probably influenced by the air pollutants associated with the wildland fire.As can be seen in 10 were selected as biomass burning tracers, because, during combustion, large quantities of these compounds are emitted in the smoke aerosol. The daily concentrations of these three tracers in the rural site F (near fire) and in the urban site T are reported in The influence of the wildland fire on the air quality of rural and urban areas was further investigated considering the concentrations of specific biomass burning tracers in PM . In partTaking together, the air pollution data confirm that wildland fires are associated with an increase of PM as reported by previous studies ,43,44,45Since the airborne pollution induced by wildland fires can mainly affect the respiratory system, the cell viability and the genotoxicity were investigated using human bronchial epithelial cells (BEAS-2B).t-test vs. negative control p > 0.05, t-test vs. negative control p > 0.05, 10 organic extracts collected in the Brazilian Amazon during wildland fires did not induce any cytotoxic effect in human alveolar epithelial cells (A549). However, these results differ from other published studies [Despite the different doses and the different exposure times, the extracts did not induce any cytotoxic effect in the WST-1 assay  ,8,12,13.Salmonella typhimurium strains  with and without metabolic activation (±S9) was performed to assess the mutagenicity of the PM organic extracts. The results are presented in The Ames test using different For both the strains, the effect was higher without metabolic activation (−S9), suggesting that the mutagenicity was mainly due to the presence of direct mutagenic substances. Moreover, the extracts induced a higher mutagenicity on TA98 than on TA100 strain, hence the mutagenic effect was induced mainly by frame-shift mutations instead of replacement of nitrogenous bases. 3/plate, 3/plate the urban extracts induced a significantly higher number of TA98 revertants than the rural extracts . This result is in accordance with a recent study in which urban and rural samples (collected in the Piedmont region) were analyzed using the Ames test [Considering the results obtained on TA98 strain, the samples collected in the two urban sites (T and N) induced a number of total revertants that was at least twice than mean spontaneous revertants for all tested doses . This result confirmed the influence of the wildland fire on the air quality of this site.Overall, all the extracts were mutagenic; however, comparing the mutagenic effect of the different sites, the highest TMF was induced by the PMc levels e. The mu10 extracts, the rural site located near the fire (F1) induced a less mutagenic effect than the organic extract collected in the site T but a more mutagenic effect than the extract collected in the other rural site (C) . This result suggests that the rural site near the fire may be influenced by some mutagenic substances released by the fire itself that are responsible for the higher mutagenic effect with respect to the effect induced by the other rural site, located more distantly from fires. It is important to highlight that the organic extract collected near the fire (F1) induced a mutagenic effect which was similar to the effect measured in the site T in a period not characterized by fires , suggesting that the fire was able to deteriorate the air quality of this rural site. Moreover, it is also important to notice that, although the mutagenicity measured in C was low, the presence of this effect is unexpected in this site which is located far from big cities/industrial facilities and near the Gran Paradiso National Park, suggesting an influence of the fires on this site also. Regarding the other PM10, the mutagenicity of PM2.5 organic extracts showed that the urban site N induced a higher effect than the site F (F2) (number of TA98 revertants at 20 m3/plate t-test significant for: N vs. F2 p = 0.022). However, the difference of the mutagenic effect between the urban sites and the fire site was higher for PM10 extracts than for PM2.5 extracts (T TMF—F1 TMF higher than N TMF—F2 TMF). This result could be explained considering that the two urban PM samples were collected in different cities, which may be characterized by different pollution levels [As reported for PMn levels ; howeverOverall, the results of the Ames test are consistent with the air pollution data and confirm that the wildland fires are able to influence the air quality of the sites near the wildland fire (site F) but also of distant sites (i.e. site C and site T). 3/plate, site T and site N), while no significant genotoxic effect, measured as DNA damage, was observed with the Comet assay (dose = 2.5 m3/mL) (One-way ANOVA test followed by post-hoc Dunnett test p > 0.05). These results are consistent with the study of De Oliveira Galvão et al. [0.5, PM2.5 and PM10 extracts which reported a higher effect measured using the Ames test with respect to the effect measured using the comet assay on mammalian cells [Comparing at equal doses the results of the Ames test with the results of the Comet assay, a significant mutagenic effect was detected in the Ames test , showed a significant increase of luciferase activity with respect to negative control only at 0.5 m3/mL  while a significant decrease of luciferase activity was detected at 2 m3/mL . On the contrary, the PM collected in the sites T, C and N induced an increase of luciferase activity which was significant for all the tested doses , suggesting a higher estrogenic activity with respect to the wildland fire extracts. The PM10 collected in F (wildland fire) and was equal to 1.39 luciferase activity fold induction  seems to show a non-monotonic dose response (i.e. an inverted-U shaped curve). This result might be related to a cytotoxic effect/cell proliferation inhibition induced by the extracts on MELN [3/mL). In the present study, it is interesting to notice that while the estrogenic activity of urban and rural sites increases or remains constant with the increase of the dose, the estrogenic activity of wildland fire  in combination with E2 (10−10 M). The PM collected in urban and rural sites tested with E2  showed a luciferase activity similar to the activity induced by the E2 alone (t-test p > 0.05), while the wildland fire PM tested with E2  showed a significantly lower luciferase activity with respect to the E2 alone (t-test p < 0.0001 for both samples, This hypothesis was further investigated testing the estrogenic activity of all the extracts (1 m3 of air in each site could also allow to perform a chemical characterization of the extracts, providing additional and complementary information. Further studies are needed to assess the estrogenic activity of PM collected during wildland fires; indeed the data on estrogenic activity of PM is still limited and, in this area, no estrogenic activity data collected during periods without fires are available. Moreover, to our knowledge, no study has investigated the estrogenic activity of PM during wildland fires, therefore it is not possible to compare these results with others. Future studies should be performed assessing the estrogenic activity of PM in urban and rural areas of the North-West of Italy without wildland fires in order to obtain useful data for comparison. For the same reason, in future studies it may be useful to carry out a first sampling campaign in sites affected by wildland fires and also a second sampling campaign a year later in the same areas during the same period. Moreover, for future studies, areas located even more distant than the wildland fire site should be considered, in order to collect data in sites not influenced by wildland fires. Finally, in future studies, a higher amount of each sample should be collected in order to have enough extract for the assessment of the biological effects, also using higher concentrations than the concentrations tested in this study. Moreover, the sampling of many mThe occurrence of wildland fires is growing due to climate change and specific regions, such as the Mediterranean area, will probably be more affected than others. During wildland fires, numerous hazardous pollutants are released in the atmosphere, representing a possible threat for human health. Since the increase of knowledge on air pollution induced by wildland fires is needed, it is of fundamental importance to investigate the effects induced by PM collected during wildland fire. The results of this study suggest that wildland fires worsen the air quality, increasing the concentrations of air pollutants, such as PM, and the mutagenic effect of the PM. This effect seems to influence the sites near fires, but also distant sites. Therefore fires, even if they are sporadic and short-time episodes, should be considered important human health issues. Moreover, in this study, the preliminary results on the estrogenic activity induced by PM collected during wildland fires highlighted some research gaps that have to be further investigated.


Tetradium ruticarpum can be effective inhibitors towards diabetes-related enzymes. The compounds were experimentally isolated, structurally elucidated, and tested in vitro for their inhibition effects on tyrosine phosphatase 1B (PTP1B) and α-glucosidase (3W37). Density functional theory and molecular docking techniques were utilized as computational methods to predict the stability of the ligands and simulate interaction between the studied inhibitory agents and the targeted proteins. Structural elucidation identifies two natural products: 2-heptyl-1-methylquinolin-4-one (1) and 3-[4-(4-methylhydroxy-2-butenyloxy)-phenyl]-2-propenol (2). In vitro study shows that the compounds (1 and 2) possess high potentiality for the inhibition of PTP1B  and α-glucosidase . DS values and the number of interactions obtained from docking simulation highly correlate with the experimental results yielded. Furthermore, in-depth analyses of the structure–activity relationship suggest significant contributions of amino acids Arg254 and Arg676 to the conformational distortion of PTP1B and 3W37 structures overall, thus leading to the deterioration of their enzymatic activity observed in assay-based experiments. This study encourages further investigations either to develop appropriate alternatives for diabetes treatment or to verify the role of amino acids Arg254 and Arg676.Folk experiences suggest natural products in  Bacillus cereus, can synthesize oligo-1,6-glucosidase, this enzyme is present mainly in the animal kingdom + in the fast atom bombardment mass spectrometry (FAB-MS). The presence of an N-methyl group [δH 3.72 /δC 34.3], an olefinic group /δC 154.9 (C-2) and 111.3 (C-3)), a benzene ring , 7.36 , 7.64 , and 7.49 /δC 126.8 (C-5), 123.4 (C-6), 132.2 (C-7), 115.5 (C-8), 142.1 (C-9), and 126.7 (C-10)), and a ketone group at δC 178.0 (C-4) in the 1H and 13C NMR spectra indicated 1 to be N-methyl-4(1H)-quinolone type alkaloids , 1.67 , 1.27-1.42 , 0.88 ), indicating the C7 side chain of 1 (13C NMR and heteronuclear multiple quantum correlation (HMQC) spectra of 1 showed 17 carbon signals, including one N-methyl carbon at δC 34.3 (N-CH3) and seven carbon signals of a heptyl group (δC 34.9 (C-1′), 28.7 (C-2′), 29.4 (C-3′), 29.2 (C-4′), 31.8 (C-5′), 22.8 (C-6′), and 14.2 (C-7′) -one type alkaloid derived in the Rutaceae species and was first identified from Zanthoxylum schinifolium .,25.1 wasain of 1 . The 13C2 (C-7′) ). The HM′ to C-2 . Based o2 was obtained as colorless needles. The 1H NMR and COSY spectra of 2 showed signals of a 1,4-disubstituted benzene ring  and 6.87 ), assigned to an A2B2-spin system and a trans-3-hydroxy-1-propenyl group [, 6.21 , and 4.30 ) , 5.77 , 4.09 , and 1.77  in the 1H NMR spectrum were arranged to be the signal for 4-methylhydroxy-2-butenyl group  and heteronuclear single quantum correlation (HSQC) spectra of 2 showed the presence of 14 signals, including six carbons for the benzene ring (δC 129.5 (C-1), 127.6 (C-2/C-6), 114.9 (C-3/C-5), and 158.4 (C-4)), three carbon signals for a trans-3-hydroxy-1-propenyl group (δC 130.9 (C-7), 126.2 (C-8), and 63.9 (C-9)), and five carbon signals for a 4-methylhydroxy-2-butenyl group (δC 64.3 (C-1′), 119.7 (C-2′), 140.1 (C-3′), 67.8 (C-4′), and 14.0 (C-5′)). The correlations of H-7 to C2 and C-6; H-8 to C-1; H-5′ to C-2′, C-3′, and C-4′, as well as H-2/H-6 and H-1′ to C-4, in the HMBC spectrum confirmed the structure of compound 2 ) . The sigroup see . The croroup see . The 13Cmpound 2 . The FAB1–2 potentially inhibited the PTP1B enzyme activity with IC50 values of 24.3 ± 0.8 and 47.7 ± 1.1 μM, respectively  exhibited a potential inhibitory effect on α-glucosidase enzyme activity with an IC50 value of 92.1 ± 0.8 μM, two times stronger than acarbose, the positive control used for this assay having an IC50 value of 152.4 ± 0.6 μM. Integrifoliodiol (2) showed similar activity with acarbose, whose IC50 value was 167.4 ± 0.4 μM. Genetic and biochemical studies have shown that PTP1B is a key negative regulator of leptin and insulin signaling pathways which are responsible for glucose homeostasis, control body weight, and energy expenditure . This reectively . Ursolic1 and 2 possess good inhibition activity towards α-glucosidase and protein PTP1B, given by their significant IC50 value in comparison to those of the control drugs. In detail, the corresponding values accord with the order 1 > 2 regarding either of the targeted proteins.The experimental assays reveal that Candida albicans in vivo by regulating lysosomal pathway-related genes that accelerate the metabolism and degradation of abnormal proteins [1) and intergrifoliodiol (2) did not show in vitro inhibitory effect on nitric oxide production [50 values of 33.7 ± 2.4, 21.9 ± 1.9, and 16.8 ± 2.2 μg/mL after 6, 12, and 24 h treatment, respectively, with different concentrations. The results of cell proliferative inhibition and clonogenic assay showed that SF enhanced the radio-sensitivity of A549 cells when applied before 60Co γ-irradiation, and this effect was mainly time- and concentration-dependent [Staphyloccocus aureus (ATCC25923), Staphylococcus epidermidis (ATCC12228), and Bacillus subtilis (ATCC6633) and no cytotoxicity against four human cancer cell lines  [1) and integrifoliodiol (2) possessed some biological activities, their PTP1B inhibitory activity has not been investigated to date. Our study showed that compounds 1 and 2 potentially inhibited the PTP1B enzyme activity with IC50 values of 24.3 ± 0.8 and 47.7 ± 1.1 μM, respectively. This is the first time that compound 2 has been identified from T. ruticarpum and the PTP1B inhibitory activity of these compounds has been investigated, given our reachable referencing.Schinifoline (SF) was known as a 4-quinolinone alkaloid and possessed some activities. Lu et al. reported that schinifoline inhibited the negative effects of proteins . In addioduction . Wang etependent . Other sBEL7403) , nor JurBEL7403) , using tBEL7403)  and can 1–2 are shown in 1 contains carbonyl and N-heterocyclic groups, while 2 is functionalized with groups -OH and C-O-C. These were reported to be highly conducive to polarity and solubility of the host compound [GAPE) of 1 and 2 are −6.884 and −7.102 eV, respectively. These are considered relatively low values and thus likely conducive to chemical reactivity and inhibitory stability [χ), or the chemical potential (μ) in a negative value, could be considered as a reliable inhibition indicator since it presents an electron-attracting tendency. In principle, a higher electronegativity implies a stronger attraction of electrons towards the host molecule. Therefore, the compounds seem promising for docking investigation.Geometrically optimized structures of compound ,35, thuscompound , their wcompound ,38. Furttability ,40. The 1 and 2 are virtually represented in 1 and 2  towards these potential sites are summarized in −1) and the number of interactions created (at least 3). Regarding protein PTP1B, there are no noticeable differences between the corresponding numbers of its detected sites. The lowest DS value and the corresponding number of interactions are found at site 1. In terms of 3AJ7, sites 1 and 3 are expected, providing better inhibitability than the others due to their dominant numbers of in-pose amino acids, i.e., 36 and 23, respectively. They are also the most effective inhibitory regions for 2 and 1, respectively. In addition, the comparable equivalency of the figures in comparison to those of the commercialized drug voglibose (D) justifies their inhibitory potentiality towards these diabetes-related proteins. Therefore, these sites opt for more in-depth analysis in the attempt to retrieve a structure–activity relationship between experiment-based and computer-based research studies.The quaternary structure of the targeted proteins 3W37 , PTP1B , 3AJ7 , and their approachable sites by investigated compounds 1-PTP1B and 2-PTP1B inhibitory complexes are −14.9 and −14.7 kcal·mol−1, markedly lower than those of 1-3W37 and 2-3W37, i.e., −13.2 and −13.8 kcal·mol−1, respectively. The former also contains more either hydrophilic or hydrophobic interactions between the ligands and the in-pose amino acids than the latter. In particular, the PTP1B-based inhibitions are formed by five hydrogen bond interactions and eight or nine van de Waals interactions; meanwhile, the corresponding figures for 3W37-based inhibitory systems are three and nine, respectively. All the inhibitory systems are considered biologically rigid since their RMSD value are all under 2 Å. Furthermore, the computed data regarding oligo-1,6-glucosidase (1-3AJ7 and 2-3AJ7) are included for further reference as the enzyme is well-known as an important target in diabetes-related research, despite the fact that it is not included in the experimental section in this study. These altogether can explain the experimental results that the studied compounds, i.e., 1 and 2, can inhibit enzyme tyrosine phosphatase 1B at significantly lower IC50 values than the carbohydrate-hydrolase counterpart. Although the primary parameters extracted from computational output  are highly consistent with the overall inhibition activity towards the enzymes  obtained from experimental assays, the specific orders regarding each of these proteins are still not clearly represented.The detailed docking simulation results for the inhibitory duos are summarized in 1 and 2 from T. ruticarpum on each enzyme can be reached based on certain hydrogen bonding interactions. First, the docking simulation technique expects that there exists a strong hydrogen bond formed between an oxygen atom of the ligand 1 and a nitrogen atom of amino acid Arg254 within the distance 2.92 Å in 1-PTPB1B. The bonding registers an exceptionally high value of Gibbs free energy −4.4 kcal·mol−1. While there are five predicted hydrogen bonds that contribute to the formation of the inhibitory system 2-PTPB1, the amino acid is not involved. This highly correlates with the results obtained from the enzyme assays on tyrosine phosphatase 1B inhibition; i.e., 1 (IC50 24.3 ± 0.8 µM) > 2 (IC50 47.7 ± 1.1 µM). Similarly, amino acid Arg676 is likely to play an important role in the formation of 1-3W37 inhibitory complex as it interacts with the ligand via three hydrogen-bonding interactions, whose Gibbs free energy in total is −5.9 kcal·mol−1. Meanwhile, 2 only creates one of the hydrophilic bonding with its targeted protein to form 2-3W37 inhibitory structure. The value of −2.5 kcal·mol−1 corresponds to the free Gibbs energy of this interaction. This can also be related to the differences observed between the enzyme assays on α-glucosidase inhibition. Therefore, the sufficient binding carried out on these amino acids, i.e., Arg254 and Arg676, seems to induce serious conformational changes on the enzymes PTP1B and 3W37, respectively, thus leading to the loss of their enzymatic functionality that is experimentally demonstrated in enzyme assays by the increase in IC50 values. In terms of oligo-1,6-glucosidase-based inhibitory systems (1-3AJ7 and 2-3AJ7), the concern is thought to relate to amino acid Arg442. The intuitive speculation still requires further relevant experimental work specifically targeting these amino acids to verify the proposed inhibition models.Nevertheless, a brief structure relationship for the inhibition strength of the isolated compounds D) was also screened, and the corresponding results are included in The inhibitory structures and their in-pose intermolecular interactions are visually shown in 254, 40–63 mesh) were purchased from Merck, St. Louis, MO, USA. NP and RP thin-layer chromatography (TLC) plates were from Merck . High-performance liquid chromatography  was carried out using a 1260 Agilent HPLC System: G1311C pump, G2260A auto-sampler, G1316A Thermo, and G1315D detector. Optima_Pak C18 column , RS Tech, Korea, and/or Zorbax eclipse XDB-C18  were used for purification. Proton and carbon nuclear magnetic resonance (NMR) were measured on a Varian Unity Inova 400 and/or 500 MHz spectrometers. Mass spectrometry (MS) data were obtained from a Varian FT-MS spectrometer and . Normal-phase and reverse-phase silica gels . The specimen voucher of this plant was stored at INPC, VAST, Vietnam. 2O residue, respectively. An activity-guided fractionation study resulted in the EtOAc extract being chosen for further study (The buds (1.2 kg) were extracted with methanol (5.0 L × 3 times) at 45 °C using sonication for 5 h. After filtration, the extracted solution was then evaporated by a rotary system to remove the solvent. The total methanol crude extract obtained was further suspended in distilled water (1.0 L) and partitioned with EtOAc (1.0 L × 4 times). The resulting fractions were concentrated under reduced pressure in a rotary evaporator to give the EtOAc and an Her study .1 (23.9 mg) and 2 (19.5 mg). The EtOAc fraction was thus directly chromatographed on an open silica gel column  using a stepwise gradient of hexane/acetone (from 20:1 to 0:1) to give ten fractions (TR-1 to TR-10). By the α-glucosidase activity-guided isolation , fractio1)Schinifoline ; 1H-NMR  δH (ppm): 6.21 , 8.43 , 7.36 , 7.64 , 7.49 , 2.69 , 1.67 , 1.27-1.42 , 0.88 , 3.72 ; 13C-NMR  δC (ppm): 154.9 (C-2), 111.3 (C-3), 178.0 (C-4), 126.8 (C-5), 123.4 (C-6), 132.2 (C-7), 115.5 (C-8), 142.1 (C-9), 126.7 (C-10), 34.9 (C-1′), 28.7 (C-2′), 29.4 (C-3′), 29.2 (C-4′), 31.8 (C-5′), 22.8 (C-6′), 14.2 (C-7′), 34.3 (N-CH3). Integrifoliodiol2); 1H-NMR  δH (ppm): δH: 7.32 , 6.87 , 6.55 , 6.21 , 4.30 , 4.59 , 5.77 , 4.09 , 1.77 ; 13C-NMR  δC (ppm): 129.5 (C-1), 127.6 (C-2/C-6), 114.9 (C-3/C-5), 158.4 (C-4), 130.9 (C-7), 126.2 (C-8), 63.9 (C-9), 64.3 (C-1′), 119.7 (C-2′), 140.1 (C-3′), 67.8 (C-4′), 14.0 (C-5′).p-NPP in a buffer containing 1 mM dithiothreitol, 0.1 M NaCl, 1 mM EDTA, and 50 mM citrate (pH 6.0), with or without test compounds, were added, with 100 μL final volume to each of the 96 wells. After the reaction mixture was incubated at 37 °C for 30 min, 10 M NaOH was added to quench the reaction. PTP1B enzyme activity was determined by the amount of produced p-nitrophenol at 405 nm. The nonenzymatic hydrolysis of the substrate was corrected by measuring the control, which contained no PTP1B enzyme. Ursolic acid (UA) was used as a positive control.Protein tyrosine phosphatase 1B (human recombinant), i.e., PTP1B, was purchased from Biomol International LP, Plymouth Meeting, Pennsylvania, PA, USA, and the inhibitory activities of the tested samples were evaluated using the method as described.  In a typα-glucosidase was determined according to the modified method with a readily available enzyme. In a typical procedure, α-glucosidase  was dissolved in 100 mM sodium phosphate buffer (pH 6.8) and used as an enzyme solution. A total of 1 mM p-Nitrophenyl-α-D-glucopyranoside (pNPG) in the same buffer (pH 6.8) was used as a substrate solution. Then, 95 μL of enzyme solution, 10 μL of extracts, and 10 μL of ethanol and PBS solution (1:1) were mixed in the same 96-well plate for each concentration  and measured absorbance at 405 nm after incubation for 5 min. After substrate solution (95 μL) was added, the sample solution was incubated for another 10 min at 37 °C incubator. Enzymatic activity was quantified by measuring absorbance at 405 nm using an enzyme-linked immunosorbent assay (ELISA) microplate reader . The IC50 value was defined as the concentration of α-glucosidase inhibitor that inhibited 50% of α-glucosidase activity. Acarbose was used as a positive control, and all assays were conducted in triplicate.The inhibitory activity of p < 0.05.The collected data were analyzed using SPSS 20.0 software . Average values and percentages were calculated. Differences in mean values were compared using analysis of variance (ANOVA), for normally distributed data, and the Mann–Whitney U test, for non-normally distributed data. Differences were considered significant at 1 and 2 from T. ruticarpum. Their molecular geometry was optimized using Gaussian 09 without symmetry constraints [HOMOE, represents intermolecular electron donation tendency; meanwhile, the electron-accepting ability of a molecule can be inferred from its value LUMOE . Energy gap ΔE = LUMOE − HOMOE is considered as an indicator for intermolecular reactivity since it exhibits the formation of excited-state electrons towards the molecular surface. Ionization potential (I) and electron affinity (A) were calculated using Koopmans’ theorem [HOMOI = −E and LUMOA = −E. They then were used to yield the electronegativity (χ) of a molecule via the equation χ = (I + A)/2. Regarding an N-electron system with total electronic energy (E) and external potential ν(r), electronegativity (χ) is defined as the negative value of chemical potential (μ) [χ = −μ = −(∂E/∂N)νr) was utilized to investigate quantum properties of the isolated compounds straints  at the lstraints . Calculastraints . Each rustraints . Bonding theorem , which ntial (μ) ,47. This1-2, PTP1B, and glycoside-hydrolase proteins (3W37 and 3AJ7). The simulated ligand-protein inhibitory structures were evaluated based on docking score (DS) energy as the main indicator. Root-mean-square deviation (RMSD) and intermolecular interactions were also analyzed and discussed. The typical procedure of a molecular docking simulation followed three steps [MOE 2015.10 software was responsible for molecular docking simulation. Its requisite input includes structural information of docking participants, i.e., two ligands ee steps ,20,21,22−1; max interactions = 1000; modify charge: Gasteiger–Huckel.(a) Pre-docking preparation: Structural information of the targeted proteins can be referenced at UniProtKB and Worldwide Protein Data Bank: tyrosine phosphatase protein PTP1B (entry: UniProtKB—A0A0U1XP67), alpha-glucosidase protein 3W37 (DOI: 10.2210/pdb3W37/pdb), and oligo-1,6-glucosidase protein 3AJ7 (DOI: 10.2210/pdb3AJ7/pdb). The protein structure and their 3D protonation were prepared using Quickprep tool. Their active sites were determined based on a ligand–amino acid radius of 4.5 Å. The protein structures obtained were saved in format *.pdb. The investigated adducts were optimized based on the configuration: Conj Grad for minima energy; termination for energy change = 0.0001 kcal·mol(b) Docking investigation: After input preparation, intermolecular interaction simulation was performed on MOE 2015.10 system and simulated ligand–protein inhibitory structures were saved in format *.sdf. The docking simulation parameters were configured: poses retained for intermolecular interaction probing = 10; maximum solutions per iteration = 1000; maximum solutions per fragmentation = 200.(c) Post-docking analysis: Docking score (DS) energy represents binding affinity of ligands and their targeted proteins in the site–site distance of a certain duo-system, thus considered as the primary indicator of inhibitability. Intermolecular interactions formed between the ligands and in-pose amino acids of the proteins included hydrophilic binding, e.g., electron-transferring (H-acceptor/donor), cation-arene (H-π), arene-arene (π-π), and ionic and hydrophobic interaction, also known as van der Waals forces. The simulation results in bonding amino acids, bonding lengths, and their Gibbs free energy regarding these interactions. Furthermore, static conformation of an inhibitory complex was predicted by its root-mean-square deviation (RMSD) value. This is based on the fact that RMSD represents the average between neighboring atoms; therefore, a smaller value means a more tightly bound conformation is formed. In addition, ligand conformation and orientation in its inhibited-protein active site were visualized on 2D and 3D planes. 1) and integrifoliodiol (2). Enzyme assays demonstrated that the alkaloid compound (1) is the stronger inhibitor towards the both proteins tyrosine phosphatase 1B (PTP1B) and α-glucosidase (3W37). The corresponding orders are 1 (IC50 24.3 ± 0.8 µM) > 2 (IC50 47.7 ± 1.1 µM) regarding the former and 1 (IC50 92.1 ± 0.8 µM) > 2 (IC50 167.4 ± 0.4 µM) regarding the latter. The primary parameters obtained from molecular docking simulation, also known as DS value and number of interactions, confirm these experimental yields. In particular, in-depth analyses on structure–activity relationships imply that amino acids Arg254 and Arg676 might play an important role in the overall conformational changes of PTP1B and 3W37, respectively. This speculation still needs more specifically experimental work to verify, thus encouraging further continuous research from the readership. Two isolated natural compounds were successfully identified by structural elucidation as schinifoline (


Here, we explored the competitive effects by co-expressing reteplase (rPA) with three unrelated PIs, namely NbPR4, HsTIMP, and SlCYS8, in Nicotiana benthamiana leaves. Remarkably, the accumulation of rPA was significantly repressed by each of the three PIs, suggesting that the competitive effects may be common among the PIs. The repression can be attenuated by reducing the PI inoculum dose in the co-inoculation mixtures, showing a negative correlation between the PI abundance of the PI-RP system and competitive effects. Interestingly, when a replicating vector was used to modulate the relative abundance of PI and RP in vivo, rPA was still boosted even at the maximal testing dose of PI, indicating that the competitive effects reduced to an ignorable level by this in vivo approach. Furthermore, a 7- to 12-fold increase of rPA was achieved, proving that it is a useful way for stimulating the potentials of a companion PI by overcoming competitive effects. And, this approach can be applied to molecular farming for improving the RP yields of plant expression systems.Plant-based expression platforms are currently gaining acceptance as a viable alternative for the production of recombinant proteins (RPs), but the degradation of RPs by proteases in cells hinders their superb potentials. Co-expression of a protease inhibitor (PI) shows promise as a strategy to prevent RP from proteolytic degradation in plants. However, competitive effects behind the PI-RP co-expression system may mask or obfuscate the  Agrobacterium-mediated methods were established to deliver the transgenes into the host cells for transiently producing RPs in plants  with human alpha-1-antichymotrypsin (α1ACT) in plants resulted in a marked decrease in α1ACT accumulation instead of increasing by preventing its degradation by leaf Cys proteases as expected, suggesting an apparent competitive effect between PI and RP during pre- and/or post-translational processes  in N. benthamiana leaves. Subsequently, we demonstrated a negative correlation between the PI abundance and competitive effects by reducing PI inoculum dose in mixtures. Finally, we diminished the competitive effects to an ignorable level by co-expressing a replicating vector with PI.To address this issue, three PIs, namely pression , were sepression . We explin vitro DNA synthesis and restriction enzyme-mediated cloning were employed to create the co-expressed constructs. For the construction of the non-replicating rPA expression vector, the CPMV expression cassette (via ApaI and BamHI restrict sites to generate pCPMV-EV construct. Then the N. benthamiana codon-optimized rPA (Invitrogen) with an N-terminal PR1b signal peptide (GenBank accession no. D90197.1) was synthesized and cloned into pCPMV-EV using NruI and FspI restriction sites to generate pCPMV-rPA construct  were separately cloned into plasmid pCBNoX P19 (NcoI and XbaI restriction sites generating pCB301-PIs constructs (Tobacco mosaic virus)-based vector  with gentle agitation for ~24 h at 28°C. Then, the cultures were centrifuged and re-suspended in an appropriate volume of 10 mM MES buffer  to obtain the required density . For co-expression, the suspensions were mixed in the ratio of 1:1 , and the supernatant was collected for later assays.The infiltrated zones of the leaf were sampled with a hole punch at 5 day post-inoculation (dpi) in all experiments, and nine discs from three individual plants were merged per sample. The leaf discs were weighed and then ground in pre-cooled buffer  at a ratio of 1:4 (g/ml). The homogenates were clarified in a centrifuge at 4°C . The membrane was then blocked with 5% (w/v) milk in phosphate-buffered saline (PBS) for 2 h at 25°C and immersed in 1/5,000 rabbit polyclonal anti-human tissue-type plasminogen activator (tPA) antibody (Abcam) and 1/5,000 goat anti-rabbit HRP conjugated secondary antibody (Sigma-Aldrich) successively. The specific immunoreactive proteins were visualized by enhanced chemiluminescent (ECL)-solution .Escherichia coli–expressed rPA for each plate and the extracts from the empty vector (pCPMV-EV or pTMV-EV) infiltrated leaves were used as a negative control. The relative accumulation level was described as a fold change of the amount of rPA in the absence and presence of PI. The observed values were obtained from the mean of three independent biological replicates and were used to perform Student’s t-test.The rPA accumulation was measured by using an ELISA-based assay as previously described. The ELISA plate was coated with the leaf extracts (overnight at 4°C), blocked with 5% (w/v) PBS-made milk (1 h at 37°C), and incubated in 1/10,000 rabbit polyclonal anti-tPA antibody (2 h at 37°C) and 1/5,000 goat anti-rabbit HRP conjugated secondary antibody (2 h at 37°C) successively. After each step, the plate was washed three times with PBS buffer at 5-min intervals. The plate was re-filled with 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution (Solarbio), incubated at 25°C for 15–30 min, and the reaction was halted by adding 1.0 M phosphoric acid. And then, OD values at 450 nm were read by using a microplate reader (Bio-Rad). A standard curve was generated with Cowpea mosaic virus) RNA2-based construct, which allows high-level RP expression in plants without viral replication (600 of 0.6 for each) and then inoculated into N. benthamiana leaves by agroinfiltration , and thus no enhancement of rPA accumulation. Likewise, the protection is over the repression at OD600 value of 0.2, resulting in a slight increment of rPA. We tested NbPR4 and HsTIMP, respectively, and got similar results  at a final ODmixtures ,C, indicPMV-rPA aachieved . Interes0 of 0.1 . Because was achieved upon SlCYS8 co-expression (SlCYS8 was reported to markedly decrease rather than increase α1ACT accumulation (SlCYS8 when a mutant SlCYS8 (SlCYS8-Q47P), which lacks inhibitory activity  were expressed by a single construct  results in leaf yellowing or wilting , otherwise, RP was found to increase . Nevertheless, the modulating relative abundance of PI, whether ituation , also reituation . Howevert moment . Moreove wilting . Therefoin vivo by viral replication reduces competitive effects. Those data suggest that the competitive effects may also occur during the translational processes, especially during protein synthesis. The PI mRNAs somehow are favored over the RP during translation, but this superiority is abolished when the abundance of RP mRNAs is dominant in the PI-RP system in cells.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.SW designed the experiments and wrote the manuscript. SW and ZL supervised the research. JM and XD conducted the vector construction and agroinfiltration. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This laboratory experience was implemented for students enrolled in the Introduction to Food Science course in Family and Consumer Sciences at North Carolina A&T State University.To prevent the growth of food pathogens and food spoilage organisms, antimicrobials are used to disinfect surfaces or are added to food. In light of the important role that antibiotics and antimicrobials play in food safety, food science students need a deep understanding of how these chemicals function. We describe here a combined wet lab and  Escherichia coli), the presence of even a single live cell is intolerable, while others can be tolerated at certain levels without causing illness . Therefore, how an antimicrobial interacts with a tolerable or intolerable pathogen will determine how it should be employed. We describe here a laboratory experience designed to help students visualize two biochemical concepts explaining antibiotic/antimicrobial activity: (i) bacteriolytic versus bacteriostatic activity and (ii) competitive versus noncompetitive inhibition. This laboratory experience was implemented for students enrolled in the Food Safety and Sanitation course at North Carolina A&T State University.Antibiotics are chemicals produced by microorganisms that kill other microorganisms . A hallmAntimicrobial agents can be bacteriostatic or bacteriolytic. Bacteriostatic agents inhibit the growth and reproduction of bacteria without killing them and, therefore, would be used in cases of pathogens that cannot be allowed to grow beyond a certain cutoff value. Bacteriostatic agents function by blocking protein production and DNA replication or cellular metabolism. Bacteriolytic agents, such as penicillin, kill cells outright by inducing digestion of the cell wall, leading to lysis. These agents would be used in cases where a single live pathogenic cell cannot be tolerated. Lysis of bacterial cells can be monitored by visible-range spectrophotometry, which can measure the turbidity of a solution. The level of turbidity of a bacterial culture is directly proportional to the number of intact cells. A drop in turbidity indicates lysis. Meanwhile, a constant turbidity is an indicator of a bacteriostatic mechanism. Penicillin functions by binding to proteins responsible for blocking the cross-linking of the peptidoglycan molecules in a cell wall, weakening the cell wall and leaving the cell to succumb to osmotic pressure that causes the cell to rupture.Many antibiotic and antimicrobial agents are enzyme inhibitors that function by inhibiting a key enzyme involved in cell metabolism. An enzyme is a protein molecule that catalyzes a chemical reaction and has one or more substrates. A substrate is the chemical that is modified by the enzyme. Every enzyme has an active site, which is a cavity in the structure that holds the substrates in an orientation that allows the chemical reaction to proceed. One example of an enzyme inhibitor is the choline kinase inhibitor (CKI), which blocks the activity of choline kinase , 5. CholTwo major types of enzyme inhibitors are noncompetitive and competitive inhibitors. Noncompetitive inhibitors function by binding a site on the enzyme outside the active site, which prevents the active site from functioning. Competitive inhibitors function by entering the active site of an enzyme and displacing the substrate. Docking software, such as Patchdock, can be used to assess the possibility that an inhibitor is competitive  by deterStudents came into the class having watched from home a set of YouTube videos that went over a demonstration of the wet lab activity and basic lab safety rules . Biosafety level 2 (BSL-2) safety rules were also reviewed in class (Appendix S7), followed by a lecture on antibiotics and their classifications.Streptococcus pneumoniae (Appendix S2). Based on the information provided to them about penicillin and CKI, the students were first prompted to hypothesize what the effects of each condition would be. Penicillin is a known bacteriolytic, while the mechanism of CKI was left unexplained. Students observed the optical densities of cultures treated with CKI or penicillin densities using a spectrophotometer (Appendix S2). Students were asked to analyze the data as part of a laboratory report and to come to a conclusion about how to classify penicillin and CKI .To gain an understanding of the key differences between bacteriolytic and bacteriostatic antibiotic activity, students tested the effects of penicillin, CKI, and the two combined on the nonpathogenic R6 strain of S. pneumoniae using the in silico tools PyMOL and Patchdock to determine if CKI could fit into the active site of choline kinase. The structure files in this activity (docking.zip) were generated by the instructors in PyMOL using the structural information found on the RCSB website (https://www.rcsb.org/structure/4R77).To gain an understanding of the key differences between competitive and noncompetitive inhibitors, students modeled CKI binding to the active site of the choline kinase enzyme of This activity consisted of six parts: (i) inspection of the structure of choline kinase with choline; (ii) definition of the choline kinase active site; (iii) inspection of CKI; (iv) docking of CKI to the active site of choline kinase; (v) inspection of structure of choline kinase in complex with CKI; and (vi) comparison with the structure of choline kinase in complex with its natural substrate, choline (Appendix S2 and S7).Students took a pre/postexam administered using the online service Kahoot.it. The experience took on game-show style, as students saw the percent breakdown of student answers in real time (see Appendix S1 for exam questions and Appendix S4 for a link to the Kahoot.it exam). Lab reports were also assessed (Appendix S3), as was student performance (Appendix S8 and S10).S. pneumoniae was used. Nevertheless, this organism is BSL-2 (https://www.atcc.org/Products/All/BAA-255.aspx). To increase safety, the activity could be modified by preadding the antibiotics to the cultures, leaving the student with a sealed test tube with which to measure optical densities. A BSL-1 organism also could be substituted. The in silico exercise can be carried out at home using the online tools.In the culturing activity, the nonvirulent R6 strain of This exercise teaches about the nature of bacteria and the link between controlling their growth and food safety. The instructions reinforced in the instructional videos, in-person lecture, and student activity guide were easily followed, and all participants successfully completed the activity.