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Clinical dentistry is a complex activity which the procedure of most dental treatment are almost irreversible changes. Patient safety is a major challenge in clinical dental care. This is the reason why simulation-based learning and training is emphasized in dental education. In this article, the authors presented the clinical relevant haptic simulation learning and training in tooth preparation. The practical framework to set up the simulation environment from real patients in haptic 3D virtual reality dental training simulator Simodont® (Nissin Dental Products Inc., Nieuw-Vennep, Netherlands). Then, the trainee can repeatedly practice in Simodont® before performing the clinical procedure on real patient. Taken together, the implementation of this model in dental education may not only enhance trainees' self-confidence and performance, but also facilitate patient safety during clinical dental care.

The recently discovered insecticidal protein Mpp75Aa1.1 from Brevibacillus laterosporus is a member of the ETX_MTX family of beta-pore forming proteins (β-PFPs) expressed in genetically modified (GM) maize to control western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte). In this manuscript, bioinformatic analysis establishes that although Mpp75Aa1.1 shares varying degrees of similarity to members of the ETX_MTX2 protein family, it is unlikely to have any allergenic, toxic, or otherwise adverse biological effects. The safety of Mpp75Aa1.1 is further supported by a weight of evidence approach including evaluation of the history of safe use (HOSU) of ETX_MTX2 proteins and Breviballus laterosporus. Comparisons between purified Mpp75Aa1.1 protein and a poly-histidine-tagged (His-tagged) variant of the Mpp75Aa1.1 protein demonstrate that both forms of the protein are heat labile at temperatures at or above 55°C, degraded by gastrointestinal proteases within 0.5 min, and have no adverse effects in acute mouse oral toxicity studies at a dose level of 1920 or 2120 mg/kg body weight. These results support the use of His-tagged proteins as suitable surrogates for assessing the safety of their non-tagged parent proteins. Taken together, we report that Mpp75Aa1.1 is the first ETX-MTX2 insecticidal protein from B. laterosporus and displays a similar safety profile as typical Cry proteins from Bacillus thuringiensis.

The recently discovered insecticidal protein Mpp75Aa1.1 from Brevibacillus laterosporus is one member of the ETX_MTX family of beta-pore forming proteins (β-PFPs) expressed in genetically modified (GM) maize to control western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte). In this paper, bioinformatic analysis establishes that although Mpp75Aa1.1 shares varying degrees of similarity to members of the ETX_MTX2 protein family, it is unlikely to contribute to any allergenic, toxic, or otherwise adverse biological effects. The safety of Mpp75Aa1.1 is further supported by a weight of evidence including evaluation of history of safe use (HOSU) of ETX_MTX2 proteins. Assessments using either purified Mpp75Aa1.1 protein or a poly-histidine-tagged (His-tagged) variant of the Mpp75Aa1.1 protein demonstrate that both forms of the protein are heat labile at temperatures at or above 55 °C, degraded by gastrointestinal proteases within 0.5 min, and have no adverse effects in acute mouse oral toxicity studies at a dose level of 1920 or 2120 mg/kg body weight. Results also indicate that the domain-based protein characterization should be considered as part of the weight of evidence for the safe consumption of food or feed. Furthermore, these results support the use of His-tagged proteins as suitable surrogates for assessing the safety of their non-tagged parent proteins.

Breast cancer remains the most frequently diagnosed cancer and is the leading cause of neoplastic disease burden for females worldwide, suggesting that effective therapeutic and/or diagnostic strategies are still urgently needed. In this study, a type of indocyanine green (ICG) and camptothecin (CPT) co-loaded perfluorocarbon double-layer nanocomposite named ICPNC was developed for detection and photochemotherapy of breast cancer. The ICPNCs were designed to be surface modifiable for on-demand cell targeting and can serve as contrast agents for fluorescence diffuse optical tomography (FDOT). Upon near infrared (NIR) irradiation, the ICPNCs can generate a significantly increased production of singlet oxygen compared to free ICG, and offer a comparable cytotoxicity with reduced chemo-drug dosage. Based on the results of animal study, we further demonstrated that the ICPNCs ([ICG]/[CPT] = 40-/7.5-μM) in association with 1-min NIR irradiation (808 nm, 6 W/cm2) can provide an exceptional anticancer effect to the MDA-MB-231 tumor-bearing mice whereby the tumor size was significantly reduced by 80% with neither organ damage nor systemic toxicity after a 21-day treatment. Given a number of aforementioned merits, we anticipate that the developed ICPNC is a versatile theranostic nanoagent which is highly promising to be used in the clinic.

Background Enriched electronic health records (EHRs) contain crucial information related to disease progression, and this information can help with decision-making in the health care field. Data analytics in health care is deemed as one of the essential processes that help accelerate the progress of clinical research. However, processing and analyzing EHR data are common bottlenecks in health care data analytics. Methods The dxpr R package provides mechanisms for integration, wrangling, and visualization of clinical data, including diagnosis and procedure records. First, the dxpr package helps users transform International Classification of Diseases (ICD) codes to a uniform format. After code format transformation, the dxpr package supports four strategies for grouping clinical diagnostic data. For clinical procedure data, two grouping methods can be chosen. After EHRs are integrated, users can employ a set of flexible built-in querying functions for dividing data into case and control groups by using specified criteria and splitting the data into before and after an event based on the record date. Subsequently, the structure of integrated long data can be converted into wide, analysis-ready data that are suitable for statistical analysis and visualization. Results We conducted comorbidity data processes based on a cohort of newborns from Medical Information Mart for Intensive Care-III (n = 7,833) by using the dxpr package. We first defined patent ductus arteriosus (PDA) cases as patients who had at least one PDA diagnosis (ICD, Ninth Revision, Clinical Modification [ICD-9-CM] 7470*). Controls were defined as patients who never had PDA diagnosis. In total, 381 and 7,452 patients with and without PDA, respectively, were included in our study population. Then, we grouped the diagnoses into defined comorbidities. Finally, we observed a statistically significant difference in 8 of the 16 comorbidities among patients with and without PDA, including fluid and electrolyte disorders, valvular disease, and others. Conclusions This dxpr package helps clinical data analysts address the common bottleneck caused by clinical data characteristics such as heterogeneity and sparseness.

Laser-induced fluorescence systems that are used in fluorescence research are expensive. This study demonstrated a novel, cheap, and compact fluorescence system comprising a fiber-coupled LED and pinholes to achieve a confocal arrangement. For investigating photobleaching, two model fluorescence dyes-fluorescein and calcein-were successfully tested using this platform. The results indicated that the two dyes exhibit similar self-quenching phenomena. Photobleaching by using the two dyes under normalized intensity was evaluated using a double-negative exponential model. The photobleaching rate increased with the dye concentration.

Background Periodontal disease effects 20–50% of the population worldwide, posing a global health challenge. It has been reported to be more prevalent among adults. Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) is an important organism associated with localized juvenile periodontitis. Photodynamic therapy (PDT) has been widely utilized for the treatment of periodontal disease; however, the effect of laser (light) profile on the antibacterial efficacy of PDT remains to be established. The quantitative measurement of laser profile is required to confirm the in vitro efficacy of PDT. Aim In the present study, a low cost PDT system comprising of six copper tube waveguides (CTW) was developed to provide more uniform irradiation of the culture plate. Methods The antibacterial effect of PDT, in combination with 200 μg/mL methylene blue (MB) as photosensitizer and 60 sec of irradiation, was studied on A. actinomycetemcomitans and Streptococcus mutans (S. mutans). In the present case, 660 nm laser guided with unpolished CTW, polished CTW, and optical fiber waveguide (OFW) provided radiant exposure of 0.86, 1.38, and 1.36 J/cm2, respectively, for a 24‐well culture plate. Results The designed PDT system provided antimicrobial efficacy of 98% and 91% for A. actinomycetemcomitans and S. mutans, respectively, which was significantly higher as compared to OFW guided PDT. Conclusion The results of the study highlighted the importance of laser profile as a key parameter that determines the survival rate of bacteria at the edge of the culture plate. Thus, the dose of PDT at the margin of optical profile is important for antibacterial activity for in vitro evaluation.

Liquid chromatography/mass spectrometer (LC/MS) has become one of the most popular analytical platform for metabolomics studies owing to its wide range of detectable polarity and molecular mass. However, metabolite identification remains quite costly and time-consuming in LC/MS-based metabolomics, mostly due to lower database integrity and a separated MS/MS spectra generation process. In this work, we constructed an automated, user-friendly, and freely available tool. From a peak list, the tool first groups peaks, which are usually associated with a metabolite, based on their retention time and abundance correlation across samples. In each group, different ions are annotated and the mass of the underlying metabolite is derived. Finally, the fragments are used to match with low-energy MS/MS spectra in public databases for metabolite identification. To identify metabolites without accessible MS/MS spectra, we have developed characteristic fragment and common substructure matches. Through the above approach, we anticipate facilitating the metabolite identification in LC-MS-based metabolomics studies.

The concentration of methylene blue (MB) photosensitizer could affect the eradication efficacy of antimicrobial photodynamic therapy (aPDT) in the treatment of contaminated implants, which is linked to the osseointegration of the implant. We evaluated osteoblast functions on the contaminated SLA (sandblasting, large-grit and acid-etching) Ti alloy surfaces after the concentration-dependent use of MB-aPDT. Totally 1164 SLA discs were randomly distributed for the analyses of antibacterial efficacy and osteoblast functions. Gram-negative (Aggregatibacter actinomycetemcomitans; A. actinomycetemcomitans) or Gram-positive (Streptococcus mutans; S. mutans) adhered on disc samples was subjected to aPDT with different MB concentrations (200, 250, 300, 350, and 400 μg/mL) using 660 nm diode laser with maximum output 80 mW for 1 min irradiation (4.8 J/cm2). Bactericidal effect was examined by viability, morphology, and lipopolysaccharide (LPS) assays. The disinfected disc surfaces by MB-aPDT to support osteoblast-like MG63 attachment, proliferation, differentiation, and mineralization were assessed for the predetermined culture time intervals. The statistical differences between the means were performed using a one-way analysis of variance (ANOVA) with a post hoc Scheffe test. The results of the morphology observation and bacterial survival examination consistently indicated a remarkably lower quantity of bacterial colonies on biofilm-contaminated surfaces after the aPDT treatment with higher MB concentration. Similarly, the higher MB concentration in aPDT resulted in the lower LPS amounts remaining on the A. actinomycetemcomitans-contaminated surfaces. Intriguingly, the expression of osteoblast cultured on disinfected surfaces using aPDT with higher MB concentration was comparable to the control without contamination. Within the limits of this in vitro model, this formulation of 400 μg/mL MB used in aPDT may be not only the lethal concentration against the 2 bacteria-contaminated implants, but it could also enhance the osteoblast functions on the contaminated implants. Nevertheless, the efficacy in the clinical practice for peri-implantitis therapy remains to be studied.

The study is to evaluate the effect of surface roughness of titanium implants on bacterial adhesion and then to investigate the efficacy of the three cleaning treatments for bacterial removal in titanium surfaces.Although surface debridement is the basic element for treatment of peri-implantitis to reduce bacterial adhesion, adjunctive therapies such as antiseptics and laser debridement have been proposed to improve the nonsurgical treatment options of the peri-implant infection.Titanium specimens were divided into five groups: No. 1200 grit sandpaper polishing (Grit), 50 μm (SB50), 100 μm (SB100), and 250 μm Al2O3 sandblasting (SB250), and sandblasting, large-grit, and acid-etching (SLA). Surface roughness (Ra), contact angle, and surface morphology were examined. The subsequent adhesion of Escherichia coli on the different substrates was assayed. After 8 h of bacterial culture, three different cleaning treatments, including plastic curettage, air-powder abrasive system, and Er:YAG laser debridement, were applied on the specimens.The Ra value changed from the lower value of 0.2 μm for the Grit group to the significantly higher value of 2.7 μm for the SB250 group, indicating a significant difference from the SLA group (2.0 μm). The average contact angle of SLA (101°) was significantly higher than the other groups. No significant difference in E. coli bacterial adhesion was found among the all roughened groups, except the SB50 and SB250 groups at 12 h of culture. The use of three cleaning treatments did not induce significant surface alterations. However, the E. coli adhesion was significantly reduced in the air-powder abrasive system and laser debridement in comparison with that treated with the plastic curettage.Laser debridement could be a useful cleaning method for peri-implantitis therapy.

Bacterial elimination using antimicrobial photodynamic therapy (aPDT) has been considered an alternative therapeutic modality in peri-implantitis treatment. The present in vitro study evaluated the dose-dependent and pH-dependent bactericidal effects of methylene blue (MB)-mediated aPDT at eliminating Gram-negative (P. gingivalis and A. actinomycetemcomitans) and Gram-positive (S. mutans) bacteria on sandblasting, large-grit and acid-etching (SLA)-pretreated titanium alloy. The effects of different MB concentrations (50, 100, and 200 μg/mL), the pH of the MB (4, 7, and 10), and irradiation time (0, 30, and 60 s) on the bacterial viability and residual lipopolysaccharide (LPS) levels were examined. The variations in the pH of the MB solution after aPDT for 60 s on the uncontaminated and contaminated specimens were also detected. The experimental results indicated that MB-mediated PDT could effectively kill the majority of bacteria on the titanium alloy surfaces of biofilm-contaminated implants compared with the MB alone. Of note, aPDT exhibited better antibacterial efficacy with increase in the MB concentration and irradiation time. While treated in an acidic solution on the biofilm-contaminated specimens, aPDT caused the pH to increase. By contrast, the initially high alkaline pH decreased to a value of about pH 8.5 after aPDT. Intriguingly, the neutral pH had minor changes, independent of the MB concentration and bacterial species. As expected, aPDT with higher MB concentration at higher pH environment significantly lowered the LPS concentration of A. actinomycetemcomitans and P. gingivalis. On the basis of the data, the aPDT with 200 μg/mL MB at pH 10 for 60 s of irradiation time might be an effectively treatment to eliminate bacteria and LPS adherent to titanium surface, however, the use of the multispecies biofilm model and the evaluation of in vitro osteogenesis needed to be further evaluated.

During symbiosis, organisms use a range of metabolic and protein-based signals to communicate. Of these protein signals, one class is defined as ‘effectors’, i.e., small secreted proteins (SSPs) that cause phenotypical and physiological changes in another organism. To date, protein-based effectors have been described in aphids, nematodes, fungi and bacteria. Using RNA sequencing of Populus trichocarpa roots in mutualistic symbiosis with the ectomycorrhizal fungus Laccaria bicolor, we sought to determine if host plants also contain genes encoding effector-like proteins. We identified 417 plant-encoded putative SSPs that were significantly regulated during this interaction, including 161 SSPs specific to P. trichocarpa and 15 SSPs exhibiting expansion in Populus and closely related lineages. We demonstrate that a subset of these SSPs can enter L. bicolor hyphae, localize to the nucleus and affect hyphal growth and morphology. We conclude that plants encode proteins that appear to function as effector proteins that may regulate symbiotic associations.

With predicted global changes in temperature and precipitation, drought will increasingly impose a challenge to biomass production. Most of the bioenergy crops have some degree of drought susceptibility as revealed for example through measures of low water-use efficiency (WUE). It is imperative to improve drought tolerance and WUE in bioenergy crops for sustainable biomass production in arid and semi-arid regions. Genetics and functional genomics can play critical roles in generating knowledge to inform and aid genetic improvement for drought tolerance in bioenergy crops. The molecular aspects of drought response have been extensively investigated in model plants like Arabidopsis, yet our understanding of the molecular mechanisms underlying drought tolerance in bioenergy crops is limited. Plants in general exhibit various responses to drought stress depending on species and genotype. A rational strategy for studying drought tolerance in bioenergy crops is to translate the knowledge from model plants relat...

PurposeThe purpose of this paper is to explore the social value of information in virtual investment communities and compare its effects with objective information value. A model including information quality, social comparison, and herding orientation, and their effects on decision usefulness and member satisfaction, is proposed and tested.Design/methodology/approachAn online survey with a sample of 215 members of investment communities was conducted to test the proposed model.FindingsThe opinion comparison orientation of members and information credibility are positively related to their perceived decision usefulness and satisfaction. Consistency is positively related to decision usefulness, but not to member satisfaction. Members' herding tendency moderates the effect of opinion comparison orientation on decision usefulness and the effect of ability comparison orientation on satisfaction.Research limitations/implicationsThe sample is small and not random. The proportion of students in the sample seems to be higher than it should be among virtual investment community members.Practical implicationsInvestors should be careful regarding the social influences of their communities; the effects may not always be good for investment decisions.Social implicationsVirtual communities provide members with social comparison information, which may yield positive effects for members in inspiration, self‐improvement, and self‐enhancement.Originality/valueThe virtual community can be a forum where people gain information regarding others to satisfy their needs for social comparison. Virtual communities provide special social value for their members, even for those who do not interact with others by posting in the communities.

Chemotherapeutic agents have been used as an adjunct to mechanical debridement for peri-implantitis treatment. The present in vitro study evaluated and compared the effectiveness of hypochlorous acid (HOCl), sodium hypochlorite (NaOCl), and chlorhexidine (CHX) at eliminating Gram-negative (E. coli and P. gingivalis) and Gram-positive (E. faecalis and S. sanguinis) bacteria. The effect of irrigating volume and exposure time on the antimicrobial efficacy of HOCl was evaluated, and a durability analysis was completed. Live/dead staining, morphology observation, alamarBlue assay, and lipopolysaccharide (LPS) detection were examined on grit-blasted and biofilm-contaminated titanium alloy discs after treatment with the three chemotherapeutic agents. The results indicated that HOCl exhibited better antibacterial efficacy with increasing irrigating volumes. HOCl achieved greater antibacterial efficacy as treatment time was increased. A decrease in antimicrobial effectiveness was observed when HOCl was unsealed and left in contact with the air. All the irrigants showed antibacterial activity and killed the majority of bacteria on the titanium alloy surfaces of biofilm-contaminated implants. Moreover, HOCl significantly lowered the LPS concentration of P. gingivalis when compared with NaOCl and CHX. Thus, a HOCl antiseptic may be effective for cleaning biofilm-contaminated implant surfaces.

The two head domains of the budding yeast Kinesin-14 Kar3Vik1 bind adjacent protofilaments at the start of the motility cycle, followed by release of Vik1 from one protofilament to allow the motor’s powerstroke., Kinesin-14 motors generate microtubule minus-end–directed force used in mitosis and meiosis. These motors are dimeric and operate with a nonprocessive powerstroke mechanism, but the role of the second head in motility has been unclear. In Saccharomyces cerevisiae, the Kinesin-14 Kar3 forms a heterodimer with either Vik1 or Cik1. Vik1 contains a motor homology domain that retains microtubule binding properties but lacks a nucleotide binding site. In this case, both heads are implicated in motility. Here, we show through structural determination of a C-terminal heterodimeric Kar3Vik1, electron microscopy, equilibrium binding, and motility that at the start of the cycle, Kar3Vik1 binds to or occludes two αβ-tubulin subunits on adjacent protofilaments. The cycle begins as Vik1 collides with the microtubule followed by Kar3 microtubule association and ADP release, thereby destabilizing the Vik1–microtubule interaction and positioning the motor for the start of the powerstroke. The results indicate that head–head communication is mediated through the adjoining coiled coil.

Hydroxyapatite (HA)-coated implants plasma-sprayed on metallic substrates have been widely used in load-bearing applications. In the present work, the in vitro behaviors of bioactive glass-containing HA coatings with post-deposition heat treatment in a simulated body fluid has been performed by means of electrochemical techniques. Annealing of the coatings in air at 650 °C led to recrystallization of amorphous calcium phosphate and effectively increased the conversion of non-apatite phases into apatite. The heat treatment also resulted in a reduction of layer defects associated with plasma-sprayed coatings without adversely affecting bond strength of the coatings. Moreover, the heat-treated coatings significantly increased the corrosion potential and polarization resistance value by approximately two times as compared to untreated samples. Improved corrosion resistance could be attributed to reduction of layer defects and enhancement of coating crystallinity.

Kar3Cik1 is a heterodimeric kinesin-14 from Saccharomyces cerevisiae involved in spindle formation during mitosis and karyogamy in mating cells. Kar3 represents a canonical kinesin motor domain that interacts with microtubules under the control of ATP-hydrolysis. In vivo, the localization and function of Kar3 is differentially regulated by its interacting stoichiometrically with either Cik1 or Vik1, two closely related motor homology domains that lack the nucleotide-binding site. Indeed, Vik1 structurally resembles the core of a kinesin head. Despite being closely related, Kar3Cik1 and Kar3Vik1 are each responsible for a distinct set of functions in vivo and also display different biochemical behavior in vitro. To determine a structural basis for their distinct functional abilities, we used cryo-electron microscopy and helical reconstruction to investigate the 3-D structure of Kar3Cik1 complexed to microtubules in various nucleotide states and compared our 3-D data of Kar3Cik1 with that of Kar3Vik1 and the homodimeric kinesin-14 Ncd from Drosophila melanogaster. Due to the lack of an X-ray crystal structure of the Cik1 motor homology domain, we predicted the structure of this Cik1 domain based on sequence similarity to its relatives Vik1, Kar3 and Ncd. By molecular docking into our 3-D maps, we produced a detailed near-atomic model of Kar3Cik1 complexed to microtubules in two distinct nucleotide states, a nucleotide-free state and an ATP-bound state. Our data show that despite their functional differences, heterodimeric Kar3Cik1 and Kar3Vik1 and homodimeric Ncd, all share striking structural similarities at distinct nucleotide states indicating a common mechanistic theme within the kinesin-14 family.

Minimally Invasive Surgery (MIS) has become more and more popular in recent years. An endoscopic image tracking system will assist surgeons to adjust the field of view autonomously in MIS. In this paper, we propose a novel image tracking algorithm based on natural features of surgical instruments. We suggest to use texture and geometric features in laparoscopic instrument imagery and to adopt a spiking neural network approach for object detection; considering color will be affected by lighting and the white balance conditions in the endoscope imagery. To enhance tracking performance, we further design a Kalman filter to combine with the neuro-based tracker. The instrument can be detected more robustly despite of deformation of the instrument image during surgery. A laparoscopic video has been tested to verify the developed methods. Experimental results show that two instruments can be distinguished and tracked simultaneously in the surgical video.

With an autonomous tool tracking system, surgeons do not need to handle the endoscope during surgery. This paper aims to develop an image tracking system with a stable view for minimally invasive surgery (MIS) by using monocular endoscope. In order to provide a stable view for the surgery, we propose to set a buffer zone in the center of image frame for the robotic camera holder to track the endoscope instruments. If endoscope instruments are inside the buffer zone, the endoscope robot will keep still. However, if one or both of them move away from the buffer zone, the robot will start to track the instrument. Furthermore, by calculating the distance between two instruments in the image plane, the robotic camera holder can track the depth to the instruments. The robot will therefore zoom in if the distance is too short. A safe distance from the camera lens to instrument tools can therefore be maintained during the image tracking. Preliminary experimental results show that the proposed design achieves satisfactory tracking accuracy in real time and provides a stable view simultaneously.

This paper proposes a novel planning method using genetic algorithm (GA) to achieve minimization of load shedding. The frequency of a power system declines rapidly when generator outage occurs. The general solution is to install sufficient under-frequency relays to pull frequency back to normal range. In this study, a single machine infinite bus (SMIB) is utilized to simulate system load with genetic algorithm for estimating the optimal load shedding and shedding ratio in each stage. Simulated results indicate that the proposed GA-based method is both feasible and effective to facilitate optimal load shedding planning.

Kar3Cik1 is a Saccharomyces cerevisiae kinesin-14 that functions to shorten cytoplasmic microtubules (MTs) during yeast mating yet maintains mitotic spindle stability by cross-linking anti-parallel interpolar MTs. Kar3 contains both an ATP- and a MT-binding site, yet there is no evidence of a nucleotide-binding site in Cik1. Presteady-state and steady-state kinetic experiments were pursued to define the regulation of Kar3Cik1 interactions with the MT lattice expected during interpolar MT cross-linking. The results reveal that association of Kar3Cik1 with the MT occurs at 4.9 μM(-1) s(-1), followed by a 5-s(-1) structural transition that limits ADP release from the Kar3 head. Mant-ATP binding occurred at 2.1 μM(-1) s(-1), and the pulse-chase experiments revealed an ATP-promoted isomerization at 69 s(-1). ATP hydrolysis was observed as a rapid step at 26 s(-1) and was required for the Kar3Cik1 motor to detach from MT. The conformational change at 5 s(-1) that occurred after Kar3Cik1 MT association and prior to ADP release was hypothesized to be the rate-limiting step for steady-state ATP turnover. We propose a model in which Kar3Cik1 interacts with the MT lattice through an alternating cycle of Cik1 MT collision followed by Kar3 MT binding with head-head communication between Kar3 and Cik1 modulated by the Kar3 nucleotide state and intramolecular strain.

Kar3Cik1 is a S. cerevisae Kinesin-14 motor protein that functions to shorten cytoplasmic microtubules (MTs) during yeast mating yet crosslinks interpolar MTs (ipMTs) during anaphase. Kar3 contains ATP and MT binding sites, yet Cik1 lacks the nucleotide binding site. Presteady-state kinetic and thermodynamic studies were pursued using paclitaxel-stabilized MTs to define the Kar3Cik1 interactions with the MT lattice expected during ipMT crosslinking. The results reveal that Kar3Cik1's association with the MT occurs at 4.9 μM-1s-1 followed by a 5 s-1 structural transition that limits mantADP release to 4-5 s-1. However, the intrinsic rate of mantADP release from Kar3Cik1 was observed at 109 s-1. ATP binding to nucleotide-free MT•Kar3Cik1 occurred at 2.1 μM-1s-1 followed by an ATP-promoted isomerization at 64 s-1. ATP hydrolysis was a fast step observed at 25 s-1, yet the reduced burst amplitude indicates reversals at this step. ATP hydrolysis was required for ATP-promoted Kar3Cik1 detachment from the MT at 12.7 s-1. The rate constant of phosphate release at 10 s-1 was similar to the rate constant of ATP-promoted MT•Kar3Cik1 dissociation, suggesting that these two steps are coupled. The rate-limiting step for steady-state ATP turnover at 5 s-1 is hypothesized to be the conformational change after Cik1 collision with the MT leading to Kar3 association with the MT followed by ADP release. These findings provide a model in which Kar3Cik1 interacts with the MT through an alternating cycle of Cik1 MT association followed by Kar3 binding with regulation through Kar3 nucleotide state and mechanical strain between the Kar3-Cik1 heads. Supported by NIH grant R01-GM54141 to SPG.

Kar3Cik1 is a S. cerevisae Kinesin-14 motor protein that functions to shorten cytoplasmic microtubule (MT) during yeast mating for nuclear fusion, yet crosslinks interpolar MTs (ipMTs) during anaphase. The Kar3 head contains both an ATP and MT binding site, yet the Cik1 head lacks an ATP catalytic site. Presteady-state and steady state experiments were conducted to define the mechanochemical pathway by which Kar3Cik1 stabilizes anti-parallel ipMTs for its mitotic function. To initiate the cycle, we used a high ADP strategy to promote MT-binding by the Cik1 head at 4.9 ∈1/4M-1s-1. The initial association is then followed by a 4-5 s-1 conformational change to induce Kar3 head binding to the MT with rapid ADP release from the active site at 109 s-1. MantATP binding to the nucleotide free MT•Kar3Cik1 is fast at 2.1 ∈1/4M-1s-1 with koff = 16.6 s-1. Pulse-chase methodology further reveals that MgATP binding to MT•Kar3Cik1 follows a two step process, formation of a collision complex followed by a 64 s-1 isomerization step. ATP hydrolysis occurs at 26 s-1 followed by motor detachment from microtubule at 11.5 s-1. The rate-limiting step for steady-state ATP turnover at 5 s-1 is hypothesized to be the conformational change leading to Kar3 head binding to MT. These initial results suggest a model in which Kar3Cik1 interacts with the MT through an alternating cycle of Cik1 binding followed by Kar3 binding. Because Cik1 does not have a nucleotide binding site, we propose that head-head communication is mediated by a strain-dependent mechanism. Supported by NIH GM54141.