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The model moss, Physcomitrium patens, is routinely cultured on cellophane placed over a solid nutrient medium. While this culture method is convenient for moss propagation, it is not suitable for studying how topographical features and mechanical cues from the environment influence the growth and development of moss. Here, we show that P. patens can be grown on fibrous scaffolds consisting of nanoscale, randomly oriented fibers composed of polyvinylidene tri‐fluoroethylene (NRP). The moss adheres tightly to NRP in contrast to the lack of adhesion to cellophane. Adhesion to the scaffold is associated with slower tip growth of moss protonema for some time, followed by an increase in tip growth rate that is equivalent to that on cellophane. In addition, the orientation of the first subapical cell division plane differs between NRP‐grown and cellophane‐grown protonema. Nonetheless, moss colonies grown on NRP did not show signs of nutrient or photosynthetic stress and developed normal gametophores. Together, these data establish NRP as a suitable substrate for the culture of P. patens and to probe the influence of mechanical forces on tip growth and cell division of moss. [ABSTRACT FROM AUTHOR]

Zinc (Zn) aqueous batteries have gained significant research interest for large‐scale electrochemical energy storage applications due to their rich abundance of raw materials, high energy density, environmental friendliness, low capital, and maintenance expenditure. However, the progress of metallic Zn anode in the aqueous Zn battery chemistry is hindered by several critical challenges, including low Coulombic efficiency and the formation of Zn dendrites, irrespective of the pH of the electrolyte. Herein, the application of porous activated carbon (AC) derived from waste corn husk as Zn anode constituent to improve the cyclability of the aqueous Zn battery is reported. In the full cell study, it is observed that the battery containing 15 wt% of AC as anode coupled with α‐MnO2 nanorods cathode shows excellent capacity retention of 86.42% (initial discharge capacity = 211.67 mAh g−1) after 125 charge/discharge cycles at current rate of 100 mA g−1. Moreover, herein, insights into converting waste to energy by utilizing the biowaste precursor to develop stable and durable rechargeable aqueous Zn‐based batteries are provided. [ABSTRACT FROM AUTHOR]

Epidermal cells of dark-grown plant seedlings reorient their cortical microtubule arrays in response to blue light from a net lateral orientation to a net longitudinal orientation with respect to the long axis of cells. The molecular mechanism underlying this microtubule array reorientation involves katanin, a microtubule severing enzyme, and a plant-specific microtubule associated protein called SPIRAL2. Katanin preferentially severs longitudinal microtubules, generating seeds that amplify the longitudinal array. Upon severing, SPIRAL2 binds nascent microtubule minus ends and limits their dynamics, thereby stabilizing the longitudinal array while the lateral array undergoes net depolymerization. To date, no experimental structural information is available for SPIRAL2 to help inform its mechanism. To gain insight into SPIRAL2 structure and function, we determined a 1.8 Å resolution crystal structure of the Arabidopsis thaliana SPIRAL2 C-terminal domain. The domain is composed of seven core α-helices, arranged in an α-solenoid. Amino-acid sequence conservation maps primarily to one face of the domain involving helices α1, α3, α5, and an extended loop, the α6-α7 loop. The domain fold is similar to, yet structurally distinct from the C-terminal domain of Ge-1 (an mRNA decapping complex factor involved in P-body localization) and, surprisingly, the C-terminal domain of the katanin p80 regulatory subunit. The katanin p80 C-terminal domain heterodimerizes with the MIT domain of the katanin p60 catalytic subunit, and in metazoans, binds the microtubule minus-end factors CAMSAP3 and ASPM. Structural analysis predicts that SPIRAL2 does not engage katanin p60 in a mode homologous to katanin p80. The SPIRAL2 structure highlights an interesting evolutionary convergence of domain architecture and microtubule minus-end localization between SPIRAL2 and katanin complexes, and establishes a foundation upon which structure-function analysis can be conducted to elucidate the role of this domain in the regulation of plant microtubule arrays. [ABSTRACT FROM AUTHOR]

Electrochemical conversion of biomass to value‐added chemicals has gained impetus in recent years. Herein, we present a methodology for recovering biomass‐derived 2‐furoic acid from the dilute aqueous stream by reactive extraction. The reactive extraction was performed using a chemical extractant, trioctylamine (TOA), with diluents (octanol, chloroform, and diethyl ether). Equilibrium parameters influencing the recovery of 2‐furoic acid were evaluated. Using TOA in various diluents, the 2‐furoic acid was recovered with 85%–99% efficiency. A 1:1 complex of the 2‐furoic acid—TOA was formed in the organic phase, and the experimental equilibrium complexation constant was compared with that obtained from the relative basicity and Langmuir models. The equilibrium parameters were used for column design to estimate the solvent to feed ratio (S/F) and the number of theoretical stages (NTS). The NTS required is 12 to attain 99% recovery of 2‐furoic acid in counter‐current extraction. The present study sheds light on the reactive extraction process adopted for process intensification with electrochemical conversion, paving the way for the commercialization of valuable products obtained from biomass. [ABSTRACT FROM AUTHOR]

Background: COVID-19 vaccines are vital tools in the defense against infection and serious disease due to SARS-CoV-2. There are many challenges to implementing mass vaccination campaigns for large, diverse populations from crafting vaccine promotion messages to reaching individuals in a timely and effective manner. During this unprecedented period, with COVID-19 mass vaccination campaigns essential for protecting vulnerable patient populations and attaining herd immunity, health care systems were faced with the dual challenges of vaccine outreach and distribution. Objective: The aim of this cross-sectional study was to assess the effectiveness of a COVID-19 vaccine text outreach approach for patients aged 65 years and older. Our goal was to determine whether this approach was successful in scheduling patients for COVID-19 vaccine appointments. Methods: We developed SMS text messages using the Tavoca platform. These messages informed patients of their vaccine eligibility and allowed them to indicate their interest in scheduling an appointment via a specific method (email or phone) or indicate their lack of interest in the vaccine. We tracked the status of these messages and how patients responded. Messages were sent to patients aged 65 years and older (N=30,826) at a nonprofit health care system in Washington, DC. Data were collected and examined from January 14 to May 10, 2021. Data were analyzed using multivariate multinomial and binary logistic regression models in SAS (version 9.4; SAS Institute Inc). Results: Approximately 57% of text messages were delivered to patients, but many messages received no response from patients (40%). Additionally, 42.1% (12,978/30,826) of messages were not delivered. Of the patients who expressed interest in the vaccine (2938/30,826, 9.5%), Black or African American patients preferred a phone call rather than an email for scheduling their appointment (odds ratio [OR] 1.69, 95% CI 1.29-2.21) compared to White patients. Patients aged 70-74 years were more likely to schedule an appointment (OR 1.38, 95% CI 1.01-1.89) than those aged 65-69 years, and Black or African American patients were more likely to schedule an appointment (OR 2.90, 95% CI 1.72-4.91) than White patients. Conclusions: This study provides insights into some advantages and challenges of using a text messaging vaccine outreach for patients aged 65 years and older. Lessons learned from this vaccine campaign underscore the importance of using multiple outreach methods and sharing of patient vaccination status between health systems, along with a patient-centered approach to address vaccine hesitancy and access issues. [ABSTRACT FROM AUTHOR]

Spatial heterogeneity in composition and organisation of the primary cell wall affects the mechanics of cellular morphogenesis. However, directly correlating cell wall composition, organisation and mechanics has been challenging. To overcome this barrier, we applied atomic forcemicroscopy coupled with infrared (AFM-IR) spectroscopy to generate spatially correlated maps of chemical and mechanical properties for paraformaldehyde-fixed, intact Arabidopsis thaliana epidermal cell walls. AFM-IR spectra were deconvoluted by non-negative matrix factorisation (NMF) into a linear combination of IR spectral factors representing sets of chemical groups comprising different cell wall components. This approach enables quantification of chemical composition from IR spectral signatures and visualisation of chemical heterogeneity at nanometer resolution. Cross-correlation analysis of the spatial distribution of NMFs and mechanical properties suggests that the carbohydrate composition of cell wall junctions correlates with increased local stiffness. Together, our work establishes new methodology to use AFM-IR for the mechanochemical analysis of intact plant primary cell walls. [ABSTRACT FROM AUTHOR]

The expanded availability of telehealth due to the COVID-19 pandemic presents a concern that telehealth may result in an unnecessary increase in utilization. We analyzed 4,114,651 primary care encounters (939,134 unique patients) from three healthcare systems between 2019 and 2021 and found little change in utilization as telehealth became widely available. Results suggest telehealth availability is not resulting in additional primary care visits and federal policies should support telehealth use. [ABSTRACT FROM AUTHOR]

Objective: Despite a proliferation of applications (apps) to conveniently collect patient-reported outcomes (PROs) from patients, PRO data are yet to be seamlessly integrated with electronic health records (EHRs) in a way that improves interoperability and scalability. We applied the newly created PRO standards from the Office of the National Coordinator for Health Information Technology to facilitate the collection and integration of standardized PRO data. A novel multitiered architecture was created to enable seamless integration of PRO data via Substitutable Medical Apps and Reusable Technologies on Fast Healthcare Interoperability Resources apps and scaled to different EHR platforms in multiple ambulatory settings.Materials and Methods: We used a standards-based approach to deploy 2 apps that source and surface PRO data in real-time for provider use within the EHR and which rely on PRO assessments from an external center to streamline app and EHR integration.Results: The apps were developed to enable patients to answer validated assessments (eg, a Patient-Reported Outcomes Measurement Information System including using a Computer Adaptive Test format). Both apps were developed to populate the EHR in real time using the Health Level Seven FHIR standard allowing providers to view patients' data during the clinical encounter. The process of implementing this architecture with 2 different apps across 18 ambulatory care sites and 3 different EHR platforms is described.Conclusion: Our approach and solution proved feasible, secure, and time- and resource-efficient. We offer actionable guidance for this technology to be scaled and adapted to promote adoption in diverse ambulatory care settings and across different EHRs. [ABSTRACT FROM AUTHOR]

The production of bio-oil from agricultural waste is a promising route to improve the agricultural value chain. Herein, furfural (FF), a model bio-oil compound, was subjected to electrocatalytic hydrogenation (ECH) in an alkaline medium to produce economically important furfuryl alcohol (FA) and hydrofuroin (HF). The selectivity of ECH products (FA and HF) on Cu, Pt, and Ni-foam electro-catalysts showed that their generation was dependent upon the availability of Hads, which in turn varied with the choice of electrocatalyst and applied potential. Cu–NPNi/NF was obtained through dealloying Cu from a co-electrodeposited Ni–Cu electrode on a Ni-foam substrate, followed by re-electrodeposition of Cu. A porous, high-surface-area bimetallic Ni–Cu catalyst (Cu–NPNi/NF) on Ni-foam yielded high rates of FA and HF generation from furfural, e.g. 118.7 ± 8 and 176.3 ± 3.4 μmol h−1 cm−2 at −1.45 V vs. Ag/AgCl/sat KCl after 1 h of electrolysis in an alkaline electrolyte. 100% conversion of furfural was observed after 2 h of electrolysis with the same catalyst. The high rate of FA and HF formation was ascribed to enhanced adsorbed FF because of the formation of Cu-nanoplates and bimetallic Ni–Cu. We have provided a rational, high-throughput design for preparing highly active nanoporous electrodes for producing industrially relevant chemicals (furfuryl alcohol and hydrofuroin). [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic has led to the rapid expansion of telehealth services as healthcare organizations aim to mitigate community transmission while providing safe patient care. As technology adoption rapidly increases, operational telehealth teams must maintain awareness of critical information, such as patient volumes and wait times, patient and provider experience, and telehealth platform performance. Using a model of situation awareness as a conceptual foundation and a user-centered design approach we describe our process for rapidly developing and disseminating dashboard visualizations to support telehealth operations. We used a 5-step process to gain domain knowledge, identify user needs, identify data sources, design and develop visualizations, and iteratively refine these visualizations. Through this process we identified 3 distinct stakeholder groups and designed and developed visualization dashboards to meet their needs. Feedback from users demonstrated the dashboard's support situation awareness and informed important operational decisions. Lessons learned are shared to provide other organizations with insights from our process. [ABSTRACT FROM AUTHOR]

The objective of this study is to quantify how long patients took to complete their rescheduled primary care appointment pre-pandemic (2019) and during an initial pandemic period (2020). In doing so, the study evaluates telehealth's role in helping primary care patients – particularly in patients with chronic conditions – withstand COVID's significant disruption in care. Cancelled and completed primary care appointments for adult patients were extracted from the beginning of the pandemic (March 1 to July 31, 2020) and a similar period pre-pandemic (March 1 to July 31, 2019). Days to the subsequent completed visit after cancellation (through June 30, 2021) and appointment modality (in-person, phone, video) were examined. Statistical testing was done to determine statistical significance, and a linear regression was run to control for effects of other study variables. Pre-pandemic patients with chronic conditions needed 52.3 days on average to reschedule their cancelled in-person appointment. During the early pandemic period, chronic condition patients who saw their provider in-person took on average 78.8 days. During the same pre-pandemic period, patients with chronic conditions had their average wait time decrease to 51.5 days when rescheduling via telehealth. These differences were similar for patients without chronic conditions. This analysis shows that telehealth created return to care timelines comparable to the pre-pandemic period which is especially important for patients with chronic conditions. Telehealth visits (i.e., talking with a physician via phone or video call) help patients continue to receive the medical care they need – especially during disruptive periods such as the COVID pandemic. Access to telehealth is the strongest predictor in determining how soon a patient will complete their reschedule primary care appointment. Because telehealth is so important, health care providers and systems need to continue to offer patients the ability to talk with their physician via phone or video call. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease. HCV cure has been linked to improved patient outcomes. In the era of direct-acting antivirals (DAAs), HCV cure has become the goal, as defined by sustained virological response 12 weeks (SVR12) after completion of therapy. Historically, African-Americans have had lower SVR12 rates compared to White people in the interferon era, which had been attributed to the high prevalence of non-CC interleukin 28B (IL28B) type. Less is known about the association between race/ethnicity and SVR12 in DAA-treated era. The aim of the study is to evaluate the predictors of SVR12 in a diverse, singlecenter Veterans Affairs population. We conducted a retrospective study of patients undergoing HCV therapy with DAAs from 2014 to 2016 at the VA Greater Los Angeles Healthcare System. We performed a multivariable logistic regression analysis to determine predictors of SVR12, adjusting for age, HCV genotype, DAA regimen and duration, human immunodeficiency virus (HIV) status, fibrosis, nonalcoholic fatty liver disease (NAFLD) fibrosis score, homelessness, mental health, and adherence. Our cohort included 1068 patients, out of which 401 (37.5%) were White people and 400 (37.5%) were African-American. Genotype 1 was the most common genotype (83.9%, N = 896). In the adjusted models, race/ethnicity and the presence of fibrosis were statistically significant predictors of non-SVR. African-Americans had 57% lower odds for reaching SVR12 (adj.OR = 0.43, 95% CI = 1.5-4.1) compared to White people. Advanced fibrosis (adj.OR = 0.40, 95% CI = 0.26-0.68) was also a significant predictor of non-SVR. In a single-center VA population on DAAs, African-Americans were less likely than White people to reach SVR12 when adjusting for covariates. [ABSTRACT FROM AUTHOR]

Kinesins and myosins are motor proteins that can move actively along microtubules and actin filaments, respectively. Plants have evolved a unique set of motors that function as regulators and organizers of the cytoskeleton and as drivers of long-distance transport of various cellular components. Recent progress has established the full complement of motors encoded in plant genomes and has revealed valuable insights into the cellular functions of many kinesin and myosin isoforms. Interestingly, several of the motors were found to functionally connect the two cytoskeletal systems and thereby to coordinate their activities. In this review, we discuss the available genetic, cell biological, and biochemical data for each of the plant kinesin and myosin families from the context of their subcellular mechanism of action as well as their physiological function in the whole plant. We particularly emphasize work that illustrates mechanisms by which kinesins and myosins coordinate the activities of the cytoskeletal system. [ABSTRACT FROM AUTHOR]

Objective: To present user needs and usability evaluations of DataMed, a Data Discovery Index (DDI) that allows searching for biomedical data from multiple sources.Materials and Methods: We conducted 2 phases of user studies. Phase 1 was a user needs analysis conducted before the development of DataMed, consisting of interviews with researchers. Phase 2 involved iterative usability evaluations of DataMed prototypes. We analyzed data qualitatively to document researchers' information and user interface needs.Results: Biomedical researchers' information needs in data discovery are complex, multidimensional, and shaped by their context, domain knowledge, and technical experience. User needs analyses validate the need for a DDI, while usability evaluations of DataMed show that even though aggregating metadata into a common search engine and applying traditional information retrieval tools are promising first steps, there remain challenges for DataMed due to incomplete metadata and the complexity of data discovery.Discussion: Biomedical data poses distinct problems for search when compared to websites or publications. Making data available is not enough to facilitate biomedical data discovery: new retrieval techniques and user interfaces are necessary for dataset exploration. Consistent, complete, and high-quality metadata are vital to enable this process.Conclusion: While available data and researchers' information needs are complex and heterogeneous, a successful DDI must meet those needs and fit into the processes of biomedical researchers. Research directions include formalizing researchers' information needs, standardizing overviews of data to facilitate relevance judgments, implementing user interfaces for concept-based searching, and developing evaluation methods for open-ended discovery systems such as DDIs. [ABSTRACT FROM AUTHOR]

Processivity is important for kinesins that mediate intracellular transport. Structure–function analyses of N-terminal kinesins (i.e. kinesins comprising their motor domains at the N-terminus) have identified several non-motor regions that affect processivity in vitro. However, whether these structural elements affect kinesin processivity and function in vivo is not known. Here, we used an Arabidopsis thaliana kinesin-4, called Fragile Fiber 1 (FRA1, also known as KIN4A), which is thought to mediate vesicle transport, to test whether mutations that alter processivity in vitro lead to similar changes in behavior in vivo and whether processivity is important for the function of FRA1. We generated several FRA1 mutants that differed in their ‘run lengths’ in vitro and then transformed them into the fra1-5 mutant for complementation and in vivo motility analyses. Our data show that the behavior of processivity mutants in vivo can differ dramatically from in vitro properties, underscoring the need to extend structure–function analyses of kinesins in vivo. In addition, we found that a high density of processive motility is necessary for the physiological function of FRA1. [ABSTRACT FROM AUTHOR]

Cellulose, often touted as themost abundant biopolymer on Earth, is a critical component of the plant cell wall and is synthesized by plasma membrane-spanning cellulose synthase (CESA) enzymes, which in plants are organized into rosette-like CESA complexes (CSCs). Plants construct two types of cell walls, primary cell walls (PCWs) and secondary cell walls (SCWs), which differ in composition, structure, and purpose. Cellulose in PCWs and SCWs is chemically identical but has different physical characteristics. During PCW synthesis, multiple dispersed CSCsmove along a shared linear track in opposing directions while synthesizing cellulose microfibrils with low aggregation. In contrast, during SCW synthesis, we observed swaths of densely arranged CSCs that moved in the same direction along tracks while synthesizing cellulose microfibrils that became highly aggregated. Our data support a model in which distinct spatiotemporal features of active CSCs during PCW and SCW synthesis contribute to the formation of cellulose with distinct structure and organization in PCWs and SCWs of Arabidopsis thaliana. This study provides a foundation for understanding differences in the formation, structure, and organization of cellulose in PCWs and SCWs. [ABSTRACT FROM AUTHOR]

One of the key enablers of shape and growth in plant cells is the cortical microtubule (CMT) system, which is a polymer array that forms an appropriately-structured scaffolding in each cell. Plant biologists have shown that stochastic dynamics and simple rules of interactions between CMTs can lead to a coaligned CMT array structure. However, the mechanisms and conditions that cause CMT arrays to become organized are not well understood. It is prohibitively time-consuming to use actual plants to study the effect of various genetic mutations and environmental conditions on CMT self-organization. In fact, even computer simulations with multiple replications are not fast enough due to the spatio-temporal complexity of the system. To redress this shortcoming, we develop analytical models and methods for expeditiously computing CMT system metrics that are related to self-organization and array structure. In particular, we formulate a mean-field model to derive sufficient conditions for the organization to occur. We show that growth-prone dynamics itself is sufficient to lead to organization in presence of interactions in the system. In addition, for such systems, we develop predictive methods for estimation of system metrics such as expected average length and number of CMTs over time, using a stochastic fluid-flow model, transient analysis, and approximation algorithms tailored to our problem. We illustrate the effectiveness of our approach through numerical test instances and discuss biological insights. [ABSTRACT FROM AUTHOR]

The cell wall consists of cellulose microfibrils embedded within a matrix of hemicellulose and pectin. Cellulose microfibrils are synthesized at the plasma membrane, whereas matrix polysaccharides are synthesized in the Golgi apparatus and secreted. The trafficking of vesicles containing cell wall components is thought to depend on actin-myosin. Here, we implicate microtubules in this process through studies of the kinesin-4 family member, Fragile Fiber1 (FRA1). In anfra1-5 knockout mutant, the expansion rate of the inflorescence stem is halved compared with the wild type along with the thickness of both primary and secondary cell walls. Nevertheless, cell walls in fra1-5 have an essentially unaltered composition and ultrastructure. A functional triple green fluorescent protein-tagged FRA1 fusion protein moves processively along cortical microtubules, and its abundance and motile density correlate with growth rate. Motility of FRA1 and cellulose synthase complexes is independent, indicating that FRA1 is not directly involved in cellulose biosynthesis; however, the secretion rate of fucose-alkyne-labeled pectin is greatly decreased in fra1-5, and the mutant has Golgi bodies with fewer cisternae and enlarged vesicles. Based on our results, we propose that FRA1 contributes to cell wall production by transporting Golgi-derived vesicles along cortical microtubules for secretion. [ABSTRACT FROM AUTHOR]

The interphase cortical microtubules ( CMTs) of plant cells form strikingly ordered arrays in the absence of a dedicated microtubule-organizing center. Considerable research effort has focused on activities such as bundling and severing that occur after CMT nucleation and are thought to be important for generating and maintaining ordered arrays. In this review, we focus on how nucleation affects CMT array organization. The bulk of CMTs are initiated from γ-tubulin-containing nucleation complexes localized to the lateral walls of pre-existing CMTs. These CMTs grow either at an acute angle or parallel to the pre-existing CMT. Although the impact of microtubule-dependent nucleation is not fully understood, recent genetic, live-cell imaging and computer simulation studies have demonstrated that the location, timing and geometry of CMT nucleation have a considerable impact on the organization and orientation of the CMT array. These nucleation properties are defined by the composition, position and dynamics of γ-tubulin-containing nucleation complexes, which represent control points for the cell to regulate CMT array organization. [ABSTRACT FROM AUTHOR]

Plants possess a large number of microtubule-based kinesin motor proteins. While the kinesin-2, 3, 9, and 11 families are absent from land plants, the kinesin-7 and 14 families are greatly expanded. In addition, some kinesins are specifically present only in land plants. The distinctive inventory of plant kinesins suggests that kinesins have evolved to perform specialized functions in plants. Plants assemble unique microtubule arrays during their cell cycle, including the interphase cortical microtubule array, preprophase band, anastral spindle and phragmoplast. In this review, we explore the functions of plant kinesins from a microtubule array viewpoint, focusing mainly on Arabidopsis kinesins. We emphasize the conserved and novel functions of plant kinesins in the organization and function of the different microtubule arrays. [ABSTRACT FROM AUTHOR]

The Arabidopsis FRA1 kinesin contributes to the organization of cellulose microfibrils through an unknown mechanism. The cortical localization of this kinesin during interphase raises the possibility that it transports cell wall-related cargoes along cortical microtubules that either directly or indirectly influence cellulose microfibril patterning. To determine whether FRA1 is an authentic motor protein, we combined bulk biochemical assays and single molecule fluorescence imaging to analyze the motor properties of recombinant, GFP-tagged FRA1 containing the motor and coiled-coil domains (designated as FRA1(707)–GFP). We found that FRA1(707)–GFP binds to microtubules in an ATP-dependent manner and that its ATPase activity is dramatically stimulated by the presence of microtubules. Using single molecule studies, we found that FRA1(707)–GFP moves processively along microtubule tracks at a velocity of about 0.4 μm s−1. In addition, we found that FRA1(707)–GFP is a microtubule plus-end-directed motor and that it moves along microtubules as a dimer. Interestingly, our single molecule analysis shows that the processivity of FRA1(707)–GFP is at least twice the processivity of conventional kinesin, making FRA1 the most processive kinesin to date. Together, our data show that FRA1 is a bona fide motor protein that has the potential to drive long-distance transport of cargo along cortical microtubules. [ABSTRACT FROM PUBLISHER]

Neuronal development, function and repair critically depend on axonal transport of vesicles and protein complexes, which is mediated in part by the molecular motor kinesin-1. Adaptor proteins recruit kinesin-1 to vesicles via direct association with kinesin heavy chain (KHC), the force-generating component, or via the accessory light chain (KLC). Binding of adaptors to the motor is believed to engage the motor for microtubule-based transport. We report that the adaptor protein Sunday Driver (syd, also known as JIP3 or JSAP1) interacts directly with KHC, in addition to and independently of its known interaction with KLC. Using an in vitro motility assay, we show that syd activates KHC for transport and enhances its motility, increasing both KHC velocity and run length. syd binding to KHC is functional in neurons, as syd mutants that bind KHC but not KLC are transported to axons and dendrites similarly to wild-type syd. This transport does not rely on syd oligomerization with itself or other JIP family members. These results establish syd as a positive regulator of kinesin activity and motility. [ABSTRACT FROM AUTHOR]

Retrograde axonal transport of cellular signals driven by dynein is vital for neuronal survival. Mouse models with defects in the retrograde transport machinery, including the Loa mouse (point mutation in dynein) and the Tgdynamitin mouse (overexpression of dynamitin), exhibit mild neurodegenerative disease. Transport defects have also been observed in more rapidly progressive neurodegeneration, such as that observed in the SOD1G93A transgenic mouse model for familial amyotrophic lateral sclerosis (ALS). Here, we test the hypothesis that alterations in retrograde signaling lead to neurodegeneration. In vivo, in vitro, and live-cell imaging motility assays show misregulation of transport and inhibition of retrograde signaling in the SOD1G93A model. However, similar inhibition is also seen in the Loa and Tgdynamitin mouse models. Thus, slowing of retrograde signaling leads only to mild degeneration and cannot explain ALS etiology. To further pursue this question, we used a proteomics approach to investigate dynein-associated retrograde signaling. These data indicate a significant decrease in retrograde survival factors, including P-Trk (phospho-Trk) and P-Erk1/2, and an increase in retrograde stress factor signaling, including P-JNK (phosphorylated c-Jun N-terminal kinase), caspase-8, and p75NTR cleavage fragment in the SOD1G93A model; similar changes are not seen in the Loa mouse. Cocultures of motor neurons and glia expressing mutant SOD1 (mSOD1) in compartmentalized chambers indicate that inhibition of retrograde stress signaling is sufficient to block activation of cellular stress pathways and to rescue motor neurons from mSOD1-induced toxicity. Hence, a shift from survival-promoting to death-promoting retrograde signaling may be key to the rapid onset of neurodegeneration seen in ALS. [ABSTRACT FROM AUTHOR]

Microtubules are polarized polymers that exhibit dynamic instability, with alternating phases of elongation and shortening, particularly at the more dynamic plus-end. Microtubule plus-end tracking proteins (+TlPs) localize to and track with growing microtubule plus-ends in the cell. +TlPs regulate microtubule dynamics and mediate interactions with other cellular components. The molecular mechanisms responsible for the +TIP tracking activity are not well understood, however. We reconstituted the +TIP tracking of mammalian proteins EB1 and CLIP-170 in vitro at single-molecule resolution using time-lapse total internal reflection fluorescence microscopy. We found that EB1 is capable of dynamically tracking growing microtubule plus-ends. Our singlemolecule studies demonstrate that EB1 exchanges rapidly at microtubule plus-ends with a dwell time of <1 s, indicating that single EB1 molecules go through multiple rounds of binding and dissociation during microtubule polymerization. CLIP-170 exhibits lattice diffusion and fails to selectively track microtubule ends in the absence of EB1; the addition of EB1 is both necessary and sufficient to mediate plus-end tracking by CLIP-170. Single-molecule analysis of the CLIP-170-EB1 complex also indicates a short dwell time at growing plus-ends, an observation inconsistent with the copolymerization of this complex with tubulin for plus-end-specific localization. GTP hydrolysis is required for +TIP tracking, because end-specificity is lost when tubulin is polymerized in the presence of guanosine 5'-[α,β-methylene]triphosphate (GMPCPP). Together, our data provide insight into the mechanisms driving plus-end tracking by mammalian +TIPs and suggest that EB1 specifically recognizes the distinct lattice structure at the growing microtubule end. [ABSTRACT FROM AUTHOR]

The intrinsically fluorescent proteins (IFPs), such as the green, cyan and yellow fluorescent proteins, have revolutionized how we can image the dynamics of cellular events. Intrinsically fluorescent proteins have been used as reporter genes to monitor transcriptional regulation, as targeted markers for organelles and subcellular structures, in fusion proteins to directly observe protein motility and dynamics, and in sensors designed to show changes in cellular environments ranging from pH to protein kinase activity. The IFPs hold tremendous potential to reveal the dynamic processes that underlie plant cell function; however, as with all technology there are artifacts and pitfalls inherent in their use. In this review, we highlight some of the practical issues in using IFPs for live cell imaging. These include choice of the appropriate IFP, dealing with autofluorescence, photobleaching and phototoxicity, and application of approaches such as fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging (FLIM) and fluorescence recovery after photobleaching (FRAP) to gain high-resolution data about protein dynamics within the cell. We also discuss some of the more common artifacts associated with these fluorescence imaging approaches and suggest controls that should help both spot these problems and suggest their solutions. [ABSTRACT FROM AUTHOR]

Ordered cortical microtubule arrays are essential for normal plant morphogenesis, but how these arrays form is unclear. The dynamics of individual cortical microtubules are stochastic and cannot fully account for the observed order; however, using tobacco (Nicotiana tabacum) cells expressing either the MBD-DsRed (microtubule binding domain of the mammalian MAP4 fused to the Discosoma sp red fluorescent protein) or YFP-TUA6 (yellow fluorescent protein fused to the Arabidopsis α-tubulin 6 isoform) microtubule markers, we identified intermicrotubule interactions that modify their stochastic behaviors. The intermicrotubule interactions occur when the growing plus-ends of cortical microtubules encounter previously existing cortical microtubules. Importantly, the outcome of such encounters depends on the angle at which they occur: steep-angle collisions are characterized by approximately sevenfold shorter microtubule contact times compared with shallow-angle encounters, and steep-angle collisions are twice as likely to result in microtubule depolymerization. Hence, steep-angle collisions promote microtubule destabilization, whereas shallow-angle encounters promote both microtubule stabilization and coalignment. Monte Carlo modeling of the behavior of simulated microtubules, according to the observed behavior of transverse and longitudinally oriented cortical microtubules in cells, reveals that these simple rules for intermicrotubule interactions are necessary and sufficient to facilitate the self-organization of dynamic microtubules into a parallel configuration. [ABSTRACT FROM AUTHOR]

The nuclear envelope (NE) acts as a selective barrier to macromolecule trafficking between the nucleus and the cytoplasm and undergoes a complex reorganization during mitosis. Different eukaryotic kingdoms show specializations in NE function and composition. In contrast with vertebrates, the protein composition of the NE and the function of NE proteins are barely understood in plants. MFP1 attachment factor 1 (MAF1) is a plant-specific NE-associated protein first identified in tomato (Lycopersicon esculentum). Here, we demonstrate that two Arabidopsis thaliana MAF1 homologs, WPP1 and WPP2, are associated with the NE specifically in undifferentiated cells of the root tip. Reentry into cell cycle after callus induction from differentiated root segments reprograms their NE association. Based on green fluorescent protein fusions and immunogold labeling data, the proteins are associated with the outer NE and the nuclear pores in interphase cells and with the immature cell plate during cytokinesis. RNA interference-based suppression of the Arabidopsis WPP family causes shorter primary roots, a reduced number of lateral roots, and reduced mitotic activity of the root meristem. Together, these data demonstrate the existence of regulated NE targeting in plants and identify a class of plant-specific NE proteins involved in mitotic activity. [ABSTRACT FROM AUTHOR]

Offers information on cortical microtubules (CMT). Significance of CMT to plant morphogenesis; Characteristics of CMT assembly dynamics; Regulation of CMT assembly dynamics; Link between CMT and cell elongation.

The green fluorescent protein (GFP) and other intrinsically fluorescent proteins (IFPs) are popular reporters because they allow visualization of cellular constituents in living specimens. IFP technology makes it possible to view dynamic processes in living cells, but extended observation, using fluorescence microscopy (both wide-field and confocal), can result in significant light energy exposure. Therefore, it is possible that cells experience light-induced damage that alters cell physiology and confounds observations. To understand the impact that extended viewing has on cells, we obtained quantitative information about the effect of light energy dose and observation conditions on tobacco BY-2 cell physiology. Our results show a non-linear relationship between the excitation light intensity and mitotic arrest, and the frequency of mitotic arrest is dependent on the presence of an IFP that absorbs the excitation light. Moreover, fluorescence microscopy induces the production of reactive oxygen species (ROS), as assayed using BY-2 cells loaded with oxidation-sensitive dyes, and the level of ROS production increases if the cells express an IFP that absorbs the excitation light energy. The dye oxidation follows sigmoidal kinetics and is reversible if the cells are exposed to low irradiation levels. In addition, the dye oxidation rate shows a non-linear relationship to the excitation light intensity, and a good correlation exists between photobleaching, mitotic arrest, and dye oxidation. The data highlight the importance of ROS scavenging for normal mitotic progression, and provide a reference for judiciously choosing conditions that avoid photobleaching that can lead to ROS accumulation and physiological damage. [ABSTRACT FROM AUTHOR]

SummaryThe preprophase band predicts the future cell division site. However, the mechanism of how a transient preprophase band fulfils this function is unknown. We have investigated the possibility that Golgi secretion might be involved in marking the preprophase band site. Observations on living BY-2 cells labeled for microtubules and Golgi stacks indicated an increased Golgi stack frequency at the preprophase band site. However, inhibition of Golgi secretion by brefeldin A during preprophase band formation did not prevent accurate phragmoplast fusion, and subsequent cell plate formation, at the preprophase band site. The results show that Golgi secretion does not mark the preprophase band site and thus does not play an active role in determination of the cell division site. [ABSTRACT FROM AUTHOR]

In crucifers, the ability of the stigma to differentially modulate hydration of pollen grains, depending on whether the pollen is recognized to be compatible or incompatible, represents a crucial stage in pollination. Our recent analysis of the mod mutation of Brassica, which results in a breakdown of the self-incompatibility response, led to the isolation of a gene linked to the MOD locus which is expressed at low levels in mod mutants. The gene is predicted to encode a plasma membrane-localized aquaporin-like protein and has been designated MIP-MOD. We utilized reporter gene analysis to demonstrate that the MIP-MOD promoter is active in Brassica papillar cells as well as in some vegetative tissues. The encoded protein is also likely to be plasma membrane-localized based on the observation that all plasma membrane-intrinsic aquaporin-like proteins in Brassica leaves are enriched in plasma membrane fractions. The MIP-MOD protein results in a low but measurable enhancement in osmotic water permeability of Xenopus oocytes and hence represents a functional aquaporin. The results are consistent with the notion that MIP-MOD is involved in the regulation of water transport across the stigma epidermal cell membrane. [ABSTRACT FROM AUTHOR]

Transgenic broccoli ( Brassica oleracea var. italica) was produced by two Agrobacterium tumefaciens-mediated transformation methods. One used flowering stalk explants from mature plants; the other used hypocotyl and petiole explants from in vitro-grown seedlings. Several hundred transformants containing a Bacillus thuringiensis ∂-endotoxin gene (CryIA(c)-type) and the neomycin phosphotransferase gene were recovered. Rooted transformants were obtained in as little as 3 months using seedling explants. Transgenic cabbage was also obtained by the seedling explant method. Parameters important for high efficiency regeneration and transformation rates included use of a tobacco nurse cell layer, sealing of petri dishes with a porous surgical tape instead of Parafilm, preculture of seedling explants and appropriate length of co-cultivation with Agrobacterium. Advantages and disadvantages of each transformation procedure are discussed. [ABSTRACT FROM AUTHOR]

The article discusses the function of plant kinesins containing a calponin homology domain (KCH) on key cellular processes. It says that the plant cytoskeleton is composed of actin microfilaments (AFs) and microtubules (MTs) which coexist in the cell. It talks about the role of cortical KCH during interphase, its importance during interphase, and its localization during cell division. It adds that actin is a significant regulator of KHC motor activity in vivo.