Lignin is an abundant and essential polymer in land plants. It is a prime factor in the recalcitrance of lignocellulosic biomass to agricultural and industrial end-uses such as forage, pulp and papermaking, and biofuels. To better understand lignification at the molecular level, we are developing a lignin spectroscopic and imaging toolbox on one "negligible" auxiliary. Toward that end, we describe the design, synthesis, and characterization of a new designer monolignol, 3-O-propargylcaffeyl alcohol, which contains a bioorthogonal alkynyl functional group at the 3-O-position. Importantly, our data indicate that this monolignol does not alter the fidelity of lignification. We demonstrate that the designer monolignol provides a platform for multiple spectroscopic and imaging approaches to reveal temporal and spatial details of lignification, knowledge of which is critical to reap the potential of energy-rich renewable plant biomass for sustainable liquid fuels and other diverse economic applications.
The ?-1,4-glucan chains comprising cellulose are synthesized by cellulose synthases in the plasma membranes of diverse organisms including bacteria and plants. Understanding structure-function relationships in the plant enzymes involved in cellulose synthesis (CESAs) is important because cellulose is the most abundant component in the plant cell wall, a key renewable biomaterial. Here, we explored the structure and function of the region encompassing transmembrane helices (TMHs) 5 and 6 in CESA using computational and genetic tools. Ab initio computational structure prediction revealed novel bi-modal structural conformations of the region between TMH5 and 6 that may affect CESA function. Here we present our computational findings on this region in three CESAs of Arabidopsis thaliana (AtCESA1, 3, and 6), the Atcesa3 (ixr1-2) mutant, and a novel missense mutation in AtCESA1. A newly engineered point mutation in AtCESA1 (Atcesa1 (F954L) ) that altered the structural conformation in silico resulted in a protein that was not fully functional in the temperature-sensitive Atcesa1 (rsw1-1) mutant at the restrictive temperature. The combination of computational and genetic results provides evidence that the ability of the TMH5-6 region to adopt specific structural conformations is important for CESA function.
The constituents of large, multisubunit protein complexes dictate their functions in cells, but determining their precise molecular makeup in vivo is challenging. One example of such a complex is the cellulose synthesis complex (CSC), which in plants synthesizes cellulose, the most abundant biopolymer on Earth. In growing plant cells, CSCs exist in the plasma membrane as six-lobed rosettes that contain at least three different cellulose synthase (CESA) isoforms, but the number and stoichiometry of CESAs in each CSC are unknown. To begin to address this question, we performed quantitative photobleaching of GFP-tagged AtCESA3-containing particles in living Arabidopsis thaliana cells using variable-angle epifluorescence microscopy and developed a set of information-based step detection procedures to estimate the number of GFP molecules in each particle. The step detection algorithms account for changes in signal variance due to changing numbers of fluorophores, and the subsequent analysis avoids common problems associated with fitting multiple Gaussian functions to binned histogram data. The analysis indicates that at least 10 GFP-AtCESA3 molecules can exist in each particle. These procedures can be applied to photobleaching data for any protein complex with large numbers of fluorescently tagged subunits, providing a new analytical tool with which to probe complex composition and stoichiometry.
We investigate how students connect explanations and arguments from evidence about plant growth and metabolism-two key practices described by the Next Generation Science Standards. This study reports analyses of interviews with 22 middle and high school students postinstruction, focusing on how their sense-making strategies led them to interpret-or misinterpret-scientific explanations and arguments from evidence. The principles of conservation of matter and energy can provide a framework for making sense of phenomena, but our results show that some students reasoned about plant growth as an action enabled by water, air, sunlight, and soil rather than a process of matter and energy transformation. These students reinterpreted the hypotheses and results of standard investigations of plant growth, such as van Helmont's experiment, to match their own understanding of how plants grow. Only the more advanced students consistently interpreted mass changes in plants or soil as evidence of movement of matter. We also observed that a higher degree of scaffolding during some of the interview questions allowed mid-level students to improve their responses. We describe our progress and challenges developing teaching materials with scaffolding to improve students' understanding of plant growth and metabolism.
Plant cell walls define cell shape during development and are composed of interlaced carbohydrate and protein networks. Fluorescent dyes have long been used to label plant cell walls, enabling optical microscopy-based interrogation of cell wall structure and composition. However, the specific cell wall components to which these dyes bind are often poorly defined. The availability of fluorescent compound libraries provides the potential to screen for and identify new fluorescent compounds that interact with specific plant cell wall components, enabling the study of cell wall architecture in intact, living tissues. Here, we describe a technique for screening fluorescent compound libraries for enhanced fluorescence upon interaction with plant cell walls, a secondary screening method to identify which cell wall components interact with a given dye, and a protocol for staining and observing Arabidopsis seedlings using a fluorescent cell wall-labeling dye. These methods have the potential to be applied to screening for differences in cell wall structure and composition among genetically diverse plant varieties or species.
Siganus insomnis sp. nov. is described from the Maldives, Sri Lanka and southern India. It most closely resembles S. lineatus (Valenciennes) from the Western Pacific but differs in coloration, principally in that most if not all of the bronze bands on its mid and upper sides continue horizontally and unbroken through to the nape and opercular slit. By contrast, in S. lineatus, typically the anterior area below the spinous dorsal fin down to the mid-sides is irregularly marked with golden bronze spots, commas, or a maze of contorted lines. S. guttatus (Bloch) is the third member of this group of sibling species; its sides are covered with orange to bronze-gold spots. It is distributed throughout S.E. Asia, i.e., it occupies a geographic position between the areas inhabited by S. lineatus and S. insomnis. Thus the gene pools of S. lineatus and S. insomnis are quarantined from one another by distance and the intervening presence of S. guttatus in S.E. Asia. The geographical separation of the populations of S. lineatus and S. insomnis from one another is reinforced by the absence of suitable, coralline habitats for these species in the western half of the Bay of Bengal.
Pectins are acidic carbohydrates that comprise a significant fraction of the primary walls of eudicotyledonous plant cells. They influence wall porosity and extensibility, thus controlling cell and organ growth during plant development. The regulated degradation of pectins is required for many cell separation events in plants, but the role of pectin degradation in cell expansion is poorly defined. Using an activation tag screen designed to isolate genes involved in wall expansion, we identified a gene encoding a putative polygalacturonase that, when overexpressed, resulted in enhanced hypocotyl elongation in etiolated Arabidopsis thaliana seedlings. We named this gene POLYGALACTURONASE INVOLVED IN EXPANSION1 (PGX1). Plants lacking PGX1 display reduced hypocotyl elongation that is complemented by transgenic PGX1 expression. PGX1 is expressed in expanding tissues throughout development, including seedlings, roots, leaves, and flowers. PGX1-GFP (green fluorescent protein) localizes to the apoplast, and heterologously expressed PGX1 displays in vitro polygalacturonase activity, supporting a function for this protein in apoplastic pectin degradation. Plants either overexpressing or lacking PGX1 display alterations in total polygalacturonase activity, pectin molecular mass, and wall composition and also display higher proportions of flowers with extra petals, suggesting PGX1's involvement in floral organ patterning. These results reveal new roles for polygalacturonases in plant development.
Permanent junctional reciprocating tachycardia (PJRT) is an uncommon form of supraventricular tachycardia in children. Treatment of this arrhythmia has been considered difficult because of a high medication failure rate and risk of cardiomyopathy. Outcomes in the current era of interventional treatment with catheter ablation have not been published.
Pectins are acidic sugar-containing polysaccharides that are universally conserved components of the primary cell walls of plants and modulate both tip and diffuse cell growth. However, many of their specific functions and the evolution of the genes responsible for producing and modifying them are incompletely understood. The moss Physcomitrella patens is emerging as a powerful model system for the study of plant cell walls. To identify deeply conserved pectin-related genes in Physcomitrella, we generated phylogenetic trees for 16 pectin-related gene families using sequences from ten plant genomes and analyzed the evolutionary relationships within these families.
Under Food and Drug Administration investigational device exemption, the Prospective Randomized On-X Anticoagulation Clinical Trial (PROACT) has been testing the safety of less aggressive anticoagulation than recommended by the American College of Cardiology/American Heart Association guidelines after implantation of an approved bileaflet mechanical valve.
Accurate identification of units for conservation is particularly challenging for marine species as obvious barriers to gene flow are generally lacking. Brydes whales (Balaenoptera spp.) are subject to multiple human-mediated stressors, including fisheries bycatch, ship strikes, and scientific whaling by Japan. For effective management, a clear understanding of how populations of each Brydes whale species/subspecies are genetically structured across their range is required. We conducted a population-level analysis of mtDNA control region sequences with 56 new samples from Oman, Maldives, and Bangladesh, plus published sequences from off Java and the Northwest Pacific. Nine diagnostic characters in the mitochondrial control region and a maximum parsimony phylogenetic analysis identified 2 genetically recognized subspecies of Brydes whale: the larger, offshore form, Balaenoptera edeni brydei, and the smaller, coastal form, Balaenoptera edeni edeni. Genetic diversity and differentiation indices, combined with a reconstructed maximum parsimony haplotype network, indicate strong differences in the genetic diversity and population structure within each subspecies. Discrete population units are identified for B. e. brydei in the Maldives, Java, and the Northwest Pacific and for B. e. edeni between the Northern Indian Ocean (Oman and Bangladesh) and the coastal waters of Japan.
Habitat selection is a fundamental aspect of animal ecology, the understanding of which is critical to management and conservation. Global positioning system data from animals allow fine-scale assessments of habitat selection and typically are analyzed in a use availability framework, whereby animal locations are contrasted with random locations (the availability sample). Although most use-availability methods are in fact spatial point process models, they often are fit using logistic regression. This framework offers numerous methodological challenges, for which the literature provides little guidance. Specifically, the size and spatial extent of the availability sample influences coefficient estimates potentially causing interpretational bias. We examined the influence of availability on statistical inference through simulations and analysis of serially correlated mule deer GPS data. Bias in estimates arose from incorrectly assessing and sampling the spatial extent of availability. Spatial autocorrelation in covariates, which is common for landscape characteristics, exacerbated the error in availability sampling leading to increased bias. These results have strong implications for habitat selection analyses using GPS data, which are increasingly prevalent in the literature. We recommend that researchers assess the sensitivity of their results to their availability sample and, where bias is likely, take care with interpretations and use cross validation to assess robustness.
Clathrin-mediated endocytosis (CME) is the best-characterized type of endocytosis in eukaryotic cells. Plants appear to possess all of the molecular components necessary to carry out CME; however, functional characterization of the components is still in its infancy. A yeast two-hybrid screen identified ?2 as a putative interaction partner of CELLULOSE SYNTHASE6 (CESA6). Arabidopsis (Arabidopsis thaliana) ?2 is homologous to the medium subunit 2 of the mammalian ADAPTOR PROTEIN COMPLEX2 (AP2). In mammals, the AP2 complex acts as the central hub of CME by docking to the plasma membrane while concomitantly recruiting cargo proteins, clathrin triskelia, and accessory proteins to the sites of endocytosis. We confirmed that ?2 interacts with multiple CESA proteins through the ?-homology domain of ?2, which is involved in specific interactions with endocytic cargo proteins in mammals. Consistent with its role in mediating the endocytosis of cargos at the plasma membrane, ?2-YELLOW FLUORESCENT PROTEIN localized to transient foci at the plasma membrane, and loss of ?2 resulted in defects in bulk endocytosis. Furthermore, loss of ?2 led to increased accumulation of YELLOW FLUORESCENT PROTEIN-CESA6 particles at the plasma membrane. Our results suggest that CESA represents a new class of CME cargo proteins and that plant cells might regulate cellulose synthesis by controlling the abundance of active CESA complexes at the plasma membrane through CME.
Numerous proteins contain domains that are enriched in glutamine and asparagine residues, and aggregation of some of these proteins has been linked to both prion formation in yeast and a number of human diseases. Unfortunately, predicting whether a given glutamine/asparagine-rich protein will aggregate has proven difficult. Here we describe a recently developed algorithm designed to predict the aggregation propensity of glutamine/asparagine-rich proteins. We discuss the basis for the algorithm, its limitations, and usage of recently developed online and downloadable versions of the algorithm.
Although it is well established that many glutamatergic neurons sequester Zn(2+) within their synaptic vesicles, the physiological significance of synaptic Zn(2+) remains poorly understood. In experiments performed in a Zn(2+)-enriched auditory brainstem nucleus--the dorsal cochlear nucleus--we discovered that synaptic Zn(2+) and GPR39, a putative metabotropic Zn(2+)-sensing receptor (mZnR), are necessary for triggering the synthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). The postsynaptic production of 2-AG, in turn, inhibits presynaptic probability of neurotransmitter release, thus shaping synaptic strength and short-term synaptic plasticity. Zn(2+)-induced inhibition of transmitter release is absent in mutant mice that lack either vesicular Zn(2+) or the mZnR. Moreover, mass spectrometry measurements of 2-AG levels reveal that Zn(2+)-mediated initiation of 2-AG synthesis is absent in mice lacking the mZnR. We reveal a previously unknown action of synaptic Zn(2+): synaptic Zn(2+) inhibits glutamate release by promoting 2-AG synthesis.
The systemic failure to detect early-stage ovarian cancer may be attributed to a significant amount of pelvic serous cancers arising from the fallopian tube rather than the ovarian surface epithelium. This article reviews the possibility of applying risk-reducing salpingectomy as a new paradigm for the prevention of pelvic serous cancer in both high- and low-risk women.
VAR2CSA, a member of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family, is a leading candidate for use in vaccines to protect first-time mothers from placental malaria (PM). VAR2CSA, which is comprised of a series of six Duffy binding-like (DBL) domains, binds chondroitin sulfate A (CSA) on placental syncytiotrophoblast. Several recombinant DBL domains have been shown to bind CSA. In order to identify and develop recombinant proteins suitable for clinical development, DBL2X and DBL3X, as well as their respective third subdomain (S3) from the FCR3 parasite clone, were expressed in Escherichia coli, refolded, and purified. All but DBL3X-S3 recombinant proteins bound to CSA expressed on Chinese hamster ovary (CHO)-K1 cells but not to CHO-pgsA745 cells, which are CSA negative as determined by flow cytometry. All but DBL3X-S3 bound to CSA on chondroitin sulfate proteoglycan (CSPG) as determined by surface plasmon resonance (SPR) analysis. Purified IgG from rats and rabbits immunized with these four recombinant proteins bound homologous and some heterologous parasite-infected erythrocytes (IE). Using a novel flow cytometry inhibition-of-binding assay (flow-IBA), antibodies against DBL3X-S3 inhibited 35% and 45% of IE binding to CSA on CHO-K1 cells compared to results for soluble CSA (sCSA) and purified multigravida (MG) IgG, respectively, from areas in Tanzania to which malaria is endemic. Antibodies generated against the other domains provided little or no inhibition of IE binding to CSA on CHO-K1 cells as determined by the flow cytometry inhibition-of-binding assay. These results demonstrate for the first time the ability to identify antibodies to VAR2CSA DBL domains and subdomains capable of inhibiting VAR2CSA parasite-IE binding to CSA by flow cytometry. The flow cytometry inhibition-of-binding assay was robust and provided an accurate, reproducible, and reliable means to identify blocking of IE binding to CSA and promises to be significant in the development of a vaccine to protect pregnant women.
Pectin is a component of the cell walls of plants that is composed of acidic sugar-containing backbones with neutral sugar-containing side chains. It functions in cell adhesion and wall hydration, and pectin crosslinking influences wall porosity and plant morphogenesis. Despite its low abundance in the secondary cell walls that make up the majority of lignocellulosic biomass, recent results have indicated that pectin influences secondary wall formation in addition to its roles in primary wall biosynthesis and modification. This mini-review will examine these and other recent results in the context of biomass yield and digestibility and discuss how these traits might be enhanced by the genetic and molecular modification of pectin. The utility of pectin as a high-value, renewable biomass co-product will also be highlighted.
Migration is an adaptive strategy that enables animals to enhance resource availability and reduce risk of predation at a broad geographic scale. Ungulate migrations generally occur along traditional routes, many of which have been disrupted by anthropogenic disturbances. Spring migration in ungulates is of particular importance for conservation planning, because it is closely coupled with timing of parturition. The degree to which oil and gas development affects migratory patterns, and whether ungulate migration is sufficiently plastic to compensate for such changes, warrants additional study to better understand this critical conservation issue.
Local hyperconnectivity in the neocortex is a hypothesized pathophysiological state in autism spectrum disorder (ASD). MET, a receptor tyrosine kinase that regulates dendrite and spine morphogenesis, has been established as a risk gene for ASD. Here, we analyzed the synaptic circuit organization of identified pyramidal neurons in the anterior frontal cortex of mice with a dorsal pallium-derived, conditional knock-out (cKO) of Met. Synaptic mapping by glutamate uncaging identified layer 2/3 as the main source of local excitatory input to layer 5 projection neurons in controls. In both cKO and heterozygotes, this pathway was stronger by a factor of approximately 2. This increase was both sublayer and projection-class specific, restricted to corticostriatal neurons in upper layer 5B and not neighboring corticopontine neurons. Paired recordings in cKO slices demonstrated increased unitary connectivity. We propose that excitatory hyperconnectivity in specific neocortical microcircuits constitutes a physiological basis for Met-mediated ASD risk.
Eleven channels of EEG were recorded from a subject performing four mental tasks. A time-embedded representation of the untransformed EEG samples was constructed. Classification of the time-embedded samples was performed by linear and quadratic discriminant analysis and by an artificial neural network. A classifiers output for consecutive samples is combined to increase reliability. A new performance measure is defined as the number of correct selections that would be made by a brain-computer interface (BCI) user of the system, accounting for the need for an incorrect selection to be followed by a correct one to delete the previous selection. A best result of 0.32 correct selections s(-1) (about 3 s per BCI decision) was obtained with a neural network using a time-embedding dimension of 50.
This paper reviews several critical issues facing signal processing for brain-computer interfaces (BCIs) and suggests several recent approaches that should be further examined. The topics were selected based on discussions held during the 4th International BCI Meeting at a workshop organized to review and evaluate the current state of, and issues relevant to, feature extraction and translation of field potentials for BCIs. The topics presented in this paper include the relationship between electroencephalography and electrocorticography, novel features for performance prediction, time-embedded signal representations, phase information, signal non-stationarity, and unsupervised adaptation.
Objective. To compare Visual Analogue Scale (VAS) scores with overall postoperative pain medication requirements including cumulative dose and patterns of medication utilization and to determine whether VAS scores predict pain medication utilization. Methods. VAS scores and pain medication data were collected from participants in a randomized trial of the utility of phenazopyridine for improved pain control following gynecologic surgery. Results. The mean age of the 219 participants was 54 (range19 to 94). We did not detect any association between VAS and pain medication utilization for patient-controlled anesthesia (PCA) or RN administered (intravenous or oral) medications. We also did not detect any association between the number of VAS scores recorded and mean pain scores. Conclusion. Postoperative VAS scores do not predict pain medication use in catheterized women inpatients following gynecologic surgery. Increased pain severity, as reflected by higher VAS scores, is not associated with an increase in pain assessment. Our findings suggest that VAS scores are of limited utility for optimal pain control. Alternative or complimentary methods may improve pain management.
Inhibition of parasite growth is a major objective of blood-stage malaria vaccines. The in vitro assay of parasite growth inhibitory activity (GIA) is widely used as a surrogate marker for malaria vaccine efficacy in the down-selection of candidate blood-stage vaccines. Here we report the first study to examine the relationship between in vivo Plasmodium falciparum growth rates and in vitro GIA in humans experimentally infected with blood-stage malaria.
Current recommendations discourage elective radiofrequency ablation in patients <5 years old and/or weighing <15 kg, primarily because of the greater complication rate. To describe the current use, complications, and immediate outcomes of cryoablation in this patient population, a multicenter retrospective review of all patients <5 years old and/or weighing <15 kg who were treated with cryoablation for arrhythmia was performed. Eleven centers contributed data for 68 procedures on 61 patients. Of those, 34% were elective and 24% (n = 16) were both cryoablation and radiofrequency ablation. The median age and weight at ablation was 3.5 years (range 8 days to 9.9 years) and 15.2 kg (range 2.3 to 23), respectively. Congenital heart disease was present in 23% of the patients. The immediate success rate of cryoablation alone was 74%. No major complications occurred with cryoablation only; however, 2 of the 16 patients who underwent cryoablation and radiofrequency ablation had major complications. Of the 50 patients receiving cryoablation, 8 (16%) had variable degrees of transient atrioventricular block. The recurrence rate was 20% after cryoablation and 30% after cryoablation plus radiofrequency ablation. In conclusion, cryoablation appears to have a high safety profile in these patients. Compared to older and larger patients, the efficacy of cryoablation in this small, young population was lower and the recurrence rates were higher. Cryoablations effect on the coronary arteries has not been fully elucidated and requires additional research.
Over a 2-year study period, aerobic capacity was measured at time of hire for 1419 delivery drivers. Injury experience and tenure were then monitored for these new-hires during that same period. Number of strain injuries, time to first strain and time to termination were regressed on aerobic capacity adjusting for tenure. Statistically significant, monotonically changing relationships were found for all three outcome variables. A unit increase in aerobic capacity was predicted to result in a 3.7% decrease in injury rate and a 1.1% decrease in risk of termination. When age was included in the model for time to termination, aerobic capacity was no longer a significant predictor. The findings regarding injury experience and aerobic capacity support National Institute for Occupational Safety and Health recommendations that individuals should work at no more than 21-30% of their aerobic capacity. STATEMENT OF RELEVANCE: Knowledge of the nature of the relationship between aerobic capacity, injury experience and retention allows the ergonomist to determine whether there is a point of diminishing returns in intervention effectiveness for higher levels of aerobic capacity.
The sphingosine kinases (sphingosine kinase-1 and -2) have been implicated in a variety of physiological functions. Discerning their mechanism of action is complicated because in addition to producing the potent lipid second messenger sphingosine-1-phosphate, sphingosine kinases, both by producing sphingosine-1-phosphate and consuming sphingosine, have profound effects on sphingolipid metabolism. Sphingosine kinase-1 translocates to the plasma membrane upon agonist stimulation and this translocation is essential for the pro-oncogenic properties of this enzyme. Many of the enzymes of sphingolipid metabolism, including the enzymes that degrade sphingosine-1-phosphate, are membrane bound with restricted subcellular distributions. In the work described here we explore how subcellular localization of sphingosine kinase-1 affects the downstream metabolism of sphingosine-1-phosphate and the access of sphingosine kinase to its substrates. We find, surprisingly, that restricting sphingosine kinase to either the plasma membrane or the endoplasmic reticulum has a negligible effect on the rate of degradation of the sphingosine-1-phosphate that is produced. This suggests that sphingosine-1-phosphate is rapidly transported between membranes. However we also find that cytosolic or endoplasmic-reticulum targeted sphingosine kinase expressed at elevated levels produces extremely high levels of dihydrosphingosine-1-phosphate. Dihydrosphingosine is a proximal precursor in ceramide biosynthesis. Our data indicate that sphingosine kinase can divert substrate from the ceramide de novo synthesis pathway. However plasma membrane-restricted sphingosine kinase cannot access the pool of dihydrosphingosine. Therefore whereas sphingosine kinase localization does not affect downstream metabolism of sphingosine-1-phosphate, localization has an important effect on the pools of substrate to which this key signaling enzyme has access.
Filamentous fungi secrete a wide range of enzymes, including cellulases and hemicellulases, with potential applications in the production of lignocellulosic biofuels. Of the cellulolytic fungi, Hypocrea jecorina (anamorph Trichoderma reesei) is the best characterized in terms of cellulose degradation, but other cellulolytic fungi, such as the model filamentous fungus Neurospora crassa, can serve a crucial role in building our knowledge about the fungal response to biomass due to the many molecular and genetic tools available for this organism. Here we cloned and expressed GH5-1 (NCU00762), a secreted endoglucanase in N. crassa. The protein was produced using a ccg-1 promoter under conditions in which no other cellulases are present. Native GH5-1 (nGH5-1) and this recombinant GH5-1 (rGH5-1) were purified to gauge differences in glycosylation and activity; both rGH5-1 and nGH5-1 were similarly glycosylated, with an estimated molecular weight of 52kDa. On azo-carboxymethylcellulose, rGH5-1 activity was equal to that of nGH5-1, and on cellulose (Avicel) rGH5-1 was 20% more active. The activity of a GH5-1-GFP fusion protein (rGH5-1-GFP-6xHis) was similar to rGH5-1 and nGH5-1. To determine the binding pattern of catalytically active rGH5-1-GFP-6xHis to plant cell walls, Arabidopsis seedlings were incubated with rGH5-1-GFP-6xHis or Pontamine Fast Scarlet 4B (S4B), a cellulose-specific dye. Confocal microscopy showed that rGH5-1-GFP-6xHis bound in linear, longitudinal patterns on seedling roots, similar to S4B. The functional expression and characterization of rGH5-1 and its GFP fusion derivative set important precedents for further investigation of biomass degradation by filamentous fungi, especially N. crassa, with applications for characterization and manipulation of novel enzymes.
Development of a fully effective vaccine against the pre-erythrocytic stage of malaria infection will likely require induction of both humoral and cellular immune responses. Protein based vaccines can elicit such broad-based immunity depending on the adjuvant and how the protein is formulated. Here to assess these variables, non human primates (NHP) were immunized three times with Plasmodium falciparum (Pf) circumsporozoite protein (CSP) or CSP cloned into MG38, a monoclonal antibody that targets DEC-205 (?DEC-CSP), an endocytic receptor on dendritic cells (DCs). Both vaccines were administered with or without poly(I:C) as adjuvant. Following three immunizations, the magnitude and quality of cytokine secreting CD4+ T cells were comparable between CSP+poly(I:C) and ?DEC-CSP+poly(I:C) groups with both regimens eliciting multi-functional cytokine responses. However, NHP immunized with CSP+poly(I:C) had significantly higher serum titers of CSP-specific IgG antibodies and indirect immunofluorescent antibody (IFA) titers against Pf sporozoites. Furthermore, sera from both CSP or ?DEC-CSP+poly(I:C) immunized animals limited sporozoite invasion of a hepatocyte cell line (HC04) in vitro. To determine whether CSP-specific responses could be enhanced, all NHP primed with CSP or ?DEC-CSP+poly(I:C) were boosted with a single dose of 150,000 irradiated Pf sporozoites (PfSPZ) intravenously. Remarkably, boosting had no effect on the CSP-specific immunity. Finally, immunization with CSP+poly-ICLC reduced malaria parasite burden in the liver in an experimental mouse model. Taken together, these data showing that poly(I:C) is an effective adjuvant for inducing potent antibody and Th1 immunity with CSP based vaccines offers a potential alternative to the existing protein based pre-erythrocytic vaccines.
Transgender youth experience negative school environments and may not benefit directly from interventions defined to support Lesbian, Gay and Bisexual (LGB) youth. This study utilized a multi-method approach to consider the issues that transgender students encounter in school environments. Using data from two studies, survey data (total n = 2260, 68 transgender youth) from study 1 and focus groups (n = 35) from study 2, we examine transgender youths experience of school harassment, school strategies implemented to reduce harassment, the protective role of supportive school personnel, and individual responses to harassment, including dropping out and changing schools. In both studies, we found that school harassment due to transgender identity was pervasive, and this harassment was negatively associated with feelings of safety. When schools took action to reduce harassment, students reported greater connections to school personnel. Those connections were associated with greater feelings of safety. The indirect effects of school strategies to reduce harassment on feelings of safety through connection to adults were also significant. Focus group data illuminate specific processes schools can engage in to benefit youth, and how the youth experience those interventions.
Sphingosine kinase 1 (SK1) produces sphingosine-1-phosphate (S1P), a potent signaling lipid. The subcellular localization of SK1 can dictate its signaling function. Here, we use artificial targeting of SK1 to either the plasma membrane (PM) or the endoplasmic reticulum (ER) to test the effects of compartmentalization of SK1 on substrate utilization and downstream metabolism of S1P. Expression of untargeted or ER-targeted SK1, but surprisingly not PM-targeted SK1, results in a dramatic increase in the phosphorylation of dihydrosphingosine, a metabolic precursor in de novo ceramide synthesis. Conversely, knockdown of endogenous SK1 diminishes both dihydrosphingosine-1-phosphate and S1P levels. We tested the effects of SK1 localization on degradation of S1P by depletion of the ER-localized S1P phosphatases and lyase. Remarkably, S1P produced at the PM was degraded to the same extent as that produced in the ER. This indicates that there is an efficient mechanism for the transport of S1P from the PM to the ER. In acute labeling experiments, we find that S1P degradation is primarily driven by lyase cleavage of S1P. Counterintuitively, when S1P-specific phosphatases are depleted, acute labeling of S1P is significantly reduced, indicative of a phosphatase-dependent recycling process. We conclude that the localization of SK1 influences the substrate pools that it has access to and that S1P can rapidly translocate from the site where it is synthesized to other intracellular sites.
Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel that is present in a variety of epithelial cell types, and usually expressed in the luminal membrane. In contrast, prestin (SLC26A5) is a voltage-dependent motor protein, which is present in the basolateral membrane of cochlear outer hair cells (OHCs), and plays an important role in the frequency selectivity and sensitivity of mammalian hearing. By using in situ hybridization and immunofluorescence, we found that both mRNA and protein of CFTR are present in OHCs, and that CFTR localizes in both the apical and the lateral membranes. CFTR was not detected in the lateral membrane of inner hair cells (IHCs) or in that of OHCs derived from prestin-knockout mice, i.e., in instances where prestin is not expressed. These results suggest that prestin may interact physically with CFTR in the lateral membrane of OHCs. Immunoprecipitation experiments confirmed a prestin-CFTR interaction. Because chloride is important for prestin function and for the efferent-mediated inhibition of cochlear output, the prestin-directed localization of CFTR to the lateral membrane of OHCs has a potential physiological significance. Aside from its role as a chloride channel, CFTR is known as a regulator of multiple protein functions, including those of the solute carrier family 26 (SLC26). Because prestin is in the SLC26 family, several members of which interact with CFTR, we explored the potential modulatory relationship associated with a direct, physical interaction between prestin and CFTR. Electrophysiological experiments demonstrated that cAMP-activated CFTR is capable of enhancing voltage-dependent charge displacement, a signature of OHC motility, whereas prestin does not affect the chloride conductance of CFTR.
It was recently reported that when mosquitoes infected with P. berghei sporozoites feed on mice, they deposit approximately 100-300 sporozoites in the dermis. When we inoculate P. yoelii (Py) sporozoites intravenously (IV) into BALB/c mice, the 50% infectious dose (ID(50)) is often less than 3 sporozoites, indicating that essentially all Py sporozoites in salivary glands are infectious. Thus, it should only take the bite of one infected mosquito to infect 100% of mice. In human subjects, it takes the bite of at least 5 P. falciparum-infected mosquitoes to achieve 100% blood stage infection. Exposure to 1-2 infected mosquitoes only leads to blood stage infection in approximately 50% of subjects. If mosquitoes carrying Py sporozoites inoculate 100-300 sporozoites per bite, and 1 to 2 mosquito bites achieve 50% blood stage infection rates, then this would suggest that the majority of sporozoites inoculated by mosquitoes into the dermis are not responsible for a productive infection, or that a significant number of sporozoite-infected mosquitoes do not inoculate any sporozoites. The objective of this study was to determine if this is the case. We therefore studied the infectivity to mice of the bites of 1, 2, 4, or 5-8 Py-infected mosquitoes. The bite of one Py sporozoite-infected mosquito caused blood stage infection in 41.4% (12/29) of mice, two bites infected 66.7% (22/33), four bites infected 75% (18/24), and five to eight bites infected 100% (21/21). These findings demonstrate that inoculation of sporozoites by mosquito bite is much less efficient than IV inoculation of Py sporozoites by needle and syringe. Such data may have implications for determining the best route and dose of administration to humans of our attenuated P. falciparum sporozoite vaccine, the scientific basis of which is immunity by bites from irradiated infected mosquitoes, and suggest that the challenge is to develop a method of administration that approximates IV inoculation, not one that mimics mosquito bite.
The mammalian motor system is organized around distinct subcortical subsystems, suggesting that the intracortical circuits immediately upstream of spinal cord and basal ganglia might be functionally differentiated as well. We found that the main excitatory pathway in mouse motor cortex, layer 2/3-->5, is fractionated into distinct pathways targeting corticospinal and corticostriatal neurons, which are involved in motor control. However, connections were selective for neurons in certain sublayers: corticospinal neurons in upper layer 5B and corticostriatal neurons in lower 5A. A simple structural combinatorial principle accounts for this highly specific functional circuit architecture: potential connectivity is established by neuronal sublayer positioning and actual connectivity in this framework is determined by long-range axonal projection targets. Thus, intracortical circuits of these pyramidal neurons are specified not only by their long-range axonal targets or their layer or sublayer positions, but by both, in specific combinations.
Patterns in electroencephalogram (EEG) signals are analyzed for a Brain Computer Interface (BCI). An important aspect of this analysis is the work on transformations of high dimensional EEG data to low dimensional spaces in which we can classify the data according to mental tasks being performed. In this research we investigate how a Neural Network (NN) in an auto-encoder with bottleneck configuration can find such a transformation. We implemented two approximate second-order methods to optimize the weights of these networks, because the more common first-order methods are very slow to converge for networks like these with more than three layers of computational units. The resulting non-linear projections of time embedded EEG signals show interesting separations that are related to tasks. The bottleneck networks do indeed discover nonlinear transformations to low-dimensional spaces that capture much of the information present in EEG signals. However, the resulting low-dimensional representations do not improve classification rates beyond what is possible using Quadratic Discriminant Analysis (QDA) on the original time-lagged EEG.
Cellulose forms the major load-bearing network of the plant cell wall, which simultaneously protects the cell and directs its growth. Although the process of cellulose synthesis has been observed, little is known about the behavior of cellulose in the wall after synthesis. Using Pontamine Fast Scarlet 4B, a dye that fluoresces preferentially in the presence of cellulose and has excitation and emission wavelengths suitable for confocal microscopy, we imaged the architecture and dynamics of cellulose in the cell walls of expanding root cells. We found that cellulose exists in Arabidopsis (Arabidopsis thaliana) cell walls in large fibrillar bundles that vary in orientation. During anisotropic wall expansion in wild-type plants, we observed that these cellulose bundles rotate in a transverse to longitudinal direction. We also found that cellulose organization is significantly altered in mutants lacking either a cellulose synthase subunit or two xyloglucan xylosyltransferase isoforms. Our results support a model in which cellulose is deposited transversely to accommodate longitudinal cell expansion and reoriented during expansion to generate a cell wall that is fortified against strain from any direction.
A chimera is a genetic composite containing a unique mix of cells derived from more than one zygote. This mouse model allows one to learn how cells of contrasting genotype functionally interact in vivo. Here, we investigate the effect that different proportions of prestin-containing outer hair cells (OHC) have on cochlear amplification. To address this issue, we developed a prestin chimeric mouse in which both ROSA26 wild-type (WT) and prestin knock-out (KO) genotypes are present in a single cochlea. The WT ROSA26 mice express a cell marker, allowing one to identify cells originating from the WT genome. Examination of cochlear tissue indicated that prestin chimeric mice demonstrate a mosaic in which mutant and normal OHCs interleave along the cochlear partition, similar to all other chimeric mouse models. The anatomical distribution of prestin-containing OHCs was compared with physiological data including thresholds and tuning curves for the compound action potential (CAP) recorded in anesthetized mice. Analysis of these measures did not reveal mixed phenotypes in which the distribution of prestin-containing OHCs impacted sensitivity and frequency selectivity to different degrees. However, by reducing the number of prestin-containing OHCs, phenotypes intermediate between WT and KO response patterns were obtained. Accordingly, we demonstrate a proportional reduction in sensitivity and in the tip length of CAP tuning curves as the number of OHCs derived from the KO genome increases; i.e., genotype ratio and phenotype are closely related.
Control of infection caused by Leishmania major requires the development of IFN-gamma+CD4+ lymphocytes for the induction of microbicidal activity in host macrophages. We recently reported on the inability of conventionally resistant C57BL/6 mice to successfully resolve infection by an isolate of L. major, despite a strong IFN-gamma response by the host. Susceptibility was caused by Ag-specific IL-10 from CD4+ cells that were also producing IFN-gamma. In the present studies, we have explored the role for IL-27 in the regulation of IL-10 from Th1 cells in leishmaniasis. Cytokine analysis of CD4+ cells in the lesions and draining lymph nodes of infected IL-27R-deficient (WSX-1(-/-)) mice revealed diminished IL-10 from IFN-gamma+ CD4+ cells, which was accompanied by a reduction in total IFN-gamma+CD4+ cells and an increase in IL-4. Despite the inhibition of IL-10 from CD4+ cells, no significant change in parasite numbers was observed, due both to the shift in the Th1/Th2 balance and to residual levels of IL-10. Strikingly, infected WSX-1(-/-) mice developed more severe lesions that were associated with the appearance of IL-17+ CD4+ cells, demonstrating a function for IL-27 in blocking the development of inappropriate Th17 cells during L. major infection. The results demonstrate the pleiotropic effects that IL-27 has on L. major-driven Th1, Th2, and Th17 development, and reinforce its function as a key regulatory cytokine that controls the balance between immunity and pathology.
A multiplicative combination of tuning to interaural time difference (ITD) and interaural level difference (ILD) contributes to the generation of spatially selective auditory neurons in the owls midbrain. Previous analyses of multiplicative responses in the owl have not taken into consideration the frequency-dependence of ITD and ILD cues that occur under natural listening conditions. Here, we present a model for the responses of ITD- and ILD-sensitive neurons in the barn owls inferior colliculus which satisfies constraints raised by experimental data on frequency convergence, multiplicative interaction of ITD and ILD, and response properties of afferent neurons. We propose that multiplication between ITD- and ILD-dependent signals occurs only within frequency channels and that frequency integration occurs using a linear-threshold mechanism. The model reproduces the experimentally observed nonlinear responses to ITD and ILD in the inferior colliculus, with greater accuracy than previous models. We show that linear-threshold frequency integration allows the system to represent multiple sound sources with natural sound localization cues, whereas multiplicative frequency integration does not. Nonlinear responses in the owls inferior colliculus can thus be generated using a combination of cellular and network mechanisms, showing that multiple elements of previous theories can be combined in a single system.
We dissociated the contributions to learning of four corticostriatal "loops" (interacting striatal and cortical regions): motor (putamen and motor cortex), visual (posterior caudate and visual cortex), executive (anterior caudate and prefrontal cortex), and motivational (ventral striatum and ventromedial frontal cortex). Subjects learned to categorize individual repeated images into one of two arbitrary categories via trial and error. We identified (1) regions sensitive to correct categorization, categorization learning, and feedback valence; (2) regions sensitive to prediction error (violation of feedback expectancy) and reward prediction (expected feedback associated with category response) using reinforcement learning modeling; and (3) directed influences between regions using Granger causality modeling. Each loop showed a unique pattern of sensitivity to each of these factors. Both the motor and visual loops were involved in acquisition of categorization ability: activity during correct categorization increased across learning and was sensitive to reward prediction. However, the posterior caudate received directed influence from visual cortex, whereas the putamen exerted directed influence on motor cortex. The motivational and executive loops were involved in feedback processing: both regions were sensitive to feedback valence, which interacted with learning across scans. However, the motivational loop activity reflected prediction error, whereas executive loop activity reflected reward prediction, consistent with the executive loop role in integrating reward and action. Granger causality modeling found directed influences between striatal and cortical regions within each loop. Across loops, the motor loop exerted directed influence on the executive loop which is consistent with the role of the executive loop in integrating feedback with stimulus-response history.
Although outer hair cells (OHCs) play a key role in cochlear amplification, it is not fully understood how they amplify sound signals by more than 100 fold. Two competing or possibly complementary mechanisms, stereocilia-based and somatic electromotility-based amplification, have been considered. Lacking knowledge about the exceptionally rich protein networks in the OHC plasma membrane, as well as related protein-protein interactions, limits our understanding of cochlear function. Therefore, we focused on finding protein partners for two important membrane proteins: Cadherin 23 (cdh23) and prestin. Cdh23 is one of the tip-link proteins involved in transducer function, a key component of mechanoelectrical transduction and stereocilia-based amplification. Prestin is a basolateral membrane protein responsible for OHC somatic electromotility.
Primary cilia are microtubule-based sensory organelles that play important roles in development and disease . They are required for Sonic hedgehog (Shh) and platelet-derived growth factor (PDGF) signaling. Primary cilia grow from the older of the two centrioles of the centrosome, referred to as the mother centriole. In cycling cells, the cilium typically grows in G1 and is lost before mitosis, but the regulation of its growth is poorly understood. Centriole duplication at G1/S results in two centrosomes, one with an older mother centriole and one with a new mother centriole, that are segregated in mitosis. Here we report that primary cilia grow asynchronously in sister cells resulting from a mitotic division and that the sister cell receiving the older mother centriole usually grows a primary cilium first. We also show that the signaling proteins inversin and PDGFRalpha localize asynchronously to sister cell primary cilia and that sister cells respond asymmetrically to Shh. These results suggest that the segregation of differently aged mother centrioles, an asymmetry inherent to every animal cell division, can influence the ability of sister cells to respond to environmental signals, potentially altering the behavior or fate of one or both sister cells.
In this study, we have improved upon the P300 speller Brain-Computer Interface paradigm by introducing a new character encoding method. Our concept in detection of the intended character is not based on a classification of target and nontarget responses, but based on an identifaction of the character which maximize the difference between P300 amplitudes in target and nontarget stimuli. Each bit included in the code corresponds to flashing character, 1, and non-flashing, 0. Here, the codes were constructed in order to maximize the minimum hamming distance between the characters. Electroencephalography was used to identify the characters using a waveform calculated by adding and subtracting the response of the target and non-target stimulus according the codes respectively. This stimulus presentation method was applied to a 3×3 character matrix, and the results were compared with that of a conventional P300 speller of the same size. Our method reduced the time until the correct character was obtained by 24%.
Large numbers of the Globe Skimmer dragonfly (Pantala flavescens) appear in the Maldives every October-December. Since they cannot breed on these largely waterless islands, it has recently been suggested that they are "falling out" during a trans-oceanic flight from India to East Africa. In addition, it has been suggested that this trans-oceanic crossing is just one leg of a multi-generational migratory circuit covering about 14,000-18,000 km. The dragonflies are presumed to accomplish this remarkable feat by riding high-altitude winds associated with the Inter-tropical Convergence Zone (ITCZ). While there is considerable evidence for this migratory circuit, much of that evidence is circumstantial. Recent developments in the application of stable isotope analyses to track migratory dragonflies include the establishment of direct associations between dragonfly wing chitin ?(2)H values with those derived from long-term ?(2)H precipitation isoscapes. We applied this approach by measuring wing chitin ?(2)H values in 49 individual Pantala flavescens from the November-December migration through the Maldives. Using a previously established spatial calibration algorithm for dragonflies, the mean wing ?(2)H value of -117±16 ‰ corresponded to a predicted mean natal ambient water source of -81 ‰, which resulted in a probabilistic origin of northern India, and possibly further north and east. This strongly suggests that the migratory circuit of this species in this region is longer than previously suspected, and could possibly involve a remarkable trans-Himalayan high-altitude traverse.
Pre-erythrocytic malaria vaccines target Plasmodium during its sporozoite and liver stages, and can prevent progression to blood-stage disease, which causes a million deaths each year. Whole organism sporozoite vaccines induce sterile immunity in animals and humans and guide subunit vaccine development. A recombinant protein-in-adjuvant pre-erythrocytic vaccine called RTS,S reduces clinical malaria without preventing infection in field studies and additional antigens may be required to achieve sterile immunity. Although few vaccine antigens have progressed to human testing, new insights into parasite biology, expression profiles and immunobiology have offered new targets for intervention. Future advances require human trials of additional antigens, as well as platforms to induce the durable antibody and cellular responses including CD8(+) T cells that contribute to sterile protection.
The Animal Industry Division of the Michigan Department of Agriculture and Rural Development (MDARD) has been challenged with assisting farmers with modifying farm practices to reduce potential for exposure to Mycobacterium bovis from wildlife to cattle. The MDARD recommendations for on-farm risk mitigation practices were developed from experiences in the US, UK and Ireland and a review of the scientific literature. The objectives of our study were to review the present state of knowledge on M. bovis excretion, transmission, and survival in the environment and the interactions of wildlife and cattle with the intention of determining if the current recommendations by MDARD on farm practices are adequate and to identify additional changes to farm practices that may help to mitigate the risk of transmission. This review will provide agencies with a comprehensive summary of the scientific literature on mitigation of disease transmission between wildlife and cattle and to identify lacunae in published research.
Methods to individually mark and identify free-ranging wildlife without trapping and handling would be useful for a variety of research and management purposes. The use of Passive Integrated Transponder technology could be an efficient method for collecting data for mark-recapture analysis and other strategies for assessing characteristics about populations of various wildlife species. Passive Integrated Transponder tags (PIT) have unique numbered frequencies and have been used to successfully mark and identify mammals. We tested for successful injection of PIT and subsequent functioning of PIT into gelatin blocks using 4 variations of a prototype dart. We then selected the prototype dart that resulted in the least depth of penetration in the gelatin block to assess the ability of PIT to be successfully implanted into muscle tissue of white-tailed deer (Odocoileus virginianus) post-mortem and long-term in live, captive Rocky Mountain elk (Cervus elaphus). The prototype dart with a 12.7 mm (0.5 inch) needle length and no powder charge resulted in the shallowest mean (± SD) penetration depth into gelatin blocks of 27.0 mm (± 5.6 mm) with 2.0 psi setting on the Dan-Inject CO(2)-pressured rifle. Eighty percent of PIT were successfully injected in the muscle mass of white-tailed deer post-mortem with a mean (± SD) penetration depth of 22.2 mm (± 3.8 mm; n = 6). We injected PIT successfully into 13 live, captive elk by remote delivery at about 20 m that remained functional for 7 months. We successfully demonstrated that PIT could be remotely delivered in darts into muscle mass of large mammals and remain functional for >6 months. Although further research is warranted to fully develop the technique, remote delivery of PIT technology to large mammals is possible using prototype implant darts.
Supraventricular tachycardia (SVT) is one of the most common conditions requiring emergent cardiac care in children, yet its management has never been subjected to a randomized controlled clinical trial. The purpose of this study was to compare the efficacy and safety of the 2 most commonly used medications for antiarrhythmic prophylaxis of SVT in infants: digoxin and propranolol.
The architecture of dendritic arbors determines circuit connectivity, receptive fields, and computational properties of neurons, and dendritic structure is impaired in several psychiatric disorders. While apical and basal dendritic compartments of pyramidal neurons are functionally specialized and differentially regulated, little is known about mechanisms that selectively maintain basal dendrites. Here we identified a role for the Ras/Epac2 pathway in maintaining basal dendrite complexity of cortical neurons. Epac2 is a guanine nucleotide exchange factor (GEF) for the Ras-like small GTPase Rap, and it is highly enriched in the adult mouse brain. We found that in vivo Epac2 knockdown in layer 2/3 cortical neurons via in utero electroporation reduced basal dendritic architecture, and that Epac2 knockdown in mature cortical neurons in vitro mimicked this effect. Overexpression of an Epac2 rare coding variant, found in human subjects diagnosed with autism, also impaired basal dendritic morphology. This mutation disrupted Epac2s interaction with Ras, and inhibition of Ras selectively interfered with basal dendrite maintenance. Finally, we observed that components of the Ras/Epac2/Rap pathway exhibited differential abundance in the basal versus apical dendritic compartments. These findings define a role for Epac2 in enabling crosstalk between Ras and Rap signaling in maintaining basal dendrite complexity, and exemplify how rare coding variants, in addition to their disease relevance, can provide insight into cellular mechanisms relevant for brain connectivity.
Plant cell walls are composed of interlinked polymer networks consisting of cellulose, hemicelluloses, pectins, proteins, and lignin. The ordered deposition of these components is a dynamic process that critically affects the development and differentiation of plant cells. However, our understanding of cell wall synthesis and remodeling, as well as the diverse cell wall architectures that result from these processes, has been limited by a lack of suitable chemical probes that are compatible with live-cell imaging. In this review, we summarize the currently available molecular toolbox of probes for cell wall polysaccharide imaging in plants, with particular emphasis on recent advances in small molecule-based fluorescent probes. We also discuss the potential for further development of small molecule probes for the analysis of cell wall architecture and dynamics.
Plant cell walls are the most abundant biomaterials on Earth and serve a multitude of purposes in human society. These complex extracellular matrices are mainly composed of polysaccharides, including cellulose, hemicelluloses, and pectins, which cannot be cytologically examined using conventional techniques. Click chemistry, which exploits a bio-orthogonal cycloaddition reaction between alkynyl and azido groups, has proven to be useful for the metabolic incorporation and detection of modified sugars in polysaccharides in animals, fungi, and bacteria, but its use to interrogate the biosynthesis or dynamics of plant cell walls has not been previously reported. Recently, we found that an alkynylated analog of fucose can be metabolically incorporated into Arabidopsis thaliana cell walls and click labeled with fluorescent probes, facilitating imaging of cell wall carbohydrates. Despite the presence of fucose in several classes of wall polysaccharides, fucose-alkyne was primarily incorporated into rhamnogalacturonan-I, a type of pectin. Using timecourse and pulse-labeling experiments, we observed the dynamics of pectin delivery and reorganization in expanding cell walls. The use of click chemistry to investigate plant cell wall architecture should help bridge the gap between biochemical characterization of isolated cell wall components and an understanding of how those components interact in intact cell walls.
Primary cilia, solitary microtubule-based structures that grow from the centriole and extend into the extracellular space, have increasingly been implicated as sensors of a variety of biochemical and biophysical signals. Mutations in primary cilium-related genes have been linked to a number of rare developmental disorders as well as dysregulation of cell proliferation. We propose that primary cilia are also important in mechanically regulated bone formation in adults and that their malfunction could play a role in complex multi-factorial bone diseases, such as osteoporosis. In this study, we generated mice with an osteoblast- and osteocyte-specific knockout of Kif3a, a subunit of the kinesin II intraflagellar transport (IFT) protein; IFT is required for primary cilia formation, maintenance, and function. These Col?1(I) 2.3-Cre;Kif3a(fl/fl) mice exhibited no obvious morphological skeletal abnormalities. Skeletally mature Col?1(I) 2.3-Cre;Kif3a(fl/fl) and control mice were exposed to 3 consecutive days of cyclic axial ulna loading, which resulted in a significant increase in bone formation in both the conditional knockouts and controls. However, Col?1(I) 2.3-Cre;Kif3a(fl/fl) mice did exhibit decreased formation of new bone in response to mechanical ulnar loading compared to control mice. These results suggest that primary cilia act as cellular mechanosensors in bone and that their function may be critical for the regulation of bone physiology due to mechanical loading in adults.
We present a diagnostic question cluster (DQC) that assesses undergraduates thinking about photosynthesis. This assessment tool is not designed to identify individual misconceptions. Rather, it is focused on students abilities to apply basic concepts about photosynthesis by reasoning with a coordinated set of practices based on a few scientific principles: conservation of matter, conservation of energy, and the hierarchical nature of biological systems. Data on students responses to the cluster items and uses of some of the questions in multiple-choice, multiple-true/false, and essay formats are compared. A cross-over study indicates that the multiple-true/false format shows promise as a machine-gradable format that identifies students who have a mixture of accurate and inaccurate ideas. In addition, interviews with students about their choices on three multiple-choice questions reveal the fragility of students understanding. Collectively, the data show that many undergraduates lack both a basic understanding of the role of photosynthesis in plant metabolism and the ability to reason with scientific principles when learning new content. Implications for instruction are discussed.
Compounds exhibiting low non-specific intracellular binding or non-stickiness are concomitant with rapid clearing and in high demand for live-cell imaging assays because they allow for intracellular receptor localization with a high signal/noise ratio. The non-stickiness property is particularly important for imaging intracellular receptors due to the equilibria involved.
Polysaccharide-rich cell walls are a defining feature of plants that influence cell division and growth, but many details of cell-wall organization and dynamics are unknown because of a lack of suitable chemical probes. Metabolic labeling using sugar analogs compatible with click chemistry has the potential to provide new insights into cell-wall structure and dynamics. Using this approach, we found that an alkynylated fucose analog (FucAl) is metabolically incorporated into the cell walls of Arabidopsis thaliana roots and that a significant fraction of the incorporated FucAl is present in pectic rhamnogalacturonan-I (RG-I). Time-course experiments revealed that FucAl-containing RG-I first localizes in cell walls as uniformly distributed punctae that likely mark the sites of vesicle-mediated delivery of new polysaccharides to growing cell walls. In addition, we found that the pattern of incorporated FucAl differs markedly along the developmental gradient of the root. Using pulse-chase experiments, we also discovered that the pectin network is reoriented in elongating root epidermal cells. These results reveal previously undescribed details of polysaccharide delivery, organization, and dynamics in cell walls.
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