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Background Synapse density is an essential indicator of development and functioning of the central nervous system. It is estimated indirectly through the accumulation of pre and postsynaptic proteins in tissue sections. 3D reconstruction of the electron microscopic images in serial sections is one of the most definitive means of estimating the formation of active synapses in the brain. It is tedious and highly skill-dependent. Confocal imaging of whole mounts or thick sections of the brain provides a natural alternative for rapid gross estimation of the synapse density in large areas. The optical resolution and other deep-tissue imaging aberrations limit the quantitative scope of this technique. Results Here we demonstrate a simple sample preparation method that could enhance the clarity of the confocal images of the neuropil regions of the ventral nerve cord of Drosophila larvae, providing a clear view of synapse distributions. We estimated the gross volume occupied by the synaptic junctions using 3D object counter plug-in of Fiji/ImageJ ® . It gave us a proportional estimate of the number of synaptic junctions in the neuropil region. The method is corroborated by correlated super-resolution imaging analysis and through genetic perturbation of synaptogenesis in the larval brain. Conclusions The method provides a significant improvement in the relative estimate of region-specific synapse density in the central nervous system. Also, it reduced artifacts in the super-resolution images obtained using the stimulated emission depletion microscopy technique.

γ-Tubulin ring complex (γ-TuRC) is the major microtubule-nucleating factor. After nucleation, microtubules can be released from γ-TuRC and stabilized by other proteins, such as CAMSAPs, but the biochemical cross-talk between minus-end regulation pathways is poorly understood. Here we reconstituted this process in vitro using purified components. We found that all CAMSAPs could bind to the minus ends of γ-TuRC-attached microtubules. CAMSAP2 and CAMSAP3, which decorate and stabilize growing minus ends but not the minus-end tracking protein CAMSAP1, induced microtubule release from γ-TuRC. CDK5RAP2, a γ-TuRC-interactor, and CLASP2, a regulator of microtubule growth, strongly stimulated γ-TuRC-dependent microtubule nucleation, but only CDK5RAP2 suppressed CAMSAP binding to γ-TuRC-anchored minus ends and their release. CDK5RAP2 also improved selectivity of γ-tubulin-containing complexes for 13- rather than 14-protofilament microtubules in microtubule-capping assays. Knockout and overexpression experiments in cells showed that CDK5RAP2 inhibits the formation of CAMSAP2-bound microtubules detached from the microtubule-organizing centre. We conclude that CAMSAPs can release newly nucleated microtubules from γ-TuRC, whereas nucleation-promoting factors can differentially regulate this process. Rai et al. report that CAMSAPs can bind to minus ends of microtubules attached to γ-tubulin ring complex (γ-TuRC) and drive microtubule release. They show that CDK5RAP2, but not CLASP2, inhibits CAMSAP-mediated microtubule release from γ-TuRC.

Microtubules are cytoskeleton components with unique mechanical and dynamic properties. They are rigid polymers that alternate phases of growth and shrinkage. Nonetheless, the cells can display a subset of stable microtubules, but it is unclear whether microtubule dynamics and mechanical properties are related. Recent in vitro studies suggest that microtubules have mechano-responsive properties, being able to stabilize their lattice by self-repair on physical damage. Here we study how microtubules respond to cycles of compressive forces in living cells and find that microtubules become distorted, less dynamic and more stable. This mechano-stabilization depends on CLASP2, which relocates from the end to the deformed shaft of microtubules. This process seems to be instrumental for cell migration in confined spaces. Overall, these results demonstrate that microtubules in living cells have mechano-responsive properties that allow them to resist and even counteract the forces to which they are subjected, being a central mediator of cellular mechano-responses.Microtubules respond to mechanical compression by deforming, becoming more stable, which results in CLASP2 recruitment to the distorted shaft—a process crucial for cell migration through confined spaces.

There is a need to compare greenhouse gases (GHGs) measurements methods for data harmonization. The objective of this study was to compare Gas Chromatography (GC) and a Photo-Acoustic Analyzer System (PAS) as methods for measuring carbon dioxide (CO2) and nitrous oxide (N2O) emissions in a corn/soybean field. The study was conducted on a Waldron silt loam soil at Freeman farm of Lincoln University of Missouri. Sixteen cylindrical polyvinylchloride static chambers measuring 0.30 m in height and 0.20 m in diameter were permanently inserted into the soil to a depth of 0.05 m for GHGs measurements. Soil air samples were collected in each chamber with a 60 ml syringe and stored in a Tedlar bag for CO2 and N2O analysis by GC within 2 hours of sampling. CO2 and N2O measurements with PAS were done directly in-situ. Significant, stronger and positive correlations were found between PAS and GC in 11 out of 15 months - measurement cycles for CO2. The most significant correlation was observed in April 2015 (r = 0.90, p = 0.0001). For N2O, however, only 5 out of 15 measurements between PAS and GC had positive, but weak correlations. The highest correlation between PAS and GC measurements for N2O was also found in April 2015 (r = 0.71, p = 0.0020). Continuous monitoring of GHGs emissions is necessary to better understand their dynamics and how they are exacerbated by agricultural management practices. However, accurate measurement techniques are also needed.

Prescribed burning is a common management strategy employed by land managers in Florida native rangelands. Fires can have major effects on soil properties, with subsequent impacts on soil C and N cycling. However, there is not only limited information on the effects of prescribed burning on soil microbial community in subtropical rangelands, but it is also unknown how potential shifts in microbial community are related to fire-induced changes in key soil properties. This research assessed soil nutrients, microbial activity, and microbial community responses immediately after fire. Our study showed that there were effects of prescribed burning on soil properties including soil pH, TC, TN, C:N ratio, NH4+, extractable P and Ca concentrations. Fire affected microbial response including BGA activity and NAG activity which was likely due to the inactivation of enzyme by high soil temperature. Increasing CO2 fluxes following fire were attributed to seasonal shifts rather direct effect of burning. Bacterial and fungal diversity remained unchanged in the short-term after the burning but a significant effect on beta-diversity was observed among the sampling sites. Bacterial and fungal relative abundance revealed no changes after fire at the phylum level. However, at order level, Desulfurellales and Rhizobiales increased while Eurotiales and Pleosporales decreased significantly after fire. Our data indicated that the prescribed burning had a minimal effect on soil microbial abundance and diversity immediately after the burning. Spatial heterogeneity across the sites can be considered as a crucial factor for the variation in microbial community composition than the burning but soil microbial groups could not always be linked to key soil properties. Our meta-analysis results showed a significant effect of wildfire on soil bacterial diversity compared to prescribed burning. Significant effect of fire frequency interval of 4-7 years was observed in overall soil microbial communities with high heterogeneity. However, long term assessment of fire is needed to determine if the effect takes place at different time scales and to evaluate the suitability of prescribed burning in subtropical regions for rangeland management.

Japanese encephalitis (JE) virus is the leading cause of vaccine-preventable encephalitis and a significant cause of disability in Asia and the western Pacific. JE is endemic in many parts of India and the state of Uttar Pradesh (UP) contributed approximately 75% of the cases in India over the last three decades. Since 2006, UP has used the live attenuated SA-14-14-2 vaccine (CD-JEV, Chengdu Institute of Biological Products) to prevent JE. To measure the impact of vaccine introduction in UP, the number of confirmed JE cases in 1- to 15-year-old children and vaccination coverage were analyzed by year for 40 districts in UP. Vaccine-induced protection against JE seems to have come primarily from campaign coverage, with the only distinct downward trend in disease occurring in the initial 7 high-risk districts where a second high-coverage catch-up campaign targeting children aged 1- 15 years occurred 4 years after the first campaign in 2006 (p-value <0.005). There was 25% reduction in JE cases for every 10% increase in vaccine coverage in these districts. However, there was no statistically significant impact of vaccine on the number of JE cases in a combined analysis for all districts over the study time period. Limited routine immunization coverage, especially in areas where the susceptible populations were large, was one potential reason behind inefficient vaccine impact. There is a need for catch-up campaigns in districts with poor routine immunization coverage as well as for robust routine immunization programs to sustain protection in each year’s new birth cohort.

Rab4 GTPase, essential for endosomal sorting and trafficking, is implicated in synaptic atrophy and dementia. To uncover the underlying mechanism, we studied the correlation between Rab4 vesicle transport in axons and episodic remodeling of synapses in the central nervous system (CNS) of Drosophila larvae. We found that synapse-bound traffic and presynaptic enrichment of Rab4 vesicles increase during the programmed, transient contraction of synapses in the ventral neuropil region at a specific larval stage. This coincides with the episodic activation of insulin and Vps34-mediated signaling, which elevates phosphatidylinositol-3-phosphate levels on Rab4 vesicles. The presence of this phospholipid on Rab4-associated vesicles recruit a PX-domain-containing motor protein, Klp98A, accelerating synapse-directed traffic. This, in turn, increases presynaptic enrichment of Rab4 during the developmentally programmed synapse contraction phase. Our findings elucidate the molecular mechanism that regulates developmental synaptic plasticity in the CNS via insulin signaling and directed axonal transport of endosomes.Competing Interest StatementThe authors have declared no competing interest.