Experience shapes chandelier cell function and structure in the visual cortex.

Detailed characterization of interneuron types in primary visual cortex (V1) has greatly contributed to understanding visual perception, yet the role of chandelier cells (ChCs) in visual processing remains poorly characterized. Using viral tracing we found that V1 ChCs predominantly receive monosynaptic input from local layer 5 pyramidal cells and higher-order cortical regions. Two-photon calcium imaging and convolutional neural network modeling revealed that ChCs are visually responsive but weakly selective for stimulus content. In mice running in a virtual tunnel, ChCs respond strongly to events known to elicit arousal, including locomotion and visuomotor mismatch. Repeated exposure of the mice to the virtual tunnel was accompanied by reduced visual responses of ChCs and structural plasticity of ChC boutons and axon initial segment length. Finally, ChCs only weakly inhibited pyramidal cells. These findings suggest that ChCs provide an arousal-related signal to layer 2/3 pyramidal cells that may modulate their activity and/or gate plasticity of their axon initial segments during behaviorally relevant events.

Discrepant expressive language lateralization in children and adolescents with epilepsy.

Neuronavigated transcranial magnetic stimulation (nTMS) has emerged as a presurgical language mapping tool distinct from the widely used functional magnetic resonance imaging (fMRI). We report fMRI and nTMS language-mapping results in 19 pediatric-epilepsy patients and compare those to definitive testing by electrical cortical stimulation, Wada test, and/or neuropsychological testing. Most discordant results occurred when fMRI found right-hemispheric language. In those cases, when nTMS showed left-hemispheric or bilateral language representation, left-hemispheric language was confirmed by definitive testing. Therefore, we propose nTMS should be considered for pediatric presurgical language-mapping when fMRI shows right-hemispheric language, with nTMS results superseding fMRI results in those scenarios.

Physical structure and biological composition of canopies in tropical secondary and old-growth forests.

The area of tropical secondary forests is increasing rapidly, but data on the physical and biological structure of the canopies of these forests are limited. To obtain such data and to measure the ontogeny of canopy structure during tropical rainforest succession, we studied patch-scale (5 m2) canopy structure in three areas of 18-36 year-old secondary forest in Costa Rica, and compared the results to data from old-growth forest at the same site. All stands were sampled with a stratified random design with complete harvest from ground level to the top of the canopy from a modular portable tower. All canopies were organized into distinct high- and low-leaf-density layers (strata), and multiple strata developed quickly with increasing patch height. The relation of total Leaf Area Index (LAI, leaf area per area of ground) to patch canopy height, the existence of distinct high and low leaf- density layers (strata and free air spaces), the depth and LAI of the canopy strata and free air spaces, and the relation of the number of strata to patch canopy height were remarkably constant across the entire successional gradient. Trees were the most important contributor to LAI at all stages, while contribution of palm LAI increased through succession. We hypothesize that canopy physical structure at the patch scale is driven by light competition and discuss how this hypothesis could be tested. That canopy physical structure was relatively independent of the identity of the species present suggests that canopy physical structure may be conserved even as canopy floristics shift due to changing climate.

Effect of a Lower vs Higher Positive End-Expiratory Pressure Strategy on Ventilator-Free Days in ICU Patients Without ARDS: A Randomized Clinical Trial.

Importance: It is uncertain whether invasive ventilation can use lower positive end-expiratory pressure (PEEP) in critically ill patients without acute respiratory distress syndrome (ARDS). Objective: To determine whether a lower PEEP strategy is noninferior to a higher PEEP strategy regarding duration of mechanical ventilation at 28 days. Design, Setting, and Participants: Noninferiority randomized clinical trial conducted from October 26, 2017, through December 17, 2019, in 8 intensive care units (ICUs) in the Netherlands among 980 patients without ARDS expected not to be extubated within 24 hours after start of ventilation. Final follow-up was conducted in March 2020. Interventions: Participants were randomized to receive invasive ventilation using either lower PEEP, consisting of the lowest PEEP level between 0 and 5 cm H2O (n = 476), or higher PEEP, consisting of a PEEP level of 8 cm H2O (n = 493). Main Outcomes and Measures: The primary outcome was the number of ventilator-free days at day 28, with a noninferiority margin for the difference in ventilator-free days at day 28 of -10%. Secondary outcomes included ICU and hospital lengths of stay; ICU, hospital, and 28- and 90-day mortality; development of ARDS, pneumonia, pneumothorax, severe atelectasis, severe hypoxemia, or need for rescue therapies for hypoxemia; and days with use of vasopressors or sedation. Results: Among 980 patients who were randomized, 969 (99%) completed the trial (median age, 66 [interquartile range {IQR}, 56-74] years; 246 [36%] women). At day 28, 476 patients in the lower PEEP group had a median of 18 ventilator-free days (IQR, 0-27 days) and 493 patients in the higher PEEP group had a median of 17 ventilator-free days (IQR, 0-27 days) (mean ratio, 1.04; 95% CI, 0.95-∞; P = .007 for noninferiority), and the lower boundary of the 95% CI was within the noninferiority margin. Occurrence of severe hypoxemia was 20.6% vs 17.6% (risk ratio, 1.17; 95% CI, 0.90-1.51; P = .99) and need for rescue strategy was 19.7% vs 14.6% (risk ratio, 1.35; 95% CI, 1.02-1.79; adjusted P = .54) in patients in the lower and higher PEEP groups, respectively. Mortality at 28 days was 38.4% vs 42.0% (hazard ratio, 0.89; 95% CI, 0.73-1.09; P = .99) in patients in the lower and higher PEEP groups, respectively. There were no statistically significant differences in other secondary outcomes. Conclusions and Relevance: Among patients in the ICU without ARDS who were expected not to be extubated within 24 hours, a lower PEEP strategy was noninferior to a higher PEEP strategy with regard to the number of ventilator-free days at day 28. These findings support the use of lower PEEP in patients without ARDS. Trial Registration: ClinicalTrials.gov Identifier: NCT03167580.

Pilot Implementation of Adapted-Family Diabetes Self-Management Education Into a Clinical Setting.

Objectives: Marshallese are a Pacific Islander community that experience a disproportionate rate of type 2 diabetes. The purpose of this study is to evaluate the preliminary effectiveness and feasibility of an Adapted-Family Diabetes Self-Management Education (DSME) intervention among Marshallese adults diagnosed with type 2 diabetes and their family members when delivered in a clinical setting. Methods: Marshallese patients (primary participants) with type 2 diabetes (n = 10) and their family members (n = 10) enrolled in a pilot study deigned to evaluate an Adapted-Family DSME curriculum conducted by community health workers and a certified diabetes educator in a clinical setting. Primary and family participants' health information and biometric data (HbA1c, blood pressure, cholesterol, and body mass index) were collected at preintervention and 12 weeks postintervention. Results: All 10 primary participants and 8 of the family members received all 10 hours of the education intervention. Nine of the 10 primary participants and 8 of the 10 family members completed the pre- and postintervention data collection events. Primary participants demonstrated a mean decrease in HbA1c of 0.7%, from pre- to postintervention, as well as improved blood pressure and cholesterol. Family members demonstrated minor improvements in HbA1c and blood pressure. Conclusions: Results suggest preliminary effectiveness and feasibility of the Adapted-Family DSME in a clinic setting and will inform implementation of a fully powered study.

Shotgun metagenomic analysis of microbial communities from the Loxahatchee nature preserve in the Florida Everglades.

BACKGROUND: Currently, much is unknown about the taxonomic diversity and the mechanisms of methane metabolism in the Florida Everglades ecosystem. The Loxahatchee National Wildlife Refuge is a section of the Florida Everglades that is almost entirely unstudied in regard to taxonomic profiling. This short report analyzes the metagenome of soil samples from this Refuge to investigate the predominant taxa, as well as the abundance of genes involved in environmentally significant metabolic pathways related to methane production (nitrogen fixation and dissimilatory sulfite reduction). METHODS: Shotgun metagenomic sequencing using the Illumina platform was performed on 17 soil samples from four different sites within the Loxahatchee National Wildlife Refuge, and underwent quality control, assembly, and annotation. The soil from each sample was tested for water content and concentrations of organic carbon and nitrogen. RESULTS: The three most common phyla of bacteria for every site were Actinobacteria, Acidobacteria, and Proteobacteria; however, there was variation in relative phylum composition. The most common phylum of Archaea was Euryarchaeota for all sites. Alpha and beta diversity analyses indicated significant congruity in taxonomic diversity in most samples from Sites 1, 3, and 4 and negligible congruity between Site 2 and the other sites. Shotgun metagenomic sequencing revealed the presence of biogeochemical biomarkers of particular interest (e.g., mrcA, nifH, and dsrB) within the samples. The normalized abundances of mcrA, nifH, and dsrB exhibited a positive correlation with nitrogen concentration and water content, and a negative correlation with organic carbon concentration. CONCLUSION: This Everglades soil metagenomic study allowed examination of wetlands biological processes and showed expected correlations between measured organic constituents and prokaryotic gene frequency. Additionally, the taxonomic profile generated gives a basis for the diversity of prokaryotic microbial life throughout the Everglades.

Redescription of Milnesium alpigenum Ehrenberg, 1853 (Tardigrada: Apochela) and a description of Milnesium inceptum sp. nov., a tardigrade laboratory model.

Intra- and interspecific variability, being at the very core of alpha taxonomy, has been a long-standing topic of debate among tardigrade taxonomists. Early studies tended to assume that tardigrades exhibit wide intraspecific variation. However, with more careful morphological studies, especially those incorporating molecular tools that allow for an independent verification of species identifications based on phenotypic traits, we now recognise that ranges of tardigrade intraspecific variability are narrower, and that differences between species may be more subtle than previously assumed. The taxonomic history of the genus Milnesium, and more specifically that of the nominal species, M. tardigradum described by Doyère in 1840, is a good illustration of the evolution of views on intraspecific variability in tardigrades. The assumption of wide intraspecific variability in claw morphology led Marcus (1928) to synonymise two species with different claw configurations, M. alpigenum and M. quadrifidum, with M. tardigradum. Currently claw configuration is recognised as one of the key diagnostic traits in the genus Milnesium, and the two species suppressed by Marcus have recently been suggested to be valid. In this study, we clarify the taxonomic status of M. alpigenum, a species that for nearly a century was considered invalid. We redescribe M. alpigenum, using a population collected from the locus typicus, by the means of integrative taxonomy, i.e. including light microscopy, scanning electron microscopy, ontogenetic observations, and genetic barcoding. Moreover, the redescription of M. alpigenum allowed us to verify the uncertain taxonomic status of two popular laboratory models that were originally considered to be M. tardigradum; though one was recently reidentified as M. cf. alpigenum. Our analysis showed that both laboratory strains, despite being morphologically and morphometrically nearly identical to M. alpigenum, in fact represent a new species, M. inceptum sp. nov.  The two species, being disnguishable only by statistical morphometry and/or DNA sequences, are the first example of pseudocryptic species in tardigrades.

Design of a comparative effectiveness randomized controlled trial testing a faith-based Diabetes Prevention Program (WORD DPP) vs. a Pacific culturally adapted Diabetes Prevention Program (PILI DPP) for Marshallese in the United States.

BACKGROUND: Pacific Islander populations, including Marshallese, face a disproportionately high burden of health disparities relative to the general population. OBJECTIVES: A community-based participatory research (CBPR) approach was utilized to engage Marshallese participants in a comparative effectiveness trial testing 2 Diabetes Prevention Program (DPP) interventions designed to reduce participant's weight, lower HbA1c, encourage healthy eating, and increase physical activity. DESIGN: To compare the effectiveness of the faith-based (WORD) DPP to the culturally adapted (Pacific Culturally Adapted Diabetes Prevention Program [PILI]) DPP, a clustered randomized controlled trial (RCT) with 384 Marshallese participants will be implemented in 32 churches located in Arkansas, Kansas, Missouri, and Oklahoma. Churches will be randomly assigned to WORD DPP arm or to PILI DPP arm. METHODS: WORD DPP focuses on connecting faith and health to attain a healthy weight, eat healthy, and be more physically active. In contrast, PILI DPP is a family and community focused DPP curriculum specifically adapted for implementation in Pacific Islander communities. PILI focuses on engaging social support networks to maintain a healthy weight, eat healthy, and be more physically active. All participants are assessed at baseline, immediate post intervention, and 12 months post intervention. SUMMARY: Both interventions aim to cause weight loss through improving physical activity and healthy eating, with the goal of preventing the development of T2D. The clustered RCT will determine which intervention is most effective with the Marshallese population. The utilization of a CBPR approach that involves local stakeholders and engages faith-based institutions in Marshallese communities will increase the potential for success and sustainability. This study is registered at clinicaltrials.gov (NCT03270436).

Drivers of advanced stage at breast cancer diagnosis in the multicountry African breast cancer - disparities in outcomes (ABC-DO) study.

Breast cancer (BC) survival rates in sub-Saharan Africa (SSA) are low in part due to advanced stage at diagnosis. As one component of a study of the entire journey of SSA women with BC, we aimed to identify shared and setting-specific drivers of advanced stage BC. Women newly diagnosed in the multicountry African Breast Cancer-Disparities in Outcomes (ABC-DO) study completed a baseline interview and their stage information was extracted from medical records. Ordinal logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CI) for advanced stage (I, II, III, IV) in relation to individual woman-level, referral and biological factors. A total of 1795 women were included from Nigeria, Uganda, Zambia, and the multiracial populations of Namibia and South Africa, 1091 of whom (61%) were stage III/IV. Stage was lower in women with greater BC knowledge (OR 0.77 (95% CI: 0.70, 0.85) per point on a 6 point scale). More advanced stage was associated with being black (4.00 (2.79, 5.74)), having attended

Leveraging community-based participatory research capacity to recruit Pacific Islanders into a genetics study.

Pacific Islanders face many health disparities, including higher rates of cardiovascular disease, cancer, obesity, and diabetes compared to other racial and ethnic groups. Specifically, the Marshallese population suffers disproportionately from type 2 diabetes, with rates 400% higher than the general US population. As part of an ongoing community-based participatory research (CBPR) partnership, 148 participants were recruited for a study examining genetic variants to better understand diabetes. Participants provided a saliva specimen in an Oragene® DNA self-collection kit. Each participant provided approximately 2 mL volume of saliva and was asked qualitative questions about their experience. The study yielded a recruitment rate of 95.5%. Among the 148 persons who participated, 143 (96.6%) agreed to be contacted for future studies; 142 (95.9%) agreed to have their samples used for future IRB-approved studies; and 144 (97.3%) gave permission for the researchers to link information from this study to other studies in which they had participated. Qualitative responses showed that the majority of participants were willing to participate because of their desire to contribute to the health of their community and to understand the genetic influence related to diabetes. This study demonstrates willingness to participate in genetic research among Marshallese living in Arkansas. Willingness was likely enhanced because the feasibility study was part of a larger CBPR effort. This study is important to community stakeholders who have voiced a desire to collaboratively conduct genetic research related to diabetes, perinatal outcomes, and cancer.

Multidirectional abundance shifts among North American birds and the relative influence of multifaceted climate factors.

Shifts in species distributions are major fingerprint of climate change. Examining changes in species abundance structures at a continental scale enables robust evaluation of climate change influences, but few studies have conducted these evaluations due to limited data and methodological constraints. In this study, we estimate temporal changes in abundance from North American Breeding Bird Survey data at the scale of physiographic strata to examine the relative influence of different components of climatic factors and evaluate the hypothesis that shifting species distributions are multidirectional in resident bird species in North America. We quantify the direction and velocity of the abundance shifts of 57 permanent resident birds over 44 years using a centroid analysis. For species with significant abundance shifts in the centroid analysis, we conduct a more intensive correlative analysis to identify climate components most strongly associated with composite change of abundance within strata. Our analysis focus on two contrasts: the relative importance of climate extremes vs. averages, and of temperature vs. precipitation in strength of association with abundance change. Our study shows that 36 species had significant abundance shifts over the study period. The average velocity of the centroid is 5.89 km·yr-1 . The shifted distance on average covers 259 km, 9% of range extent. Our results strongly suggest that the climate change fingerprint in studied avian distributions is multidirectional. Among 6 directions with significant abundance shifts, the northwestward shift was observed in the largest number of species (n = 13). The temperature/average climate model consistently has greater predictive ability than the precipitation/extreme climate model in explaining strata-level abundance change. Our study shows heterogeneous avian responses to recent environmental changes. It highlights needs for more species-specific approaches to examine contributing factors to recent distributional changes and for comprehensive conservation planning for climate change adaptation.

Scope and Limitations of Fmoc Chemistry SPPS-Based Approaches to the Total Synthesis of Insulin Lispro via Ester Insulin.

We have systematically explored three approaches based on 9-fluorenylmethoxycarbonyl (Fmoc) chemistry solid phase peptide synthesis (SPPS) for the total chemical synthesis of the key depsipeptide intermediate for the efficient total chemical synthesis of insulin. The approaches used were: stepwise Fmoc chemistry SPPS; the "hybrid method", in which maximally protected peptide segments made by Fmoc chemistry SPPS are condensed in solution; and, native chemical ligation using peptide-thioester segments generated by Fmoc chemistry SPPS. A key building block in all three approaches was a Glu[O-ß-(Thr)] ester-linked dipeptide equipped with a set of orthogonal protecting groups compatible with Fmoc chemistry SPPS. The most effective method for the preparation of the 51 residue ester-linked polypeptide chain of ester insulin was the use of unprotected peptide-thioester segments, prepared from peptide-hydrazides synthesized by Fmoc chemistry SPPS, and condensed by native chemical ligation. High-resolution X-ray crystallography confirmed the disulfide pairings and three-dimensional structure of synthetic insulin lispro prepared from ester insulin lispro by this route. Further optimization of these pilot studies could yield an efficient total chemical synthesis of insulin lispro (Humalog) based on peptide synthesis by Fmoc chemistry SPPS.

Spaceborne potential for examining taiga-tundra ecotone form and vulnerability.

In the taiga-tundra ecotone (TTE), site-dependent forest structure characteristics can influence the subtle and heterogeneous structural changes that occur across the broad circumpolar extent. Such changes may be related to ecotone form, described by the horizontal and vertical patterns of forest structure (e.g., tree cover, density and height) within TTE forest patches, driven by local site conditions, and linked to ecotone dynamics. The unique circumstance of subtle, variable and widespread vegetation change warrants the application of spaceborne data including high-resolution (< 5m) spaceborne imagery (HRSI) across broad scales for examining TTE form and predicting dynamics. This study analyzes forest structure at the patch-scale in the TTE to provide a means to examine both vertical and horizontal components of ecotone form. We demonstrate the potential of spaceborne data for integrating forest height and density to assess TTE form at the scale of forest patches across the circumpolar biome by (1) mapping forest patches in study sites along the TTE in northern Siberia with a multi-resolution suite of spaceborne data, and (2) examining the uncertainty of forest patch height from this suite of data across sites of primarily diffuse TTE forms. Results demonstrate the opportunities for improving patch-scale spaceborne estimates of forest height, the vertical component of TTE form, with HRSI. The distribution of relative maximum height uncertainty based on prediction intervals is centered at ~40%, constraining the use of height for discerning differences in forest patches. We discuss this uncertainty in light of a conceptual model of general ecotone forms, and highlight how the uncertainty of spaceborne estimates of height can contribute to the uncertainty in identifying TTE forms. A focus on reducing the uncertainty of height estimates in forest patches may improve depiction of TTE form, which may help explain variable forest responses in the TTE to climate change and the vulnerability of portions of the TTE to forest structure change.

Synthesis and Thorough Investigation of Discrete Organotin Telluride Clusters.

Systematic experimental and theoretical investigations of reactions of R(1)SnCl3 (R(1) = CMe2CH2C(Me)O) with (Me3Si)2Te allowed for the stepwise formation and single-crystalline isolation of the first tin sesquitelluride clusters with functional organic ligands. Subsequent derivatization of the latter took place under reorganization of the inorganic core, affording clusters with complex hybrid architectures.

Behaviour of the plathelminth Symsagittifera roscoffensis under different light conditions and the consequences for the symbiotic algae Tetraselmis convolutae.

Symsagittifera roscoffensis is a plathelminth living in symbiosis with the green algae Tetraselmis convolutae. Host and symbiont are a model system for the study of endosymbiosis, which has so far mainly focused on their biochemical interactions. Symsagittifera roscoffensis is well known for its positive phototaxis that is hypothesized to optimize the symbiont's light perception for photosynthesis. In this study, we conducted a detailed analysis of phototaxis using light sources of different wavelength and brightness by videotracking. Furthermore, we compared the behavioural data with the electron transfer rate of the photosystem from cultured symbiotic cells. The symbiotic algae is adapted to low light conditions, showing a positive electron transfer rate at a photosynthetically active radiation of 0.112 µmol photons m(-2) s(-1), and S. roscoffensis showed a positive phototactic behaviour for light intensities up to 459.17 µmol photons m(-2) s(-1), which is not optimal regarding the needs of the symbiotic cells and may even harm host and symbiont. Red light cannot be detected by the animals and therefore their eyes seem not to be suitable for measuring the exact photosynthetically active radiation to the benefit of the photosymbionts.

Comparison of seeding methods for visualization of the corticospinal tracts using single tensor tractography.

OBJECTIVES: To compare five different seeding methods to delineate hand, foot, and lip components of the corticospinal tract (CST) using single tensor tractography. METHODS: We studied five healthy subjects and 10 brain tumor patients. For each subject, we used five different seeding methods, from (1) cerebral peduncle (CP), (2) posterior limb of the internal capsule (PLIC), (3) white matter subjacent to functional MRI activations (fMRI), (4) whole brain and then selecting the fibers that pass through both fMRI and CP (WBF-CP), and (5) whole brain and then selecting the fibers that pass through both fMRI and PLIC (WBF-PLIC). Two blinded neuroradiologists rated delineations as anatomically successful or unsuccessful tractography. The proportions of successful trials from different methods were compared by Fisher's exact test. RESULTS: To delineate hand motor tract, seeding through fMRI activation areas was more effective than through CP (p<0.01), but not significantly different from PLIC (p>0.1). WBF-CP delineated hand motor tracts in a larger proportion of trials than CP alone (p<0.05). Similarly, WBF-PLIC depicted hand motor tracts in a larger proportion of trials than PLIC alone (p<0.01). Foot motor tracts were delineated in all trials by either PLIC or whole brain seeding (WBF-CP and WBF-PLIC). Seeding from CP or fMRI activation resulted in foot motor tract visualization in 87% of the trials (95% confidence interval: 60-98%). The lip motor tracts were delineated only by WBF-PLIC and in 36% of trials (95% confidence interval: 11-69%). CONCLUSIONS: Whole brain seeding and then selecting the tracts that pass through two anatomically relevant ROIs can delineate more plausible hand and lip motor tracts than seeding from a single ROI. Foot motor tracts can be successfully delineated regardless of the seeding method used.

Passive fMRI mapping of language function for pediatric epilepsy surgical planning: validation using Wada, ECS, and FMAER.

In this study we validate passive language fMRI protocols designed for clinical application in pediatric epilepsy surgical planning as they do not require overt participation from patients. We introduced a set of quality checks that assess reliability of noninvasive fMRI mappings utilized for clinical purposes. We initially compared two fMRI language mapping paradigms, one active in nature (requiring participation from the patient) and the other passive in nature (requiring no participation from the patient). Group-level analysis in a healthy control cohort demonstrated similar activation of the putative language centers of the brain in the inferior frontal (IFG) and temporoparietal (TPG) regions. Additionally, we showed that passive language fMRI produced more left-lateralized activation in TPG (LI=+0.45) compared to the active task; with similarly robust left-lateralized IFG (LI=+0.24) activations using the passive task. We validated our recommended fMRI mapping protocols in a cohort of 15 pediatric epilepsy patients by direct comparison against the invasive clinical gold-standards. We found that language-specific TPG activation by fMRI agreed to within 9.2mm to subdural localizations by invasive functional mapping in the same patients, and language dominance by fMRI agreed with Wada test results at 80% congruency in TPG and 73% congruency in IFG. Lastly, we tested the recommended passive language fMRI protocols in a cohort of very young patients and confirmed reliable language-specific activation patterns in that challenging cohort. We concluded that language activation maps can be reliably achieved using the passive language fMRI protocols we proposed even in very young (average 7.5 years old) or sedated pediatric epilepsy patients.

A transcriptome approach to ecdysozoan phylogeny.

The monophyly of Ecdysozoa, which comprise molting phyla, has received strong support from several lines of evidence. However, the internal relationships of Ecdysozoa are still contended. We generated expressed sequence tags from a priapulid (penis worm), a kinorhynch (mud dragon), a tardigrade (water bear) and five chelicerate taxa by 454 transcriptome sequencing. A multigene alignment was assembled from 63 taxa, which comprised after matrix optimization 24,249 amino acid positions with high data density (2.6% gaps, 19.1% missing data). Phylogenetic analyses employing various models support the monophyly of Ecdysozoa. A clade combining Priapulida and Kinorhyncha (i.e. Scalidophora) was recovered as the earliest branch among Ecdysozoa. We conclude that Cycloneuralia, a taxon erected to combine Priapulida, Kinorhyncha and Nematoda (and others), are paraphyletic. Rather Arthropoda (including Onychophora) are allied with Nematoda and Tardigrada. Within Arthropoda, we found strong support for most clades, including monophyletic Mandibulata and Pancrustacea. The phylogeny within the Euchelicerata remained largely unresolved. There is conflicting evidence on the position of tardigrades: While Bayesian and maximum likelihood analyses of only slowly evolving genes recovered Tardigrada as a sister group to Arthropoda, analyses of the full data set, and of subsets containing genes evolving at fast and intermediate rates identified a clade of Tardigrada and Nematoda. Notably, the latter topology is also supported by the analyses of indel patterns.

Towards decrypting cryptobiosis--analyzing anhydrobiosis in the tardigrade Milnesium tardigradum using transcriptome sequencing.

BACKGROUND: Many tardigrade species are capable of anhydrobiosis; however, mechanisms underlying their extreme desiccation resistance remain elusive. This study attempts to quantify the anhydrobiotic transcriptome of the limno-terrestrial tardigrade Milnesium tardigradum. RESULTS: A prerequisite for differential gene expression analysis was the generation of a reference hybrid transcriptome atlas by assembly of Sanger, 454 and Illumina sequence data. The final assembly yielded 79,064 contigs (>100 bp) after removal of ribosomal RNAs. Around 50% of them could be annotated by SwissProt and NCBI non-redundant protein sequences. Analysis using CEGMA predicted 232 (93.5%) out of the 248 highly conserved eukaryotic genes in the assembly. We used this reference transcriptome for mapping and quantifying the expression of transcripts regulated under anhdydrobiosis in a time-series during dehydration and rehydration. 834 of the transcripts were found to be differentially expressed in a single stage (dehydration/inactive tun/rehydration) and 184 were overlapping in two stages while 74 were differentially expressed in all three stages. We have found interesting patterns of differentially expressed transcripts that are in concordance with a common hypothesis of metabolic shutdown during anhydrobiosis. This included down-regulation of several proteins of the DNA replication and translational machinery and protein degradation. Among others, heat shock proteins Hsp27 and Hsp30c were up-regulated in response to dehydration and rehydration. In addition, we observed up-regulation of ployubiquitin-B upon rehydration together with a higher expression level of several DNA repair proteins during rehydration than in the dehydration stage. CONCLUSIONS: Most of the transcripts identified to be differentially expressed had distinct cellular function. Our data suggest a concerted molecular adaptation in M. tardigradum that permits extreme forms of ametabolic states such as anhydrobiosis. It is temping to surmise that the desiccation tolerance of tradigrades can be achieved by a constitutive cellular protection system, probably in conjunction with other mechanisms such as rehydration-induced cellular repair.