Temporal sorting of microdroplets can identify productivity differences of itaconic acid from libraries of Yarrowia lipolytica.

Microdroplet screening of microorganisms can improve the rate of strain selection and characterization within the canonical design-build-test paradigm. However, a full analysis of the microdroplet environment and how well these conditions translate to culturing conditions and techniques is lacking in the field. Quantification of three different biosensor/analyte combinations at 12 hour timepoints reveals the potential for extended dose-response ranges as compared to traditional in vitro conditions. Using these dynamics, we present an application and analysis of microfluidic droplet screening utilizing whole-cell biosensors, ultimately identifying an altered productivity profile of itaconic acid in a Yarrowia lipolytica-based piggyBac transposon library. Specifically, we demonstrate that the timepoint for microdroplet selection can influence the outcome of the selection and thus shift the identified strain productivity and final titer. In this case, strains selected at earlier timepoints showed increased early productivity in flask scale, with the converse true as well. Differences in response indicate microdroplet assays require tailored development to more accurately sort for phenotypes that are scalable to larger incubation volumes. Likewise, these results further highlight that screening conditions are critical parameters for success in high-throughput applications.

Atypical Presentations of Extraparenchymal Neurocysticercosis.

BACKGROUND: Neurocysticercosis (NCC) is the most common parasitic infection of the central nervous system and is typically diagnosed through visualization of the cysts in the cerebral parenchyma by neuro-imaging. However, neuro-imaging may not detect extraparenchymal neurocysticercosis (EPNCC), which is a rare manifestation of the disease involving the subarachnoid, meningeal, and intraventricular spaces. We report 2 cases of extraparenchymal neurocysticercosis, and discuss the diagnostic challenges and management of this entity. METHODS: Two cases were identified through clinical records. RESULTS: Both patients had an insidious onset with slow progression of disease, and presented with papilledema and cerebrospinal fluid (CSF) eosinophilia. One case was diagnosed with spinal cord biopsy. The other was diagnosed with CSF serology and next-generation sequencing-based pathogen analysis. Both patients were treated with ventriculoperitoneal shunt, systemic antiparasitic agents, and immunosuppression. CONCLUSIONS: EPNCC is less common than parenchymal NCC. A high level of clinical suspicion is required given its rarity, long incubation period, and slow progression. Diagnosis and treatment can be challenging and requires a multidisciplinary approach.

Chromosome Numbers and Genome Sizes of All 36 Duckweed Species (Lemnaceae).

Usually, chromosome sets (karyotypes) and genome sizes are rather stable for distinct species and therefore of diagnostic value for taxonomy. In combination with (cyto)genomics, both features provide essential cues for genome evolution and phylogenetic relationship studies within and between taxa above the species level. We present for the first time a survey on chromosome counts and genome size measurement for one or more accessions from all 36 duckweed species and discuss the evolutionary impact and peculiarities of both parameters in duckweeds.

The Ribosomal DNA Loci of the Ancient Monocot Pistia stratiotes L. (Araceae) Contain Different Variants of the 35S and 5S Ribosomal RNA Gene Units.

The freshwater plant water lettuce (Pistia stratiotes L.) grows in warm climatic zones and is used for phytoremediation and biomass production. P. stratiotes belongs to the Araceae, an ecologically and structurally diverse early monocot family, but the phylogenetic relationships among Araceae members are poorly understood. Ribosomal DNAs (rDNAs), including the 35S and 5S rDNA, encode the RNA components of ribosomes and are widely used in phylogenetic and evolutionary studies of various plant taxa. Here, we comprehensively characterized the chromosomal locations and molecular organization of 35S and 5S rDNA genes in water lettuce using karyological and molecular methods. Fluorescence in situ hybridization revealed a single location for the 35S and 5S rDNA loci, each on a different pair of the species' 28 chromosomes. Molecular cloning and nucleotide sequencing of 35S rDNA of P. stratiotes, the first representative Araceae sensu stricto in which such a study was performed, displayed typical structural characteristics. The full-length repeat showed high sequence conservation of the regions producing the 18S, 5.8S, and 25S rRNAs and divergence of the internal transcribed spacers ITS1 and ITS2 as well as the large intergenic spacer (IGS). Alignments of the deduced sequence of 18S rDNA with the sequences available for other Araceae and representatives of other clades were used for phylogenetic analysis. Examination of 11 IGS sequences revealed significant intra-genomic length variability due to variation in subrepeat number, with four types of units detected within the 35S rDNA locus of the P. stratiotes genome (estimated size 407 Mb/1C). Similarly, the 5S rDNA locus harbors gene units comprising a conserved 119-bp sequence encoding 5S rRNA and two types of non-transcribed spacer (NTS) sequences. Type I was classified into four subtypes, which apparently originated via progressive loss of subrepeats within the duplicated NTS region containing the 3' part of the 5S rRNA gene. The minor Type II NTS is shorter than Type I and differs in nucleotide composition. Some DNA clones containing two or three consecutive 5S rDNA repeats harbored 5S rDNA genes with different types of NTSs, confirming the mosaic composition of the 5S rDNA locus.

Single-cell transcriptomic analysis of the adult mouse spinal cord reveals molecular diversity of autonomic and skeletal motor neurons.

The spinal cord is a fascinating structure that is responsible for coordinating movement in vertebrates. Spinal motor neurons control muscle activity by transmitting signals from the spinal cord to diverse peripheral targets. In this study, we profiled 43,890 single-nucleus transcriptomes from the adult mouse spinal cord using fluorescence-activated nuclei sorting to enrich for motor neuron nuclei. We identified 16 sympathetic motor neuron clusters, which are distinguishable by spatial localization and expression of neuromodulatory signaling genes. We found surprising skeletal motor neuron heterogeneity in the adult spinal cord, including transcriptional differences that correlate with electrophysiologically and spatially distinct motor pools. We also provide evidence for a novel transcriptional subpopulation of skeletal motor neuron (γ*). Collectively, these data provide a single-cell transcriptional atlas ( http://spinalcordatlas.org ) for investigating the organizing molecular logic of adult motor neuron diversity, as well as the cellular and molecular basis of motor neuron function in health and disease.

Limitation of current probe design for oligo-cross-FISH, exemplified by chromosome evolution studies in duckweeds.

Duckweeds represent a small, free-floating aquatic family (Lemnaceae) of the monocot order Alismatales with the fastest growth rate among flowering plants. They comprise five genera (Spirodela, Landoltia, Lemna, Wolffiella, and Wolffia) varying in genome size and chromosome number. Spirodela polyrhiza had the first sequenced duckweed genome. Cytogenetic maps are available for both species of the genus Spirodela (S. polyrhiza and S. intermedia). However, elucidation of chromosome homeology and evolutionary chromosome rearrangements by cross-FISH using Spirodela BAC probes to species of other duckweed genera has not been successful so far. We investigated the potential of chromosome-specific oligo-FISH probes to address these topics. We designed oligo-FISH probes specific for one S. intermedia and one S. polyrhiza chromosome (Fig. 1a). Our results show that these oligo-probes cross-hybridize with the homeologous regions of the other congeneric species, but are not suitable to uncover chromosomal homeology across duckweeds genera. This is most likely due to too low sequence similarity between the investigated genera and/or too low probe density on the target genomes. Finally, we suggest genus-specific design of oligo-probes to elucidate chromosome evolution across duckweed genera.

Chromosome-scale genome assembly for the duckweed Spirodela intermedia, integrating cytogenetic maps, PacBio and Oxford Nanopore libraries.

Duckweeds are small, free-floating, morphologically highly reduced organisms belonging to the monocot order Alismatales. They display the most rapid growth among flowering plants, vary ~ 14-fold in genome size and comprise five genera. Spirodela is the phylogenetically oldest genus with only two mainly asexually propagating species: S. polyrhiza (2n = 40; 160 Mbp/1C) and S. intermedia (2n = 36; 160 Mbp/1C). This study combined comparative cytogenetics and de novo genome assembly based on PacBio, Illumina and Oxford Nanopore (ON) reads to obtain the first genome reference for S. intermedia and to compare its genomic features with those of the sister species S. polyrhiza. Both species' genomes revealed little more than 20,000 putative protein-coding genes, very low rDNA copy numbers and a low amount of repetitive sequences, mainly Ty3/gypsy retroelements. The detection of a few new small chromosome rearrangements between both Spirodela species refined the karyotype and the chromosomal sequence assignment for S. intermedia.

Variation in genome size, cell and nucleus volume, chromosome number and rDNA loci among duckweeds.

Duckweeds are small, free-floating, largely asexual and highly neotenous organisms. They display the most rapid growth among flowering plants and are of growing interest in aquaculture and genome biology. Genomic and chromosomal data are still rare. Applying flow-cytometric genome size measurement, microscopic determination of frond, cell and nucleus morphology, as well as fluorescence in situ hybridization (FISH) for localization of ribosomal DNA (rDNA), we compared eleven species, representative for the five duckweed genera to search for potential correlations between genome size, cell and nuclei volume, simplified body architecture (neoteny), chromosome numbers and rDNA loci. We found a ~14-fold genome size variation (from 160 to 2203 Mbp), considerable differences in frond size and shape, highly variable guard cell and nucleus size, chromosome number (from 2n = 36 to 82) and number of 5S and 45S rDNA loci. In general, genome size is positively correlated with guard cell and nucleus volume (p < 0.001) and with the neoteny level and inversely with the frond size. In individual cases these correlations could be blurred for instance by particular body and cell structures which seem to be linked to specific floating styles. Chromosome number and rDNA loci variation between the tested species was independent of the genome size. We could not confirm previously reported intraspecific variation of chromosome numbers between individual clones of the genera Spirodela and Landoltia.

Effect of periodic health exam on provider management of preventive services.

RATIONALE AND OBJECTIVE: To evaluate the relationship between receipt of annual physicals and the receipt of provider recommendation for preventive services, during a period when Medicare did not cover annual physicals (before 2011). METHODS: Electronic medical records of patients aged 65 years and older from a US health care system were extracted for the 2001 to 2007 period. A fixed-effects logistic model was used to assess the relationship between receipt of periodic health examination (PHE) and receipt of provider recommendation for mammogram screening for 6466 female Medicare beneficiaries. Logistic regression models were used to assess the relationship between receipt of PHE and receipt of provider recommendation for colonoscopy screening and pneumococcal vaccination for 10 318 Medicare beneficiaries. Nine primary care providers from the network were also interviewed, selected by random sampling stratified by care model. RESULTS: Electronic medical record analyses suggest that patients with a PHE were more likely to obtain provider recommendations for mammogram screening (OR = 2.17, P < 0.0001), colonoscopy screening (OR = 1.54, P < 0.0001), and pneumococcal vaccination (OR = 1.10, P < 0.0001). Providers suggested that prevention care quality improves with the PHE because certain screening measures (eg, skin cancer screening, breast exam) would be neglected without it, and healthy patients could miss recommended preventive services entirely. Without the PHE, some providers reported having tried to incorporate discussions of preventive services by scheduling more frequent follow-up chronic care visits than they would have otherwise, and some routinely charged Medicare for a more complex follow-up visit than they would have charged without the preventive service discussions. CONCLUSION: Periodic health examination is important in connecting patients to recommended preventive services. Provider interviews suggested that, indirectly, Medicare ended up paying for the PHE via greater frequency of follow-up visits or higher visit charges from providers integrating the services with other visits.

Subtype Diversification and Synaptic Specificity of Stem Cell-Derived Spinal Interneurons.

Neuronal diversification is a fundamental step in the construction of functional neural circuits, but how neurons generated from single progenitor domains acquire diverse subtype identities remains poorly understood. Here we developed an embryonic stem cell (ESC)-based system to model subtype diversification of V1 interneurons, a class of spinal neurons comprising four clades collectively containing dozens of molecularly distinct neuronal subtypes. We demonstrate that V1 subtype diversity can be modified by extrinsic signals. Inhibition of Notch and activation of retinoid signaling results in a switch to MafA clade identity and enriches differentiation of Renshaw cells, a specialized MafA subtype that mediates recurrent inhibition of spinal motor neurons. We show that Renshaw cells are intrinsically programmed to migrate to species-specific laminae upon transplantation and to form subtype-specific synapses with motor neurons. Our results demonstrate that stem cell-derived neuronal subtypes can be used to investigate mechanisms underlying neuronal subtype specification and circuit assembly.

Reconstruction of chromosome rearrangements between the two most ancestral duckweed species Spirodela polyrhiza and S. intermedia.

The monophyletic duckweeds comprising five genera within the monocot order Alismatales are neotenic, free-floating, aquatic organisms with fast vegetative propagation. Some species are considered for efficient biomass production, for life stock feeding, and for (simultaneous) wastewater phytoremediation. The ancestral genus Spirodela consists of only two species, Spirodela polyrhiza and Spirodela intermedia, both with a similar small genome (~160 Mbp/1C). Reference genome drafts and a physical map of 96 BACs on the 20 chromosome pairs of S. polyrhiza strain 7498 are available and provide useful tools for further evolutionary studies within and between duckweed genera. Here we applied sequential comparative multicolor fluorescence in situ hybridization (mcFISH) to address homeologous chromosomes in S. intermedia (2n = 36), to detect chromosome rearrangements between both species and to elucidate the mechanisms which may have led to the chromosome number alteration after their evolutionary separation. Ten chromosome pairs proved to be conserved between S. polyrhiza and S. intermedia, the remaining ones experienced, depending on the assumed direction of evolution, translocations, inversion, and fissions, respectively. These results represent a first step to unravel karyotype evolution among duckweeds and are anchor points for future genome assembly of S. intermedia.

Cost effectiveness of tobacco control policies in Vietnam: the case of population-level interventions.

BACKGROUND: Tobacco smoking is one of the leading public health problems in the world. It is also possible to prevent and/or reduce the harm from tobacco use through the use of cost-effective tobacco control measures. However, most of this evidence comes from developed countries and little research has been conducted on this issue in developing countries. OBJECTIVE: The objective of this study was to analyse the cost effectiveness of four population-level tobacco control interventions in Vietnam. METHODS: Four tobacco control interventions were evaluated: excise tax increase; graphic warning labels on cigarette packs; mass media campaigns; and smoking bans (in public or in work places). A multi-state life table model was constructed in Microsoft® Excel to examine the cost effectiveness of the tobacco control intervention options. A government perspective was adopted, with costing conducted using a bottom-up approach. Health improvement was considered in terms of disability-adjusted life-years (DALYs) averted. All assumptions were subject to sensitivity and uncertainty analysis. RESULTS: All the interventions fell within the definition of being very cost effective according to the threshold level suggested by the WHO (i.e.

The neurosurvival factor Humanin inhibits beta-cell apoptosis via signal transducer and activator of transcription 3 activation and delays and ameliorates diabetes in nonobese diabetic mice.

Pancreatic beta-cell apoptosis is important in the pathogenesis and potential treatment of type 1 diabetes mellitus. We investigated whether Humanin, a recently described survival factor for neurons, could improve the survival of beta-cells and delay or treat diabetes in the nonobese diabetic (NOD) model. Humanin reduced apoptosis induced by serum starvation in NIT-1 cells and decreased apoptosis induced by cytokine treatment. Humanin induced signal transducer and activator of transcription 3 and extracellular signal-regulated kinase phosphorylation over a 24-hour time course. Specific inhibition of signal transducer and activator of transcription 3 resulted in nullifying the protective effect of Humanin. Humanin normalized glucose tolerance in NOD mice treated for 6 weeks, and their pancreata revealed decreased lymphocyte infiltration and severity. In addition, Humanin delayed/prevented the onset of diabetes in NOD mice treated for 20 weeks. In summary, Humanin treatment decreases cytokine-induced apoptosis in beta-cells in vitro and improved glucose tolerance and onset of diabetes in NOD mice in vivo. This indicates that Humanin may be useful for islet protection and survival in a spectrum of diabetes-related therapeutics.