A Handbook of Learner-Centered Projects to Suppliment Fifth Grade, Social Studies Curriculum

This handbook has been created in an effort to assist teachers with establishing a hands-on, learner-centered environment. The purpose of this handbook is to assist educators with teaching fifth grade, Social Studies curriculum. This handbook will help teachers to organize fifth grade expectations into thematic, learner-centered, intrinsically motivated projects that will allow for a well-planned, teacher facilitated, and educational experience. This is more than just a how-to manual; it includes the theoretical and empirical basis thesis for using thematic, cross-curricular projects, to enhance student retention, motivation and independent learning

Application of multiplexed ion mobility spectrometry towards the identification of host protein signatures of treatment effect in pulmonary tuberculosis.

RationaleThe monitoring of TB treatments in clinical practice and clinical trials relies on traditional sputum-based culture status indicators at specific time points. Accurate, predictive, blood-based protein markers would provide a simpler and more informative view of patient health and response to treatment.ObjectiveWe utilized sensitive, high throughput multiplexed ion mobility-mass spectrometry (IM-MS) to characterize the serum proteome of TB patients at the start of and at 8 weeks of rifamycin-based treatment. We sought to identify treatment specific signatures within patients as well as correlate the proteome signatures to various clinical markers of treatment efficacy.MethodsSerum samples were collected from 289 subjects enrolled in CDC TB Trials Consortium Study 29 at time of enrollment and at the end of the intensive phase (after 40 doses of TB treatment). Serum proteins were immunoaffinity-depleted of high abundant components, digested to peptides and analyzed for data acquisition utilizing a unique liquid chromatography IM-MS platform (LC-IM-MS). Linear mixed models were utilized to identify serum protein changes in the host response to antibiotic treatment as well as correlations with culture status end points.ResultsA total of 10,137 peptides corresponding to 872 proteins were identified, quantified, and used for statistical analysis across the longitudinal patient cohort. In response to TB treatment, 244 proteins were significantly altered. Pathway/network comparisons helped visualize the interconnected proteins, identifying up regulated (lipid transport, coagulation cascade, endopeptidase activity) and down regulated (acute phase) processes and pathways in addition to other cross regulated networks (inflammation, cell adhesion, extracellular matrix). Detection of possible lung injury serum proteins such as HPSE, significantly downregulated upon treatment. Analyses of microbiologic data over time identified a core set of serum proteins (TTHY, AFAM, CRP, RET4, SAA1, PGRP2) which change in response to treatment and also strongly correlate with culture status. A similar set of proteins at baseline were found to be predictive of week 6 and 8 culture status.ConclusionA comprehensive host serum protein dataset reflective of TB treatment effect is defined. A repeating set of serum proteins (TTHY, AFAM, CRP, RET4, SAA1, PGRP2, among others) were found to change significantly in response to treatment, to strongly correlate with culture status, and at baseline to be predictive of future culture conversion. If validated in cohorts with long term follow-up to capture failure and relapse of TB, these protein markers could be developed for monitoring of treatment in clinical trials and in patient care

Production of ent-kaurene from lignocellulosic hydrolysate in Rhodosporidium toruloides.

BACKGROUND:Rhodosporidium toruloides has emerged as a promising host for the production of bioproducts from lignocellulose, in part due to its ability to grow on lignocellulosic feedstocks, tolerate growth inhibitors, and co-utilize sugars and lignin-derived monomers. Ent-kaurene derivatives have a diverse range of potential applications from therapeutics to novel resin-based materials. RESULTS:The Design, Build, Test, and Learn (DBTL) approach was employed to engineer production of the non-native diterpene ent-kaurene in R. toruloides. Following expression of kaurene synthase (KS) in R. toruloides in the first DBTL cycle, a key limitation appeared to be the availability of the diterpene precursor, geranylgeranyl diphosphate (GGPP). Further DBTL cycles were carried out to select an optimal GGPP synthase and to balance its expression with KS, requiring two of the strongest promoters in R. toruloides, ANT (adenine nucleotide translocase) and TEF1 (translational elongation factor 1) to drive expression of the KS from Gibberella fujikuroi and a mutant version of an FPP synthase from Gallus gallus that produces GGPP. Scale-up of cultivation in a 2 L bioreactor using a corn stover hydrolysate resulted in an ent-kaurene titer of 1.4 g/L. CONCLUSION:This study builds upon previous work demonstrating the potential of R. toruloides as a robust and versatile host for the production of both mono- and sesquiterpenes, and is the first demonstration of the production of a non-native diterpene in this organism

Urinary virome perturbations in kidney transplantation

The human microbiome is important for health and plays a role in essential metabolic functions and protection from certain pathogens. Conversely, dysbiosis of the microbiome is seen in the context of various diseases. Recent studies have highlighted that a complex microbial community containing hundreds of bacteria colonizes the healthy urinary tract, but little is known about the human urinary viruses in health and disease. To evaluate the human urinary virome in the context of kidney transplantation (tx), variations in the composition of the urinary virome were evaluated in urine samples from normal healthy volunteers as well as patients with kidney disease after they had undergone kidney tx. Liquid chromatography-mass spectrometry/mass spectrometry analysis was undertaken on a selected cohort of 142 kidney tx patients and normal healthy controls, from a larger biobank of 770 kidney biopsy matched urine samples. In addition to analysis of normal healthy control urine, the cohort of kidney tx patients had biopsy confirmed phenotype classification, coincident with the urine sample analyzed, of stable grafts (STA), acute rejection, BK virus nephritis, and chronic allograft nephropathy. We identified 37 unique viruses, 29 of which are being identified for the first time in human urine samples. The composition of the human urinary virome differs in health and kidney injury, and the distribution of viral proteins in the urinary tract may be further impacted by IS exposure, diet and environmental, dietary, or cutaneous exposure to various insecticides and pesticides

Potential for nitrogen fixation in fungus-growing termite symbiosis

Termites host a gut microbiota of diverse and essential symbionts that enable specialization on dead plant material; an abundant, but nutritionally imbalanced food source. To supplement the severe shortage of dietary nitrogen (N), some termite species make use of diazotrophic bacteria to fix atmospheric nitrogen (N2). Fungus-growing termites (subfamily Macrotermitinae) host a fungal exosymbiont (genus Termitomyces) that provides digestive services and the main food source for the termites. This has been thought to obviate the need for N2-fixation by bacterial symbionts. Here we challenge this notion by performing acetylene reduction assays of live colony material to show that N2 fixation is present in two major genera (Macrotermes and Odontotermes) of fungus-growing termites. We compare and discuss fixation rates in relation to those obtained from other termites, and suggest avenues of research that may lead to a better understanding of N2 fixation in fungus-growing and other termites

MPLEx: a Robust and Universal Protocol for Single-Sample Integrative Proteomic, Metabolomic, and Lipidomic Analyses

ABSTRACT Integrative multi-omics analyses can empower more effective investigation and complete understanding of complex biological systems. Despite recent advances in a range of omics analyses, multi-omic measurements of the same sample are still challenging and current methods have not been well evaluated in terms of reproducibility and broad applicability. Here we adapted a solvent-based method, widely applied for extracting lipids and metabolites, to add proteomics to mass spectrometry-based multi-omics measurements. The m etabolite, p rotein, and l ipid ex traction (MPLEx) protocol proved to be robust and applicable to a diverse set of sample types, including cell cultures, microbial communities, and tissues. To illustrate the utility of this protocol, an integrative multi-omics analysis was performed using a lung epithelial cell line infected with Middle East respiratory syndrome coronavirus, which showed the impact of this virus on the host glycolytic pathway and also suggested a role for lipids during infection. The MPLEx method is a simple, fast, and robust protocol that can be applied for integrative multi-omic measurements from diverse sample types (e.g., environmental, in vitro , and clinical). IMPORTANCE In systems biology studies, the integration of multiple omics measurements (i.e., genomics, transcriptomics, proteomics, metabolomics, and lipidomics) has been shown to provide a more complete and informative view of biological pathways. Thus, the prospect of extracting different types of molecules (e.g., DNAs, RNAs, proteins, and metabolites) and performing multiple omics measurements on single samples is very attractive, but such studies are challenging due to the fact that the extraction conditions differ according to the molecule type. Here, we adapted an organic solvent-based extraction method that demonstrated broad applicability and robustness, which enabled comprehensive proteomics, metabolomics, and lipidomics analyses from the same sample

Engineering Rhodosporidium toruloides for production of 3-hydroxypropionic acid from lignocellulosic hydrolysate

Microbial production of valuable bioproducts is a promising route towards green and sustainable manufacturing. The oleaginous yeast, Rhodosporidium toruloides, has emerged as an attractive host for the production of biofuels and bioproducts from lignocellulosic hydrolysates. 3-hydroxypropionic acid (3HP) is an attractive platform molecule that can be used to produce a wide range of commodity chemicals. This study focuses on establishing and optimizing the production of 3HP in R. toruloides. As R. toruloides naturally has a high metabolic flux towards malonyl-CoA, we exploited this pathway to produce 3HP. Upon finding the yeast capable of catabolizing 3HP, we then implemented functional genomics and metabolomic analysis to identify the catabolic pathways. Deletion of a putative malonate semialdehyde dehydrogenase gene encoding an oxidative 3HP pathway was found to significantly reduce 3HP degradation. We further explored monocarboxylate transporters to promote 3HP transport and identified a novel 3HP transporter in Aspergillus pseudoterreus by RNA-seq and proteomics. Combining these engineering efforts with media optimization in a fed-batch fermentation resulted in 45.4 g/L 3HP production. This represents one of the highest 3HP titers reported in yeast from lignocellulosic feedstocks. This work establishes R. toruloides as a host for 3HP production from lignocellulosic hydrolysate at high titers, and paves the way for further strain and process optimization towards enabling industrial production of 3HP in the future

Proteomic and Transcriptomic Analyses of “Candidatus Pelagibacter ubique” Describe the First P[subscript II]-Independent Response to Nitrogen Limitation in a Free-Living Alphaproteobacterium

Nitrogen is one of the major nutrients limiting microbial productivity in the ocean, and as a result, most marine microorganisms have evolved systems for responding to nitrogen stress. The highly abundant alphaproteobacterium “Candidatus Pelagibacter ubique,” a cultured member of the order Pelagibacterales (SAR11), lacks the canonical GlnB, GlnD, GlnK, and NtrB/NtrC genes for regulating nitrogen assimilation, raising questions about how these organisms respond to nitrogen limitation. A survey of 266 Alphaproteobacteria genomes found these five regulatory genes nearly universally conserved, absent only in intracellular parasites and members of the order Pelagibacterales, including “Ca. Pelagibacter ubique.” Global differences in mRNA and protein expression between nitrogen-limited and nitrogen-replete cultures were measured to identify nitrogen stress responses in “Ca. Pelagibacter ubique” strain HTCC1062. Transporters for ammonium (AmtB), taurine (TauA), amino acids (YhdW), and opines (OccT) were all elevated in nitrogen-limited cells, indicating that they devote increased resources to the assimilation of nitrogenous organic compounds. Enzymes for assimilating amine into glutamine (GlnA), glutamate (GltBD), and glycine (AspC) were similarly upregulated. Differential regulation of the transcriptional regulator NtrX in the two-component signaling system NtrY/NtrX was also observed, implicating it in control of the nitrogen starvation response. Comparisons of the transcriptome and proteome supported previous observations of uncoupling between transcription and translation in nutrient-deprived “Ca. Pelagibacter ubique” cells. Overall, these data reveal a streamlined, P[subscript II]-independent response to nitrogen stress in “Ca. Pelagibacter ubique,” and likely other Pelagibacterales, and show that they respond to nitrogen stress by allocating more resources to the assimilation of nitrogen-rich organic compounds.This is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by the American Society for Microbiology. The published article can be found at: http://mbio.asm.org/