Distributed representations of prediction error signals across the cortical hierarchy are synergistic.

A relevant question concerning inter-areal communication in the cortex is whether these interactions are synergistic. Synergy refers to the complementary effect of multiple brain signals conveying more information than the sum of each isolated signal. Redundancy, on the other hand, refers to the common information shared between brain signals. Here, we dissociated cortical interactions encoding complementary information (synergy) from those sharing common information (redundancy) during prediction error (PE) processing. We analyzed auditory and frontal electrocorticography (ECoG) signals in five common awake marmosets performing two distinct auditory oddball tasks and investigated to what extent event-related potentials (ERP) and broadband (BB) dynamics encoded synergistic and redundant information about PE processing. The information conveyed by ERPs and BB signals was synergistic even at lower stages of the hierarchy in the auditory cortex and between auditory and frontal regions. Using a brain-constrained neural network, we simulated the synergy and redundancy observed in the experimental results and demonstrated that the emergence of synergy between auditory and frontal regions requires the presence of strong, long-distance, feedback, and feedforward connections. These results indicate that distributed representations of PE signals across the cortical hierarchy can be highly synergistic.

Mycobacterial RNase E cleaves with a distinct sequence preference and controls the degradation rates of most Mycolicibacterium smegmatis mRNAs.

The mechanisms and regulation of RNA degradation in mycobacteria have been subject to increased interest following the identification of interplay between RNA metabolism and drug resistance. Mycobacteria encode multiple ribonucleases predicted to participate in mRNA degradation and/or processing of stable RNAs. RNase E is hypothesized to play a major role in mRNA degradation because of its essentiality in mycobacteria and its role in mRNA degradation in gram-negative bacteria. Here, we defined the impact of RNase E on mRNA degradation rates transcriptome-wide in the nonpathogenic model Mycolicibacterium smegmatis. RNase E played a rate-limiting role in degradation of the transcripts encoded by at least 89% of protein-coding genes, with leadered transcripts often being more affected by RNase E repression than leaderless transcripts. There was an apparent global slowing of transcription in response to knockdown of RNase E, suggesting that M. smegmatis regulates transcription in responses to changes in mRNA degradation. This compensation was incomplete, as the abundance of most transcripts increased upon RNase E knockdown. We assessed the sequence preferences for cleavage by RNase E transcriptome-wide in M. smegmatis and Mycobacterium tuberculosis and found a consistent bias for cleavage in C-rich regions. Purified RNase E had a clear preference for cleavage immediately upstream of cytidines, distinct from the sequence preferences of RNase E in gram-negative bacteria. We furthermore report a high-resolution map of mRNA cleavage sites in M. tuberculosis, which occur primarily within the RNase E-preferred sequence context, confirming that RNase E has a broad impact on the M. tuberculosis transcriptome.

A research program-linked, course-based undergraduate research experience that allows undergraduates to participate in current research on mycobacterial gene regulation.

Undergraduate instructional biology laboratories are typically taught within two paradigms. Some labs focus on protocols and techniques delivered in "cookbook" format with defined experimental outcomes. There is increasing momentum to alternatively employ student-driven, open-ended, and discovery-based strategies, often via course-based undergraduate research experiences (CUREs) using crowd-sourcing initiatives. A fraction of students also participate in funded research in faculty research labs, where they have opportunities to work on projects designed to expand the frontiers of human knowledge. These experiences are widely recognized as valuable but are not scalable, as most institutions have many more undergraduates than research lab positions. We sought to address this gap through our department's curriculum by creating an opportunity for students to participate in the real-world research process within a laboratory course. We conceived, developed, and delivered an authentic, guided research experience to students in an upper-level molecular biology laboratory course. We refer to this model as a "research program-linked CURE." The research questions come directly from a faculty member's research lab and evolve along with that research program. Students study post-transcriptional regulation in mycobacteria. We use current molecular biology methodologies to test hypotheses like "UTRs affect RNA and protein expression levels," "there is functional redundancy among RNA helicases," and "carbon starvation alters mRNA 5' end chemistries." We conducted standard assessments and developed a customized "Skills and Concepts Inventory" survey to gauge how well the course met our student learning outcomes. We report the results of our assessments and describe challenges addressed during development and execution of the course, including organizing activities to fit within an instructional lab, balancing breadth with depth, and maintaining authenticity while giving students the experience of obtaining interpretable and novel results. Our data suggest student learning was enhanced through this truly authentic research approach. Further, students were able to perceive they were participants and contributors within an active research paradigm. Students reported increases in their self-identification as scientists, and a positive impact on their career trajectories. An additional benefit was reciprocation back to the funded research laboratory, by funneling course alumni, results, materials, and protocols.

The Impact of Leadered and Leaderless Gene Structures on Translation Efficiency, Transcript Stability, and Predicted Transcription Rates in Mycobacterium smegmatis.

Regulation of gene expression is critical for Mycobacterium tuberculosis to tolerate stressors encountered during infection and for nonpathogenic mycobacteria such as Mycobacterium smegmatis to survive environmental stressors. Unlike better-studied models, mycobacteria express ∼14% of their genes as leaderless transcripts. However, the impacts of leaderless transcript structures on mRNA half-life and translation efficiency in mycobacteria have not been directly tested. For leadered transcripts, the contributions of 5' untranslated regions (UTRs) to mRNA half-life and translation efficiency are similarly unknown. In M. tuberculosis and M. smegmatis, the essential sigma factor, SigA, is encoded by a transcript with a relatively short half-life. We hypothesized that the long 5' UTR of sigA causes this instability. To test this, we constructed fluorescence reporters and measured protein abundance, mRNA abundance, and mRNA half-life and calculated relative transcript production rates. The sigA 5' UTR conferred an increased transcript production rate, shorter mRNA half-life, and decreased apparent translation rate compared to a synthetic 5' UTR commonly used in mycobacterial expression plasmids. Leaderless transcripts appeared to be translated with similar efficiency as those with the sigA 5' UTR but had lower predicted transcript production rates. A global comparison of M. tuberculosis mRNA and protein abundances failed to reveal systematic differences in protein/mRNA ratios for leadered and leaderless transcripts, suggesting that variability in translation efficiency is largely driven by factors other than leader status. Our data are also discussed in light of an alternative model that leads to different conclusions and suggests leaderless transcripts may indeed be translated less efficiently.IMPORTANCE Tuberculosis, caused by Mycobacterium tuberculosis, is a major public health problem killing 1.5 million people globally each year. During infection, M. tuberculosis must alter its gene expression patterns to adapt to the stress conditions it encounters. Understanding how M. tuberculosis regulates gene expression may provide clues for ways to interfere with the bacterium's survival. Gene expression encompasses transcription, mRNA degradation, and translation. Here, we used Mycobacterium smegmatis as a model organism to study how 5' untranslated regions affect these three facets of gene expression in multiple ways. We furthermore provide insight into the expression of leaderless mRNAs, which lack 5' untranslated regions and are unusually prevalent in mycobacteria.

Match and mismatch between dietary switches and microbial partners in plant sap-feeding insects.

Some animal groups associate with the same vertically transmitted microbial symbionts over extended periods of evolutionary time, punctuated by occasional symbiont switches to different microbial taxa. Here we test the oft-repeated suggestion that symbiont switches are linked with host diet changes, focusing on hemipteran insects of the suborder Auchenorrhyncha. These insects include the only animals that feed on plant xylem sap through the life cycle, as well as taxa that feed on phloem sap and plant parenchyma cells. Ancestral state reconstruction provides strong statistical support for a xylem feeding auchenorrhynchan ancestor bearing the dual symbiosis with the primary symbiont Sulcia (Bacteroidetes) and companion symbiont 'ß-Sym' (ß-proteobacteria). We identified seven dietary transitions from xylem feeding (six to phloem feeding, one to parenchyma feeding), but no reversions to xylem feeding; five evolutionary losses of Sulcia, including replacements by yeast symbionts, exclusively in phloem/parenchyma-feeding lineages; and 14-15 losses of ß-Sym, including nine transitions to a different bacterial companion symbiont. Our analysis indicates that, although companion symbiont switching is not associated with shifts in host diet, Sulcia is probably required for xylem-feeding. Furthermore, the ancestral auchenorrhynchan bearing Sulcia and ß-Sym probably represents the sole evolutionary origin of xylem feeding in the animal kingdom.

Rhodiola crenulata induces death and inhibits growth of breast cancer cell lines.

Diverse compounds from many different chemical classes are currently targeted in preclinical analyses for their ability to act as both chemopreventive and chemotherapeutic agents. Phenolic phytochemicals from Rhodiola crenulata has such potential. This Rhodiola species is a perennial plant that grows in the Tundra, Siberia, and high-elevation regions of Tibet. The phenolic secondary metabolites isolated from R. crenulata were recently analyzed in a preclinical setting for their ability to treat lymphosarcomas and superficial bladder cancers. However, the effects of R. crenulata have yet to be examined for its implications in breast cancer prevention or for its chemotherapeutic abilities. Therefore this study investigated the effects of R. crenulata on breast cancer both in vivo and in vitro. Experiments using aggressive human-derived MDA-MB-231 and mouse-derived V14 breast cancer cell lines demonstrated that phenolic-enriched R. crenulata extract was capable of inhibiting the proliferation, motility, and invasion of these cells. In addition, the extracts induced autophagic-like vesicles in all cell lines, eventually leading to death of the tumor cell lines but not the immortal or normal human mammary epithelial cells. Finally, an in vivo experiment showed that phenolic-enriched dietary R. crenulata is effective in preventing the initiation of tumors and slowing down the tumor growth in mice bearing tumor grafts, thereby further demonstrating its possible potential for treatment of breast cancer progression and metastasis.

Mutations in Arabidopsis yellow stripe-like1 and yellow stripe-like3 reveal their roles in metal ion homeostasis and loading of metal ions in seeds.

Here, we describe two members of the Arabidopsis (Arabidopsis thaliana) Yellow Stripe-Like (YSL) family, AtYSL1 and AtYSL3. The YSL1 and YSL3 proteins are members of the oligopeptide transporter family and are predicted to be integral membrane proteins. YSL1 and YSL3 are similar to the maize (Zea mays) YS1 phytosiderophore transporter (ZmYS1) and the AtYSL2 iron (Fe)-nicotianamine transporter, and are predicted to transport metal-nicotianamine complexes into cells. YSL1 and YSL3 mRNAs are expressed in both root and shoot tissues, and both are regulated in response to the Fe status of the plant. Beta-glucuronidase reporter expression, driven by YSL1 and YSL3 promoters, reveals expression patterns of the genes in roots, leaves, and flowers. Expression was highest in senescing rosette leaves and cauline leaves. Whereas the single mutants ysl1 and ysl3 had no visible phenotypes, the ysl1ysl3 double mutant exhibited Fe deficiency symptoms, such as interveinal chlorosis. Leaf Fe concentrations are decreased in the double mutant, whereas manganese, zinc, and especially copper concentrations are elevated. In seeds of double-mutant plants, the concentrations of Fe, zinc, and copper are low. Mobilization of metals from leaves during senescence is impaired in the double mutant. In addition, the double mutant has reduced fertility due to defective anther and embryo development. The proposed physiological roles for YSL1 and YSL3 are in delivery of metal micronutrients to and from vascular tissues.

Application of colorimetric assays to assess viability, growth and metabolism of hydrogel-encapsulated cells.

The applicability of the colorimetric 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays to measure cell growth and viability in hydrogel encapsulation systems was investigated using HepG2 liver cells encapsulated in alginate matrices. The MTT assay was effective in measuring viable cell density in alginate-encapsulated cell systems, demonstrating less variance and higher throughput capability than hemocytometry. The LDH assay was effective in measuring dead cell density in monolayer cultures and in alginate-encapsulated cells simply by measuring the LDH concentration secreted into the medium. Further validation of these assays was shown in two additional cell lines (rat muscle and mouse embryonic fibroblasts). The MTT and LDH assays are particularly significant in the rapid evaluation of in vitro cell encapsulation device design.

Arabidopsis Yellow Stripe-Like2 (YSL2): a metal-regulated gene encoding a plasma membrane transporter of nicotianamine-metal complexes.

The Yellow Stripe-Like (YSL) family of proteins has been identified based on sequence similarity to maize Yellow Stripe1 (YS1), the transporter responsible for the primary uptake of iron from the soil. YS1 transports iron that is complexed by specific plant-derived Fe(III) chelators called phytosiderophores (PS). Non-grass species of plants neither make nor use PS, yet YSL family members are found in non-grass species (monocot, dicot, gymnosperm, and moss species) including Arabidopsis thaliana. YSLs in non-grasses have been hypothesized to transport metals complexed by nicotianamine (NA), an iron chelator that is structurally similar to PS and which is found in all higher plants. Here we show that Arabidopsis YSL2 (At5g24380) transports iron and copper when these metals are chelated by NA. YSL2 is expressed in many cell types in both roots and shoots, suggesting that diverse cell types obtain metals as metal-NA complexes. YSL2 transcription is regulated by the levels of iron and copper in the growth medium. Based on its expression pattern, a major function of the YSL2 appears to be in the lateral movement of metals in the vasculature.

Yellow stripe1. Expanded roles for the maize iron-phytosiderophore transporter.

Graminaceous monocots, including most of the world's staple grains (i.e. rice, corn, and wheat) use a chelation strategy (Strategy II) for primary acquisition of iron from the soil. Strategy II plants secrete phytosiderophores (PS), compounds of the mugineic acid family that form stable Fe(III) chelates in soil. Uptake of iron-PS chelates, which occurs through specific transporters at the root surface, thus represents the primary route of iron entry into Strategy II plants. The gene Yellow stripe1 (Ys1) encodes the Fe(III)-PS transporter of maize (Zea mays). Here the physiological functions performed by maize YS1 were further defined by examining the pattern of Ys1 mRNA and protein accumulation and by defining YS1 transport specificity in detail. YS1 is able to translocate iron that is bound either by PS or by the related compound, nicotianamine; thus, the role of YS1 may be to transport either of these complexes. Ys1 expression at both the mRNA and protein levels responds rapidly to changes in iron availability but is not strongly affected by limitation of copper or zinc. Our data provide no support for the idea that YS1 is a transporter of zinc-PS, based on YS1 biochemical activity and Ys1 mRNA expression patterns in response to zinc deficiency. YS1 is capable of transporting copper-PS, but expression data suggest that the copper-PS uptake has limited significance in primary uptake of copper.

Cdc42 and RhoA are differentially regulated during arachidonate-mediated HeLa cell adhesion.

Cell adhesion to extracellular matrix requires stimulation of an eicosanoid signaling pathway through the metabolism of arachidonate by 5-lipoxygenase to leukotrienes and cyclooxygenase-1/2 to prostaglandins, as well as activation of the small GTPase signaling pathway involving Cdc42 and Rho. These signaling pathways direct remodeling of the actin cytoskeleton during the adhesion process, specifically the polymerization of actin during cell spreading and the bundling of actin filaments when cells migrate. However, few studies linking these signaling pathways have been described in the literature. We have previously shown that HeLa cell adhesion to collagen requires oxidation of arachidonic acid (AA) by lipoxygenase for actin polymerization and cell spreading, and cyclooxygenase for bundling actin filaments during cell migration. We demonstrate that small GTPase activity is required for HeLa cell spreading upon gelatin, and that Cdc42 is activated while Rho is downregulated during the spreading process. Using constitutively active and dominant negative expression studies, we show that Cdc42 is required for HeLa cell spreading and migration, while activated RhoA is antagonistic towards spreading. Constitutively active RhoA promotes cell migration and increases the degree of actin bundling in HeLa cells. Further, we demonstrate that activation of either the AA oxidation pathway or the small GTPase pathway cannot rescue inhibition of spreading when the alternate pathway is blocked. Our results suggest (1) both the eicosanoid signaling pathway and small GTPase activation are required during HeLa cell adhesion, and (2) these signaling pathways converge to properly direct remodeling of the actin cytoskeleton during HeLa cell spreading and migration upon collagen.

Mcm3 is polyubiquitinated during mitosis before establishment of the pre-replication complex.

To ensure fidelity in genome duplication, eukaryotes restrict DNA synthesis to once every cell division by a cascade of regulated steps. Central to this cascade is the periodic assembly of the hexameric MCM2-7 complex at replication origins. However, in Saccharomyces cerevisiae, only a fraction of each MCM protein is able to assemble into hexamers and associate with replication origins during M phase, suggesting that MCM complex assembly and recruitment may be regulated post-translationally. Here we show that a small fraction of Mcm3p is polyubiquitinated at the onset of MCM complex assembly. Reducing the rate of ubiquitination by uba1-165, a suppressor of mcm3-10, restored the interaction of Mcm3-10p with subunits of the MCM complex and its recruitment to the replication origin. Possible roles for ubiquitinated Mcm3p in the assembly of the MCM complex at replication origins are discussed.

Two mcm3 mutations affect different steps in the initiation of DNA replication.

Mcm3 is a subunit of the hexameric MCM2-7 complex required for the initiation and elongation of DNA replication in eukaryotes. We have characterized two mutant alleles, mcm3-1 and mcm3-10, in Saccharomyces cerevisiae and showed that they are defective at different steps of the replication initiation process. Mcm3-10 contains a P118L substitution that compromises its interaction with Mcm5 and the recruitment of Mcm3 and Mcm7 to a replication origin. P118 is conserved between Mcm3, Mcm4, Mcm5, and Mcm7. An identical substitution of this conserved residue in Mcm5 (P83L of mcm5-bob1) strengthens the interaction between Mcm3 and Mcm5 and allows cells to enter S phase independent of Cdc7-Dbf4 kinase (Hardy, C. F., Dryga, O., Pahl, P. M. B., and Sclafani, R. A. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3151-3155). Mcm3-1 contains a G246E mutation that diminishes the efficiency of replication initiation (Yan, H., Merchant, A. M., and Tye, B. K. (1993) Genes Dev. 7, 2149-2160) but not its interaction with Mcm5 or recruitment of the MCM2-7 complex to replication origin. These observations indicate that Mcm3-10 is defective in a step before, and Mcm3-1 is defective in a step after the recruitment of the MCM2-7 complex to replication origins.