Responsive Nanogel Probe for Ratiometric Fluorescent Sensing of pH and Strain in Hydrogels

In this study a new pH-responsive nanogel probe containing a complementary nonradiative resonance energy transfer (NRET) fluorophore pair is investigated and its ability to act as a versatile probe of network-related changes in three hydrogels demonstrated. Fluorescent sensing using NRET is a powerful method for studying relationships between Angstrom length-scale structure and macroscopic properties of soft matter. Unfortunately, inclusion of NRET fluorophores into such materials requires material-specific chemistry. Here, low concentrations of preformed nanogel probes were included into hydrogel hosts. Ratiometric photoluminescence (PL) data for the gels labeled with the nanogel probes enabled pH-triggered swelling and deswelling to be studied as well as Ca2+-triggered collapse and solute release. PL measurements during compression of a nanogel probe-labeled nanocomposite gel demonstrated mechanochromic behavior and strain sensing. The new nanogel probes have excellent potential for investigating the internal structures of gels and provide a versatile ratiometric fluorescent platform for studying pH and strain

PmoB subunit of particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath): The Cu^I sponge and its function

In this study, we describe efforts to clarify the role of the copper cofactors associated with subunit B (PmoB) of the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) (M. capsulatus). This subunit exhibits strong affinity toward Cu^I ions. To elucidate the high copper affinity of the subunit, the full-length PmoB, and the N-terminal truncated mutants PmoB_(33–414) and PmoB_(55–414), each fused to the maltose-binding protein (MBP), are cloned and over-expressed into Escherichia coli (E. coli) K12 TB1 cells. The Y374F, Y374S and M300L mutants of these protein constructs are also studied. When this E. coli is grown with the pmoB gene in 1.0 mM Cu^(II), it behaves like M. capsulatus (Bath) cultured under high copper stresswith abundant membrane accumulation and high CuI content. The recombinantPmoB proteins are verified by Western blotting of antibodies directed against the MBP sub-domain in each of the copper-enriched PmoB proteins. Cu K-edge X-rayabsorption near edge spectroscopy (XANES) of the copper ions confirms that all the PmoB recombinants are Cu^I proteins. All the PmoB proteins show evidence of a “dicopper site” according to analysis of the Cu extended X-ray absorption edge fine structure (EXAFS) of the membranes. No specific activities toward methane and propene oxidation are observed with the recombinant membrane-bound PmoB proteins. However, significant production of hydrogen peroxide is observed in the case of the PmoB_(33–414) mutant. Reaction of the dicopper site with dioxygenproduces hydrogen peroxide and leads to oxidation of the CuI ions residing in the C-terminal sub-domain of the PmoB subunit

Complement receptor 1 is the human erythrocyte receptor for Plasmodium vivax erythrocyte binding protein

The discovery that Africans were resistant to infection by Plasmodium vivax ( P. vivax) led to the conclusion that P. vivax invasion relied on the P. vivax Duffy Binding Protein (PvDBP) interacting with the Duffy Antigen Receptor for Chemokines (DARC) expressed on erythrocytes. However, the recent reporting of P. vivax infections in DARC-negative Africans suggests that the parasite might use an alternate invasion pathway to infect DARC-negative reticulocytes. To identify the parasite ligands and erythrocyte receptors that enable P. vivax invasion of both DARC-positive and -negative erythrocytes, we expressed region II containing the Duffy Binding-Like (DBL) domain of P. vivax erythrocyte binding protein (PvEBP-RII) and verified that the DBL domain binds to both DARC-positive and -negative erythrocytes. Furthermore, an AVidity-based EXtracelluar Interaction Screening (AVEXIS) was used to identify the receptor for PvEBP among over 750 human cell surface receptor proteins, and this approach identified only Complement Receptor 1 (CR1, CD35, or C3b/C4b receptor) as a PvEBP receptor. CR1 is a well-known receptor for P. falciparum Reticulocyte binding protein Homology 4 (PfRh4) and is present on the surfaces of both reticulocytes and normocytes, but its expression decreases as erythrocytes age. Indeed, PvEBP-RII bound to a subpopulation of both reticulocytes and normocytes, and this binding was blocked by the addition of soluble CR1 recombinant protein, indicating that CR1 is the receptor of PvEBP. In addition, we found that the Long Homology Repeat A (LHR-A) subdomain of CR1 is the only subdomain responsible for mediating the interaction with PvEBP-RII

PmoB subunit of particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath): The Cu^I sponge and its function

In this study, we describe efforts to clarify the role of the copper cofactors associated with subunit B (PmoB) of the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) (M. capsulatus). This subunit exhibits strong affinity toward Cu^I ions. To elucidate the high copper affinity of the subunit, the full-length PmoB, and the N-terminal truncated mutants PmoB_(33–414) and PmoB_(55–414), each fused to the maltose-binding protein (MBP), are cloned and over-expressed into Escherichia coli (E. coli) K12 TB1 cells. The Y374F, Y374S and M300L mutants of these protein constructs are also studied. When this E. coli is grown with the pmoB gene in 1.0 mM Cu^(II), it behaves like M. capsulatus (Bath) cultured under high copper stresswith abundant membrane accumulation and high CuI content. The recombinantPmoB proteins are verified by Western blotting of antibodies directed against the MBP sub-domain in each of the copper-enriched PmoB proteins. Cu K-edge X-rayabsorption near edge spectroscopy (XANES) of the copper ions confirms that all the PmoB recombinants are Cu^I proteins. All the PmoB proteins show evidence of a “dicopper site” according to analysis of the Cu extended X-ray absorption edge fine structure (EXAFS) of the membranes. No specific activities toward methane and propene oxidation are observed with the recombinant membrane-bound PmoB proteins. However, significant production of hydrogen peroxide is observed in the case of the PmoB_(33–414) mutant. Reaction of the dicopper site with dioxygenproduces hydrogen peroxide and leads to oxidation of the CuI ions residing in the C-terminal sub-domain of the PmoB subunit

Optical Control of Metabotropic Glutamate Receptors

G-protein coupled receptors (GPCRs), the largest family of membrane signaling proteins, respond to neurotransmitters, hormones and small environmental molecules. The neuronal function of many GPCRs has been difficult to resolve because of an inability to gate them with subtype-specificity, spatial precision, speed and reversibility. To address this, we developed an approach for opto-chemical engineering native GPCRs. We applied this to the metabotropic glutamate receptors (mGluRs) to generate light-agonized and light-antagonized “LimGluRs”. The light-agonized “LimGluR2”, on which we focused, is fast, bistable, and supports multiple rounds of on/off switching. Light gates two of the primary neuronal functions of mGluR2: suppression of excitability and inhibition of neurotransmitter release. The light-antagonized “LimGluR2block” can be used to manipulate negative feedback of synaptically released glutamate on transmitter release. We generalize the optical control to two additional family members: mGluR3 and 6. The system works in rodent brain slice and in zebrafish in vivo, where we find that mGluR2 modulates the threshold for escape behavior. These light-gated mGluRs pave the way for determining the roles of mGluRs in synaptic plasticity, memory and disease

Sequencing of diverse mandarin, pummelo and orange genomes reveals complex history of admixture during citrus domestication

Cultivated citrus are selections from, or hybrids of, wild progenitor species whose identities and contributions to citrus domestication remain controversial. Here we sequence and compare citrus genomes-a high-quality reference haploid clementine genome and mandarin, pummelo, sweet-orange and sour-orange genomes-and show that cultivated types derive from two progenitor species. Although cultivated pummelos represent selections from one progenitor species, Citrus maxima, cultivated mandarins are introgressions of C. maxima into the ancestral mandarin species Citrus reticulata. The most widely cultivated citrus, sweet orange, is the offspring of previously admixed individuals, but sour orange is an F1 hybrid of pure C. maxima and C. reticulata parents, thus implying that wild mandarins were part of the early breeding germplasm. A Chinese wild 'mandarin' diverges substantially from C. reticulata, thus suggesting the possibility of other unrecognized wild citrus species. Understanding citrus phylogeny through genome analysis clarifies taxonomic relationships and facilitates sequence-directed genetic improvement

Landscape of somatic single nucleotide variants and indels in colorectal cancer and impact on survival

Colorectal cancer (CRC) is a biologically heterogeneous disease. To characterize its mutational profile, we conduct targeted sequencing of 205 genes for 2,105 CRC cases with survival data. Our data shows several findings in addition to enhancing the existing knowledge of CRC. We identify PRKCI, SPZ1, MUTYH, MAP2K4, FETUB, and TGFBR2 as additional genes significantly mutated in CRC. We find that among hypermutated tumors, an increased mutation burden is associated with improved CRC-specific survival (HR=0.42, 95% CI: 0.21-0.82). Mutations in TP53 are associated with poorer CRC-specific survival, which is most pronounced in cases carrying TP53 mutations with predicted 0% transcriptional activity (HR=1.53, 95% CI: 1.21-1.94). Furthermore, we observe differences in mutational frequency of several genes and pathways by tumor location, stage, and sex. Overall, this large study provides deep insights into somatic mutations in CRC, and their potential relationships with survival and tumor features. Large scale sequencing study is of paramount importance to unravel the heterogeneity of colorectal cancer. Here, the authors sequenced 205 cancer genes in more than 2000 tumours and identified additional mutated driver genes, determined that mutational burden and specific mutations in TP53 are associated with survival odds

Three Novel Downstream Promoter Elements Regulate MHC Class I Promoter Activity in Mammalian Cells

BACKGROUND: MHC CLASS I TRANSCRIPTION IS REGULATED BY TWO DISTINCT TYPES OF REGULATORY PATHWAYS: 1) tissue-specific pathways that establish constitutive levels of expression within a given tissue and 2) dynamically modulated pathways that increase or decrease expression within that tissue in response to hormonal or cytokine mediated stimuli. These sets of pathways target distinct upstream regulatory elements, have distinct basal transcription factor requirements, and utilize discrete sets of transcription start sites within an extended core promoter. METHODOLOGY/PRINCIPAL FINDINGS: We studied regulatory elements within the MHC class I promoter by cellular transfection and in vitro transcription assays in HeLa, HeLa/CIITA, and tsBN462 of various promoter constructs. We have identified three novel MHC class I regulatory elements (GLE, DPE-L1 and DPE-L2), located downstream of the major transcription start sites, that contribute to the regulation of both constitutive and activated MHC class I expression. These elements located at the 3' end of the core promoter preferentially regulate the multiple transcription start sites clustered at the 5' end of the core promoter. CONCLUSIONS/SIGNIFICANCE: Three novel downstream elements (GLE, DPE-L1, DPE-L2), located between +1 and +32 bp, regulate both constitutive and activated MHC class I gene expression by selectively increasing usage of transcription start sites clustered at the 5' end of the core promoter upstream of +1 bp. Results indicate that the downstream elements preferentially regulate TAF1-dependent, relative to TAF1-independent, transcription

Effect of surgical experience and spine subspecialty on the reliability of the {AO} Spine Upper Cervical Injury Classification System

OBJECTIVE The objective of this paper was to determine the interobserver reliability and intraobserver reproducibility of the AO Spine Upper Cervical Injury Classification System based on surgeon experience (< 5 years, 5–10 years, 10–20 years, and > 20 years) and surgical subspecialty (orthopedic spine surgery, neurosurgery, and "other" surgery). METHODS A total of 11,601 assessments of upper cervical spine injuries were evaluated based on the AO Spine Upper Cervical Injury Classification System. Reliability and reproducibility scores were obtained twice, with a 3-week time interval. Descriptive statistics were utilized to examine the percentage of accurately classified injuries, and Pearson’s chi-square or Fisher’s exact test was used to screen for potentially relevant differences between study participants. Kappa coefficients (κ) determined the interobserver reliability and intraobserver reproducibility. RESULTS The intraobserver reproducibility was substantial for surgeon experience level (< 5 years: 0.74 vs 5–10 years: 0.69 vs 10–20 years: 0.69 vs > 20 years: 0.70) and surgical subspecialty (orthopedic spine: 0.71 vs neurosurgery: 0.69 vs other: 0.68). Furthermore, the interobserver reliability was substantial for all surgical experience groups on assessment 1 (< 5 years: 0.67 vs 5–10 years: 0.62 vs 10–20 years: 0.61 vs > 20 years: 0.62), and only surgeons with > 20 years of experience did not have substantial reliability on assessment 2 (< 5 years: 0.62 vs 5–10 years: 0.61 vs 10–20 years: 0.61 vs > 20 years: 0.59). Orthopedic spine surgeons and neurosurgeons had substantial intraobserver reproducibility on both assessment 1 (0.64 vs 0.63) and assessment 2 (0.62 vs 0.63), while other surgeons had moderate reliability on assessment 1 (0.43) and fair reliability on assessment 2 (0.36). CONCLUSIONS The international reliability and reproducibility scores for the AO Spine Upper Cervical Injury Classification System demonstrated substantial intraobserver reproducibility and interobserver reliability regardless of surgical experience and spine subspecialty. These results support the global application of this classification system