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Here we describe a method for the detection of Clostridium difficile from stool using a novel low-complexity and rapid extraction process called Heat Elution (HE). The HE method is two-step and takes just 10 minutes, no specialist instruments are required and there is minimal hands-on time. A test method using HE was developed in conjunction with Loop-mediated Isothermal Amplification (LAMP) combined with the real-time bioluminescent reporter system known as BART targeting the toxin B gene (tcdB). The HE-LAMP-BART method was evaluated in a pilot study on clinical fecal samples (tcdB(+), n = 111; tcdB(-), n= 107). The HE-LAMP-BART method showed 95.5% sensitivity and 100% specificity against a gold standard reference method using cytotoxigenic culture and also a silica-based robotic extraction followed by tcdB PCR to control for storage. From sample to result, the HE-LAMP-BART method typically took 50 minutes, whereas the PCR method took >2.5 hours. In a further study (tcdB(+), n = 47; tcdB(-), n= 28) HE-LAMP-BART was compared to an alternative commercially available LAMP-based method, Illumigene (Meridian Bioscience, OH), and yielded 87.2% sensitivity and 100% specificity for the HE-LAMP-BART method compared to 76.6% and 100%, respectively, for Illumigene against the reference method. A subset of 27 samples (tcdB(+), n = 25; tcdB(-), n= 2) were further compared between HE-LAMP-BART, Illumigene, GeneXpert (Cepheid, Sunnyvale, CA) and RIDA®QUICK C. difficile Toxin A/B lateral flow rapid test (R-Biopharm, Darmstadt, Germany) resulting in sensitivities of HE-LAMP-BART 92%, Illumigene 72% GeneXpert 96% and RIDAQuick 76% against the reference method. The HE-LAMP-BART method offers the advantages of molecular based approaches without the cost and complexity usually associated with molecular tests. Further, the rapid time-to-result and simple protocol means the method can be applied away from the centralized laboratory settings.

Background: Group B streptococci (GBS) are a leading cause of sepsis and meningitis in newborns. We previously developed a rapid diagnostic system for GBS detection from vaginal/anal samples obtained from pregnant women during delivery. To facilitate the adaptation of this method for point-of-care testing, we have developed a specific and efficient GBS DNA capture method that is compatible with both PCR and nonamplification detection technologies. Methods: Superparamagnetic beads were functionalized with oligonucleotide capture probes of different lengths and used to capture GBS genomic DNA (gDNA). A rapid extraction procedure was used to provide DNA from GBS cultures or vaginal/anal samples with added GBS. Hybridization reactions consisting of functionalized beads and target DNA in 30 μL of hybridization buffer were performed for 1 h at room temperature, followed by washing and resuspension in water. Captured DNA was then detected using quantitative PCR. Results: A 25-mer capture probe allowed detection of 1000 genome copies of purified GBS DNA. The ability to detect GBS was improved by use of a 50-mer (100 copies) and a 70-mer capture probe (10 copies). Detection of approximately 1250 CFU/mL was achieved for diluted GBS broth culture and for vaginal/anal swab samples with added GBS. Conclusion: Oligonucleotide-functionalized superparamagnetic microbeads efficiently capture GBS gDNA from both bacterial cultures and vaginal/anal samples with added GBS. Efficiency of gDNA capture increases with oligonucleotide length. This technology could be combined with sample preparation and detection technologies in a microfluidic system to allow point-of-care testing for GBS.

Here we describe a method for the detection of Clostridium difficile from stool using a novel low-complexity and rapid extraction process called Heat Elution (HE). The HE method is two-step and takes just 10 minutes, no specialist instruments are required and there is minimal hands-on time. A test method using HE was developed in conjunction with Loop-mediated Isothermal Amplification (LAMP) combined with the real-time bioluminescent reporter system known as BART targeting the toxin B gene (tcdB). The HE-LAMP-BART method was evaluated in a pilot study on clinical fecal samples (tcdB+, n = 111; tcdB-, n = 107). The HE-LAMP-BART method showed 95.5% sensitivity and 100% specificity against a gold standard reference method using cytotoxigenic culture and also a silica-based robotic extraction followed by tcdB PCR to control for storage. From sample to result, the HE-LAMP-BART method typically took 50 minutes, whereas the PCR method took >2.5 hours. In a further study (tcdB+, n = 47; tcdB-, n = 28) HE-LAMP-BART was compared to an alternative commercially available LAMP-based method, Illumigene (Meridian Bioscience, OH), and yielded 87.2% sensitivity and 100% specificity for the HE-LAMP-BART method compared to 76.6% and 100%, respectively, for Illumigene against the reference method. A subset of 27 samples (tcdB+, n = 25; tcdB-, n = 2) were further compared between HE-LAMP-BART, Illumigene, GeneXpert (Cepheid, Sunnyvale, CA) and RIDA registered QUICK C. difficile Toxin A/B lateral flow rapid test (R-Biopharm, Darmstadt, Germany) resulting in sensitivities of HE-LAMP-BART 92%, Illumigene 72% GeneXpert 96% and RIDAQuick 76% against the reference method. The HE-LAMP-BART method offers the advantages of molecular based approaches without the cost and complexity usually associated with molecular tests. Further, the rapid time-to-result and simple protocol means the method can be applied away from the centralized laboratory settings.

The physics of doped Mott insulators remains controversial after decades of active research, hindered by the interplay among competing orders and fluctuations. It is thus highly desired to distinguish the intrinsic characters of the Mott-metal crossover from those of other origins. Here we investigate the evolution of electronic structure and dynamics of the hole-doped pseudospin-1/2 Mott insulator Sr 2 IrO 4 . The effective hole doping is achieved by replacing Ir with Rh atoms, with the chemical potential immediately jumping to or near the top of the lower Hubbard band. The doped iridates exhibit multiple iconic low-energy features previously observed in doped cuprates—pseudogaps, Fermi arcs and marginal-Fermi-liquid-like electronic scattering rates. We suggest these signatures are most likely an integral part of the material’s proximity to the Mott state, rather than from many of the most claimed mechanisms, including preformed electron pairing, quantum criticality or density-wave formation. The physics of Mott insulators is obscured by the interplay between competing orders and fluctuations. Here, the authors track the evolution of the electronic structure of Mott insulator strontium iridate as the iridium atoms are replaced by rhodium, providing insight into this exotic state of matter.

While our understanding of the genetics underlying the Brassica - Leptosphaeria pathosystem has advanced greatly in the last decade, differences in molecular responses due to interaction between resistance genes and host genetic background has not been studied. We applied RNAseq technology to monitor the transcriptome profiles of Brassica napus ( Bn ) lines carrying one of four blackleg R genes ( Rlm2 , Rlm3 , LepR1 & LepR2 ) in Topas or Westar background, during the early stages of infection by a Leptosphaeria maculans ( Lm ) isolate carrying the corresponding Avr genes. We observed upregulation of host genes involved in hormone signalling, cell wall thickening, response to chitin and glucosinolate production in all R gene lines at 3 day after inoculation (dai) albeit having higher level of expression in LepR1 and Rlm2 than in Rlm3 and LepR2 lines. Bn-SOBIR1 (Suppressor Of BIR1-1), a receptor like kinase (RLK) that forms complex receptor like proteins (RLPs) was highly expressed in LepR1 and Rlm2 at 3 dai. In contrast Bn-SOBIR1 induction was low in Rlm3 line, which could indicate that Rlm3 may function independent of SOBIR1 . Expression of Salicylic acid (SA) related defense was enhanced in LepR1 and Rlm2 at 3 dai. In contrast to SA, expression of Bn genes with homology to PDF1.2 , a jasmonic acid (JA) pathway marker, were increased in all Rlm and LepR lines at 6 and 9 dai. Effect of host genetic background on induction of defense, was determined by comparison of LepR1 and LepR2 in Topas vs Westar genotype (i.e. T- LepR1 vs W- LepR1 and T- LepR2 vs W- LepR2 ). In both cases (regardless of R gene) overall number of defense related genes at the earliest time point (3 dai) was higher in Tops compared to Westar. SA and JA markers genes such as PR1 and PDF1.2 were more induced in Topas compared to Westar introgression lines at this time point. Even in the absence of any R gene, effect of Topas genotype in enhanced defense, was also evident by the induction of PDF1.2 that started at a low level at 3 dai and peaked at 6 and 9 dai, while no induction in Westar genotype was observed at any of these time points. Overall, variation in time and intensity of expression of genes related to defense, was clearly dependent on both R gene and the host genotype.

Genes coding for nucleotide-binding leucine-rich repeat (LRR) receptors (NLRs) control resistance against intracellular (cell-penetrating) pathogens. However, evidence for a role of genes coding for proteins with LRR domains in resistance against extracellular (apoplastic) fungal pathogens is limited. Here, the distribution of genes coding for proteins with eLRR domains but lacking kinase domains was determined for the Brassica napus genome. Predictions of signal peptide and transmembrane regions divided these genes into 184 coding for receptor-like proteins (RLPs) and 121 coding for secreted proteins (SPs). Together with previously annotated NLRs, a total of 720 LRR genes were found. Leptosphaeria maculans-induced expression during a compatible interaction with cultivar Topas differed between RLP, SP and NLR gene families; NLR genes were induced relatively late, during the necrotrophic phase of pathogen colonization. Seven RLP, one SP and two NLR genes were found in Rlm1 and Rlm3/Rlm4/Rlm7/Rlm9 loci for resistance against L. maculans on chromosome A07 of B. napus. One NLR gene at the Rlm9 locus was positively selected, as was the RLP gene on chromosome A10 with LepR3 and Rlm2 alleles conferring resistance against L. maculans races with corresponding effectors AvrLm1 and AvrLm2, respectively. Known loci for resistance against L. maculans (extracellular hemi-biotrophic fungus), Sclerotinia sclerotiorum (necrotrophic fungus) and Plasmodiophora brassicae (intracellular, obligate biotrophic protist) were examined for presence of RLPs, SPs and NLRs in these regions. Whereas loci for resistance against P. brassicae were enriched for NLRs, no such signature was observed for the other pathogens. These findings demonstrate involvement of (i) NLR genes in resistance against the intracellular pathogen P. brassicae and a putative NLR gene in Rlm9-mediated resistance against the extracellular pathogen L. maculans.

Brassica napus (oilseed rape, canola) seedling resistance to Leptosphaeria maculans, the causal agent of blackleg (stem canker) disease, follows a gene‐for‐gene relationship. The avirulence genes AvrLmS and AvrLep2 were described to be perceived by the resistance genes RlmS and LepR2, respectively, present in B. napus ‘Surpass 400’. Here we report cloning of AvrLmS and AvrLep2 using two independent methods. AvrLmS was cloned using combined in vitro crossing between avirulent and virulent isolates with sequencing of DNA bulks from avirulent or virulent progeny (bulked segregant sequencing). AvrLep2 was cloned using a biparental cross of avirulent and virulent L. maculans isolates and a classical map‐based cloning approach. Taking these two approaches independently, we found that AvrLmS and AvrLep2 are the same gene. Complementation of virulent isolates with this gene confirmed its role in inducing resistance on Surpass 400, Topas‐LepR2, and an RlmS‐line. The gene, renamed AvrLmS‐Lep2, encodes a small cysteine‐rich protein of unknown function with an N‐terminal secretory signal peptide, which is a common feature of the majority of effectors from extracellular fungal plant pathogens. The AvrLmS‐Lep2/LepR2 interaction phenotype was found to vary from a typical hypersensitive response through intermediate resistance sometimes towards susceptibility, depending on the inoculation conditions. AvrLmS‐Lep2 was nevertheless sufficient to significantly slow the systemic growth of the pathogen and reduce the stem lesion size on plant genotypes with LepR2, indicating the potential efficiency of this resistance to control the disease in the field. AvrLmS‐Lep2, the 10th cloned Leptosphaeria maculans AVR gene, was easily identified by bulk sequencing, and induces a fluctuating phenotype on rapeseed cotyledons that nevertheless limits stem canker severity.

Recent debates in the literature over the relationship between topology and Extreme Magnetoresistance (XMR) have drawn attention to the Lanthanum Monopnictide family of binary compounds. Angle resolved photoemission spectroscopy (ARPES) is used to measure the electronic structure of the XMR topological semimetal candidates LaBi, LaSb, and LaAs. The orbital content of the near- E F states in LaBi and LaSb are extracted using varying photon polarizations and both dominant d and p bands are observed near X . The measured bulk bands are shifted in energy when compared to the results of Density Functional Calculations. This disagreement is minor in LaBi, but large in LaSb and LaAs. The measured bulk band structure of LaBi shows a clear band inversion and puts LaBi in the υ  = 1 class of Topological Insulators (or semimetals), as predicted by calculations and consistent with the measured Dirac-like surface states. LaSb is on the verge of a band inversion with a less-clear case for any distinctly topological surface states and in disagreement with calculations. Lastly, these same bands in LaAs are clearly non-inverted implying its topological triviality and demonstrating a topological phase transition in the Lanthanum monopnictides. Using a wide range of photon energies the true bulk states are cleanly disentangled from the various types of surface states which are present. These surface states exist due to surface projections of bulk states in LaSb and for topological reasons in LaBi. ARPES: investigating the link between topology and extreme magnetoresistance Lanthanum monopnictides are drawing interest because they are topological semimetal candidates and they exhibit extreme magnetoresistance, that is, they transition from being good metals to having high resistance when a magnetic field is applied. Extreme magnetoresistance is very interesting in view of applications, but the mechanism behind it is not understood yet. Daniel Dessau at the University of Colorado and collaborators performed angle-resolved photoemission spectroscopy measurements and density functional theory calculations to investigate the electronic structure of lanthanum monopnictides and its connection with extreme magnetoresistance. LaBi shows band inversion and is thus a topological material, whereas the case is less clear for LaSb, which is close to band inversion. LaAs is topologically trivial. The results suggest that the connection between topology and extreme magnetoresistance might be less clear than previously thought.

Nutrient deprivation during ischemia leads to severe insult to neurons causing widespread excitotoxic damage in specific brain regions such as the hippocampus. One possible strategy for preventing neurodegeneration is to express therapeutic proteins in the brain to protect against excitotoxicity. We investigated the utility of equine infectious anemia virus (EIAV)-based vectors as genetic tools for delivery of therapeutic proteins in an in vivo excitotoxicity model. The efficacy of these vectors at preventing cellular loss in target brain areas following excitotoxic insult was also assessed. EIAV vectors generated to overexpress the human antiapoptotic Bcl-2 or growth factor glial-derived neurotrophic factor (GDNF) genes protected against glutamate-induced toxicity in cultured hippocampal neurons. In an in vivo excitotoxicity model, adult Wistar rats received a unilateral dose of the glutamate receptor agonist N-methyl-d-aspartate to the hippocampus that induced a large lesion in the CA1 region. Neuronal loss could not be protected by prior transduction of a control vector expressing β-galactosidase. In contrast, EIAV-mediated expression of Bcl-2 and GDNF significantly reduced lesion size thus protecting the hippocampus from excitotoxic damage. These results demonstrate that EIAV vectors can be effectively used to deliver putative neuroprotective genes to target brain areas and prevent cellular loss in the event of a neurological insult. Therefore these lentiviral vectors provide potential therapeutic tools for use in cases of acute neurotrauma such as cerebral ischemia.