Whole genome sequencing of Canadian Saccharomyces cerevisiae strains isolated from spontaneous wine fermentations reveals a new Pacific West Coast Wine clade.

Vineyards in wine regions around the world are reservoirs of yeast with oenological potential. Saccharomyces cerevisiae ferments grape sugars to ethanol and generates flavor and aroma compounds in wine. Wineries place a high-value on identifying yeast native to their region to develop a region-specific wine program. Commercial wine strains are genetically very similar due to a population bottleneck and in-breeding compared to the diversity of S. cerevisiae from the wild and other industrial processes. We have isolated and microsatellite-typed hundreds of S. cerevisiae strains from spontaneous fermentations of grapes from the Okanagan Valley wine region in British Columbia, Canada. We chose 75 S. cerevisiae strains, based on our microsatellite clustering data, for whole genome sequencing using Illumina paired-end reads. Phylogenetic analysis shows that British Columbian S. cerevisiae strains cluster into 4 clades: Wine/European, Transpacific Oak, Beer 1/Mixed Origin, and a new clade that we have designated as Pacific West Coast Wine. The Pacific West Coast Wine clade has high nucleotide diversity and shares genomic characteristics with wild North American oak strains but also has gene flow from Wine/European and Ecuadorian clades. We analyzed gene copy number variations to find evidence of domestication and found that strains in the Wine/European and Pacific West Coast Wine clades have gene copy number variation reflective of adaptations to the wine-making environment. The "wine circle/Region B", a cluster of 5 genes acquired by horizontal gene transfer into the genome of commercial wine strains is also present in the majority of the British Columbian strains in the Wine/European clade but in a minority of the Pacific West Coast Wine clade strains. Previous studies have shown that S. cerevisiae strains isolated from Mediterranean Oak trees may be the living ancestors of European wine yeast strains. This study is the first to isolate S. cerevisiae strains with genetic similarity to nonvineyard North American Oak strains from spontaneous wine fermentations.

Faecal immunochemical testing (FIT): sources of result variation based on three years of routine testing of symptomatic patients in English primary care.

Introduction: We aimed to determine the analytical capabilities of a commonly used faecal immunochemical test (FIT) to detect faecal haemoglobin (Hb) in symptomatic people attending primary care in the context of the English NICE DG30 guidance.Materials and Methods: Data obtained from independent verification studies and clinical testing of the HM-JACKarc FIT method in routine primary care practice were analysed to derive performance characteristics.Results: Detection capabilities for the FIT method were 0.5 µg/g (limit of blank), 1.3 µg/g (limit of detection) and 3.0 µg/g (limit of quantitation). Of 33 non-homogenized specimens, 31 (93.9%) analysed in triplicate were consistently categorized relative to 10 µg/g, compared to all 33 (100%) homogenized specimens. Imprecision was higher (median 27.8%, (range 20.5% to 48.6%)) in non-homogenized specimens than in homogenized specimens (10.2%, (7.0 to 13.5%)). Considerable variation was observed in sequential clinical specimens from individual patients but no positive or negative trend in specimen degradation was observed over time (p = 0.26).Discussion: The FIT immunoassay evaluated is capable of detecting faecal Hb at concentrations well below the DG30 threshold of 10 µg/g and is suitable for application in this context. The greatest practical challenge to FIT performance is reproducible sampling, the pre-analytical step associated with most variability. Further research should focus on reducing sampling variability, particularly as post-COVID-19 guidance recommends greater FIT utilization.

CRAGE-mediated insertion of fluorescent chromosomal markers for accurate and scalable measurement of co-culture dynamics in Escherichia coli.

Monitoring population dynamics in co-culture is necessary in engineering microbial consortia involved in distributed metabolic processes or biosensing applications. However, it remains difficult to measure strain-specific growth dynamics in high-throughput formats. This is especially vexing in plate-based functional screens leveraging whole-cell biosensors to detect specific metabolic signals. Here, we develop an experimental high-throughput co-culture system to measure and model the relationship between fluorescence and cell abundance, combining chassis-independent recombinase-assisted genome engineering (CRAGE) and whole-cell biosensing with a PemrR-green fluorescent protein (GFP) monoaromatic reporter used in plate-based functional screening. CRAGE was used to construct Escherichia coli EPI300 strains constitutively expressing red fluorescent protein (RFP) and the relationship between RFP expression and optical density (OD600) was determined throughout the EPI300 growth cycle. A linear equation describing the increase of normalized RFP fluorescence during deceleration phase was derived and used to predict biosensor strain dynamics in co-culture. Measured and predicted values were compared using flow cytometric detection methods. Induction of the biosensor lead to increased GFP fluorescence normalized to biosensor cell abundance, as expected, but a significant decrease in relative abundance of the biosensor strain in co-culture and a decrease in bulk GFP fluorescence. Taken together, these results highlight sensitivity of population dynamics to variations in metabolic activity in co-culture and the potential effect of these dynamics on the performance of functional screens in plate-based formats. The engineered strains and model used to evaluate these dynamics provide a framework for optimizing growth of synthetic co-cultures used in screening, testing and pathway engineering applications.

Cardiac arrest in an obstetric patient using remifentanil patient-controlled analgesia.

This case report describes the management of a patient, diagnosed with an intrauterine death at 31 weeks' gestation, who suffered a cardiorespiratory arrest during her induced labour while using a remifentanil PCA. She made a full recovery from resuscitation which included a peri-mortem caesarean section.

F-spondin gene transfer improves memory performance and reduces amyloid-ß levels in mice.

Alzheimer's disease (AD) is the most prevalent form of dementia affecting the elderly. Evidence has emerged signifying that stimulation of the reelin pathway should promote neural plasticity and suppress molecular changes associated with AD, suggesting a potential therapeutic application to the disease. This was explored through the use of lentiviral vector-mediated overexpression of the reelin homolog, F-spondin, which is an activator of the reelin pathway. Intrahippocampal gene transfer of F-spondin improved spatial learning/memory in the Morris Water Maze and increased exploration of the novel object in the Novel Object Recognition test in wild-type mice. F-spondin overexpression also suppressed endogenous levels of amyloid beta (Aß(42)) in these mice and reduced Aß plaque deposition while improving synaptophysin expression in transgenic mouse models of AD. These data demonstrate pathologic and cognitive improvements in mice through F-spondin overexpression.

NEP-like endopeptidases and Alzheimer's disease [corrected].

The accumulation of the amyloid-beta peptide (Abeta) continues to emerge as a central factor in Alzheimer's disease (AD). In recent years attention has been drawn to clearance mechanisms of Abeta as evidence suggests reduced clearance may be linked to late-onset AD. Direct degradation of Abeta by endopeptidases has emerged as one critical pathway of clearance. Of particular interest are endopeptidases that are sensitive to the neprilysin inhibitors thiorphan and phosphoramidon (i.e. "NEP-like") as these inhibitors induce a dramatic increase in Abeta levels resulting in rapid plaque formation in wild-type rodents. This review focuses on neprilysin (NEP) and on another NEP-like endopeptidase termed neprilysin-2 (NEP2). The involvement of these endopeptidases in AD and the state of their therapeutic development are discussed.

Human membrane metallo-endopeptidase-like protein degrades both beta-amyloid 42 and beta-amyloid 40.

Beta-amyloid (Abeta) degrading endopeptidases are thought to protect against Alzheimer's disease (AD) and are potentially therapeutic. Of particular interest are endopeptidases that are blocked by thiorphan and phosphoramidon (T/P), as these inhibitors rapidly induce Abeta deposition in rodents. Neprilysin (NEP) is the best known target of T/P; however neprilysin knockout results in only modest Abeta increases insufficient to induce deposition. Therefore, other endopeptidases targeted by T/P must be critical for Abeta catabolism. Another candidate is the T/P sensitive membrane metallo-endopeptidase-like protein (MMEL), a close homolog of neprilysin. The endopeptidase properties of beta and gamma splice forms of human MMEL were determined in HEK293T cells transduced with the human cDNAs for the two splice forms; this showed degradation of both Abeta(42) and Abeta(40) by hMMEL-beta but not hMMEL-gamma. hMMEL-beta activity was found at the extracellular surface with no significant secreted activity. hMMEL-gamma was not expressed at the extracellular surface. Finally, it was found that hMMEL cleaves Abeta near the alpha-secretase site (producing Abeta(1-17)>>Abeta(1-16)). These data establish hMMEL as a mediator of Abeta catabolism and raise the possibility of its involvement in the etiology of AD and as a target for intervention.

Stabilisation of crosslinked ultra-high molecular weight polyethylene (UHMW-PE)-acetabular components with alpha-tocopherol.

A stabilisation of crosslinked ultra-high molecular weight polyethylene (UHMW-PE) with alpha-tocopherol (vitamin E) used for endoprostheses can increase its resistance against oxidative degradation remarkably. However, the method used for conventional UHMW-PE of adding alpha-tocopherol to the UHMW-PE powder before processing can not be applied for crosslinked UHMW-PE, since the alpha-tocopherol hinders the crosslinking process, which would be accompanied by a heavy degradation of this vitamin. The alpha-tocopherol has therefore to be added after the crosslinking process. This paper presents two methods for a stabilisation of finished products with alpha-tocopherol. In method 1, UHMW-PE-cubes (20 x 20 x 20 mm3) were stored in pure alpha-tocopherol under inert atmosphere at temperatures from 100 degrees C to 150 degrees C resulting in a high mass fraction of alpha-tocopherol in the edge zones. For further homogenisation, the cubes were stored in inert atmosphere at temperatures from 160 degrees C to 200 degrees C. In method 2, supercritical CO2 was used to incorporate the vitamin into the UHMW-PE. In an autoclave vessel, the cubes were treated with alpha-tocopherol dissolved in supercritical CO2 for several hours at temperatures from 100 degrees C to 170 degrees C. In both cases, the mass fraction of alpha-tocopherol was detected with the help of a FTIR-microscope. Both methods are well suited to stabilise crosslinked UHMW-PE with alpha-tocopherol. A stabilisation of the sensitive edge layer as well as a nearly homogenous distribution with varying alpha-tocopherol content may be realised by varying the process parameters. Using method 2, standard hip cups were stabilized nearly homogeneously with varying mass fraction of alpha-tocopherol. No oxidation of the UHMW-PE could be detected by infrared spectroscopy (FTIR) and HPLC studies showed a very low degradation of the alpha-tocopherol for both processes.

An antiaggregation gene therapy strategy for Lewy body disease utilizing beta-synuclein lentivirus in a transgenic model.

Current experimental gene therapy approaches for Parkinson's disease (PD) and dementia with Lewy bodies (DLB) include the use of viral vectors expressing antiapoptosis genes, neurotrophic factors and dopaminergic system enzymes. However, since increasing evidence favors a role for alpha-synuclein accumulation in the pathogenesis of these disorders, an alternative therapy might require the transfer of genes that might block alpha-synuclein accumulation. beta-Synuclein, the nonamyloidogenic homologue of alpha-synuclein, has recently been identified as a potential candidate. Thus, in vivo transfer of genes encoding beta-synuclein might provide a novel approach to the development of experimental treatments for PD and DLB. To assess this possibility and to better understand the mechanisms involved, a lentiviral vector expressing human (h) beta-synuclein (lenti-beta-synuclein) was tested in a transgenic (tg) mouse model of halpha-synuclein aggregation. This study showed that unilateral intracerebral injection of lenti-beta-synuclein reduced the formation of halpha-synuclein inclusions and the accumulation of halpha-synuclein in synapses and ameliorated the neurodegenerative alterations in the tg mice. Both in vivo and in vitro coimmunoprecipitation and immunoblot experiments show that the mechanisms of beta-synuclein neuroprotection involve binding of this molecule to halpha-synuclein and Akt, resulting in the decreased aggregation and accumulation of halpha-synuclein in the synaptic membrane. Together, these data further support a role for beta-synuclein in regulating the conformational state of alpha-synuclein and suggest that this gene transfer approach might have potential for the development of alternative therapies for PD and DLB.

A p75 tumor necrosis factor receptor-specific mutant of murine tumor necrosis factor alpha expressed from an adenovirus vector induces an antitumor response with reduced toxicity.

The toxic effects of tumor necrosis factor alpha (TNFalpha) have greatly limited its use in tumor therapy. Recently, clear evidence has been obtained linking the p55 TNF receptor (TNFR) to the induction of systemic toxicity. We have generated a p75 murine TNFR (mTNFR)-specific mutant of mTNFalpha (D142N-A144R), cloned this gene into a recombinant adenovirus vector (Ad-75), and studied its efficacy for tumor immunotherapy of a murine transgenic breast cancer model. Cell culture supernatants from Ad-75-transduced cells showed no cytotoxic activity on L929 cells, but retained the ability to induce proliferation of a murine T-cell line (CT6); this activity was not blocked by soluble p55 mTNFR. Furthermore, it was shown that the mutant form of mTNFalpha was able to coimmunoprecipitate only with the p75 mTNFR and not with the p55 mTNFR. Tumors injected with Ad-75 became necrotic, and mice injected with < or =1 x 10(9) plaque-forming units showed no mortality, whereas both wild-type murine and human TNF vectors induced lethality at doses of 1 and 5 x 10(8) plaque-forming units. All Ad-TNF vectors induced partial or permanent tumor regressions, with cured mice showing immune memory against the tumor. These results demonstrate that a p75 mTNFR agonist expressed from a recombinant adenovirus vector does not induce mortality at doses that cause tumor regression.

Transfer of tumor necrosis factor-alpha to rat lung induces severe pulmonary inflammation and patchy interstitial fibrogenesis with induction of transforming growth factor-beta1 and myofibroblasts.

Tumor necrosis factor-alpha is up-regulated in a variety of different human immune-inflammatory and fibrotic pulmonary pathologies. However, its precise role in these pathologies and, in particular, the mechanism(s) by which it may induce fibrogenesis are not yet elucidated. Using a replication-deficient adenovirus to transfer the cDNA of tumor necrosis factor-alpha to rat lung, we have been able to study the effect of transient but prolonged (7 to 10 days) overexpression of tumor necrosis factor-alpha in normal adult pulmonary tissue. We have demonstrated that local overexpression resulted in severe pulmonary inflammation with significant increases in neutrophils, macrophages, and lymphocytes and, to a lesser extent, eosinophils, with a peak at day 7. By day 14, the inflammatory cell accumulation had declined, and fibrogenesis became evident, with fibroblast accumulation and deposition of extracellular matrix proteins. Fibrotic changes were patchy but persisted to beyond day 64. To elucidate the mechanism underlying this fibrogenesis, we examined bronchoalveolar fluids for the presence of the fibrogenic cytokine transforming growth factor-beta1 and tissues for induction of alpha-smooth muscle actin-rich myofibroblasts. Transforming growth factor-beta1 was transiently elevated from day 7 (peak at day 14) immediately preceding the onset of fibrogenesis. Furthermore, there was a striking accumulation of myofibroblasts from day 7, with the most extensive and intense immunostaining at day 14, ie, coincident with the up-regulation of transforming growth factor-beta1 and onset of fibrogenesis. Thus, we have provided a model of tumor necrosis factor-alpha-mediated pulmonary inflammation and fibrosis in normal adult lung, and we suggest that the fibrogenesis may be mediated by the secondary up-regulation of transforming growth factor-beta1 and induction of pulmonary myofibroblasts.