Validation of DNA methylation profiling in formalin-fixed paraffin-embedded samples using the Infinium HumanMethylation450 Microarray

A formalin-fixed paraffin-embedded (FFPE) sample usually yields highly degraded DNA, which limits the use of techniques requiring high-quality DNA, such as Infinium Methylation microarrays. To overcome this restriction, we have applied an FFPE restoration procedure consisting of DNA repair and ligation processes in a set of paired fresh-frozen (FF) and FFPE samples. We validated the FFPE results in comparison with matched FF samples, enabling us to use FFPE samples on the Infinium HumanMethylation450 Methylation array.     

Antibody responses to galectin-8, TARP and TRAP1 in prostate cancer patients treated with a GM-CSF-secreting cellular immunotherapy

A critical factor in clinical development of cancer immunotherapies is the identification of tumor-associated antigens that may be related to immunotherapy potency. In this study, protein microarrays containing >8,000 human proteins were screened with serum from prostate cancer patients (N = 13) before and after treatment with a granulocyte–macrophage colony-stimulating factor (GM-CSF)-secreting whole cell immunotherapy. Thirty-three proteins were identified that displayed significantly elevated (P ≤ 0.05) signals in post-treatment samples, including three proteins that have previously been associated with prostate carcinogenesis, galectin-8, T-cell alternative reading frame protein (TARP) and TNF-receptor-associated protein 1 (TRAP1). Expanded analysis of antibody induction in metastatic, castration-resistant prostate cancer (mCRPC) patients (N = 92) from two phase 1/2 trials of prostate cancer immunotherapy, G-9803 and G-0010, indicated a significant (P = 0.03) association of TARP antibody induction and median survival time (MST). Antibody induction to TARP was also significantly correlated (P = 0.036) with an increase in prostate-specific antigen doubling time (PSADT) in patients with a biochemical (PSA) recurrence following prostatectomy or radiation therapy (N = 19) from in a previous phase 1/2 trial of prostate cancer immunotherapy, G-9802. RNA and protein encoding TARP and TRAP1 was up-regulated in prostate cancer tissue compared to matched normal controls. These preliminary findings suggest that antibody induction to TARP may represent a possible biomarker for treatment response to GM-CSF secreting cellular immunotherapy in prostate cancer patients and demonstrates the utility of using protein microarrays for the high-throughput screening of patient-derived antibody responses.     

Hydrolysis of fish oil by hyperactivated rhizomucor miehei lipase immobilized by multipoint anion exchange

Rhizomucor miehei lipase (RML) is greatly hyperactivated (around 20‐ to 25‐fold toward small substrates) in the presence of sucrose laurate. Hyperactivation appears to be an intramolecular process because it is very similar for soluble enzymes and covalently immobilized derivatives. The hyperactivated enzyme was immobilized (in the presence of sucrose laurate) on cyanogen bromide‐activated Sepharose (very mild covalent immobilization through the amino terminal residue), on glyoxyl Sepharose (intense multipoint covalent immobilization through the region with the highest amount of Lys residues), and on different anion exchangers (by multipoint anionic exchange through the region with the highest density of negative charges). Covalent immobilization does not promote the fixation of the hyperactivated enzyme, but immobilization on Sepharose Q retains the hyperactivated enzyme even in the absence of a detergent. The hydrolysis of fish oils by these hyperactivated enzyme derivatives was sevenfold faster than by covalently immobilized derivatives and three and a half times faster than by the enzyme hyperactivated on octyl‐Sepharose. The open structure of the hyperactivated lipase is fairly exposed to the medium, and no steric hindrance should interfere with the hydrolysis of large substrates. These new hyperactivated derivatives seem to be more suitable for hydrolysis of oils by RML immobilized inside porous supports. In addition, the hyperactivated derivatives are fairly stable against heat and organic cosolvents. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Profiling SARS-CoV-2 HLA-I peptidome reveals T cell epitopes from out-of-frame ORFs

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Habitat preference in the critically endangered yellow‐tailed woolly monkey (Lagothrix flavicauda) at La Esperanza,Peru

Habitat loss is one of the main threats to wildlife. Therefore, knowledge of habitat use and preference is essential for the design of conservation strategies and identification of priority sites for the protection of endangered species. The yellow‐tailed woolly monkey (Lagothrix flavicauda Humboldt, 1812), categorized as Critically Endangered on the IUCN Red List, is endemic to montane forests in northern Peru where its habitat is greatly threatened. We assessed how habitat use and preference in L. flavicauda are linked to forest structure and composition. The study took place near La Esperanza, in the Amazonas region, Peru. Our objective was to identify characteristics of habitat most utilized by L. flavicauda to provide information that will be useful for the selection of priority sites for conservation measures. Using presence records collected from May 2013 to February 2014 for one group of L. flavicauda, we classified the study site into three different use zones: low‐use, medium‐use, and high‐use. We assessed forest structure and composition for all use zones using 0.1 ha Gentry vegetation transects. Results show high levels of variation in plant species composition across the three use zones. Plants used as food resources had considerably greater density, dominance, and ecological importance in high‐use zones. High‐use zones presented similar structure to medium‐ and low‐use zones; thus it remains difficult to assess the influence of forest structure on habitat preference. We recommend focusing conservation efforts on areas with a similar floristic composition to the high‐use zones recorded in this study and suggest utilizing key alimentation species for reforestation efforts.     

An initial top-down proteomic analysis of the standard cuprizone mouse model of multiple sclerosis

An initial proteomic analysis of the cuprizone mouse model to characterise the breadth of toxicity by assessing cortex, skeletal muscle, spleen and peripheral blood mononuclear cells. Cuprizone treated vs. control mice for an initial characterisation. Select tissues from each group were pooled, analysed in triplicate using two-dimensional gel electrophoresis (2DE) and deep imaging and altered protein species identified using liquid chromatography tandem mass spectrometry (LC/MS/MS). Forty-three proteins were found to be uniquely detectable or undetectable in the cuprizone treatment group across the tissues analysed. Protein species identified in the cortex may potentially be linked to axonal damage in this model, and those in the spleen and peripheral blood mononuclear cells to the minimal peripheral immune cell infiltration into the central nervous system during cuprizone mediated demyelination. Primary oligodendrocytosis has been observed in type III lesions in multiple sclerosis. However, the underlying mechanisms are poorly understood. Cuprizone treatment results in oligodendrocyte apoptosis and secondary demyelination. This initial analysis identified proteins likely related to axonal damage; these may link primary oligodendrocytosis and secondary axonal damage. Furthermore, this appears to be the first study of the cuprizone model to also identify alterations in the proteomes of skeletal muscle, spleen and peripheral blood mononuclear cells. Notably, protein disulphide isomerase was not detected in the cuprizone cohort; its absence has been linked to reduced major histocompatibility class I assembly and reduced antigen presentation. Overall, the results suggest that, like experimental autoimmune encephalomyelitis, results from the standard cuprizone model should be carefully considered relative to clinical multiple sclerosis.     

Behavioural modification of a social spider by a parasitoid wasp

1. Manipulation of host behaviour by parasitoids has long captured the imagination of ecologists. Parasitoid wasps in the Polysphincta group of genera develop as external parasitoids of spiders. 2. In the present study, the previously undescribed interaction between a Zatypota sp. wasp (Ichneumonidae) and a social spider Anelosimus eximius (Theridiidae) is described. The larva of this Zatypota wasp is found to induce its host to disperse from their communal web and build an entirely enclosed web consisting of densely spun silk. 3. The wasp is observed to target primarily immature A. eximius individuals, with 37.5–44% of nests in a given area being parasitised. Of those nests, approximately 1.3–2.0% of individuals are hosts to the parasitoid larvae. Larger spider colonies had a significantly higher probability of harbouring parasitoids. 4. This interaction results in unusual behaviours for A. eximius induced by the parasitoid: (i) leaving the protection of the social nest and (ii) building a unique, altered web that it would not otherwise build. It is suggested that the wasp may be tapping into ancestral dispersal behaviours in its host and that a social species provides this wasp an evolutionary advantage by allowing a stable host source.