The secreted proteins (secretome) of fungi play a key role in interactions of pathogenic and symbiotic fungi with plants. Using the plant pathogenic fungus Leptosphaeria maculans and symbiont ...Laccaria bicolor grown in culture, we have established a proteomic protocol for extraction, concentration and resolution of the fungal secretome. As no proteomic data were available on mycelium tissues from both L. maculans and L. bicolor, mycelial proteins were studied; they also helped verifying the purity of secretome samples. The quality of protein extracts was initially assessed by both 1-DE and 2-DE using first a broad pH range for IEF, and then narrower acidic and basic pH ranges, prior to 2-DE. Compared with the previously published protocols for which only dozens of 2-D spots were recovered from fungal secretome samples, up to approximately 2000 2-D spots were resolved by our method. MS identification of proteins along several pH gradients confirmed this high resolution, as well as the presence of major secretome markers such as endopolygalacturonases, β-glucanosyltransferases, pectate lyases and endoglucanases. Shotgun proteomic experiments evidenced the enrichment of secreted protein within the liquid medium. This is the first description of the proteome of L. maculans and L. bicolor, and the first application of liquid-phase IEF to any fungal extracts.
Styrene-maleic acid (SMA) copolymers have attracted interest in membrane research because they allow the solubilization and purification of membrane-spanning proteins from biological membranes in the ...form of native-like nanodisks. However, our understanding of the underlying SMA-lipid interactions is hampered by the fact that SMA preparations are very polydisperse. Here, we obtained fractions of the two most commonly used SMA preparations: SMA 2:1 and SMA 3:1 (both with specified Mw ∼10 kD), with different number-average molecular weight (Mn) and styrene content. The fractionation is based on the differential solubility of styrene-maleic anhydride (SMAnh) in hexane and acetone mixtures. SMAnh fractions were hydrolyzed to SMA and added to lipid self-assemblies. It was found that SMA fractions inserted in monolayers and solubilized vesicles to a different extent, with the highest efficiency being observed for low-Mn SMA polymers. Electron microscopy and dynamic light scattering size analyses confirmed the presence of nanodisks independent of the Mn of the SMA polymers forming the belt, and it was shown that the nanodisks all have approximately the same size. However, nanodisks bounded by high-Mn SMA polymers were more stable than those bounded by low-Mn polymers, as indicated by a better retention of the native lipid thermotropic properties and by slower exchange rates of lipids between nanodisks. In conclusion, we here present a simple method to separate SMAnh molecules based on their Mn from commercial SMAnh blends, which allowed us to obtain insights into the importance of SMA length for polymer-lipid interactions.
To define a set of core patient-reported domains and respective instruments for use in idiopathic inflammatory myopathies (IIM). Previously, we reported a systematic literature review on ...patient-reported outcomes (PRO) in IIM followed by conducting international focus groups to elicit patient perspectives of myositis symptoms and effects.
Based on qualitative content analysis of focus groups, an initial list of 26 candidate domains was constructed. We subsequently conducted an international modified Delphi survey to identify the importance of each of the 26 domains. Participants were asked to rate each domain on a scale of 0-10 (0 = not important, 10 = very important).
In this first round of the Delphi survey, 643 patients participated from the United States (n = 543), Sweden (n = 49), and South Korea (n = 51). Of the 26 domains, 19 (73%) were rated of high importance (≥ 7/10). The top 5 domains were muscle symptoms, fatigue, interactions with healthcare, medication side effects, and pain. During Outcome Measures in Rheumatology (OMERACT) 2016, we discussed the goal for ultimate reduction in the number of domains and the importance of considering representation of healthcare providers from other specialties, caregivers, representatives of pharmaceutical industries, and regulatory authorities in the next rounds of Delphi to represent broader perspectives on IIM.
Further prioritization and a reduction in the number of domains will be needed for the next Delphi. At the next biennial OMERACT meeting, we aim to present and seek voting on a Myositis Preliminary PRO Core Set to enable ultimate measure selection and development.
Breast cancer is a common disease partially caused by genetic risk factors. Germline pathogenic variants in DNA repair genes
,
,
,
, and
are associated with breast cancer risk.
, which encodes for a ...DNA translocase, has been proposed as a breast cancer predisposition gene, with greater effects for the ER-negative and triple-negative breast cancer (TNBC) subtypes. We tested the three recurrent protein-truncating variants
:p.Arg658*, p.Gln1701*, and p.Arg1931* for association with breast cancer risk in 67,112 cases, 53,766 controls, and 26,662 carriers of pathogenic variants of
or
. These three variants were also studied functionally by measuring survival and chromosome fragility in
patient-derived immortalized fibroblasts treated with diepoxybutane or olaparib. We observed that
:p.Arg658* was associated with increased risk of ER-negative disease and TNBC (OR = 2.44,
= 0.034 and OR = 3.79;
= 0.009, respectively). In a country-restricted analysis, we confirmed the associations detected for
:p.Arg658* and found that also
:p.Arg1931* was associated with ER-negative breast cancer risk (OR = 1.96;
= 0.006). The functional results indicated that all three variants were deleterious affecting cell survival and chromosome stability with
:p.Arg658* causing more severe phenotypes. In conclusion, we confirmed that the two rare
deleterious variants p.Arg658* and p.Arg1931* are risk factors for ER-negative and TNBC subtypes. Overall our data suggest that the effect of truncating variants on breast cancer risk may depend on their position in the gene. Cell sensitivity to olaparib exposure, identifies a possible therapeutic option to treat
-associated tumors.
Background: Cellulases, which catalyze the hydrolysis of glycosidic bonds in cellulose, can be classified into several different protein families. Endoglucanase CelA is a member of glycosyl hydrolase ...family 8, a family for which no structural information was previously available.
Results The crystal structure of CelA was determined by multiple isomorphous replacement and refined to 1.65 å resolution. The protein folds into a regular (
α/
α)
6 barrel formed by six inner and six outer
α helices. Cello-oligosaccharides bind to an acidic cleft containing at least five
D
-glucosyl-binding subsites (A–E) such that the scissile glycosidic linkage lies between subsites C and D. The strictly conserved residue Glu95, which occupies the center of the substrate-binding cleft and is hydrogen bonded to the glycosidic oxygen, has been assigned the catalytic role of proton donor.
Conclusion The present analysis provides a basis for modeling homologous family 8 cellulases. The architecture of the active-site cleft, presenting at least five glucosyl-binding subsites, explains why family 8 cellulases cleave cello-oligosaccharide polymers that are at least five
D
-glycosyl subunits long. Furthermore, the structure of CelA allows comparison with (
α/
α)
6 barrel glycosidases that are not related in sequence, suggesting a possible, albeit distant, evolutionary relationship between different families of glycosyl hydrolases.
Heterozygous mutations in the granulin (GRN) gene are a leading cause of frontotemporal lobar degeneration with TDP-43 aggregates (FTLD-TDP). Polymorphisms in TMEM106B have been associated with ...disease risk in GRN mutation carriers and protective TMEM106B variants associated with reduced levels of TMEM106B, suggesting that lowering TMEM106B might be therapeutic in the context of FTLD. Here, we tested the impact of full deletion and partial reduction of TMEM106B in mouse and iPSC-derived human cell models of GRN deficiency. TMEM106B deletion did not reverse transcriptomic or proteomic profiles in GRN-deficient microglia, with a few exceptions in immune signaling markers. Neither homozygous nor heterozygous Tmem106b deletion normalized disease-associated phenotypes in Grn −/−mice. Furthermore, Tmem106b reduction by antisense oligonucleotide (ASO) was poorly tolerated in Grn −/−mice. These data provide novel insight into TMEM106B and GRN function in microglia cells but do not support lowering TMEM106B levels as a viable therapeutic strategy for treating FTD-GRN.
Display omitted
•TMEM106B KO does not rescue GRN KO human microglia omic profile•Homozygous deletion of Tmem106b induces neuropathology and early mortality in Grn KO mice•Heterozygous deletion of Tmem106b does not alter phenotypes in Grn KO mice•Reduction of Tmem106b with antisense oligonucleotides is toxic in Grn KO mice
Molecular medicine; Neuroscience; Cellular neuroscience
Gap junctions are membrane structures composed of connexins (Cx) that allow diffusion of small molecules between cells. They are involved in tissue homeostasis, and various organ dysfunctions have ...been associated with gap junction defects. To verify their possible involvement in thyroid pathologies, the expression of connexin43 (Cx43), the major Cx in the human thyroid, was evaluated in a variety of diseases including cancer.
There were 122 samples from various thyroid pathologies that were collected to analyze the presence of Cx43 by immunofluorescence. Through confocal microscopy, different patterns of Cx43 localization were identified as normal (membrane) or abnormal (cytoplasmic or lack of detection). The analysis of Cx43 expression was further performed by quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry in a subset of 25 papillary carcinomas and compared with nontumoral thyroid tissues.
The presence of Cx43 was commonly altered in thyroid cancer, as abnormal Cx43 staining was detected in 94.1% of cancer, 47.4% of adenomas, 45.7% of multinodular goiter, 16.7% of Graves' disease, and 25% of thyroiditis. In papillary carcinoma samples, the deregulation of Cx43 expression was mostly the consequence of a decrease of Cx43 mRNA (68% of cases) when compared with normal tissue. When Cx43 mRNA was not downregulated (32% of cases), both loss of membrane staining and aberrant cytoplasmic distribution of the protein were observed.
These results show that aberrations of Cx43 expression are associated with thyroid papillary carcinoma.
PDF
