The complete and specific proteolytic cleavage of protein samples into peptides is crucial for the success of every shotgun LC–MS/MS experiment. In particular, popular peptide-based label-free and ...targeted mass spectrometry approaches rely on efficient generation of fully cleaved peptides to ensure accurate and sensitive protein quantification. In contrast to previous studies, we globally and quantitatively assessed the efficiency of different digestion strategies using a yeast cell lysate, label-free quantification, and statistical analysis. Digestion conditions include double tryptic, surfactant-assisted, and tandem-combinatorial Lys-C/trypsin digestion. In comparison to tryptic digests, Lys-C/trypsin digests were found most efficient to yield fully cleaved peptides while reducing the abundance of miscleaved peptides. Subsequent sequence context analysis revealed improved digestion performances of Lys-C/trypsin for miscleaved sequence stretches flanked by charged basic and particulary acidic residues. Furthermore, targeted MS analysis demonstrated a more comprehensive protein cleavage only after Lys-C/trypsin digestion, resulting in a more accurrate absolute protein quantification and extending the number of peptides suitable for SRM assay development. Therefore, we conclude that a serial Lys-C/trypsin digestion is highly attractive for most applications in quantitative MS-based proteomics building on in-solution digestion schemes.
During cell division, the mitotic spindle segregates replicated chromosomes to opposite poles of the cell, while the position of the spindle determines the plane of cleavage. Spindle positioning and ...chromosome segregation depend on pulling forces on microtubules extending from the centrosomes to the cell cortex. Critical in pulling force generation is the cortical anchoring of cytoplasmic dynein by a conserved ternary complex of Gα, GPR-1/2, and LIN-5 proteins in C. elegans (Gα-LGN-NuMA in mammals). Previously, we showed that the polarity kinase PKC-3 phosphorylates LIN-5 to control spindle positioning in early C. elegans embryos. Here, we investigate whether additional LIN-5 phosphorylations regulate cortical pulling forces, making use of targeted alteration of in vivo phosphorylated residues by CRISPR/Cas9-mediated genetic engineering. Four distinct in vivo phosphorylated LIN-5 residues were found to have critical functions in spindle positioning. Two of these residues form part of a 30 amino acid binding site for GPR-1, which we identified by reverse two-hybrid screening. We provide evidence for a dual-kinase mechanism, involving GSK3 phosphorylation of S659 followed by phosphorylation of S662 by casein kinase 1. These LIN-5 phosphorylations promote LIN-5-GPR-1/2 interaction and contribute to cortical pulling forces. The other two critical residues, T168 and T181, form part of a cyclin-dependent kinase consensus site and are phosphorylated by CDK1-cyclin B in vitro. We applied a novel strategy to characterize early embryonic defects in lethal T168,T181 knockin substitution mutants, and provide evidence for sequential LIN-5 N-terminal phosphorylation and dephosphorylation in dynein recruitment. Our data support that phosphorylation of multiple LIN-5 domains by different kinases contributes to a mechanism for spatiotemporal control of spindle positioning and chromosome segregation.
Aims:
To present an authoritative, universal, easy-to-use morphologic classification of diabetic maculopathy based on spectral domain optical coherence tomography.
Methods:
The first draft of the ...project was developed based on previously published classifications and a literature search regarding the spectral domain optical coherence tomography quantitative and qualitative features of diabetic maculopathy. This draft was sent to an international panel of retina experts for a first revision. The panel met at the European School for Advanced Studies in Ophthalmology headquarters in Lugano, Switzerland, and elaborated the final document.
Results:
Seven tomographic qualitative and quantitative features are taken into account and scored according to a grading protocol termed TCED-HFV, which includes foveal thickness (T), corresponding to either central subfoveal thickness or macular volume, intraretinal cysts (C), the ellipsoid zone (EZ) and/or external limiting membrane (ELM) status (E), presence of disorganization of the inner retinal layers (D), number of hyperreflective foci (H), subfoveal fluid (F), and vitreoretinal relationship (V). Four different stages of the disease, that is, early diabetic maculopathy, advanced diabetic maculopathy, severe diabetic maculopathy, and atrophic maculopathy, are based on the first four variables, namely the T, C, E, and D. The different stages reflect progressive severity of the disease.
Conclusion:
A novel grading system of diabetic maculopathy is hereby proposed. The classification is aimed at providing a simple, direct, objective tool to classify diabetic maculopathy (irrespective to the treatment status) even for non-retinal experts and can be used for therapeutic and prognostic purposes, as well as for correct evaluation and reproducibility of clinical investigations.
A synergistic combination of two next-generation sequencing platforms with a detailed comparative BAC physical contig map provided a cost-effective assembly of the genome sequence of the domestic ...turkey (Meleagris gallopavo). Heterozygosity of the sequenced source genome allowed discovery of more than 600,000 high quality single nucleotide variants. Despite this heterozygosity, the current genome assembly (∼1.1 Gb) includes 917 Mb of sequence assigned to specific turkey chromosomes. Annotation identified nearly 16,000 genes, with 15,093 recognized as protein coding and 611 as non-coding RNA genes. Comparative analysis of the turkey, chicken, and zebra finch genomes, and comparing avian to mammalian species, supports the characteristic stability of avian genomes and identifies genes unique to the avian lineage. Clear differences are seen in number and variety of genes of the avian immune system where expansions and novel genes are less frequent than examples of gene loss. The turkey genome sequence provides resources to further understand the evolution of vertebrate genomes and genetic variation underlying economically important quantitative traits in poultry. This integrated approach may be a model for providing both gene and chromosome level assemblies of other species with agricultural, ecological, and evolutionary interest.
Cross-linking mass spectrometry (MS) has substantially matured as a method over the past 2 decades through parallel development in multiple labs, demonstrating its applicability to protein structure ...determination, conformation analysis, and mapping protein interactions in complex mixtures. Cross-linking MS has become a much-appreciated and routinely applied tool, especially in structural biology. Therefore, it is timely that the community commits to the development of methodological and reporting standards. This white paper builds on an open process comprising a number of events at community conferences since 2015 and identifies aspects of Cross-linking MS for which guidelines should be developed as part of a Cross-linking MS standards initiative.
Leitner et al. propose steps toward the development of best practices in Cross-linking mass spectrometry in this white paper. This community-driven effort represents a consensus of approximately 30 groups from academia and industry and, if successfully implemented, will increase transparency and facilitate data reuse.
To evaluate the efficacy and safety of photodynamic therapy with verteporfin combined with intravitreal triamcinolone in choroidal neovascularization secondary to age-related macular degeneration ...(AMD).
Prospective, noncomparative, interventional case series.
One hundred eighty-four patients undergoing treatment for neovascular AMD at one retinal referral center.
One hundred eighty-four eyes of 184 consecutive patients (63.6% female, 36.4% male) with a mean age of 76.5 years and a follow-up of a median of 38.8 weeks (range, 12-103) were included in a case series. One hundred forty-eight (80.4%) patients had subfoveal choroidal neovascularization, 19 patients (10.3%) had juxtafoveal choroidal neovascularization, and 17 patients (9.2%) had extrafoveal choroidal neovascularization. Verteporfin photodynamic therapy was performed using the recommended standard procedure. A solution containing 25 mg of triamcinolone was injected intravitreally 16 hours after photodynamic therapy in 184 patients. The combined therapy procedure was repeated at the 3-month follow-up visits whenever persistent choroidal neovascularization leakage was documented angiographically.
Mean change in best-refracted visual acuity (VA) between baseline and the last visit, and number of treatments necessary to achieve absence of leakage.
Visual acuity improved in the majority of patients (baseline VA, mean 20/125) by a mean increase of 1.22 Snellen lines and 1.43 lines using laser interferometry (P<0.01). The mean number of required treatments was 1.21. Twenty-three eyes (12.5%) required 2 treatments, 6 eyes (3.26%) required 3 treatments, and 1 eye (0.5%) required 4 treatments. The combination treatment including laser and intravitreal steroid administration was well tolerated. Forty-six patients (25%) required glaucoma therapy due to a transient steroid-induced intraocular pressure (IOP) increase. Twelve patients (6.5%) were on topical medication for preexisting glaucoma. Two patients (1%) whose IOP increase could not be controlled with topical therapy required surgery.
Verteporfin photodynamic therapy combined with intravitreal triamcinolone may improve the outcome of standard verteporfin photodynamic therapy in the treatment of choroidal neovascularization secondary to AMD. A significant improvement in VA was observed in a majority of treated patients and was maintained during the maximum follow-up. In addition, retreatment rates were lower than anticipated.
To evaluate the efficacy and safety of photodynamic therapy (PDT) with verteporfin combined with intravitreal triamcinolone (IVTA) in occult choroidal neovascularization (CNV) secondary to ...age-related macular degeneration (AMD).
Single center, nonrandomized interventional case series.
A prospective, noncomparative, interventional case series of 41 eyes of 41 patients with a two-year follow-up period. Verteporfin PDT was performed using the recommended standard procedure for approved forms of AMD. A solution containing 25 mg of crystalline triamcinolone acetonide was injected intravitreally 16 hours post PDT. The procedure was repeated after three months in case of persistent CNV leakage.
The mean number of treatments needed was 1.8. Thirty-four eyes (82.9%) required one retreatment at three months. No additional retreatments were necessary. Visual acuity improved gradually in most of the patients with mean values of 20/133 and 20/115 at baseline and three months; 20/101 and 20/84 at six and twelve months; and 20/83 and 20/81 at eighteen and twenty-four months. Eleven of 41 treated study eyes (26.8%) underwent cataract surgery between six and fifteen months after the first treatment. Nine patients required local or systemic glaucoma therapy because of a transient steroid induced intraocular pressure increase.
Verteporfin PDT combined with intravitreal triamcinolone may improve the outcome of standard verteporfin PDT in the treatment of occult CNV secondary to AMD. An improvement in visual acuity was observed in most of the treated patients and was maintained during a two-year follow-up period. Retreatment numbers were lower than expected from monotherapy trials.
The process development and the kilogram-scale synthesis of linrodostat (BMS-986205, 1) are described. The synthesis features several highly efficient telescoped processes and the use of Evans ...auxiliary to install a methyl-bearing stereocenter. The target was prepared in 12 steps with 7 isolations in an overall yield of 31%.
Regulatory T cells (Tregs) control key events of immune tolerance, primarily by suppression of effector T cells. We previously revealed that Tregs rapidly suppress T cell receptor (TCR)-induced ...calcium store depletion in conventional CD4
CD25
T cells (Tcons) independently of IP
levels, consequently inhibiting NFAT signaling and effector cytokine expression. Here, we study Treg suppression mechanisms through unbiased phosphoproteomics of primary human Tcons upon TCR stimulation and Treg-mediated suppression, respectively. Tregs induced a state of overall decreased phosphorylation as opposed to TCR stimulation. We discovered novel phosphosites (T595_S597) in the DEF6 (SLAT) protein that were phosphorylated upon TCR stimulation and conversely dephosphorylated upon coculture with Tregs. Mutation of these DEF6 phosphosites abrogated interaction of DEF6 with the IP
receptor and affected NFAT activation and cytokine transcription in primary Tcons. This novel mechanism and phosphoproteomics data resource may aid in modifying sensitivity of Tcons to Treg-mediated suppression in autoimmune disease or cancer.
A search for instability of nucleons bound in Xe136 nuclei is reported with 223 kg·yr exposure of Xe136 in the EXO-200 experiment. Lifetime limits of 3.3×1023 and 1.9×1023 yr are established for ...nucleon decay to Sb133 and Te133, respectively. These are the most stringent to date, exceeding the prior decay limits by a factor of 9 and 7, respectively.
PDF
