Deciphering the Complex Molecular Pathogenesis of Myotonic Dystrophy Type 1 through Omics Studies

Omics studies are crucial to improve our understanding of myotonic dystrophy type 1 (DM1), the most common muscular dystrophy in adults. Employing tissue samples and cell lines derived from patients and animal models, omics approaches have revealed the myriad alterations in gene and microRNA expression, alternative splicing, 3′ polyadenylation, CpG methylation, and proteins levels, among others, that contribute to this complex multisystem disease. In addition, omics characterization of drug candidate treatment experiments provides crucial insight into the degree of therapeutic rescue and off-target effects that can be achieved. Finally, several innovative technologies such as single-cell sequencing and artificial intelligence will have a significant impact on future DM1 research.     

Mining the Wheat Grain Proteome

Bread wheat is the most widely cultivated crop worldwide, used in the production of food products and a feed source for animals. Selection tools that can be applied early in the breeding cycle are needed to accelerate genetic gain for increased wheat production while maintaining or improving grain quality if demand from human population growth is to be fulfilled. Proteomics screening assays of wheat flour can assist breeders to select the best performing breeding lines and discard the worst lines. In this study, we optimised a robust LC–MS shotgun quantitative proteomics method to screen thousands of wheat genotypes. Using 6 cultivars and 4 replicates, we tested 3 resuspension ratios (50, 25, and 17 µL/mg), 2 extraction buffers (with urea or guanidine-hydrochloride), 3 sets of proteases (chymotrypsin, Glu-C, and trypsin/Lys-C), and multiple LC settings. Protein identifications by LC–MS/MS were used to select the best parameters. A total 8738 wheat proteins were identified. The best method was validated on an independent set of 96 cultivars and peptides quantities were normalised using sample weights, an internal standard, and quality controls. Data mining tools found particularly useful to explore the flour proteome are presented (UniProt Retrieve/ID mapping tool, KEGG, AgriGO, REVIGO, and Pathway Tools).     

蛋白质组学在反式脂肪酸和心血管疾病关系研究中的进展

近年来,众多研究发现反式脂肪酸对人体危害极大,其对心血管的影响已得到充分解释,然而仍有一些否定数据需要得到解释。蛋白质组学作为一种有效的蛋白质分析技术,已广泛用于人类疾病的研究中。鉴于其在营养素研究中的成功应用,将其与反式脂肪酸的研究相结合,有利于找到新的突破点。本文就反式脂肪酸的来源、危害及蛋白质组学的研究进行综述。     

Micro-RNA and Proteomic Profiles of Plasma-Derived Exosomes from Irradiated Mice Reveal Molecular Changes Preventing Apoptosis in Neonatal Cerebellum

Cell communication via exosomes is capable of influencing cell fate in stress situations such as exposure to ionizing radiation. In vitro and in vivo studies have shown that exosomes might play a role in out-of-target radiation effects by carrying molecular signaling mediators of radiation damage, as well as opposite protective functions resulting in resistance to radiotherapy. However, a global understanding of exosomes and their radiation-induced regulation, especially within the context of an intact mammalian organism, has been lacking. In this in vivo study, we demonstrate that, compared to sham-irradiated (SI) mice, a distinct pattern of proteins and miRNAs is found packaged into circulating plasma exosomes after whole-body and partial-body irradiation (WBI and PBI) with 2 Gy X-rays. A high number of deregulated proteins (59% of WBI and 67% of PBI) was found in the exosomes of irradiated mice. In total, 57 and 13 miRNAs were deregulated in WBI and PBI groups, respectively, suggesting that the miRNA cargo is influenced by the tissue volume exposed to radiation. In addition, five miRNAs (miR-99b-3p, miR-200a-3p, miR-200a, miR-182-5p, miR-182) were commonly overexpressed in the exosomes from the WBI and PBI groups. In this study, particular emphasis was also given to the determination of the in vivo effect of exosome transfer by intracranial injection in the highly radiosensitive neonatal cerebellum at postnatal day 3. In accordance with a major overall anti-apoptotic function of the commonly deregulated miRNAs, here, we report that exosomes from the plasma of irradiated mice, especially in the case of WBI, prevent radiation-induced apoptosis, thus holding promise for exosome-based future therapeutic applications against radiation injury.     

Application of Proteomics in Pancreatic Ductal Adenocarcinoma Biomarker Investigations: A Review

Pancreatic ductal adenocarcinoma (PDAC), a highly aggressive malignancy with a poor prognosis is usually detected at the advanced stage of the disease. The only US Food and Drug Administration-approved biomarker that is available for PDAC, CA 19-9, is most useful in monitoring treatment response among PDAC patients rather than for early detection. Moreover, when CA 19-9 is solely used for diagnostic purposes, it has only a recorded sensitivity of 79% and specificity of 82% in symptomatic individuals. Therefore, there is an urgent need to identify reliable biomarkers for diagnosis (specifically for the early diagnosis), ascertain prognosis as well as to monitor treatment response and tumour recurrence of PDAC. In recent years, proteomic technologies are growing exponentially at an accelerated rate for a wide range of applications in cancer research. In this review, we discussed the current status of biomarker research for PDAC using various proteomic technologies. This review will explore the potential perspective for understanding and identifying the unique alterations in protein expressions that could prove beneficial in discovering new robust biomarkers to detect PDAC at an early stage, ascertain prognosis of patients with the disease in addition to monitoring treatment response and tumour recurrence of patients.     

Comparison of Different Mass Spectrometry Workflows for the Proteomic Analysis of Tear Fluid

The tear film is a multi-layer fluid that covers the corneal and conjunctival epithelia of the eye and provides lubrication, nutrients, and protection from the outside environment. Tear fluid contains a high concentration of proteins and has thus been recognized as a potential source of biomarkers for ocular disorders due to its proximity to disease sites on the ocular surface and the non-invasive nature of its collection. This is particularly true in the case of dry eye disease, which directly impacts the tear film and its components. Proteomic analysis of tear fluid is challenging mainly due to the wide dynamic range of proteins and the small sample volumes. However, recent advancements in mass spectrometry have revolutionized the field of proteomics enabling unprecedented depth, speed, and accuracy, even with small sample volumes. In this study using the Orbitrap Fusion Tribrid mass spectrometer, we compared four different mass spectrometry workflows for the proteomic analysis of tear fluid collected via Schirmer strips. We were able to establish a method of in-strip protein digestion that identified >3000 proteins in human tear samples from 11 healthy subjects. Our method offers a significant improvement in the number of proteins identified compared to previously reported methods without pooling samples.     

Pediatric Brain Tumors: Signatures from the Intact Proteome

The present investigation aimed to explore the intact proteome of tissues of pediatric brain tumors of different WHO grades and localizations, including medulloblastoma, pilocytic astrocytoma, and glioblastoma, in comparison with the available data on ependymoma, to contribute to the understanding of the molecular mechanisms underlying the onset and progression of these pathologies. Tissues have been homogenized in acidic water–acetonitrile solutions containing proteases inhibitors and analyzed by LC–high resolution MS for proteomic characterization and label-free relative quantitation. Tandem MS spectra have been analyzed by either manual inspection or software elaboration, followed by experimental/theoretical MS fragmentation data comparison by bioinformatic tools. Statistically significant differences in protein/peptide levels between the different tumor histotypes have been evaluated by ANOVA test and Tukey’s post-hoc test, considering a p-value > 0.05 as significant. Together with intact protein and peptide chains, in the range of molecular mass of 1.3–22.8 kDa, several naturally occurring fragments from major proteins, peptides, and proteoforms have been also identified, some exhibiting proper biological activities. Protein and peptide sequencing allowed for the identification of different post-translational modifications, with acetylations, oxidations, citrullinations, deamidations, and C-terminal truncations being the most frequently characterized. C-terminal truncations, lacking from two to four amino acid residues, particularly characterizing the β-thymosin peptides and ubiquitin, showed a different modulation in the diverse tumors studied. With respect to the other tumors, medulloblastoma, the most frequent malignant brain tumor of the pediatric age, was characterized by higher levels of thymosin β4 and β10 peptides, the latter and its des-IS form particularly marking this histotype. The distribution pattern of the C-terminal truncated forms was also different in glioblastoma, particularly underlying gender differences, according to the definition of male and female glioblastoma as biologically distinct diseases. Glioblastoma was also distinguished for the peculiar identification of the truncated form of the α-hemoglobin chain, lacking the C-terminal arginine, and exhibiting oxygen-binding and vasoconstrictive properties different from the intact form. The proteomic characterization of the undigested proteome, following the top-down approach, was challenging to originally investigate the post-translational events that differently characterize pediatric brain tumors. This study provides a contribution to elucidate the molecular profiles of the solid tumors most frequently affecting the pediatric age, and which are characterized by different grades of aggressiveness and localization.     

Low Light Increases the Abundance of Light Reaction Proteins: Proteomics Analysis of Maize (Zea mays L.) Grown at High Planting Density

Maize (Zea mays L.) is usually planted at high density, so most of its leaves grow in low light. Certain morphological and physiological traits improve leaf photosynthetic capacity under low light, but how light absorption, transmission, and transport respond at the proteomic level remains unclear. Here, we used tandem mass tag (TMT) quantitative proteomics to investigate maize photosynthesis-related proteins under low light due to dense planting, finding increased levels of proteins related to photosystem II (PSII), PSI, and cytochrome b₆f. These increases likely promote intersystem electron transport and increased PSI end electron acceptor abundance. OJIP transient curves revealed increases in some fluorescence parameters under low light: quantum yield for electron transport (φE_o), probability that an electron moves beyond the primary acceptor Q_A⁻ (ψ_o), efficiency/probability of electron transfer from intersystem electron carriers to reduction end electron acceptors at the PSI acceptor side (δR_o), quantum yield for reduction of end electron acceptors at the PSI acceptor side (φR_o), and overall performance up to the PSI end electron acceptors (PI_total). Thus, densely planted maize shows elevated light utilization through increased electron transport efficiency, which promotes coordination between PSII and PSI, as reflected by higher apparent quantum efficiency (AQE), lower light compensation point (LCP), and lower dark respiration rate (R_d).