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Lipidomic samples are complex mixtures of structurally different species of a wide range of concentrations providing challenges in their characterization. In this work, we present a proof of concept for the application of a simple microgradient liquid chromatography device on the detailed analysis of lipid classes. Our lipidomic analysis is based on a lipid class microgradient fractionation of a total lipid extract using an in-house-prepared hydrophilic interaction liquid chromatography microcolumn followed by RP-LC/MS of the collected lipid class fractions. The final fractionation method uses a 40-mm-long microcolumn of 500 μm ID with silica stationary phase obtained from a commercially available chromatographic column and the microgradient of the mobile phase prepared in a microsyringe using methyl tert-butyl ether (MTBE) - methanol - water - ammonium acetate mixtures of various elution strengths. MTBE total lipid extract is directly separated by microgradient elution into lipid classes according to their polarity, which enables the collection of isolated fractions of most lipid classes. The method has been applied to the fractionation of porcine brain extract into nonpolar lipids, hexosylceramides, phosphoethanolamines, phosphocholines, sphingomyelins, and lysophosphocholines classes. Achieved repeatability, recovery, and advanced lipid coverage prove the applicability of the microgradient fractionation of total lipid extract for the comprehensive lipidomic analysis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The native dimeric Petroselinum crispum (Mill.) Fuss protein Pet c 1.0201 and a monomeric xyloglucan endotransglycosylase enzyme (Garajova et al., 2008) isolated from the root cells co-purify and share similar molecular masses and acidic isoelectric points. In this work, we determined the complete primary structure of the parsley Pet c 1.0201 protein, based on tryptic and chymotryptic peptides followed by the manual micro-gradient chromatographic separation coupled with offline MALDI-TOF/TOF mass spectrometry. The bioinformatics approach enabled us to include the parsley protein into the PR-10 family, as it exhibited the highest protein sequence identity with the Apium graveolens Api g 1.0201 allergen and the major Daucus carota allergen Dau c 1.0201. Hence, we designated the Petroselinum crispum protein as Pet c 1.0201 and deposited it in the UniProt Knowledgebase under the accession C0HKF5. 3D protein homology modelling and molecular dynamics simulations of the Pet c 1.0201 dimer confirmed the typical structure of the Bet v 1 family allergens, and the potential of the Pet c 1.0201 protein to dimerize in water. However, the behavioural properties of Pet c 1.0201 and the celery allergen Api g 1.0101 differed in the presence of salts due to transiently and stably formed dimeric forms of Pet c 1.0201 and Api g 1.0101, respectively., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Although the role of high-risk human papillomaviruses (hrHPVs) as etiological agents in cancer development has been intensively studied during the last decades, there is still the necessity of understanding the impact of the HPV E6 and E7 oncogenes on host cells, ultimately leading to malignant transformation. Here, we used newly established immortalized human keratinocytes with a well-defined HPV16 E6E7 expression cassette to get a more complete and less biased overview of global changes induced by HPV16 by employing transcriptome sequencing (RNA-Seq) and stable isotope labeling by amino acids in cell culture (SILAC). This is the first study combining transcriptome and proteome data to characterize the impact of HPV oncogenes in human keratinocytes in comparison with their virus-negative counterparts. To enhance the informative value and accuracy of the RNA-Seq data, four different bioinformatic workflows were used. We identified potential novel upstream regulators (e.g., CNOT7, SPDEF, MITF, and PAX5) controlling distinct clusters of genes within the HPV-host cell network as well as distinct factors (e.g., CPPED1, LCP1, and TAGLN) with essential functions in cancer. Validated results in this study were compared to data sets from The Cancer Genome Atlas (TCGA), demonstrating that several identified factors were also differentially expressed in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) and HPV-positive head and neck squamous cell carcinomas (HNSCs). This highly integrative approach allows the identification of novel HPV-induced cellular changes that are also reflected in cancer patients, providing a promising omics data set for future studies in both basic and translational research. IMPORTANCE Human papillomavirus (HPV)-associated cancers still remain a big health problem, especially in developing countries, despite the availability of prophylactic vaccines. Although HPV oncogenes have been intensively investigated for decades, a study applying recent advances in RNA-Seq and quantitative proteomic approaches to a precancerous model system with well-defined HPV oncogene expression alongside HPV-negative parental cells has been missing until now. Here, combined omics analyses reveal global changes caused by the viral oncogenes in a less biased way and allow the identification of novel factors and key cellular networks potentially promoting malignant transformation. In addition, this system also provides a basis for mechanistic research on novel key factors regulated by HPV oncogenes, especially those that are confirmed in vivo in cervical cancer as well as in head and neck cancer patient samples from TCGA data sets., (Copyright © 2019 Yang et al.)

α-Galactosidases are assigned to the class of hydrolases and the subclass of glycoside hydrolases (GHs). They belong to six GH families and include the only characterized α-galactosidases from yeasts (GH 27, Saccharomyces cerevisiae). The present study focuses on an investigation of the lactose-inducible α-galactosidase produced by Papiliotrema flavescens. The enzyme was present on the surface of cells and in the cytosol. Its temperature optimum was about 60 °C and the pH optimum was 4.8; the pH stability ranged from 3.2 to 6.6. This α-galactosidase also exhibited transglycosylation activity. The cytosol α-galactosidase with a molecular weight about 110 kDa, was purified using a combination of liquid chromatography techniques. Three intramolecular peptides were determined by the partial structural analysis of the sequences of the protein isolated, using MALDI-TOF/TOF mass spectrometry. The data obtained recognized the first yeast α-galactosidase, which belongs to the GH 36 family. The bioinformatics analysis and homology modeling of a 210 amino acids long C-terminal sequence (derived from cDNA) confirmed the correctness of these findings. The study was also supplemented by the screening of capsular cryptococcal yeasts, which produce the surface lactose-inducible α- and β-galactosidases. The production of the lactose-inducible α-galactosidases was not found to be a general feature within the yeast strains examined and, therefore, the existing hypothesis on the general function of this enzyme in cryptococcal capsule rearrangement cannot be confirmed.

In contrast to the well defined mechanism of merocrine exocytosis, the mechanism of apocrine secretion, which was first described over 180 years ago, remains relatively uncharacterized. We identified apocrine secretory activity in the late prepupal salivary glands of Drosophila melanogaster just prior to the execution of programmed cell death (PCD). The excellent genetic tools available in Drosophila provide an opportunity to dissect for the first time the molecular and mechanistic aspects of this process. A prerequisite for such an analysis is to have pivotal immunohistochemical, ultrastructural, biochemical and proteomic data that fully characterize the process. Here we present data showing that the Drosophila salivary glands release all kinds of cellular proteins by an apocrine mechanism including cytoskeletal, cytosolic, mitochondrial, nuclear and nucleolar components. Surprisingly, the apocrine release of these proteins displays a temporal pattern with the sequential release of some proteins (e.g. transcription factor BR-C, tumor suppressor p127, cytoskeletal β-tubulin, non-muscle myosin) earlier than others (e.g. filamentous actin, nuclear lamin, mitochondrial pyruvate dehydrogenase). Although the apocrine release of proteins takes place just prior to the execution of an apoptotic program, the nuclear DNA is never released. Western blotting indicates that the secreted proteins remain undegraded in the lumen. Following apocrine secretion, the salivary gland cells remain quite vital, as they retain highly active transcriptional and protein synthetic activity.

Nearly all cervical cancer cases are caused by infection with high-risk human papillomavirus (HR-HPV) types. The mechanism of cervical cell transformation is related to the powerful action of viral oncoproteins and cellular gene alterations. Transcriptomic data from cervical cancer and normal cervical cells were utilized to identify upregulated genes and their associated pathways. The laminin subunit beta-3 (LAMB3) mRNAwas overexpressed in cervical cancer and was chosen for functional analysis. The LAMB3 was predominantly expressed in the extracellular region and the plasma membrane, which play a role in protein binding and cell adhesion molecule binding, leading to cell migration and tissue development. LAMB3 was found to be implicated in the pathway in cancer and the PI3K-AKT signaling pathway. LAMB3 knockdown decreased cell migration, invasion, anchorage-dependent and anchorage-independent cell growth and increased the number of apoptotic cells. These effects were linked to a decrease in protein levels involved in the PI3K-AKT signaling pathway and an increase in p53 protein. This study demonstrated that LAMB3 could promote cervical cancer cell migration, invasion and survival. [ABSTRACT FROM AUTHOR]

Lipids represent a large group of biomolecules that are responsible for various functions in organisms. Diseases such as diabetes, chronic inflammation, neurological disorders, or neurodegenerative and cardiovascular diseases can be caused by lipid imbalance. Due to the different stereochemical properties and composition of fatty acyl groups of molecules in most lipid classes, quantification of lipids and development of lipidomic analytical techniques are problematic. Identification of different lipid species from complex matrices is difficult, and therefore individual analytical steps, which include extraction, separation, and detection of lipids, must be chosen properly. This review critically documents recent strategies for lipid analysis from sample pretreatment to instrumental analysis and data interpretation published in the last five years (2019 to 2023). The advantages and disadvantages of various extraction methods are covered. The instrumental analysis step comprises methods for lipid identification and quantification. Mass spectrometry (MS) is the most used technique in lipid analysis, which can be performed by direct infusion MS approach or in combination with suitable separation techniques such as liquid chromatography or gas chromatography. Special attention is also given to the correct evaluation and interpretation of the data obtained from the lipid analyses. Only accurate, precise, robust and reliable analytical strategies are able to bring complex and useful lipidomic information, which may contribute to clarification of some diseases at the molecular level, and may be used as putative biomarkers and/or therapeutic targets. [ABSTRACT FROM AUTHOR]

The ectopic expression of cellular retinoic acid binding protein 2 (CRABP2) is associated with various tumorigenesis. However, the effects of CRABP2 on the progression of cervical cancer are still unclear. The current study aimed to investigate the role of CRABP2 in the malignant phenotypes of cervical cancer cells. CRABP2 was artificially regulated in CaSki, SiHa, and C-33A cells. CCK-8 assay and flow cytometry were used to assess the cell proliferation and apoptosis abilities, respectively. Wound healing assay and transwell assay were employed to measure the cell migration and invasion abilities, respectively. The results showed that CRABP2 was highly expressed in cervical carcinoma tissues and cell lines, and its high expression was associated with poor overall survival. Knockdown of CRABP2 promoted the cell apoptosis and inhibited cell proliferation, migration, and invasion in cervical carcinoma cells, whereas CRABP2 overexpression exhibited the opposite results. Mechanically, CRABP2 silencing suppressed the Integrin β1/FAK/ERK signaling via HuR. Treatment with siITGB1 or a FAK inhibitor PF-562271 or an ERK inhibitor FR180204 reversed the promoting effects of CRABP2 on cell proliferation, migration, and invasion. Moreover, the overexpression of CRABP2 reverted the HPV16 E6/E7 knockdown-induced inhibition of cell proliferation, migration, and invasion in cervical cancer cells. These results suggested that HPV16 E6/E7 promoted the malignant phenotypes of cervical cancer by upregulating the expression of CRABP2. In conclusion, CRABP2, upregulated by HPV E6/E7, promoted the progression of cervical cancer through activating the Integrin β1/FAK/ERK signaling pathway via HuR., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Proteomics in respiratory allergic diseases has such a battery of techniques and programs that one would almost think there is nothing impossible to find, invent or mold. All the resources that we document here are involved in solving problems in allergic diseases, both diagnostic and prognostic treatment, and immunotherapy development. The main perspectives, according to this version, are in three strands and/or a lockout immunological system: (1) Blocking the diapedesis of the cells involved, (2) Modifications and blocking of paratopes and epitopes being understood by modifications to antibodies, antagonisms, or blocking them, and (3) Blocking FcεRI high-affinity receptors to prevent specific IgEs from sticking to mast cells and basophils. These tools and targets in the allergic landscape are, in our view, the prospects in the field. However, there are still many allergens to identify, including some homologies between allergens and cross-reactions, through the identification of structures and epitopes. The current vision of using proteomics for this purpose remains a constant; this is also true for the basis of diagnostic and controlled systems for immunotherapy. Ours is an open proposal to use this vision for treatment. [ABSTRACT FROM AUTHOR]

Flax, or linseed, is considered a "superfood", which means that it is a food with diverse health benefits and potentially useful bioactive ingredients. It is a multi-purpose crop that is prized for its seed oil, fibre, nutraceutical, and probiotic qualities. It is suited to various habitats and agro-ecological conditions. Numerous abiotic and biotic stressors that can either have a direct or indirect impact on plant health are experienced by flax plants as a result of changing environmental circumstances. Research on the impact of various stresses and their possible ameliorators is prompted by such expectations. By inducing the loss of specific alleles and using a limited number of selected varieties, modern breeding techniques have decreased the overall genetic variability required for climate-smart agriculture. However, gene banks have well-managed collectionns of landraces, wild linseed accessions, and auxiliary Linum species that serve as an important source of novel alleles. In the past, flax-breeding techniques were prioritised, preserving high yield with other essential traits. Applications of molecular markers in modern breeding have made it easy to identify quantitative trait loci (QTLs) for various agronomic characteristics. The genetic diversity of linseed species and the evaluation of their tolerance to abiotic stresses, including drought, salinity, heavy metal tolerance, and temperature, as well as resistance to biotic stress factors, viz., rust, wilt, powdery mildew, and alternaria blight, despite addressing various morphotypes and the value of linseed as a supplement, are the primary topics of this review. [ABSTRACT FROM AUTHOR]

Plants are constantly faced with biotic or abiotic stress, which affects their growth and development. Yield reduction due to biotic and abiotic stresses on economically important crop species causes substantial economic loss at a global level. Breeding for stress tolerance to create elite and superior genotypes has been a common practice for many decades, and plant tissue culture can be an efficient and cost-effective method. Tissue culture is a valuable tool to develop stress tolerance, screen stress tolerance, and elucidate physiological and biochemical changes during stress. In vitro selection carried out under controlled environment conditions in confined spaces is highly effective and cheaper to maintain. This review emphasizes the relevance of plant tissue culture for screening major abiotic stresses, drought, and salinity, and the development of disease resistance. Further emphasis is given to screening metal hyperaccumulators and transgenic technological applications for stress tolerance. [ABSTRACT FROM AUTHOR]

Beer is a beverage that contains gluten and cannot be consumed by people with celiac disease. In this context, the enzyme prolyl endoprotease (PEP) can be used to reduce the gluten content in beer. The present study aimed to produce the PEP from Aspergillus sp. FSDE 16 using solid-state fermentation with 5 conditions and comparing with a similar commercial enzyme produced from Aspergillus niger in the production of a gluten-free beer. The results of the performed cultures showed that during the culture, the most increased protease activity (54.46 U/mL) occurred on the 4th day. In contrast, for PEP, the highest activity (0.0356 U/mL) was obtained on the 3rd day of culture in condition. Regarding beer production, cell growth, pH, and total soluble solids showed similar behavior over the 7 days for beers produced without enzyme addition or with the addition of commercial enzyme and with the addition of the enzyme extract produced. The addition of the enzyme and the enzyme extract did not promote changes, and all the beers produced showed similar and satisfactory results, with acid pH between 4 and 5, total soluble solids ranging from 4.80 to 5.05, alcohol content ranging from 2.83% to 3.08%, and all beers having a dark character with deep amber and light copper color. Gluten removal was effectively using the commercial enzyme and the enzyme produced according to condition (v) reaching gluten concentrations equal to 17 ± 5.31 and 21.19 ± 11.28 ppm, respectively. In this way, the production of the enzyme by SSF and its application in the removal of gluten in beer was efficient., (© 2024 International Union of Biochemistry and Molecular Biology, Inc.)

Schizochytrium sp. is a heterotrophic microorganism capable of accumulating polyunsaturated fatty acids and has achieved industrial production of docosahexaenoic acid (DHA). It also has the potential for eicosapentaenoic acid (EPA) production. In this study, it was found that the cell growth, lipid synthesis and fatty acid composition of Schizochytrium sp. were significantly affected by the level of cobalamin in the medium, especially with regard to the content of EPA in the fatty acids. The content of EPA in the fatty acids increased 17.91 times, reaching 12.00%, but cell growth and lipid synthesis were significantly inhibited under cobalamin deficiency. The response mechanism for this phenomenon was revealed through combined lipidomic and transcriptomic analysis. Although cell growth was inhibited under cobalamin deficiency, the genes encoding key enzymes in central carbon metabolism were still up-regulated to provide precursors (Acetyl-CoA) and reducing power (NADPH) for the synthesis and accumulation of fatty acids. Moreover, the main lipid subclasses observed during cobalamin deficiency were glycerolipids (including glycerophospholipids), with EPA primarily distributed in them. The genes involved in the biosynthesis of these lipid subclasses were significantly up-regulated, such as the key enzymes in the Kennedy pathway for the synthesis of triglycerides. Thus, this study provided insights into the specific response of Schizochytrium sp. to cobalamin deficiency and identified a subset of new genes that can be engineered for modification., (© 2024 Wiley-VCH GmbH.)

The water-soluble glue substance of the capture threads in Trichonephila clavata is solely produced from two pairs of aggregate silk glands. During the web glue production, secretory vesicles were synthesized via the extensive rough endoplasmic reticulum of epithelial cells. Unlike the clearly described fibrous web production in spiders, the process of aqueous web glue production appears to involve either a condensing or a packaging step by the Golgi complex. In particular, the fine structure of secretory vesicles varies from cell to cell and may represent the secretory cycle. The electron-dense multivesicular bodies were clearly visible as discrete droplets, and the mature secretory product in the glandular epithelium appeared as a spherical vacuole grown by fusion with surrounding small vesicles. Our fine structural observation reveals that the secretion occurs when the release of secreted material involves the loss of part of the cytoplasm. The bleb along the luminal surface of the secretory cells and membrane-bound extracellular vesicles which pinched off from the cell suggests that the secretory product is released by the mechanism of apocrine secretion. [ABSTRACT FROM AUTHOR]

SUMMARY: Formate dehydrogenase (FDH; EC 1.2.1.2.) has been implicated in plant responses to a variety of stresses, including aluminum (Al) stress in acidic soils. However, the role of this enzyme in Al tolerance is not yet fully understood, and how FDH gene expression is regulated is unknown. Here, we report the identification and functional characterization of the tomato (Solanum lycopersicum) SlFDH gene. SlFDH encodes a mitochondria‐localized FDH with Km values of 2.087 mm formate and 29.1 μm NAD+. Al induced the expression of SlFDH in tomato root tips, but other metals did not, as determined by quantitative reverse transcriptase‐polymerase chain reaction. CRISPR/Cas9‐generated SlFDH knockout lines were more sensitive to Al stress and formate than wild‐type plants. Formate failed to induce SlFDH expression in the tomato root apex, but NAD+ accumulated in response to Al stress. Co‐expression network analysis and interaction analysis between genomic DNA and transcription factors (TFs) using PlantRegMap identified seven TFs that might regulate SlFDH expression. One of these TFs, SlSTOP1, positively regulated SlFDH expression by directly binding to its promoter, as demonstrated by a dual‐luciferase reporter assay and electrophoretic mobility shift assay. The Al‐induced expression of SlFDH was completely abolished in Slstop1 mutants, indicating that SlSTOP1 is a core regulator of SlFDH expression under Al stress. Taken together, our findings demonstrate that SlFDH plays a role in Al tolerance and reveal the transcriptional regulatory mechanism of SlFDH expression in response to Al stress in tomato. Significance Statement: Formate dehydrogenase (FDH; EC 1.2.1.2.) catalyzes the degradation of formate and the reduction of NAD+ to NADH. Here, we demonstrate that the FDH gene SlFDH is induced by aluminum stress in tomato, which relies on the transcription factor SlSTOP1, to enhance tolerance to aluminum stress via SlFDH‐catalyzed reduction of NAD+. [ABSTRACT FROM AUTHOR]

Cadmium is one of the most toxic heavy metal pollutants, and its accumulation in the soil is harmful to agriculture. Plants have a higher cadmium tolerance than animals, and some species can be used for phytoremediation. Flax (Linum usitatissimum L.) can accumulate high amounts of cadmium, but the molecular mechanism behind its tolerance is unknown. Here, we employed four genotypes representing two fiber cultivars, an oilseed breeding line, and a transgenic line overexpressing the metallothionein domain for improved cadmium tolerance. We analyzed the proteome of suspensions and the proteome and metabolome of seedling roots in response to cadmium. We identified more than 1400 differentially abundant proteins representing putative mechanisms in cadmium tolerance, including metal-binding proteins and transporters, enzymes of flavonoid, jasmonate, polyamine, glutathione metabolism, and HSP70 proteins. Our data indicated the role of the phytohormone cytokinin in the observed responses. The metabolome profiling found that pipecolinic acid could be a part of the cadmium accumulation mechanism, and the observed accumulation of putrescine, coumaric acid, cinnamic acid, and coutaric acid confirmed the role of polyamines and flavonoids in tolerance to cadmium. In conclusion, our data provide new insight into cadmium tolerance and prospective targets for improving cadmium tolerance in other plants. [ABSTRACT FROM AUTHOR]

Sorghum and cowpea are very compatible for intercropping in hot and dry environments, and they also have complementary nutritional compositions. Thus, the crops have the potential to improve food security in regions threatened by climate change. The aim of this study was to investigate different enzymes (carbohydrate-degrading, proteases and phytases) and lactic acid bacteria (LAB) fermentation to improve the techno-functional properties of sorghum and cowpea flours. Results show that sorghum carbohydrates were very resistant to hydrolysis induced by bioprocessing treatments. Most of the protease treatments resulted in low or moderate protein solubilization (from ca. 6.5% to 10%) in sorghum, while the pH adjustment to 8 followed by alkaline protease increased solubility to 40%. With cowpea, protease treatment combined with carbohydrate-degrading enzymes increased the solubility of proteins from 37% up to 61%. With regard to the techno-functional properties, LAB and amylase treatment decreased the sorghum peak paste viscosities (from 504 to 370 and 325 cPa, respectively), while LAB and chemical acidification increased cowpea viscosity (from 282 to 366 and 468 cPa, respectively). When the bioprocessed sorghum and cowpea were tested in breadmaking, only moderate effects were observed, suggesting that the modifications by enzymes and fermentation were not strong enough to improve breadmaking. [ABSTRACT FROM AUTHOR]

Flax (Linum usitatissimum L.) or linseed is one of the important industrial crops grown all over the world for seed oil and fiber. Besides oil and fiber, flax offers a wide range of nutritional and therapeutic applications as a feed and food source owing to high amount of α-linolenic acid (omega-3 fatty acid), lignans, protein, minerals, and vitamins. Periodic losses caused by unpredictable environmental stresses such as drought, heat, salinity-alkalinity, and diseases pose a threat to meet the rising market demand. Furthermore, these abiotic and biotic stressors have a negative impact on biological diversity and quality of oil/ fiber. Therefore, understanding the interaction of genetic and environmental factors in stress tolerance mechanism and identification of underlying genes for economically important traits is critical for flax improvement and sustainability. In recent technological era, numerous omics techniques such as genomics, transcriptomics, metabolomics, proteomics, phenomics, and ionomics have evolved. The advancements in sequencing technologies accelerated development of genomic resources which facilitated finer genetic mapping, quantitative trait loci (QTL) mapping, genome-wide association studies (GWAS), and genomic selection in major cereal and oilseed crops including flax. Extensive studies in the area of genomics and transcriptomics have been conducted post flax genome sequencing. Interestingly, research has been focused more for abiotic stresses tolerance compared to disease resistance in flax through transcriptomics, while the other areas of omics such as metabolomics, proteomics, ionomics, and phenomics are in the initial stages in flax and several key questions remain unanswered. Little has been explored in the integration of omic-scale data to explain complex genetic, physiological and biochemical basis of stress tolerance in flax. In this review, the current status of various omics approaches for elucidation of molecular pathways underlying abiotic and biotic stress tolerance in flax have been presented and the importance of integrated omics technologies in future research and breeding have been emphasized to ensure sustainable yield in challenging environments. [ABSTRACT FROM AUTHOR]

Simple Summary: Like many hematophagous parasites, the poultry red mite Dermanyssus gallinae may release some material during the biting process. This investigation evidenced that small amounts of mite DNA may be found in chicken skin after D. gallinae infestation. Since the retrieved DNA is both of nuclear and mitochondrial origin, it is possible to hypothesize that, while biting, the mite releases cellular material. Dermanyssus gallinae is a hematophagous ectoparasitic mite that usually infests poultry, but is also known for occasionally attacking other animals and humans. It represents a major problem for poultry systems all over the world, with detrimental effects for both production and animal welfare. Despite the significance of D. gallinae, very little is known about the biting process to date. Therefore, this study has aimed to verify if mite DNA is injected into the host skin during the blood meal. Mite DNA has been detected by seminested PCR from infested chicken skin and quantified by real-time PCR. Furthermore, its localization within the host tissue has been checked by fluorescent in situ hybridization. Results showed that a very little amount of D. gallinae DNA can be released by mites, suggesting that the latter do not introduce whole or partially destroyed cells into the host, but rather it injects traces of nucleic acids, possibly together with merocrine secretions. [ABSTRACT FROM AUTHOR]

Background: The decapods are a major group of crustaceans that includes shrimps, prawns, crayfishes, lobsters, and crabs. Several studies focused on the study of the digestive system of the decapods, constituted by the oesophagus, stomach, midgut tract, midgut gland, and hindgut. Nevertheless, in the midgut tract there are associated a set of organs called "midgut caeca", which are among the most controversial and less studied digestive organs of this group. This work used the common spider crab Maja brachydactyla Balss, 1922 as a model to resolve the origin, development, and potential role of the midgut caeca. Such organs were studied in the larvae (zoea I, zoea II, megalopa), first juveniles, and adult phases, being employed traditional and modern techniques: dissection, micro-computed tomography (Micro-CT), and light and electron microscopical analyses (TEM and SEM). Results: The common spider crab has a pair of anterior midgut caeca and a single posterior caecum that originate from the endoderm germ layer: they develop from the midgut tract, and their epithelium is composed by secretory cells while lacking a cuticle lining. The midgut caeca are small buds in the newly hatched larvae, enlarge linearly during the larval development, and then continue growing until became elongated and coiled blind-tubules in adults. The adult midgut caeca are internally folded to increase their inner surface. The electron microscopy observations showed that the midgut caeca are highly active organs with important macroapocrine and microapocrine secretory activity. Our results suggest that the role of the caeca might be related to the digestive enzyme secretion. The secretory activity should increase as the animal grows in size. Conclusion: The present study resolves the embryonic origin of the midgut caeca (endoderm derived organs), development (general lengthening starting from small buds), and role (active secretory organs). The secretory activity of the midgut caeca should be incorporated in the current models of the digestive physiology in different decapod taxa. [ABSTRACT FROM AUTHOR]

Proteins play a major role in the three-dimensional organization of nuclear genome and its function. While histones arrange DNA into a nucleosome fiber, other proteins contribute to higher-order chromatin structures in interphase nuclei, and mitotic/meiotic chromosomes. Despite the key role of proteins in maintaining genome integrity and transferring hereditary information to daughter cells and progenies, the knowledge about their function remains fragmentary. This is particularly true for the proteins of condensed chromosomes and, in particular, chromosomes of plants. Here, we purified barley mitotic metaphase chromosomes by a flow cytometric sorting and characterized their proteins. Peptides from tryptic protein digests were fractionated either on a cation exchanger or reversed-phase microgradient system before liquid chromatography coupled to tandem mass spectrometry. Chromosomal proteins comprising almost 900 identifications were classified based on a combination of software prediction, available database localization information, sequence homology, and domain representation. A biological context evaluation indicated the presence of several groups of abundant proteins including histones, topoisomerase 2, POLYMERASE 2, condensin subunits, and many proteins with chromatin-related functions. Proteins involved in processes related to DNA replication, transcription, and repair as well as nucleolar proteins were found. We have experimentally validated the presence of FIBRILLARIN 1, one of the nucleolar proteins, on metaphase chromosomes, suggesting that plant chromosomes are coated with proteins during mitosis, similar to those of human and animals. These results improve significantly the knowledge of plant chromosomal proteins and provide a basis for their functional characterization and comparative phylogenetic analyses. [ABSTRACT FROM AUTHOR]

SUMMARY: Certain transglucanases can covalently graft cellulose and mixed‐linkage β‐glucan (MLG) as donor substrates onto xyloglucan as acceptor substrate and thus exhibit cellulose:xyloglucan endotransglucosylase (CXE) and MLG:xyloglucan endotransglucosylase (MXE) activities in vivo and in vitro. However, missing information on factors that stimulate or inhibit these hetero‐transglucosylation reactions limits our insight into their biological functions. To explore factors that influence hetero‐transglucosylation, we studied Equisetum fluviatile hetero‐trans‐β‐glucanase (EfHTG), which exhibits both CXE and MXE activity, exceeding its xyloglucan:xyloglucan homo‐transglucosylation (XET) activity. Enzyme assays employed radiolabelled and fluorescently labelled oligomeric acceptor substrates, and were conducted in vitro and in cell walls (in situ). With whole denatured Equisetum cell walls as donor substrate, exogenous EfHTG (extracted from Equisetum or produced in Pichia) exhibited all three activities (CXE, MXE, XET) in competition with each other. Acting on pure cellulose as donor substrate, the CXE action of Pichia‐produced EfHTG was up to approximately 300% increased by addition of methanol‐boiled Equisetum extracts; there was no similar effect when the same enzyme acted on soluble donors (MLG or xyloglucan). The methanol‐stable factor is proposed to be expansin‐like, a suggestion supported by observations of pH dependence. Screening numerous low‐molecular‐weight compounds for hetero‐transglucanase inhibition showed that cellobiose was highly effective, inhibiting the abundant endogenous CXE and MXE (but not XET) action in Equisetum internodes. Furthermore, cellobiose retarded Equisetum stem elongation, potentially owing to its effect on hetero‐transglucosylation reactions. This work provides insight and tools to further study the role of cellulose hetero‐transglucosylation in planta by identifying factors that govern this reaction. Significance Statement: The enzyme HTG can graft segments of cellulose molecules onto xyloglucan (a hemicellulose), thereby re‐structuring the cell wall via hetero‐transglycosylation. In native walls, two endogenous hemicelluloses competed with cellulose (and with each other) as substrate. HTG more readily selected cellulose as substrate if an expansin‐enriched preparation was added. Hetero‐transglycosylation was inhibited by cellobiose – a potential tool for exploring HTG's biological functions. Interestingly, cellobiose retarded Equisetum stem elongation, suggesting a role for HTG in growth. [ABSTRACT FROM AUTHOR]

The Glycine max xyloglucan endotransglycosylase/hydrolase (EC 2.4.1.207), GmXTH43, has been identified through RNA sequencing of RNA isolated through laser microdissection of Heterodera glycines-parasitized root cells (syncytia) undergoing the process of defense. Experiments reveal that genetically increasing XTH43 transcript abundance in the H. glycines-susceptible genotype G. max[Williams 82/PI 518671] decreases parasitism. Experiments presented here show decreasing XTH43 transcript abundance through RNA interference (RNAi) in the H. glycines-resistant G. max[Peking/PI 548402] increases susceptibility, but it is unclear what role XTH43 performs. The experiments presented here show XTH43 overexpression decreases the relative length of xyloglucan (XyG) chains, however, there is an increase in the amount of those shorter chains. In contrast, XTH43 RNAi increases XyG chain length. The experiments show that XTH43 has the capability to function, when increased in its expression, to limit XyG chain extension. This outcome would likely impair the ability of the cell wall to expand. Consequently, XTH43 could provide an enzymatically-driven capability to the cell that would allow it to limit the ability of parasitic nematodes like H. glycines to develop a feeding structure that, otherwise, would facilitate parasitism. The experiments presented here provide experimentally-based proof that XTHs can function in ways that could be viewed as being able to limit the expansion of the cell wall. [ABSTRACT FROM AUTHOR]

Mass spectrometry‐based proteomics experiments typically start with the digestion of proteins using trypsin, chosen because of its high specificity, availability, and ease of use. It has become apparent that the sole use of trypsin may impose certain limits on our ability to grasp the full proteome, missing out particular sites of post‐translational modifications, protein segments, or even subsets of proteins. To tackle this problem, alternative proteases have been introduced and shown to lead to an increase in the detectable (phospho)proteome. Here, we argue that there may be further room for improvement and explore the protease EndoPro. For optimal peptide identification rates, we explored multiple peptide fragmentation techniques (HCD, ETD, and EThcD) and employed Byonic as search algorithm. We obtain peptide IDs for about 40% of the MS2 spectra (66% for trypsin). EndoPro cleaves with high specificity at the C‐terminal site of Pro and Ala residues and displays activity in a broad pH range, where we focused on its performance at pH = 2 and 5.5. The proteome coverage of EndoPro at these two pH values is rather distinct, and also complementary to the coverage obtained with trypsin. As about 40% of mammalian protein phosphorylations are proline‐directed, we also explored the performance of EndoPro in phosphoproteomics. EndoPro extends the coverable phosphoproteome substantially, whereby both the, at pH = 2 and 5.5, acquired phosphoproteomes are complementary to each other and to the phosphoproteome obtained using trypsin. Hence, EndoPro is a powerful tool to exploit in (phospho)proteomics applications. [ABSTRACT FROM AUTHOR]

Flax (Linum usitatissimum L.), one of the oldest cultivated crops, continues to be widely grown for oil, fiber and food. Furthermore, the plants show a metal tolerance dependent on species so is ideal for research. Present study was conducted to find out the influence of copper (Cu) toxicity on plant biomass, growth, chlorophyll content, malondialdehyde (MDA) contents, proline production, antioxidative enzymes and metal up taken by L. usitatissimum from the soil grown under mixing of Cu-contaminated soil with natural soil by 0:1 (control), 1:0, 1:1, 1:2 and 1:4. Results revealed that, high concentration of Cu in the soil affected plant growth and development by reducing plant height, plant diameter and plant fresh and dry biomass and chlorophyll contents in the leaves compared with the control. Furthermore, Cu in excess causes generation of reactive oxygen species (ROS) such as superoxide radical (O–) and hydroxyl radicals (OH), which is manifested by high malondialdehyde (MDA) and proline contents also. The increasing activities of superoxidase dismutase (SOD) and peroxidase (POD) in the roots and leaves of L. usitatissimum are involved in the scavenging of ROS. Results also showed that L. usitatissimum also has capability to revoke large amount of Cu from the contaminated soil. As Cu concentration in the soil increases, the final uptake of Cu concentration by L. usitatissimum increases. Furthermore, the soil chemical parameters (pH, electrical conductivity and cation exchange capacity) were increasing to highest levels as the ratio of Cu concentration to the natural soil increases. Thus, Cu-contaminated soil is amended with the addition of natural soil significantly reduced plant growth and biomass, while L. usitatissimum is able to revoke large amount of Cu from the soil and could be grown as flaxseed and a potential candidate for phytoremediation of Cu. [ABSTRACT FROM AUTHOR]

Formate dehydrogenase (FDH; EC 1.2.1.2.) has been implicated in plant responses to a variety of stresses, including aluminum (Al) stress in acidic soils. However, the role of this enzyme in Al tolerance is not yet fully understood, and how FDH gene expression is regulated is unknown. Here, we report the identification and functional characterization of the tomato (Solanum lycopersicum) SlFDH gene. SlFDH encodes a mitochondria-localized FDH with K m values of 2.087 mm formate and 29.1 μm NAD + . Al induced the expression of SlFDH in tomato root tips, but other metals did not, as determined by quantitative reverse transcriptase-polymerase chain reaction. CRISPR/Cas9-generated SlFDH knockout lines were more sensitive to Al stress and formate than wild-type plants. Formate failed to induce SlFDH expression in the tomato root apex, but NAD + accumulated in response to Al stress. Co-expression network analysis and interaction analysis between genomic DNA and transcription factors (TFs) using PlantRegMap identified seven TFs that might regulate SlFDH expression. One of these TFs, SlSTOP1, positively regulated SlFDH expression by directly binding to its promoter, as demonstrated by a dual-luciferase reporter assay and electrophoretic mobility shift assay. The Al-induced expression of SlFDH was completely abolished in Slstop1 mutants, indicating that SlSTOP1 is a core regulator of SlFDH expression under Al stress. Taken together, our findings demonstrate that SlFDH plays a role in Al tolerance and reveal the transcriptional regulatory mechanism of SlFDH expression in response to Al stress in tomato., (© 2022 Society for Experimental Biology and John Wiley & Sons Ltd.)

Key message: The knowledge of substrate specificity of XET enzymes is important for the general understanding of metabolic pathways to challenge the established notion that these enzymes operate uniquely on cellulose-xyloglucan networks. Xyloglucan xyloglucosyl transferases (XETs) (EC 2.4.1.207) play a central role in loosening and re-arranging the cellulose-xyloglucan network, which is assumed to be the primary load-bearing structural component of plant cell walls. The sequence of mature TmXET6.3 from Tropaeolum majus (280 residues) was deduced by the nucleotide sequence analysis of complete cDNA by Rapid Amplification of cDNA Ends, based on tryptic and chymotryptic peptide sequences. Partly purified TmXET6.3, expressed in Pichia occurred in N-glycosylated and unglycosylated forms. The quantification of hetero-transglycosylation activities of TmXET6.3 revealed that (1,3;1,4)-, (1,6)- and (1,4)-β-d-glucooligosaccharides were the preferred acceptor substrates, while (1,4)-β-d-xylooligosaccharides, and arabinoxylo- and glucomanno-oligosaccharides were less preferred. The 3D model of TmXET6.3, and bioinformatics analyses of identified and putative plant xyloglucan endotransglycosylases (XETs)/hydrolases (XEHs) of the GH16 family revealed that H94, A104, Q108, K234 and K237 were the key residues that underpinned the acceptor substrate specificity of TmXET6.3. Compared to the wild-type enzyme, the single Q108R and K237T, and double-K234T/K237T and triple-H94Q/A104D/Q108R variants exhibited enhanced hetero-transglycosylation activities with xyloglucan and (1,4)-β-d-glucooligosaccharides, while those with (1,3;1,4)- and (1,6)-β-d-glucooligosaccharides were suppressed; the incorporation of xyloglucan to (1,4)-β-d-glucooligosaccharides by the H94Q variant was influenced most extensively. Structural and biochemical data of non-specific TmXET6.3 presented here extend the classic XET reaction mechanism by which these enzymes operate in plant cell walls. The evaluations of TmXET6.3 transglycosylation activities and the incidence of investigated residues in other members of the GH16 family suggest that a broad acceptor substrate specificity in plant XET enzymes could be more widespread than previously anticipated. [ABSTRACT FROM AUTHOR]

Reproducibility has been one of the biggest hurdles faced when attempting to develop quantitative protocols for MALDI mass spectrometry. The heterogeneous nature of sample recrystallization has made automated sample acquisition somewhat “hit and miss” with manual intervention needed to ensure that all sample spots have been analyzed. In this review, we explore the last 30 years of literature and anecdotal evidence that has attempted to address and improve reproducibility in MALDI MS. Though many methods have been attempted, we have discovered a significant publication history surrounding the use of nitrocellulose as a substrate to improve homogeneity of crystal formation and therefore reproducibility. We therefore propose that this is the most promising avenue of research for developing a comprehensive and universal preparation protocol for quantitative MALDI MS analysis. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 37:217–228, 2018. [ABSTRACT FROM AUTHOR]

The order Thelyphonida (Chelicerata: Arachnida) contains more than 100 species of whipscorpions that live in warm tropical and xeric environments. All whipscorpions possess a pair of pygidial glands in their opisthosoma that secrete and actively discharge a defensive spray composed mostly of acetic acid, hence their common name, vinegaroon. To date, the structure of the glands remains poorly understood and has not been subject to detailed analysis for more than a century. Here, we provide the first description of gland structure in the North American vinegaroon, Mastigoproctus giganteus. We used paraffin histology to study general gland structure in juveniles (2nd instar) and adults. We supplement these observations with histochemical stains, confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). We found the glands to be composed of numerous long strips of multinucleated syncytium that rest on a basal lamina that lacks an external muscle coat. The gland syncytia contain abundant glycogen granules and secretion drops of different sizes and staining qualities. The adult epithelium is organized into three general patterns: as simple epithelia, as simple acinar glands, and as compound acinar glands. The juvenile gland epithelium is less distinctly organized. A cuticular intima lines the apical (lumenal) surface of adult and juvenile glands, is multilaminate, contains chitin, and may be permeated by irregular pores. The basal side of the intima consists of cuticular, finger-like projections that protrude into a subcuticular space. The underlying epithelium possesses an apical brush border of microvilli that appears to engage in Kurosumi’s type III extrusion, but the contents (cuticle or acids) of the vesicles are unknown. Curiously, the cells possess apical nuclei that also suggest a basal route of secretion, but details on this process are undetermined. Together, these results reveal that vinegaroon pygidial glands are unlike most other arachnid exocrine glands in their structure, methods of secretion, and mechanisms of discharge. [ABSTRACT FROM AUTHOR]

Rationale: Matrix-assisted laser desorption ionisation with mass spectrometry imaging (MSI) has seen rapid development in recent years and as such is becoming an important technique for the mapping of biomolecules from the surface of tissues. One key area of development is the optimisation of analyte extraction by using modified matrices or mixes of common ones., Methods: A series of serial sections were prepared for lipid MSI by either dry coating (sublimation) or by wet spray application of several matrices. These samples were then evaluated for analyte extraction, delocalisation and dynamic range., Results: We have shown that the spraying and sublimation methods of matrix application can be used complementarily. This creates large datasets, with each preparation method applied narrowly and then interpreted as a 'fraction' of the whole. Once combined, the dynamic range is significantly increased. We have dubbed this technique 'matrix phase fractionation'., Conclusions: We have found that, by utilising matrix phase fractionation for the detection of lipids in brain tissue, it is possible to create a significantly more comprehensive dataset than would otherwise be possible with traditional 'single-run' workflows., (© 2021 John Wiley & Sons Ltd.)

Many aquatic invertebrates are associated with surfaces, using adhesives to attach to the substratum for locomotion, prey capture, reproduction, building or defence. Their intriguing and sophisticated biological glues have been the focus of study for decades. In all but a couple of specific taxa, however, the precise mechanisms by which the bioadhesives stick to surfaces underwater and (in many cases) harden have proved to be elusive. Since the bulk components are known to be based on proteins in most organisms, the opportunities provided by advancing 'omics technologies have revolutionised bioadhesion research. Time-consuming isolation and analysis of single molecules has been either replaced or augmented by the generation of massive data sets that describe the organism's translated genes and proteins. While these new approaches have provided resources and opportunities that have enabled physiological insights and taxonomic comparisons that were not previously possible, they do not provide the complete picture and continued multi-disciplinarity is essential. This review covers the various ways in which 'omics have contributed to our understanding of adhesion by aquatic invertebrates, with new data to illustrate key points. The associated challenges are highlighted and priorities are suggested for future research., (© 2021 The Authors. Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society.)

Key points Xanthine oxidoreductase (XOR) modulates milk lipid secretion and lactation initiation., XOR is required for butyrophilin1a1 clustering in the membrane during milk lipid secretion., XOR mediates apical membrane reorganization during milk lipid secretion., Loss of XOR delays milk fat globule secretion., XOR loss alters the proteome of milk fat globules., Abstract Apocrine secretion is utilized by epithelial cells of exocrine glands. These cells bud off membrane-bound particles into the lumen of the gland, losing a portion of the cytoplasm in the secretion product. The lactating mammary gland secretes milk lipid by this mechanism, and xanthine oxidoreductase (XOR) has long been thought to be functionally important. We generated mammary-specific XOR knockout (MGKO) mice, expecting lactation to fail. Histology of the knockout glands showed very large lipid droplets enclosed in the mammary alveolar cells, but milk analysis showed that these large globules were secreted. Butyrophilin, a membrane protein known to bind to XOR, was clustered at the point of contact of the cytoplasmic lipid droplet with the apical plasma membrane, in the wild-type gland but not in the knockout, suggesting that XOR mediates 'docking' to this membrane. Secreted milk fat globules were isolated from mouse milk of wild-type and XOR MGKO dams, and subjected to LC-MS/MS for analysis of protein component. Proteomic results showed that loss of XOR leads to an increase in cytoplasmic, cytoskeletal, Golgi apparatus and lipid metabolism proteins associated with the secreted milk fat globule. Association of XOR with the lipid droplet results in membrane docking and more efficient retention of cytoplasmic components by the secretory cell. Loss of XOR then results in a reversion to a more rudimentary, less efficient, apocrine secretion mechanism, but does not prevent milk fat globule secretion. [ABSTRACT FROM AUTHOR]

The Drosophila salivary glands ( SGs) were well known for the puffing patterns of their polytene chromosomes and so became a tissue of choice to study sequential gene activation by the steroid hormone ecdysone. One well-documented function of these glands is to produce a secretory glue, which is released during pupariation to fix the freshly formed puparia to the substrate. Over the past two decades SGs have been used to address specific aspects of developmentally-regulated programmed cell death ( PCD) as it was thought that they are doomed for histolysis and after pupariation are just awaiting their fate. More recently, however, we have shown that for the first 3-4 h after pupariation SGs undergo tremendous endocytosis and vacuolation followed by vacuole neutralization and membrane consolidation. Furthermore, from 8 to 10 h after puparium formation ( APF) SGs display massive apocrine secretion of a diverse set of cellular proteins. Here, we show that during the period from 11 to 12 h APF, the prepupal glands are very active in calcium oxalate (CaOx) extrusion that resembles renal or nephridial excretory activity. We provide genetic evidence that Prestin, a Drosophila homologue of the mammalian electrogenic anion exchange carrier SLC26A5, is responsible for the instantaneous production of CaOx by the late prepupal SGs. Its positive regulation by the protein kinases encoded by fray and wnk lead to increased production of CaOx. The formation of CaOx appears to be dependent on the cooperation between Prestin and the vATPase complex as treatment with bafilomycin A1 or concanamycin A abolishes the production of detectable CaOx. These data demonstrate that prepupal SGs remain fully viable, physiologically active and engaged in various cellular activities at least until early pupal period, that is, until moments prior to the execution of PCD. [ABSTRACT FROM AUTHOR]

Polysaccharide endotransglycosylases (PETs) are the cell wall-modifying enzymes of fungi and plants. They catalyze random endo-splitting of the polysaccharide donor molecule and transfer of the newly formed reducing sugar residue to the nonreducing end of an acceptor molecule which can be a polysaccharide or an oligosaccharide. Owing to their important role in the cell wall formation, the inhibition of PETs represents an attractive strategy in the fight against fungal infections. We have elaborated two variants of a versatile high-throughput microplate fluorimetric assay that could be used for effective identification of PETs and screening of their inhibitors. Both assays use the respective polysaccharides as the donors and sulforhodamine-labeled oligosaccharides as the acceptors but differ from each other by mode of how the labeled polysaccharide products of transglycosylation are separated from the unreacted oligosaccharide acceptors. In the first variant, the reactions take place in a layer of agar gel laid on the bottoms of the wells of a microtitration plate. After the reaction, the high-M transglycosylation products are precipitated with 66 % ethanol and retained within the gel while the low-M products and the unreacted acceptors are washed out. In the second variant, the donor polysaccharides are adsorbed to the surface of a microplate well and remain adsorbed there also after becoming labeled in the course of the transglycosylation reaction whereas the unused low-M acceptors are washed out. As a proof of versatility, assays of heterologously expressed transglycosylases ScGas1, ScCrh1, and ScCrh2 from the yeast Saccharomyces cerevisiae, CaPhr1 and CaPhr2 from Candida albicans, and of a plant xyloglucan endotransglycosylase (XET) are demonstrated. [ABSTRACT FROM AUTHOR]

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A central function of the Drosophila salivary glands ( SGs), historically known for their polytene chromosomes, is to produce and then release during pupariation the secretory glue used to affix a newly formed puparium to a substrate. This essential event in the life history of Drosophila is regulated by the steroid hormone ecdysone in the late-larval period. Ecdysone triggers a cascade of sequential gene activation that leads to glue secretion and initiates the developmentally-regulated programmed cell death ( PCD) of the larval salivary glands, which culminates 16 h after puparium formation ( APF). We demonstrate here that, even after the larval salivary glands have completed what is perceived to be one of their major biological functions - glue secretion during pupariation - they remain dynamic and physiologically active up until the execution phase of PCD. We have used specific metabolic inhibitors and genetic tools, including mutations or transgenes for shi, Rab5, Rab11, vha55, vha68-2, vha36-1, syx1A, syx4, and Vps35 to characterize the dramatic series of cellular changes occurring in the SG cells between pupariation and 7-8 h APF. Early in the prepupal period, they are remarkably active in endocytosis, forming acidic vacuoles. Midway through the prepupal period, there is abundant late endosomal trafficking and vacuole growth, which is followed later by vacuole neutralization and disappearance via membrane consolidation. This work provides new insights into the function of Drosophila SGs during the early- to mid-prepupal period. [ABSTRACT FROM AUTHOR]

Plant xyloglucan:xyloglucosyl transferases, known as xyloglucan endo-transglycosylases (XETs) are the key players that underlie plant cell wall dynamics and mechanics. These fundamental roles are central for the assembly and modifications of cell walls during embryogenesis, vegetative and reproductive growth, and adaptations to living environments under biotic and abiotic (environmental) stresses. XET enzymes (EC 2.4.1.207) have the β-sandwich architecture and the β-jelly-roll topology, and are classified in the glycoside hydrolase family 16 based on their evolutionary history. XET enzymes catalyse transglycosylation reactions with xyloglucan (XG)-derived and other than XG-derived donors and acceptors, and this poly-specificity originates from the structural plasticity and evolutionary diversification that has evolved through expansion and duplication. In phyletic groups, XETs form the gene families that are differentially expressed in organs and tissues in time- and space-dependent manners, and in response to environmental conditions. Here, we examine higher plant XET enzymes and dissect how their exclusively carbohydrate-linked transglycosylation catalytic function inter-connects complex plant cell wall components. Further, we discuss progress in technologies that advance the knowledge of plant cell walls and how this knowledge defines the roles of XETs. We construe that the broad specificity of the plant XETs underscores their roles in continuous cell wall restructuring and re-modelling. [ABSTRACT FROM AUTHOR]

We designed a concept of 3D-printed attachment with porous glass filter disks—SLIDE (Sweat sampLIng DevicE) for easy sampling of apocrine sweat. By applying advanced mass spectrometry coupled with the liquid chromatography technique, the complex lipid profiles were measured to evaluate the reproducibility and robustness of this novel approach. Moreover, our in-depth statistical evaluation of the data provided an insight into the potential use of apocrine sweat as a novel and diagnostically relevant biofluid for clinical analyses. Data transformation using probabilistic quotient normalization (PQN) significantly improved the analytical characteristics and overcame the 'sample dilution issue' of the sampling. The lipidomic content of apocrine sweat from healthy subjects was described in terms of identification and quantitation. A total of 240 lipids across 15 classes were identified. The lipid concentrations varied from 10−10 to 10−4 mol/L. The most numerous class of lipids were ceramides (n = 61), while the free fatty acids were the most abundant ones (average concentrations of 10−5 mol/L). The main advantages of apocrine sweat microsampling include: (a) the non-invasiveness of the procedure and (b) the unique feature of apocrine sweat, reflecting metabolome and lipidome of the intracellular space and plasmatic membranes. The SLIDE application as a sampling technique of apocrine sweat brings a promising alternative, including various possibilities in modern clinical practice. [ABSTRACT FROM AUTHOR]

Simple Summary: Classical markers alone, such as HPV DNA, p16 and HPV mRNA expression, are not enough to stratify HPV-positive head and neck squamous cell carcinoma (HNSCC) patients, but when combined with serological markers, the latter are strong indicators of prognosis in oropharyngeal squamous cell carcinoma (OPSCC) patients. Specifically, HPV16 E7 oncoprotein in serum at the time of diagnosis, correlates with disease recurrence and patient overall survival. To our knowledge, this is the first study to investigate HPV E7 oncoprotein in patient serum. The E7 oncoprotein detection in serum at the time of diagnosis may be useful as a non-invasive procedure for HPV-positive OPSCC patient stratification and follow-up, helping to identify patients at risk for tumor recurrence and metastasis during follow-up, and ultimately, providing a tool for clinicians to identify patients for de-escalation treatment or those to be kept under close surveillance. Despite improved prognosis for many HPV-positive head and neck squamous cell carcinomas (HNSCCs), some cases are still marked by recurrence and metastasis. Our study aimed to identify novel biomarkers for patient stratification. Classical HPV markers: HPV-DNA, p16 and HPV mRNA expression were studied in HNSCC (n = 67) and controls (n = 58) by qPCR. Subsequently, ELISA tests were used for HPV16 L1 antibody and HPV16 E7 oncoprotein detection in serum at diagnosis and follow-up. All markers were correlated to relapse-free survival (RFS) and overall survival (OS). HPV-DNA was found in HNSCCs (29.85%), HPV16-DNA in 95% of cases, HPV16 E7 mRNA was revealed in 93.75%. p16 was overexpressed in 75% of HPV-positive HNSCC compared to negative samples and controls (p < 0.001). Classical markers correlated with improved OS (p < 0.05). Serological studies showed similar proportions of HPV16 L1 antibodies in all HNSCCs (p > 0.05). Serum E7 oncoprotein was present in 30% HPV-positive patients at diagnosis (p > 0.05) and correlated to HNSCC HPV16 E7 mRNA (p < 0.01), whereas it was associated to worse RFS and OS, especially for oropharyngeal squamous cell carcinoma (OPSCC) (p < 0.01). Detection of circulating HPV16 E7 oncoprotein at diagnosis may be useful for stratifying and monitoring HPV-positive HNSCC patients for worse prognosis, providing clinicians a tool for selecting patients for treatment de-escalation. [ABSTRACT FROM AUTHOR]

Pathogenesis-related (PR) proteins are known to play relevant roles in plant defense against biotic and abiotic stresses. In the present study, we characterize the response of transgenic faba bean (Vicia faba L.) plants encoding a PR10a gene from potato (Solanum tuberosum L.) to salinity and drought. The transgene was under the mannopine synthetase (pMAS) promoter. PR10a-overexpressing faba bean plants showed better growth than the wild-type plants after 14 days of drought stress and 30 days of salt stress under hydroponic growth conditions. After removing the stress, the PR10a-plants returned to a normal state, while the wild-type plants could not be restored. Most importantly, there was no phenotypic difference between transgenic and non-transgenic faba bean plants under well-watered conditions. Evaluation of physiological parameters during salt stress showed lower Na+-content in the leaves of the transgenic plants, which would reduce the toxic effect. In addition, PR10a-plants were able to maintain vegetative growth and experienced fewer photosystem changes under both stresses and a lower level of osmotic stress injury under salt stress compared to wild-type plants. Taken together, our findings suggest that the PR10a gene from potato plays an important role in abiotic stress tolerance, probably by activation of stress-related physiological processes. [ABSTRACT FROM AUTHOR]

The establishment of a proangiogenic phenotype and epithelial-to-mesenchymal transition (EMT) are considered as critical events that promote the induction of invasive growth in epithelial tumors, and stimulation of lymphangiogenesis is believed to confer the capacity for early dissemination to cancer cells. Recent research has revealed substantial interdependence between these processes at the molecular level as they rely on common signaling networks. Of great interest are the molecular mechanisms of (lymph-)angiogenesis and EMT associated with the earliest stages of transition from intraepithelial development to invasive growth, as they could provide the source of potentially valuable tools for targeting tumor metastasis. However, in the case of early-stage cervical cancer, the players of (lymph-)angiogenesis and EMT processes still remain substantially uncharacterized. In this study, we used RNA sequencing to compare transcriptomes of HPV(+) preinvasive neoplastic lesions and early-stage invasive carcinoma of the cervix and to identify (lymph-)angiogenesis- and EMT-related genes and pathways that may underlie early acquisition of invasive phenotype and metastatic properties by cervical cancer cells. Second, we applied flow cytometric analysis to evaluate the expression of three key lymphangiogenesis/EMT markers (VEGFR3, MET, and SLUG) in epithelial cells derived from enzymatically treated tissue specimens. Overall, among 201 differentially expressed genes, a considerable number of (lymph-)angiogenesis and EMT regulatory factors were identified, including genes encoding cytokines, growth factor receptors, transcription factors, and adhesion molecules. Pathway analysis confirmed enrichment for angiogenesis, epithelial differentiation, and cell guidance pathways at transition from intraepithelial neoplasia to invasive carcinoma and suggested immune-regulatory/inflammatory pathways to be implicated in initiation of invasive growth of cervical cancer. Flow cytometry showed cell phenotype-specific expression pattern for VEGFR3, MET, and SLUG and revealed correlation with the amount of tumor-infiltrating lymphocytes at the early stages of cervical cancer progression. Taken together, these results extend our understanding of driving forces of angiogenesis and metastasis in HPV-associated cervical cancer and may be useful for developing new treatments. [ABSTRACT FROM AUTHOR]

The growth of human papillomavirus (HPV)-transformed cells depends on the ability of the viral oncoproteins E6 and E7, especially those from high-risk HPV16/18, to manipulate the signaling pathways involved in cell proliferation, cell death, and innate immunity. Emerging evidence indicates that E6/E7 inhibition reactivates the host innate immune response, reversing what until then was an unresponsive cellular state suitable for viral persistence and tumorigenesis. Given that the disruption of distinct mechanisms of immune evasion is an attractive strategy for cancer therapy, the race is on to gain a better understanding of E6/E7-induced immune escape and cancer progression. Here, we review recent literature on the interplay between E6/E7 and the innate immune signaling pathways cGAS/STING/TBK1, RIG-I/MAVS/TBK1, and Toll-like receptors (TLRs). The overall emerging picture is that E6 and E7 have evolved broad-spectrum mechanisms allowing for the simultaneous depletion of multiple rather than single innate immunity effectors. The cGAS/STING/TBK1 pathway appears to be the most heavily impacted, whereas the RIG-I/MAVS/TBK1, still partially functional in HPV-transformed cells, can be activated by the powerful RIG-I agonist M8, triggering the massive production of type I and III interferons (IFNs), which potentiates chemotherapy-mediated cell killing. Overall, the identification of novel therapeutic targets to restore the innate immune response in HPV-transformed cells could transform the way HPV-associated cancers are treated. [ABSTRACT FROM AUTHOR]