Advancements in clinical approaches, analytical methods, and smart sampling for LC-MS-based protein determination from dried matrix spots.

Determination of proteins from dried matrix spots using MS is an expanding research area. Mainly, the collected dried matrix sample is whole blood from a finger or heal prick, resulting in dried blood spots. However as other matrices such as plasma, serum, urine, and tear fluid also can be collected in this way, the term dried matrix spot is used as an overarching term. In this review, the focus is on advancements in the field made from 2017 up to 2023. In the first part reviews concerning the subject are discussed. After this, advancements made for clinical purposes are highlighted. Both targeted protein analyses, with and without the use of affinity extractions, as well as untargeted, global proteomic approaches are discussed. In the last part, both methodological advancements are being reviewed as well as the possibility to integrate sample preparation steps during the sample handling. The focus, of this so-called smart sampling, is on the incorporation of cell separation, proteolysis, and antibody-based affinity capture.

Exploring the potential role of four Rhizophagus irregularis nuclear effectors: opportunities and technical limitations.

Arbuscular mycorrhizal fungi (AMF) are obligate symbionts that interact with the roots of most land plants. The genome of the AMF model species Rhizophagus irregularis contains hundreds of predicted small effector proteins that are secreted extracellularly but also into the plant cells to suppress plant immunity and modify plant physiology to establish a niche for growth. Here, we investigated the role of four nuclear-localized putative effectors, i.e., GLOIN707, GLOIN781, GLOIN261, and RiSP749, in mycorrhization and plant growth. We initially intended to execute the functional studies in Solanum lycopersicum, a host plant of economic interest not previously used for AMF effector biology, but extended our studies to the model host Medicago truncatula as well as the non-host Arabidopsis thaliana because of the technical advantages of working with these models. Furthermore, for three effectors, the implementation of reverse genetic tools, yeast two-hybrid screening and whole-genome transcriptome analysis revealed potential host plant nuclear targets and the downstream triggered transcriptional responses. We identified and validated a host protein interactors participating in mycorrhization in the host.S. lycopersicum and demonstrated by transcriptomics the effectors possible involvement in different molecular processes, i.e., the regulation of DNA replication, methylglyoxal detoxification, and RNA splicing. We conclude that R. irregularis nuclear-localized effector proteins may act on different pathways to modulate symbiosis and plant physiology and discuss the pros and cons of the tools used.

Development of a Gaussia luciferase immunoprecipitation assay for detecting Schistosoma japonicum infection.

Timely and accurate diagnosis of Schistosoma infection is important to adopt effective strategies for schistosomiasis control. Previously, we demonstrated that Schistosoma japonicum can secret extracellular vesicles and their cargos may serve as a novel type of biomarkers for diagnosing schistosomiasis. Here, we developed a Gaussia luciferase immunoprecipitation assay combined with S. japonicum extracellular vesicle (SjEV) protein to evaluate its potential for diagnosing schistosomiasis. A saposin-like protein (SjSLP) identified from SjEVs was fused to the Gaussia luciferase as the diagnostic antigen. The developed method showed good capability for detecting S. japonicum infection in mice and human patients. We also observed that the method could detect Schistosoma infection in mice as early as 7 days of post-infection, which showed better sensitivity than that of indirect ELISA method. Overall, the developed method showed a good potential for detecting Schistosoma infection particularly for early stage, which may provide an alternative strategy for identify Schistosoma infection for disease control.

Sodium Houttuyfonate Alleviates MCT-induced Pulmonary Hypertension by Regulating Orai1 and Orai2.

Elevated intracellular Ca2+ concentration ([Ca2+]i) is a key trigger for pulmonary arterial smooth muscle cell (PASMC) proliferation and contributes greatly to pulmonary hypertension (PH). Extracellular Ca2+ influx via a store-operated Ca2+ channel (SOCC), termed store-operated Ca2+ entry (SOCE), is a crucial mechanism for [Ca2+]i elevation in PASMCs. Calcium release-activated calcium modulator (Orai) proteins, consisting of three members (Orai1-3), are the main components of SOCC. Sodium houttuyfonate (SH) is a product of the addition reaction of sodium bisulfite and houttuynin and has antibacterial, anti-inflammatory, and other properties. In this study, we assessed the contributions of Orai proteins to MCT-enhanced SOCE, [Ca2+]i, and cell proliferation in PASMCs and determined the effect of SH on MCT-PH and the underlying mechanism, focusing on Orai proteins, SOCE, and [Ca2+]i in PASMCs. Our results showed that 1) Orai1 and Orai2 were selectively upregulated in the distal pulmonary arteries (PAs) and the PASMCs of MCT-PH rats. 2) Knockdown of Orai1 or Orai2 reduced SOCE, [Ca2+]i, and cell proliferation without affecting their expression in PASMCs in MCT-PH rats. 3) SH significantly normalized the characteristic parameters in a dose-dependent manner in the MCT-PH rat model. 4) SH decreased MCT-enhanced SOCE, [Ca2+]i and PASMC proliferation via Orai1 or Orai2. These results indicate that SH likely exerts its protective role in MCT-PH by inhibiting the Orai1,2-SOCE-[Ca2+]i signaling pathway.

Using Dimensionality Reduction to Visualize Phenotypic Changes in High-Throughput Microscopy.

High-throughput microscopy has enabled screening of cell phenotypes at unprecedented scale. Systematic identification of cell phenotype changes (such as cell morphology and protein localization changes) is a major analysis goal. Because cell phenotypes are high-dimensional, unbiased approaches to detect and visualize the changes in phenotypes are still needed. Here, we suggest that changes in cellular phenotype can be visualized in reduced dimensionality representations of the image feature space. We describe a freely available analysis pipeline to visualize changes in protein localization in feature spaces obtained from deep learning. As an example, we use the pipeline to identify changes in subcellular localization after the yeast GFP collection was treated with hydroxyurea.

The translational inhibitor and amnestic agent emetine also suppresses ongoing hippocampal neural activity similarly to other blockers of protein synthesis.

The consolidation of memory is thought to ultimately depend on the synthesis of new proteins, since translational inhibitors such as anisomycin and cycloheximide adversely affect the permanence of long-term memory. However, when applied directly in brain, these agents also profoundly suppress neural activity to an extent that is directly correlated to the degree of protein synthesis inhibition caused. Given that neural activity itself is likely to help mediate consolidation, this finding is a serious criticism of the strict de novo protein hypothesis of memory. Here, we test the neurophysiological effects of another translational inhibitor, emetine. Unilateral intra-hippocampal infusion of emetine suppressed ongoing local field and multiunit activity at ipsilateral sites as compared to the contralateral hippocampus in a fashion that was positively correlated to the degree of protein synthesis inhibition as confirmed by autoradiography. This suppression of activity was also specific to the circumscribed brain region in which protein synthesis inhibition took place. These experiments provide further evidence that ongoing protein synthesis is necessary and fundamental for neural function and suggest that the disruption of memory observed in behavioral experiments using translational inhibitors may be due, in large part, to neural suppression.

Alteration of RNA m6A methylation mediates aberrant RNA binding protein expression and alternative splicing in condyloma acuminatum.

Background: Condyloma acuminatum (CA) is caused by low-risk human papillomavirus, and is characterized by high recurrence after treatment. The RNA modification N6-methyladenosine (m6A) plays an important role during diverse viral infections, including high-risk HPV infection in cervical cancer. However, it is unclear whether low-risk HPV infection changes the RNA m6A methylation in CA. Methods: High-throughputm6A-sequencing was performed to profile the transcriptome-wide mRNA modifications of CA tissues infected by LR-HPVs and the paired normal tissues from CA patients. We further investigated the regulation of alternative splicing by RNA binding proteins (RBPs) with altered m6A modification and constructed a regulatory network among these RBPs, regulated alternative splicing events (RASEs) and regulated alternative splicing genes (RASGs) in CA. Results: The results show that the m6A level in CA tissues differed from that in the paired controls. Furthermore, cell cycle- and cell adhesion- associated genes with m6A modification were differentially expressed in CA tissues compared to the paired controls. In particular, seven RNA binding protein genes with specific m6A methylated sites, showed a higher or lower expression at the mRNA level in CA tissues than in the paired normal tissues. In addition, these differentially expressed RNA binding protein genes would regulate the alternative splicing pattern of apoptotic process genes in CA tissue. Conclusions: Our study reveals a sophisticated m6A modification profile in CA tissue that affects the response of host cells to HPV infection, and provides cues for the further exploration of the roles of m6A and the development of a novel treatment strategy for CA.

Emerging functions of the mitochondria-ER-lipid droplet three-way junction in coordinating lipid transfer, metabolism, and storage in cells.

Over the past two decades, we have witnessed a growing appreciation for the importance of membrane contact sites (CS) in facilitating direct communication between organelles. CS are tiny regions where the membranes of two organelles meet but do not fuse and allow the transfer of metabolites between organelles, playing crucial roles in the coordination of cellular metabolic activities. The significant advancements in imaging techniques and molecular and cell biology research have revealed that CS are more complex than what originally thought, and as they are extremely dynamic, they can remodel their shape, composition, and functions in accordance with metabolic and environmental changes and can occur between more than two organelles. Here, we describe how recent studies led to the identification of a three-way mitochondria-ER-lipid droplet CS and discuss the emerging functions of these contacts in maintaining lipid storage, homeostasis, and balance. We also summarize the properties and functions of key protein components localized at the mitochondria-ER-lipid droplet interface, with a special focus on lipid transfer proteins. Understanding tripartite CS is essential for unraveling the complexities of inter-organelle communication and cooperation within cells.

On the modification of plant proteins: Traditional methods and the Hofmeister effect.

With increasing consumer health awareness and demand from some vegans, plant proteins have received a lot of attention. Plant proteins have many advantages over animal proteins. However, the application of plant proteins is limited by a number of factors and there is a need to improve their functional properties to enable a wider range of applications. This paper describes the advantages and disadvantages of traditional methods of modifying plant proteins and the appropriate timing for their use, and collates and describes a method with fewer applications in the food industry: the Hofmeister effect. It is extremely simple but efficient in some respects compared to traditional methods. The paper provides theoretical guidance for the further development of plant protein-based food products and a reference value basis for improving the functional properties of proteins to enhance their applications in the food industry, pharmaceuticals and other fields.

Trypsin Encapsulation in the Zeolitic Imidazolate Framework for Low-Molecular Weight Protein Analysis with High Selectivity and Efficiency.

Low-molecular weight proteins (LWPs) are important sources of biological information in biomarkers, signaling molecules, and pathology. However, the separation and analysis of LWPs in complex biological samples are challenging, mainly due to their low abundance and the complex sample pretreatment procedure. Herein, trypsin modified by poly(acrylic acid) (PAA) was encapsulated by a zeolitic imidazolate framework (ZIF-L). Mesopores were formed on the ZIF-L with the introduction of PAA. An alternative strategy for separation and pretreatment of LWPs was developed based on the prepared ZIF-L-encapsulated trypsin with adjustable pore size. The mesoporous structure of the prepared materials selectively excluded high-molecular weight proteins from the reaction system, allowing LWPs to enter the pores and react with the internal trypsin, resulting in an improved separation efficiency. The hydrophobicity of the ZIF-L simplified the digestion process by inducing significant structural changes in substrate proteins. In addition, the enzymatic activity was significantly enhanced by the developed encapsulation method that maintained the enzyme conformation, allowed low mass transfer resistance, and possessed a high enzyme-to-substrate ratio. As a result, the ZIF-L-encapsulated trypsin can achieve highly selective separation, valid denaturation, and efficient digestion of LWPs in a short time by simply mixing with substrate proteins, greatly simplifying the separation and pretreatment process of the traditional hydrolysis method. The prepared materials and the developed strategy demonstrated an excellent size-selective assay performance in model protein mixtures, showing great potential in the application of proteomics analysis.

OPN, BSP, and Bone Quality-Structural, Biochemical, and Biomechanical Assessment in OPN-/-, BSP-/-, and DKO Mice.

Osteopontin (OPN) and Bone Sialoprotein (BSP), abundantly expressed by osteoblasts and osteoclasts, appear to have important, partly overlapping functions in bone. In gene-knockout (KO, -/-) models of either protein and their double (D)KO in the same CD1/129sv genetic background, we analyzed the morphology, matrix characteristics, and biomechanical properties of femur bone in 2 and 4 month old, male and female mice. OPN-/- mice display inconsistent, perhaps localized hypermineralization, while the BSP-/- are hypomineralized throughout ages and sexes, and the low mineralization of young DKO mice recovers with age. The higher contribution of primary bone remnants in OPN-/- shafts suggests a slow turnover, while their lower percentage in BSP-/- indicates rapid remodeling, despite FTIR-based evidence in this genotype of a high maturity of the mineralized matrix. In 3-point bending assays, OPN-/- bones consistently display higher Maximal Load, Work to Max. Load and in young mice Ultimate Stress, an intrinsic characteristic of the matrix. Young male and old female BSP-/- also display high Work to Max. Load along with low Ultimate Stress. Principal Component Analysis confirms the major role of morphological traits in mechanical competence, and evidences a grouping of the WT phenotype with the OPN-/- and of BSP-/- with DKO, driven by both structural and matrix parameters, suggesting that the presence or absence of BSP has the most profound effects on skeletal properties. Single or double gene KO of OPN and BSP thus have multiple distinct effects on skeletal phenotypes, confirming their importance in bone biology and their interplay in its regulation.

Orai1/STIMs modulators in pulmonary vascular diseases.

Calcium (Ca2+) is a secondary messenger that regulates various cellular processes. However, Ca2+ mishandling could lead to pathological conditions. Orai1 is a Ca2+channel contributing to the store-operated calcium entry (SOCE) and plays a critical role in Ca2+ homeostasis in several cell types. Dysregulation of Orai1 contributed to severe combined immune deficiency syndrome, some cancers, pulmonary arterial hypertension (PAH), and other cardiorespiratory diseases. During its activation process, Orai1 is mainly regulated by stromal interacting molecule (STIM) proteins, especially STIM1; however, many other regulatory partners have also been recently described. Increasing knowledge about these regulatory partners provides a better view of the downstream signalling pathways of SOCE and offers an excellent opportunity to decipher Orai1 dysregulation in these diseases. These proteins participate in other cellular functions, making them attractive therapeutic targets. This review mainly focuses on Orai1 regulatory partners in the physiological and pathological conditions of the pulmonary circulation and inflammation.

Age, sex and breed effect on laboratory parameters in natural Babesia canis infection.

We tested the hypothesis that age, breed, and sex are related to hematology, biochemistry, acute phase proteins (APPs), seroreactivity and level of parasitemia in dogs with an acute phase response (APR) due to Babesia canis infection. The study enrolled 61 privately owned dogs that naturally acquired B. canis infection. Groups were formed according to the age: young dogs less than one year, and adult dogs more than one year old. Moreover, the group of males was compared to females and purebred to mixed breed dogs. Seroreactivity was tested with immunofluorescence antibody test, level of parasitemia with real-time polymerase chain reaction (real-time PCR), hematology, and biochemistry with automatic analyzers, serum amyloid A with enzyme-linked immunosorbent assay, fibrinogen with heat precipitation and ceruloplasmin and paraoxonase-1 with manual spectrophotometric methods. For protein separation agarose gel electrophoresis was used. The main changes in the whole population of B. canis-infected dogs were fever, pancytopenia, and change in APPs level. One-third of young, and 96% of adult dogs were seropositive (P < 0.001). The level of parasitemia was higher in the young dogs (P < 0.001). Erythroid lineage parameters (P < 0.01), and leukocytes (P < 0.05) were lower in the young, when compared to the adult dogs. Young dogs had lower total globulins (P < 0.001), ß- and γ-globulins (P < 0.001), and higher α-globulins (P = 0.022) than adult dogs. Young dogs had higher concentrations of phosphate (P = 0.003) and cholesterol (P < 0.001) and lower amylase (P = 0.014) and lipase activity (P = 0.020) than adult ones. Male dogs had lower neutrophil count than females (P = 0.035), and purebred dogs had more band neutrophils than mixed breed dogs (P = 0.004). In conclusion, dogs with natural Babesia canis infection at a young age have more severe anemia and APR including leukopenia than adults. Male and purebred dogs might also have more severe APR than females and mix-breeds, as they have more pronounced changes related to the myeloid lineage.

Non-specific N-terminal tetrapeptide insertions disrupt the translation arrest induced by ribosome arresting peptide sequences.

The nascent polypeptide chains passing through the ribosome tunnel not only serve as an intermediate of protein synthesis but also, in some cases, act as dynamic genetic information, controlling translation through interaction with the ribosome. One notable example is Escherichia coli SecM, in which translation of the ribosome arresting peptide (RAP) sequence in SecM leads to robust elongation arrest. Translation regulations, including the SecM-induced translation arrest, play regulatory roles such as gene expression control. Recent investigations have indicated that the insertion of a peptide sequence, SKIK (or MSKIK), into the adjacent N-terminus of the RAP sequence of SecM behaves as an "arrest canceler". As the study did not provide a direct assessment of the strength of translation arrest, we conducted detailed biochemical analyses. The results revealed that the effect of SKIK insertion on weakening SecM-induced translation arrest was not specific to the SKIK sequence, that is, other tetrapeptide sequences inserted just before the RAP sequence also attenuated the arrest. Our data suggest that SKIK or other tetrapeptide insertions disrupt the context of the RAP sequence rather than canceling or preventing the translation arrest.

Dynamics of membrane tubulation coupled with fission by a two-component module.

Membrane tubulation coupled with fission (MTCF) is a widespread phenomenon but mechanisms for their coordination remain unclear, partly because of the lack of assays to monitor dynamics of membrane tubulation and subsequent fission. Using polymer cushioned bilayer islands, we analyze the membrane tubulator Bridging Integrator 1 (BIN1) mixed with the fission catalyst dynamin2 (Dyn2). Our results reveal this mixture to constitute a minimal two-component module that demonstrates MTCF. MTCF is an emergent property and arises because BIN1 facilitates recruitment but inhibits membrane binding of Dyn2 in a dose-dependent manner. MTCF is therefore apparent only at high Dyn2 to BIN1 ratios. Because of their mutual involvement in T-tubules biogenesis, mutations in BIN1 and Dyn2 are associated with centronuclear myopathies and our analysis links the pathology with aberrant MTCF. Together, our results establish cushioned bilayer islands as a facile template for the analysis of membrane tubulation and inform of mechanisms that coordinate MTCF.

Phylogenomic analysis expands the known repertoire of single-stranded DNA viruses in benthic zones of the South Indian Ocean.

Single-stranded (ss) DNA viruses are ubiquitous and constitute some of the most diverse entities on Earth. Most studies have focused on ssDNA viruses from terrestrial environments resulting in a significant deficit in benthic ecosystems including aphotic zones of the South Indian Ocean (SIO). Here, we assess the diversity and phylogeny of ssDNA in deep waters of the SIO using a combination of established viral taxonomy tools and a Hidden Markov Model based approach. Replication initiator protein-associated (Rep) phylogenetic reconstruction and sequence similarity networks were used to show that the SIO hosts divergent and as yet unknown circular Rep-encoding ssDNA viruses. Several sequences appear to represent entirely novel families, expanding the repertoire of known ssDNA viruses. Results suggest that a small proportion of these viruses may be circular genetic elements, which may strongly influence the diversity of both eukaryotes and prokaryotes in the SIO. Taken together, our data show that the SIO harbours a diverse assortment of previously unknown ssDNA viruses. Due to their potential to infect a variety of hosts, these viruses may be crucial for marine nutrient recycling through their influence of the biological carbon pump.

Rheological, thermal, and structural properties of heat-induced gluten gel: Effects of starch with varying degrees of debranching.

This study evaluated the effects of starch with varying degree of debranching on the rheological, thermal, and structural properties of heat-induced gluten gel. As the duration of starch debranching treatment increased from 0 to 8 h, the viscoelasticity of the gel containing debranched starch (DBS) improved. Compared with the gluten gel (G), the gel strength of the G + DBS (8 h) sample increased by 65.2 %. The degradation temperature of gluten was minimally affected by DBS, while the weight loss rate increased by 4.4 %. Furthermore, the α-helical structure of gluten decreased, concomitant with an increase in ß-sheet content. Notably, DBS treated for 8 h exhibited more hydrogen bonds with the tyrosine of gluten and triggered disulfide bridge conformation to transition from g-g-g to t-g-g, thereby reducing the stability of the molecular conformation of gluten proteins, as evidenced by the decreased height and width of the molecular chains observed in atomic force microscopy images. Overall, the composite gel structure induced by DBS exhibited a more continuous and homogeneous owing to the improved compatibility between DBS and gluten proteins, favoring the formation of a robust gel. These findings provide valuable insights for utilizing DBS to enhance gluten gel properties.

Screening Carbon Nano Materials for Preventing Amyloid Protein Aggregation by Adopting a Facile Method.

The soluble-to-toxic transformation of intrinsically disordered amyloidogenic proteins such as amyloid beta (Aß), α-synuclein, mutant Huntingtin Protein (mHTT) and islet amyloid polypeptide (IAPP) among others are associated with disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD) and Type 2 Diabetes (T2D), respectively. The dissolution of mature fibrils and toxic amyloidogenic intermediates, including oligomers, continues to be the pinnacle in the treatment of neurodegenerative disorders. Yet, methods to effectively and quantitatively report on the interconversion between amyloid monomers, oligomers and mature fibrils fall short. Here we describe a simplified method that implements the use of gel electrophoresis to address the transformation between soluble monomeric amyloid proteins and mature amyloid fibrils. The technique implements an optimized but well-known, simple, inexpensive, and quantitative assessment previously used to assess the oligomerization of amyloid monomers and subsequent amyloid fibrils. This method facilitates the screening of small molecules that disintegrate oligomers and fibrils into monomers, dimers, and trimers and/or retain amyloid proteins in their monomeric forms. Most importantly, our optimized method diminishes existing barriers associated with existing (alternative) techniques to evaluate fibril formation and intervention.

Exploring Edible Insects: A Review on Protein Diversity, Extraction Techniques, and Health Benefits.

This study investigates whether edible insects may be used as a sustainable protein source. This study covers insect proteins, their defatting process and extraction, health advantages, economic and environmental impacts, safety considerations, and regulatory aspects. This review also investigates the ecological benefits of insect farming and consumer acceptance of this dietary trend. A systematic search of the pertinent literature was conductedto collect data for this review. Current research examines the effects of using edible insects as functional, sustainable reservoirs of proteins, including protein screening, extraction methods, health benefits, and social recognition. The varied isolation techniques considerably affect protein amount and quality. . This review clarifies the broad spectrum of proteins in edible insects, particularly their nutritional importance and effective extraction methods. The viability of insects as a sustainable source of protein is highlighted by the safety considerations and the interaction of economic and ecological considerations with complex consumer characteristics.