A Preparative Method for the Isolation of Calponin from Molluscan Catch Muscle

We describe the development of a preparative method to isolate molluscan catch muscle, calponin. This method is based on the ability of calponin to interact with actin in a temperature-dependent manner. After extracting thin filaments, as previously described, the extract was ultracentrifuged at 2 °C. While other surface proteins of thin filaments co-precipitated with actin, calponin, along with some minor contaminants, remained in the supernatant. Calponin was purified through cation-exchange chromatography. The yield of pure protein was four-fold higher than that achieved through high-temperature extraction. To evaluate functionally isolated proteins, we determined the effect of calponin on Mg²⁺-ATPase activity of hybrid and non-hybrid actomyosin. The degree of ATPase inhibition was consistent with previously published data but strongly dependent on the environmental conditions and source of actin and myosin used. Furthermore, at low concentrations, calponin could induce the ATPase activity of hybrid actomyosin. This result was consistent with data indicating that calponin can modulate actin conformation to increase the relative content of “switched on” actin monomers in thin filaments. We assume that calponin obtained by the isolation method proposed herein is a fully functional protein that can both inhibit and induce the ATPase activity.     

Regulation of ADAM10 by the TspanC8 Family of Tetraspanins and Their Therapeutic Potential

The ubiquitously expressed transmembrane protein a disintegrin and metalloproteinase 10 (ADAM10) functions as a “molecular scissor”, by cleaving the extracellular regions from its membrane protein substrates in a process termed ectodomain shedding. ADAM10 is known to have over 100 substrates including Notch, amyloid precursor protein, cadherins, and growth factors, and is important in health and implicated in diseases such as cancer and Alzheimer’s. The tetraspanins are a superfamily of membrane proteins that interact with specific partner proteins to regulate their intracellular trafficking, lateral mobility, and clustering at the cell surface. We and others have shown that ADAM10 interacts with a subgroup of six tetraspanins, termed the TspanC8 subgroup, which are closely related by protein sequence and comprise Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33. Recent evidence suggests that different TspanC8/ADAM10 complexes have distinct substrates and that ADAM10 should not be regarded as a single scissor, but as six different TspanC8/ADAM10 scissor complexes. This review discusses the published evidence for this “six scissor” hypothesis and the therapeutic potential this offers.     

Development of New Antiproliferative Compound against Human Tumor Cells from the Marine Microalgae Nannochloropsis gaditana by Applied Proteomics

Proteomics is a crucial tool for unravelling the molecular dynamics of essential biological processes, becoming a pivotal technique for basic and applied research. Diverse bioinformatic tools are required to manage and explore the huge amount of information obtained from a single proteomics experiment. Thus, functional annotation and protein–protein interactions are evaluated in depth leading to the biological conclusions that best fit the proteomic response in the system under study. To gain insight into potential applications of the identified proteins, a novel approach named “Applied Proteomics” has been developed by comparing the obtained protein information with the existing patents database. The development of massive sequencing technology and mass spectrometry (MS/MS) improvements has allowed the application of proteomics nonmodel microorganisms, which have been deeply described as a novel source of metabolites. Between them, Nannochloropsis gaditana has been pointed out as an alternative source of biomolecules. Recently, our research group has reported the first complete proteome analysis of this microalga, which was analysed using the applied proteomics concept with the identification of 488 proteins with potential industrial applications. To validate our approach, we selected the UCA01 protein from the prohibitin family. The recombinant version of this protein showed antiproliferative activity against two tumor cell lines, Caco2 (colon adenocarcinoma) and HepG-2 (hepatocellular carcinoma), proving that proteome data have been transformed into relevant biotechnological information. From Nannochloropsis gaditana has been developed a new tool against cancer—the protein named UCA01. This protein has selective effects inhibiting the growth of tumor cells, but does not show any effect on control cells. This approach describes the first practical approach to transform proteome information in a potential industrial application, named “applied proteomics”. It is based on a novel bioalgorithm, which is able to identify proteins with potential industrial applications. From hundreds of proteins described in the proteome of N. gaditana, the bioalgorithm identified over 400 proteins with potential uses; one of them was selected as UCA01, “in vitro” and its potential was demonstrated against cancer. This approach has great potential, but the applications are potentially numerous and undefined.     

Making the Bend: DNA Tertiary Structure and Protein-DNA Interactions

DNA structure functions as an overlapping code to the DNA sequence. Rapid progress in understanding the role of DNA structure in gene regulation, DNA damage recognition and genome stability has been made. The three dimensional structure of both proteins and DNA plays a crucial role for their specific interaction, and proteins can recognise the chemical signature of DNA sequence (“base readout”) as well as the intrinsic DNA structure (“shape recognition”). These recognition mechanisms do not exist in isolation but, depending on the individual interaction partners, are combined to various extents. Driving force for the interaction between protein and DNA remain the unique thermodynamics of each individual DNA-protein pair. In this review we focus on the structures and conformations adopted by DNA, both influenced by and influencing the specific interaction with the corresponding protein binding partner, as well as their underlying thermodynamics.     

气相抽提法去除土壤中的苯和乙苯

土壤气相抽提技术（SVE）是一种安全、经济、高效的土壤治理技术,广泛应用于不饱和土壤中挥发性有机污染物的去除。以红壤为实验土样,北京潮土和吉林黑土为对照土样,选用最常见的挥发性有机污染物苯作为单一污染物,采用一维土柱通风模拟SVE过程,研究了不同土质对苯污染土壤去污过程的影响。同时选用苯与乙苯作为二元混合污染物,研究了土壤含水率对苯与乙苯污染红壤去污过程的影响。结果表明,对于二元混合物实验,在通风流量600 ml.min^-1、含水率16.8%时,苯与乙苯的净化时间分别降至最低36 h和84 h。不同类型土壤的对照实验表明,土壤质地与有机质含量也是SVE去污过程的重要影响因素。     

Folding and Biogenesis of Mitochondrial Small Tim Proteins

Correct and timely folding is critical to the function of all proteins. The importance of this is illustrated in the biogenesis of the mitochondrial intermembrane space (IMS) “small Tim” proteins. Biogenesis of the small Tim proteins is regulated by dedicated systems or pathways, beginning with synthesis in the cytosol and ending with assembly of individually folded proteins into functional complexes in the mitochondrial IMS. The process is mostly centered on regulating the redox states of the conserved cysteine residues: oxidative folding is crucial for protein function in the IMS, but oxidized (disulfide bonded) proteins cannot be imported into mitochondria. How the redox-sensitive small Tim precursor proteins are maintained in a reduced, import-competent form in the cytosol is not well understood. Recent studies suggest that zinc and the cytosolic thioredoxin system play a role in the biogenesis of these proteins. In the IMS, the mitochondrial import and assembly (MIA) pathway catalyzes both import into the IMS and oxidative folding of the small Tim proteins. Finally, assembly of the small Tim complexes is a multistep process driven by electrostatic and hydrophobic interactions; however, the chaperone function of the complex might require destabilization of these interactions to accommodate the substrate. Here, we review how folding of the small Tim proteins is regulated during their biogenesis, from maintenance of the unfolded precursors in the cytosol, to their import, oxidative folding, complex assembly and function in the IMS.     

Swift and Reliable “Easy Lab” Methods for the Sensitive Molecular Detection of African Swine Fever Virus

African swine fever (ASF) is a contagious viral hemorrhagic disease of domestic pigs and wild boars. The disease is notifiable to the World Organisation for Animal Health (OIE) and is responsible for high mortality and serious economic losses. PCR and real-time PCR (qPCR) are the OIE-recommended standard methods for the direct detection of African swine fever virus (ASFV) DNA. The aim of our work was the simplification and standardization of the molecular diagnostic workflow in the lab. For validation of this “easy lab” workflow, different sample materials from animal trials were collected and analyzed (EDTA blood, serum, oral swabs, chewing ropes, and tissue samples) to identify the optimal sample material for diagnostics in live animals. Based on our data, the EDTA blood samples or bloody tissue samples represent the best specimens for ASFV detection in the early and late phases of infection. The application of prefilled ready-to-use reagents for nucleic acid extraction or the use of a Tissue Lysis Reagent (TLR) delivers simple and reliable alternatives for the release of the ASFV nucleic acids. For the qPCR detection of ASFV, different published and commercial kits were compared. Here, a lyophilized commercial kit shows the best results mainly based on the increased template input. The good results of the “easy lab” strategy could be confirmed by the ASFV detection in field samples from wild boars collected from the 2020 ASFV outbreak in Germany. Appropriate internal control systems for extraction and PCR are key features of the “easy lab” concept and reduce the risk of false-negative and false-positive results. In addition, the use of easy-to-handle machines and software reduces training efforts and the misinterpretation of results. The PCR diagnostics based on the “easy lab” strategy can realize a high sensitivity and specificity comparable to the standard PCR methods and should be especially usable for labs with limited experiences and resources.     

In Silico Modeling of the Influence of Environment on Amyloid Folding Using FOD-M Model

The role of the environment in amyloid formation based on the fuzzy oil drop model (FOD) is discussed here. This model assumes that the hydrophobicity distribution within a globular protein is consistent with a 3D Gaussian (3DG) distribution. Such a distribution is interpreted as the idealized effect of the presence of a polar solvent—water. A chain with a sequence of amino acids (which are bipolar molecules) determined by evolution recreates a micelle-like structure with varying accuracy. The membrane, which is a specific environment with opposite characteristics to the polar aquatic environment, directs the hydrophobic residues towards the surface. The modification of the FOD model to the FOD-M form takes into account the specificity of the cell membrane. It consists in “inverting” the 3DG distribution (complementing the Gaussian distribution), which expresses the exposure of hydrophobic residues on the surface. It turns out that the influence of the environment for any protein (soluble or membrane-anchored) is the result of a consensus factor expressing the participation of the polar environment and the “inverted” environment. The ratio between the proportion of the aqueous and the “reversed” environment turns out to be a characteristic property of a given protein, including amyloid protein in particular. The structure of amyloid proteins has been characterized in the context of prion, intrinsically disordered, and other non-complexing proteins to cover a wider spectrum of molecules with the given characteristics based on the FOD-M model.     

Soybean,corn, and wheat yields with variable soil pH on Plinthic Acrisols

Liming costs have escalated since the mid 1970's in the United States. Studies of crop response to lime with irrigation are limited as well as those of crop response to soil acidifying agents. This study was conducted to determine yield response of irrigated soybean [Glycine max (L.) Merrill], corn (Zea mays L.), and wheat (Triticum aestivum L.) to lime and S and the change in soil pH, in response to lime and S. Irrigated soybean, corn, and wheat were grown on Dothan and Tifton loamy fine sand (Plinthic Acrisols) with different levels of calcite, dolomite, and S. Soil samples were collected before applying treatments and during each growing season. Soil pH and Mehlich I extractable P, K, Ca, and Mg in addition to grain yield of each crop were determined. Highest soybean yield (4.2 t ha^–1) occurred in 1984 at a soil pH of 4.9 (1:1 v/v soil—water suspension) while the yield was zero at a pH of 3.7 on S treated plots. A soil pH of 4.8 in 1985 reduced soybean yield from 3.4 to 2.7 t ha^–1 in comparison to untreated plots (pH = 5.6). In 1986, soybean yield was 0.8 t ha^–1 at pH 4.0 in comparison to 2.3 t ha^–1 at pH 5.1 and 5.9. Corn did not respond to lime with control pH of 5.3 in 1985 or 5.1 in 1986 but S at pH 4.6 reduced yield from 12.3 to 8.7 t ha^–1 in 1985 and S reduced yield from 11.0 to 0.9 t ha^–1 at pH 4.0 in 1986. Sulfur reduced wheat yield from 4.3 to 1.7 t ha^–1 in 1985 and from 2.2 to 0.9 t ha^–1 in 1987. Soil pH after cropping with S addition was 4.4 each year. Wheat did not respond to lime when unlimed soil had a pH of 5.2 or above.     

Effects of lanthanum on nitrification and ammonification in three Chinese soils

The effects of lanthanum on nitrification and ammonification in three Chinese soils were evaluated through an incubation experiment. Soils were collected from experimental plots under rice/rape rotation in Yingtan, Jiangxi province (red soil), under rice/wheat rotation in Wuxi, Jiangsu province (paddy soil), and under corn/wheat rotation in Fengqiu, Henan province (Fluvo-aquic soil). Soil nitrification was stimulated slightly by La at lower concentrations, and the stimulation rate reached about 20% in red soil at 150 mg La kg^–1 dry soil, and 14% in fluvo-aquic soil at 300 mg La kg^–1 dry soil. When more La was added in soils, nitrification was inhibited, with a maximum inhibition rate of 42, 44 and 66% in red soil, fluvo-aquic soil, and paddy soil, respectively. Soil ammonification was not significantly different between control and up to 600 mg La kg^–1 dry soil in red soil, but it was significantly reduced in doses of 900 and 1200 mg La kg^–1 dry soil. Significant reduction in soil ammonification was also found in doses from 60 to 1200 mg La kg^–1 dry soil except for 600 mg La kg^–1 dry soil in fluvo-aquic soil. In contrast the ammonification in paddy soil was strongly stimulated by La, reaching about 25 times that of control at 900 mg La kg^–1 dry soil. We assumed that application of La accelerates the transformation of nitrogen in soils at low dosage, and the currently applied dosage in agriculture in China cannot inhibit soil nitrification and ammonification even after long term successive application.     

Decreases in Colwell bicarbonate soil test P in the years after addition of superphosphate, and the residual value of superphosphate measured using plant yield and soil test P

Decreases in Colwell bicarbonate soil test P in the years after applying single (ordinary) superphosphate, and the residual value of superphosphate, was measured in a long-term field experiment on a duplex (texture contrast) soil (sand over lateritic ironstone gravel clay sand at 10–15 cm), at Wongan Hills, Western Australia, typical of many soils used to grow crops in Western Australia. Ten levels of P (0–91 kg P ha-1) were applied once only in late May to different plots in different years from 1988 to 1993. Wheat (Triticum aestivum), or lupin ( Lupinus angustifolius)) were sown in late May of each year, when the P treatments applied that year were banded (drilled) with the seed. Soil samples were collected each June to measure soil test P. Seed (grain) yields of the crops were measured each December. The residual value (RV) of P applied in previous years was calculated relative to P applied in the current year, using grain yields (RV_yield) and soil test P (RV_soil). Soil test P measured on soil samples collected in June was related to yields measured in December that year to provide soil P test calibrations. Relative to P applied in the current year, soil test P decreased by between 15 to 30% for P applied one year previously, by 25 to 30% for P applied three years previously, and by 60 to 70% for P applied six years previously. Soil test P was affected by spatial variation, and it also varied in the different years, for P applied in the current year, one year previously, two years previously, etc. Compared with P applied in the current year, mean RV_yield determined in the different years decreased by about 40% one year after P application, followed by a further 20% decrease for P applied two years previously, followed by a further 20% decrease for P applied three to five years previously. Relative to current P, RV_soil decreased by about 25% one year after P application, followed by a further 20% for P applied two years previously, followed by a further 10% for P applied three years ago, and followed by a further 6% for P applied four and five years ago. As measured in the different years, the soil P test calibration varied between years for P applied one, two etc. years previously. This was so even when the same cultivar of wheat was grown at the same site in different years.     

Amyloidogenic Peptides: New Class of Antimicrobial Peptides with the Novel Mechanism of Activity

Antibiotic-resistant bacteria are recognized as one of the leading causes of death in the world. We proposed and successfully tested peptides with a new mechanism of antimicrobial action “protein silencing” based on directed co-aggregation. The amyloidogenic antimicrobial peptide (AAMP) interacts with the target protein of model or pathogenic bacteria and forms aggregates, thereby knocking out the protein from its working condition. In this review, we consider antimicrobial effects of the designed peptides on two model organisms, E. coli and T. thermophilus, and two pathogenic organisms, P. aeruginosa and S. aureus. We compare the amino acid composition of proteomes and especially S1 ribosomal proteins. Since this protein is inherent only in bacterial cells, it is a good target for studying the process of co-aggregation. This review presents a bioinformatics analysis of these proteins. We sum up all the peptides predicted as amyloidogenic by several programs and synthesized by us. For the four organisms we studied, we show how amyloidogenicity correlates with antibacterial properties. Let us especially dwell on peptides that have demonstrated themselves as AMPs for two pathogenic organisms that cause dangerous hospital infections, and in which the minimal inhibitory concentration (MIC) turned out to be comparable to the MIC of gentamicin sulfate. All this makes our study encouraging for the further development of AAMP. The hybrid peptides may thus provide a starting point for the antibacterial application of amyloidogenic peptides.     

Structural Plasticity Is a Feature of Rheostat Positions in the Human Na+/Taurocholate Cotransporting Polypeptide (NTCP)

In the Na⁺/taurocholate cotransporting polypeptide (NTCP), the clinically relevant S267F polymorphism occurs at a “rheostat position”. That is, amino acid substitutions at this position (“S267X”) lead to a wide range of functional outcomes. This result was particularly striking because molecular models predicted the S267X side chains are buried, and thus, usually expected to be less tolerant of substitutions. To assess whether structural tolerance to buried substitutions is widespread in NTCP, here we used Rosetta to model all 19 potential substitutions at another 13 buried positions. Again, only subtle changes in the calculated stabilities and structures were predicted. Calculations were experimentally validated for 19 variants at codon 271 (“N271X”). Results showed near wildtype expression and rheostatic modulation of substrate transport, implicating N271 as a rheostat position. Notably, each N271X substitution showed a similar effect on the transport of three different substrates and thus did not alter substrate specificity. This differs from S267X, which altered both transport kinetics and specificity. As both transport and specificity may change during protein evolution, the recognition of such rheostat positions may be important for evolutionary studies. We further propose that the presence of rheostat positions is facilitated by local plasticity within the protein structure. Finally, we note that identifying rheostat positions may advance efforts to predict new biomedically relevant missense variants in NTCP and other membrane transport proteins.     

Comparative Proteomic Analysis of Mature Pollen in Triploid and Diploid Populus deltoides

Ploidy affects plant growth vigor and cell size, but the relative effects of pollen fertility and allergenicity between triploid and diploid have not been systematically examined. Here we performed comparative analyses of fertility, proteome, and abundances of putative allergenic proteins of pollen in triploid poplar ‘ZhongHuai1’ (‘ZH1’, triploid) and ‘ZhongHuai2’ (‘ZH2’, diploid) generated from the same parents. The mature pollen was sterile in triploid poplar ‘ZH1’. By applying two-dimensional gel electrophoresis (2-DE), a total of 72 differentially expressed protein spots (DEPs) were detected in triploid poplar pollen. Among them, 24 upregulated and 43 downregulated proteins were identified in triploid poplar pollen using matrix-assisted laser desorption/ionisation coupled with time of-flight tandem mass spectrometer analysis (MALDI-TOF/TOF MS/MS). The main functions of these DEPs were related with “S-adenosylmethionine metabolism”, “actin cytoskeleton organization”, or “translational elongation”. The infertility of triploid poplar pollen might be related to its abnormal cytoskeletal system. In addition, the abundances of previously identified 28 putative allergenic proteins were compared among three poplar varieties (‘ZH1’, ‘ZH2’, and ‘2KEN8‘). Most putative allergenic proteins were downregulated in triploid poplar pollen. This work provides an insight into understanding the protein regulation mechanism of pollen infertility and low allergenicity in triploid poplar, and gives a clue to improving poplar polyploidy breeding and decreasing the pollen allergenicity.     

Seminal Plasma Proteomic Biomarkers of Oxidative Stress

The prevalence of idiopathic male infertility is high, up to 75% of patients with abnormal sperm parameters. Hence, the research of its causes is mandatory. Oxidative stress (OS) can be responsible for male infertility in 30–80% of cases. In recent years, seminal plasma (SP) proteomics has developed as a useful tool to provide biomarkers of specific diseases. This systematic review aims to collect the available evidence on the changes of SP proteome in patients exposed to OS to provide possible SP biomarkers of sperm OS. To accomplish this, the following keyterms “seminal fluid proteome”, “seminal plasma proteome”, “oxidative stress”, and “sperm oxidative stress” were used and 137 records were found. Among these, 17 were finally included. Nine proteins involved with OS were found overexpressed in patients with OS. Twenty-three proteins were found differentially expressed in patients with clinical conditions associated with OS, such as varicocele, male accessory gland infection/inflammation, cigarette smoke, and obesity. These proteins do not seem to overlap among the clinical conditions taken into account. We speculate that specific SP proteins may mediate OS in different clinical conditions. Altogether, these results suggest that proteomics could help to better understand some of the molecular mechanisms involved in the pathogenesis of infertility. However, further studies are needed to identify potential biomarkers of male infertility with valuable clinical significance.     

Diagnostic Performance of Pancreatic Cytology with the Papanicolaou Society of Cytopathology System: A Systematic Review,before Shifting into the Upcoming WHO International System

The Papanicolaou Society of Cytopathology (PSC) reporting system classifies pancreatobiliary samples into six categories (I–VI), providing guidance for personalized management. As the World Health Organization (WHO) has been preparing an updated reporting system for pancreatobiliary cytopathology, this systematic review aimed to evaluate the risk of malignancy (ROM) of each PSC category, also the sensitivity and specificity of pancreatic FNA cytology using the current PSC system. Five databases were investigated with a predefined search algorithm. Inclusion and exclusion criteria were applied to select the eligible studies for subsequent data extraction. A study quality assessment was also performed. Eight studies were included in the qualitative analysis. The ROM of the PSC categories I, II, III, IV, V, VI were in the ranges of 8–50%, 0–40%, 28–100%, 0–31%, 82–100%, and 97–100%, respectively. Notably, the ROM IVB (“neoplastic—benign”) subcategory showed a 0% ROM. Four of the included studies reported separately the ROMs for the IVO subcategory (“neoplastic—other”; its overall ROM ranged from 0 to 34%) with low (LGA) and high-grade atypia (HGA). ROM for LGA ranged from 4.3 to 19%, whereas ROM for HGA from 64 to 95.2%. When the subcategory IVO with HGA was considered as cytologically positive, together with the categories V and VI, there was a higher sensitivity of pancreatic cytology, at minimal expense of the specificity. Evidence suggests the proposed WHO international system changes—shifting the IVB entities into the “benign/negative for malignancy” category and establishing two new categories, the “pancreatic neoplasm, low-risk/grade” and “pancreatic neoplasm, high-risk/grade”—could stratify pancreatic neoplasms more effectively than the current PSC system.