Molecular Weight Proteins Research Articles

Adsorption and Clearance of the Novel Fluorescent Tracer Agent MB-102 During Continuous Renal Replacement Therapy: In Vitro Results.

MB-102 is a novel fluorescent tracer agent that is exclusively removed from the body by glomerular filtration. This agent can be detected transdermally to provide a real-time measurement of glomerular filtration rate at the point-of-care and is currently in clinical studies for such. MB-102 clearance during continuous renal replacement therapy (CRRT) is unknown. Its plasma protein binding (~0%), molecular weight (~372 Da) and volume of distribution (15-20 L) suggest that it may be removed by renal replacement therapies. To determine the disposition of MB-102 during CRRT, an in vitro study assessing the transmembrane clearance (CL TM ) and adsorptive clearance of MB-102 was conducted. A validated in vitro bovine blood continuous hemofiltration (HF) and continuous hemodialysis (HD) models were performed using two types of hemodiafilters to evaluate CL TM of MB-102. For HF, three different ultrafiltration rates were evaluated. For HD, four different dialysate flow rates were evaluated. Urea was used as a control. No MB-102 adsorption to the CRRT apparatus or either of hemodiafilters was observed. MB-102 is readily removed by HF and HD. Dialysate and ultrafiltrate flow rates directly influence MB-102 CLTM. Hence MB-102 CLTM should be measurable for critically ill patients receiving CRRT.

Emulsion formation during enzymatic protein hydrolysis and its effect on protein recovery and molecular weight distribution of protein hydrolysates from sardine (Sardina pilchardus) by-products

Recovery of protein is one of the important variables to optimise enzymatic hydrolysis of fish processing by-products. This study investigated the role played by emulsion formation on protein recovery and the molecular weight distribution of the protein hydrolysates at different solids concentration of fish processing by-products with a high oil content. Solids concentration of fish processing by-products was varied between 26 and 50% at constant pH of 7.8, 60 °C hydrolysis temperature and 4% enzyme dosage using Alcalase 2.4L. The results showed that emulsion formation, more than inhibition of enzyme activity, at high solids concentration contributed to the reduction in yield of dry solids and protein recovery. Emulsion formation also led to the presence of high molecular weight lipo-protein complexes in the protein hydrolysates, which caused an increase in the average molecular weight of the hydrolysates. The findings show the negative relationship between solids concentration, protein recovery and emulsion formation in enzymatic hydrolysis, and the need for methods that eliminate or reduce emulsion formation at high solids concentration without increasing process complexity or reducing protein recovery.

Transglutaminase crosslinked fish gelatins for emulsion stabilization: From conventional to Pickering emulsions

Gelatin-stabilized emulsions, including conventional and Pickering emulsions, are gaining popularity. In this study, fish gelatins (FG) were modified with transglutaminase (TG) to prepare gelatin molecules, microgels, and their mixtures. The transition of emulsions from conventional to Pickering was observed as gelatins evolved from molecules to microgels. FG (3%) was cross-linked with TG at 0–30 U/g. FG evolved from gelatin molecules into thermo-irreversible hydrogels as TG increased. Without TG or at a low level of TG (10 U/g), FG mainly existed as gelatin molecules that were liquid at 25 °C. At high levels of TG (20 or 30 U/g), FG crosslinked and developed thermo-irreversible chemical hydrogels that were solid. As the concentration of TG increased, the protein molecular weights, zeta potentials, and surface hydrophobicity were significantly increased. Microfluidization was employed to prepare gelatin microgels. Different states of gelatins were mixed with sunflower oil to prepare conventional emulsions, Pickering emulsions, and mixtures. With increased TG, the emulsion droplets became more homogeneous, and protein networks formed around the oil droplets. Emulsions stabilized with modified FG displayed improved physical stability than those stabilized with unmodified FG. At 10 °C, the emulsions stabilized by a mixture of FG molecules and microgels presented higher physical stability. At 25 °C, Pickering emulsions stabilized by microgels showed improved anti-coalescence. The results of this work could provide valuable references for applying gelatin-stabilized conventional or Pickering emulsions with tunable properties, hence increasing the formulation flexibility of gelatin-based food products.

An Open-Source Framework for Mass Calculation of Antibody-Based Therapeutic Molecules.

Biotherapeutic masses are a means of verifying identity and structural integrity. Mass spectrometry (MS) of intact proteins or protein subunits provides an easy analytical tool for different stages of biopharmaceutical development. The protein's identity is confirmed when the experimental mass from MS is within a pre-defined mass error range of the theoretical mass. While several computational tools exist for the calculation of protein and peptide molecular weights, they either were not designed for direct application to biotherapeutic entities, have access limitations due to paid licenses, or require uploading protein sequences to host servers. We have developed a modular mass calculation routine that enables the easy determination of the average or monoisotopic masses and elemental compositions of therapeutic glycoproteins, including monoclonal antibodies (mAb), bispecific antibodies (bsAb), and antibody-drug conjugates (ADC). The modular nature of this Python-based calculation framework will allow the extension of this platform to other modalities such as vaccines, fusion proteins, and oligonucleotides in the future, and this framework could also be useful for the interrogation of top-down mass spectrometry data. By creating an open-source standalone desktop application with a graphical user interface (GUI), we hope to overcome the restrictions around use in environments where proprietary information cannot be uploaded to web-based tools. This article describes the algorithms and application of this tool, mAbScale, to different antibody-based therapeutic modalities.

Genome-Wide Identification and Expression Analysis of the Chalcone Synthase (CHS) Gene Family in Dendrobium catenatum

Flavonoids are considered to be critical metabolites in regulating plant responses to abiotic stress. Chalcone synthase (CHS) catalyzes the first key step in the flavonoid biosynthesis pathway. However, there is no in-depth information on the sequence and functional analysis of CHS genes in Dendrobium catenatum. In this study, a total of 14 DcCHS genes were identified, encoding proteins of 349–504 amino acids in length, a protein molecular weight ranging between 39.08 and 56.56 kDa, and isoelectric points from 5.64 to 9.63. The DcCHS proteins were then divided into three groups according to their phylogenetic relationships. The members of each group had similar conserved motifs and gene structures. Quantitative real-time RT-PCR (RT-qPCR) analysis revealed that the DcCHS genes exhibited variable expression patterns in the different plant tissues evaluated. Furthermore, six genes were differentially expressed following exposure to abiotic stresses: DcCHS-6, DcCHS-5/-6, DcCHS-13/-14, and DcCHS-6/-8/-9/-13, which were specifically expressed in response to drought, heat, cold, and salt stress, respectively. This is the first genome-wide analysis of the CHS genes in D. catenatum, and our findings can provide essential information for a better understanding of the function of DcCHS genes, thus facilitating further research on D. catenatum stress tolerance.

Environmental and biological significance of the action of viper poison on the homeostasis of mammals

The article examines the ecological and biological significance of the action of animal poisons as an evolutionary adaptation that maximizes the adaptation of more than 250,000 species to the conditions of their habitat. It was found that animal poisons have a significant impact on the formation and course of various forms of connections in ecosystems, perform several ecological functions and are important biotic factors of the environment. The best-studied venomous animals have been found to be snakes, of over 3,800 different species, only a small number are highly venomous. The Viperidae family includes 101 species of poisonous snakes. In Europe, Vipera ammodytes, Vipera aspis, Vipera berus, Vipera latastei, Vipera seoanei and Vipera ursinii are the most dangerous species, their bites cause severe poisoning. In Ukraine, the genus Vipera is represented by the steppe viper (Vipera renardi (Cristoph, 1861)) and two subspecies of the common viper (Vipera berus (Linnaeus, 1758)) — Vipera berus berus and Nikolsky’s viper (Vipera berus nikolskii, Vedmederja Grubant et Rudaeva, 1986). The factors that affect the diversity of the composition of snake venom are given. It is noted that snake venoms are complex mixtures of proteins, peptides and low molecular weight substances, among which enzymes and non-enzymatic components are distinguished. The biological properties of snake venom depend significantly on the properties of its components, which have a wide range of effects on the general state of homeostasis of a living organism. A generalization of scientific data on the study of the chemical composition of the venom of Vipera renardi, Vipera berus nikolskii and Vipera berus berus and the effects of these venoms on the functioning of the mammalian body has been carried out. It was found that the venom of Vipera berus nikolskii is the most, and Vipera renardi — the least toxic for mammals (mice). The effect of Vipera berus nikolskii and Vipera berus berus venom on the proteolytic balance of individual organs of rats was evaluated. It is emphasized that viper venom toxins have a great, not yet fully developed potential in many scientific fields, therefore they deserve further practical research.

Structural Characterization, Technofunctional and Rheological Properties of Sesame Proteins Treated by High-Intensity Ultrasound.

Plant-derived proteins, such as those from sesame seeds, have the potential to be used as versatile food ingredients. End-use functionality can be further improved by high-intensity ultrasound treatments. The effects of high-intensity ultrasound on the properties of sesame protein isolates from cold-pressed sesame cake were evaluated. The SDS-PAGE demonstrated no significant changes in the molecular weight of proteins. Ultrasound treatments resulted in decreased particle size with a more uniform distribution, resulting in the exposure of hydrophobicity and free -SH groups and increased zeta potential. Although FTIR spectra of proteins were similar after ultrasonication, a partial increase in the intensity of the amide A band was observed. The ultrasound significantly (p < 0.05) affected the secondary structure of proteins. While optical micrographics revealed a dispersed structure with smaller particles after treatments, microstructural observations indicated more rough and irregular surfaces. Water solubility was improved to 80.73% in the sample subjected to 6 min of ultrasonication. Sesame protein solutions treated for 4 and 6 min exhibited viscoelastic structure (storage modulus (G') > loss modulus (G'')). In addition, the gelation temperature of proteins decreased to about 60-65 °C with increasing treatment time. Overall, ultrasound is a useful technique for the modification of sesame protein isolates.

New insight into the irreversible membrane fouling in different pore-sized ultrafiltration ceramic membrane bioreactors (UCMBRs) for high-strength textile wastewater treatment

Despite great achievements in ceramic membrane bioreactor applications, membrane fouling, which decreases the permeability and separation performance of bioreactors and is associated with increased operational costs and energy consumption, remains a problem. The aim of this study was to expand our understanding of the fouling behavior in the long-term performance of ultrafiltration ceramic membrane bioreactors (UCMBRs) for high-strength textile wastewater reclamation. Using real textile wastewater effluent, the effects of ultrafiltration (UF) membrane pore sizes, cleaning strategies, and foulant distribution were systematically evaluated over more than three months of continuous operation. The results showed that UCMBR system achieved chemical oxygen demand and total nitrogen removal efficiencies as high as 91–95% and 39–43%, respectively. The high PN concentration can easily increase the viscosity of mixed liquor samples, contributing to a fouling layer on the membrane surface. In addition, the fouling layer formed on the surface of small-pore-sized ceramic UF membranes was not completely reversible but was difficult to eliminate by simple physical cleaning. Soluble extracellular polymeric substances, especially proteins and low molecular weight neutrals, remained, resulting in irreversible fouling on the UF membrane. However, saturated CO2 backwash showed great potential for enhancing the system through efficient fouling control without using environmentally unfriendly cleaning chemicals. The cake-intermediate and complete-standard models were suitable for explaining the fouling mechanism in the large- and small-pore-sized UF membranes, respectively.

Study of Physicochemical and Gelation Properties of Fish Gelatin from Different Sources

Fish gelatin has been increasingly used as a safe alternative to cattle and pig gelatin due to its similar structure, avoiding the health and socio-cultural issues associated with the use of materials of mammalian origin. Fish gelatin can be produced from processed fish products to achieve a high yield at a low cost. Recent studies show that although fish gelatin comes from a wide range of sources, the protein content and amino acid composition of fish gelatin from different sources are different, and some fish gelatin is soft and unstable transglutaminase (TGase) can catalyze the γ-amide group of glutamine residues and the ε-amino group of lysine residues in proteins to form covalent bonds to form a stable protein network structure, improve the strength of the gel so that it can be applied in a more special environment. In this experiment, after screening the raw materials of cold-water fish gelatin M06 and M08, warm-water fish gelatin M03 and M04, a strong fish gelatin was successfully prepared by catalytic modification of cold-water fish gelatin by transglutaminase (TGase), and the excellent performance of TG enzyme-catalyzed modified gelatin was proved through the application effect of chicken salt soluble protein. In this experiment, the protein content of cold-water fish M08 was the highest, which was up to 99.9%, 1.09 times that of warm-water fish. The gelatin content of cold-water fish M08 was the highest of the four kinds of fish gelatin, with a wide proportion of components and rich amino acid composition. Cold-water fish M08 gelatin-derived gel had the highest strength of 253 ± 1 g/cm at 4 °C. It was found that fish gelatin with protein molecular weight distribution and rich amino acid composition had higher gel strength. M08 gelatin is cross-linked by transglutaminase (TGase), which increases the strength of enzyme gels by approximately 200% compared to self-assembled gels. Fish gelatin catalyzed by the TG enzyme improves the gel strength of raw material and makes it more applicable. M08 gelatin also showed good application performance at low temperatures in compound chicken salt-soluble protein gel, with a water retention rate of 95.84% and gel strength of 198.5 g/cm. This study expanded the application range of fish gelatin by TG enzyme and improved the application potential of fish gelatin.

Alleviating Ultrafiltration Membrane Fouling Caused by Effluent Organic Matter Using Pre-Ozonation: A Perspective of EEM and Molecular Weight Distribution.

Wastewater reclamation has gradually become an important way to cope with the global water crisis. Ultrafiltration plays an imperative part as a safeguard for the aim but is often limited by membrane fouling. Effluent organic matter (EfOM) has been known to be a major foulant during ultrafiltration. Hence, the primary aim of this study was to investigate the effects of pre-ozonation on the membrane fouling caused by EfOM in secondary wastewater effluents. In addition, the physicochemical property changes of EfOM during pre-ozonation and the subsequent influence on membrane fouling were systemically investigated. The combined fouling model and the morphology of fouled membrane were adopted to scrutinize the fouling alleviation mechanism by pre-ozonation. It was found that membrane fouling by EfOM was dominated by hydraulically reversible fouling. In addition, an obvious fouling reduction was achieved by pre-ozonation with 1.0 mg O3/mg DOC. The resistance results showed that the normalized hydraulically reversible resistance was reduced by ~60%. The water quality analysis indicated that ozone degraded high molecular weight organics such as microbial metabolites and aromatic protein and medium molecular weight organics (humic acid-like) into smaller fractions and formed a looser fouling layer on the membrane surface. Furthermore, pre-ozonation made the cake layer foul towards pore blocking, thereby reducing fouling. In addition, there was a little degradation in the pollutant removal performance with pre-ozonation. The DOC removal rate decreased by more than 18%, while UV254 decreased by more than 20%.

Comparative analysis of three families of hygrophilid snails shows that the egg mass fluid protein haemocyanin-like 1 (Hcl-1) is unique to planorbids

ABSTRACT The egg mass fluid (EMF) of the freshwater snail Biomphalaria glabrata (Hygrophila: Planorbidae) contains haemocyanin-like 1 (Hcl-1) protein, distinct from respiratory haemocyanins. The distribution of Hcl-1 was investigated among major families of Hygrophila, Physidae and Lymnaeidae, both of which employ respiratory haemocyanins, and Planorbidae, a group that evolved haemoglobin as a respiratory pigment. Immunoblotting detected c. 150 kDa protein (molecular weight of Hcl-1) cross-reactive with anti-keyhole limpet haemocyanin antiserum in the EMF of planorbids Bulinus globosus and Planorbella duryi (from a genus closely related to Biomphalaria), but not Physella acuta (Physidae) and Ladislavella elodes (Lymnaeidae). High throughput sequence data revealed Hcl-1 homologs from Bulinus globosus and Planorbella duryi, representative species that span the range of planorbid phylogeny, but not from Physella acuta (Physidae) and Lymnaea stagnalis (Lymnaeidae). A domain architecture comprising only three functional units (FUs) and predicted secondary structures within the C-terminal FU distinguish planorbid Hcl-1 protein from molluscan respiratory haemocyanins that are natively assembled as functional didecamers. Immunoblotting confirmed a monomeric configuration of native Hcl-1. Molecular clock analysis estimated divergence of Hcl-1 proteins from gastropod respiratory haemocyanins at 267 ± 143 Ma. It is hypothesized that Hcl proteins originated in the ancestor of the planorbid lineage when evolution of respiratory haemoglobin altered selective pressures for maintaining original function, facilitating mutation and refunctionalization of the ancestral respiratory haemocyanin in Planorbidae.

Composition and Biological Activity of Fetoplacental Tissues-Derived Cryoextracts Being Differently Obtained

The protein-peptide composition of fetal tissues (FTCEs) and placenta (PCE) cryoextracts of rats was investigated by gel permeation chromatography. Cryoextracts were derived from tissue homogenates using the freeze-warming modes: 1 – single (–20°С); 2 – double (–20; –196°С) and 3 – triple (–20; –196; –196°С) ones. The biological activity of cryoextracts was in vitro evaluated by the phagocytic activity of neutrophil granulocytes (NG) of the blood of intact rats after incubation with an inactivated culture of Staphylococcus aureus (2 × 109 cells/ml) for 45 and 120 min for the concentrations of extracts in the incubation medium: 1.6; 3.1; 6 mg/ml in terms of dry substance. The use of mode 3 ensured a greater yield of total protein and low molecular weight compounds of a protein-peptide nature in the composition of cryoextracts. The content of low-molecular-weight fractions in FTCE exceeded that in PCE, regardless of the obtaining mode. Incubation of NG with cryoextracts did not significantly increase the number of neutrophils which entered into phagocytosis. However, all the samples of PCE and FTCE were characterized by a dose-dependent rise in the NG absorption activity during the 45-minute incubation compared to the control.

The Influence of Protein Charge and Molecular Weight on the Affinity of Aptamers.

Aptamers offer several advantages over antibodies. However, to ensure high affinity and specificity, a better understanding of the interactions between the nucleic-acid-based aptamers and their targets is mandatory. Therefore, we investigated the influence of two physical properties of proteins-molecular mass and charge-on the affinity of nucleic-acid-based aptamers. For this purpose, first, the affinity of two random oligonucleotides towards twelve proteins was determined. No binding was observed for proteins with a negative net charge towards the two oligonucleotides, while up to nanomolar affinity was determined for positively charged proteins with a high pI value. Second, a literature analysis comprising 369 aptamer-peptide/protein pairs was performed. The dataset included 296 different target peptides and proteins and is thus currently one of the largest databases for aptamers for proteins and peptides. The targets considered covered isoelectric points of 4.1-11.8 and a molecular weight range of 0.7-330 kDa, while the dissociation constants ranged from 50 fM to 29.5 µM. This also revealed a significant inverse correlation between the protein's isoelectric point and the affinity of aptamers. In contrast, no trend was observed between the affinity and the molecular weight of the target protein with either approach.

Characterization of commercial cricket protein powder and impact of cricket protein powder replacement on wheat dough protein composition

Background and Objectives Supplementation of foods with insect flours has been shown to be promising for improving the nutritional profile of food products. The objective of this study was to characterize two commercially available cricket protein powders and investigate their impact on molecular weight distribution when incorporated into wheat dough. Findings Characterization of commercial cricket protein powders, GrioPro® (G) and Entomo Farms (E) was carried out by size exclusion high-performance liquid chromatography (SEC-HPLC), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The functionality of the cricket protein powders was examined by measuring water holding capacity (WHC) and protein solubility across a range of pH. To see the interactions between the cricket proteins in a food-based system wheat dough samples containing 10% or 20% replacement levels of the cricket powders were collected at peak torque development and analyzed to SEC-HPLC to quantify the change in soluble polymeric proteins (SPP) and insoluble polymeric proteins (IPP) and provide information on changes to protein molecular weight distribution. SDS-PAGE analysis showed bands ranging from 40 kDa to 160 kDa for sample E while sample G had no visible bands either due to poor solubility in SDS-PAGE sample buffer and/or the presence of very high molecular proteins that did not enter the gel. Sample E absorbed approximately 2.5 times its weight in water, which was significantly lower than G (~3.0 times its weight) while sample G was significantly lower in solubility than E across all pH levels. Conclusions Both cricket protein powders increased in WHC and solubility as the pH increased. Wheat dough samples containing sample E had lower peak areas of IPP and no significant difference in SPP peak areas compared to the control. On the other hand, doughs containing sample G had a significant increase in IPP peak areas at the 20% replacement level. Significance and Novelty This study shows how the difference in processing changed the functionality of G and E which impacted their interactions when added to a wheat dough-based system.

Improving the surface tackiness of frozen cooked noodles by the addition of glutenin, gliadin, and gluten

The mechanism of gluten components on surface tackiness of frozen cooked noodles (FCNs) during processing is still unclear. The internal relationship between gluten components and the surface tackiness of FCNs during manufacturing process was investigated. Texture characteristics, surface tackiness, gelatinization degree, thermal stability, free sulfhydryl, protein molecular weight distribution, secondary structure, surface and internal microstructure of FCNs were determined. The results showed that the addition of glutenin could strengthen the backbone of gluten network, decrease free sulfhydryl content (1.81 μmol/g) of pre-cooked noodles, increase the gelatinization degree (89.71%), protein aggregation (glutenin macropolymers: 86.55%) and ordered secondary structure of recooked FCNs, and resist the damage of gluten network caused by pre-cooking. The addition of gliadin could promote the formation of membrane gluten network, improve the extensibility (tensile distance: 0.0212 m) of FCNs, resist the damage of gluten network during freezing storage for wrapping starch particles more tightly, and decrease the surface tackiness (3.60 N) of recooked FCNs. Gluten combined the advantages of glutenin and gliadin to strengthen gluten network and reduce the surface tackiness of FCNs. In the future, the synergistic effect of glutenin and gliadin will be further studied to regulate the surface tackiness and edible quality of FCNs.

Genome-wide identification and characterization of the WOX gene family in Brassica juncea

The WUSCHEL related-homeobox (WOX) family is one of the plant-specific transcription factor families, playing important roles in plant growth and development. In this study, 51 WOX gene family members were identified from the genome data of Brassica juncea by searching and screening with HUMMER, Smart and other software. Their protein molecular weight, amino acids numbers, and isoelectric point were analyzed by using Expasy online software. Furthermore, bioinformatics software was used to systematically analyze the evolutionary relationship, conservative region, and gene structure of the WOX gene family. The mustard WOX gene family was divided into three subfamilies: ancient clade, intermediate clade, and WUS clade/modern clade. Structural analysis showed that the type, organization form and gene structure of the conservative domain of WOX transcription factor family members in the same subfamily were highly consistent, while there was a certain diversity among different subfamilies. 51 WOX genes are distributed unevenly on 18 chromosomes of mustard. Most of the promoters of these genes contain cis acting elements related to light, hormone and abiotic stress. Using transcriptome data and real-time fluorescence quantitative PCR (qRT-PCR) analysis, it was found that the expression of mustard WOX gene was spatio-temporal specific, among which BjuWOX25, BjuWOX33, and BjuWOX49 might play an important role in the development of silique, and BjuWOX10, BjuWOX32, and BjuWOX11, BjuWOX23 respectively might play an important role in the response to drought and high temperature stresses. The above results may facilitate the functional study of mustard WOX gene family.

New strategy of drinking water sludge as conditioner to enhance waste activated sludge dewaterability: Collaborative disposal

Drinking water sludge (DWS) and waste activated sludge (WAS) are usually treated separately. With the continuous deepening understanding of the characteristics of two types sludge, the research and application of the collaborative disposal is worth considering. The heated modification DWS (HDWS) rich in inorganic matter and aluminum (Al2O3) can be used as a conditioner to enhance WAS dewaterability using its properties with physical skeleton and chemically catalyzed ozone (O3). The results showed that the minimum values of capillary water time (CST) and specific resistance filtration (SRF) for WAS were 20.9±2.40 s and 1.07±0.19×1013 m/kg at pH=4, O3 dosage=60 mg/g VS and HDWS dosage=700 mg/g VS, corresponding to the reduction of sludge cake water content (Wc) to 60.37±0.97 %. The mechanism of HDWS+O3 enhanced WAS dewaterability was systematically elucidated through pyridine-infrared analysis and density functional theory (DFT) calculations. The surface of Al2O3 in HDWS had more Lewis acidic sites, and the oxygen atoms of O3 combined with Al atoms to form Al-O bonds and undergo electron transfer, while O3 molecules dissociated to produce more hydroxyl radicals (·OH). With the oxidation of ·OH, the extra-microcolony/cellular polymers (EMPS/ECPS) structure were destroyed and became looser, promoting the conversion of internal moisture to free moisture. Zeta potential tended to zero, particle size increased, and the surface was more hydrophobic. Correlation analysis revealed that the component content, protein (PN) secondary structure and molecular weight (MW) in ECPS were positively and more strongly correlated with the sludge dewaterability compared to EMPS. The discovery of HDWS+O3 applied to effectively enhance WAS dewaterability provided an inspiring perspective on the emerging DWS and WAS co-processing disposition.

Genome-Wide Characterization of HSP90 Gene Family in Chinese Pumpkin (Cucurbita moschata Duch.) and Their Expression Patterns in Response to Heat and Cold Stresses

Heat shock protein 90 (HSP90) plays critical roles in plant growth and development, as well as in response to abiotic stresses such as heat and cold. To comprehensively analyze the HSP90 gene family and determine the key HSP90 gene responsive to temperature stress in pumpkin (Cucurbita moschata Duch.), bioinformatics and molecular biology techniques were used in this study. A total of 10 CmoHSP90 genes were identified from the pumpkin genome, encoding amino acids of 567–865, with protein molecular weight of 64.32–97.36 kDa. Based on the phylogenetic analysis, they were classified into four groups. The members in each group contained similar conserved motifs and gene structures. The 10 CmoHSP90 genes were distributed on the 9 chromosomes of C. moschata. Four pairs of segmental duplication genes (CmoHSP90-1/CmoHSP90-10, CmoHSP90-2/CmoHSP90-7, CmoHSP90-3/CmoHSP90-6, and CmoHSP90-4/CmoHSP90-9) were detected. Synteny analysis revealed that 10 C. maxima HSP90 genes and 10 C. moschata HSP90 genes were orthologous genes with 17 syntenic relationships. Promoter analysis detected 23 cis-acting elements including development-, light-, stress-, and hormone-related elements in the promoter regions of pumpkin HSP90 genes. Further analysis showed that the transcript levels of CmoHSP90-3 and CmoHSP90-6 were remarkably up-regulated by heat stress, while CmoHSP90-6 and CmoHSP90-10 were significantly up-regulated by cold stress, suggesting that these HSP90 genes play critical roles in response to temperature stress in pumpkins. The findings will be valuable for understanding the roles of CmoHSP90s in temperature stress response and should provide a foundation for elucidating the function of CmoHSP90s in C. moschata.

Genome-Wide Identification and Phylogenetic and Expression Analyses of the PLATZ Gene Family in Medicago sativa L.

The PLATZ family is a novel class of plant-specific zinc finger transcription factors with important roles in plant growth and development and abiotic stress responses. PLATZ members have been identified in many plants, including Oryza sativa, Zea mays, Triticum aestivum, Fagopyrum tataricum, and Arabidopsis thaliana; however, due to the complexity of the alfalfa reference genome, the members of the PLATZ gene family in alfalfa (Medicago sativa L.) have not been systematically identified and analyzed. In this study, 55 Medicago sativa PLATZ genes (MsPLATZs) were identified in the alfalfa "Xinjiangdaye" reference genome. Basic bioinformatic analysis was performed, including the characterization of sequence lengths, protein molecular weights, genomic positions, and conserved motifs. Expression analysis reveals that 7 MsPLATZs are tissue-specifically expressed, and 10 MsPLATZs are expressed in all examined tissues. The transcriptomic expression of these genes is obvious, indicating that these MsPLATZs have different functions in the growth and development of alfalfa. Based on transcriptome data analysis and real-time quantitative PCR (RT-qPCR), we identified 22, 22, and 21 MsPLATZ genes that responded to salt, cold, and drought stress, respectively, with 20 MsPLATZs responding to all three stresses. This study lays a foundation for further exploring the functions of MsPLATZs, and provides ideas for the improvement of alfalfa varieties and germplasm innovation.

Pre-analytical sample handling effects on tear fluid protein levels

Tear fluid is emerging as a source of non-invasive biomarkers, both for ocular and systemic conditions. Accurate quantification of tear proteins can be improved by standardizing methods to collect and process tear fluid. The aim of this study was to determine sample handling factors that may influence the tear protein biomarker profile. Tear fluid was collected using Schirmer’s strips. Tear proteins were extracted by elution through centrifugation. Total protein content was determined using the bicinchoninic acid assay. Key concepts that apply to the entire sample processing cycle are tear sampling, tear storage, protein extraction and data normalization. Differences in wetting or migration length were observed between Schirmer’s strips from different manufacturers, and between protein-free and protein-rich solutions. One unit of migration length (mm) did not correspond to one unit of volume (µL). A positive correlation (r = 0.6671, p < 0.0001) was observed between migration length and total tear protein content. The most beneficial storage conditions were strips that were not stored (+ 21.8%), or underwent ‘wet’ storage (+ 11.1%). Protein recovery was the highest in 400 µL extraction buffer and independent of protein molecular weight. This study helps to explain inter- and intra-variability that is often seen with tear biomarker research. This information is critical to ensure accuracy of test results, as tear biomarkers will be used for patient management and in clinical trials in the near future. This study also highlights the need for standardization of Schirmer’s strip manufacturing, tear fluid processing and analyte concentration normalization.