Total Protein Recovery Research Articles

Sulfate Nutrition Modulates the Oxidative Response against Short-Term Al3+-Toxicity Stress in Lolium perenne cv. Jumbo Shoot Tissues

Al3+-toxicity in acidic soils is among the main abiotic stress factors that generate adverse effects in plant growth; in leaves, it affects several physiological parameters such as photosynthesis and ROS balance, leading to limited crop production. On the other hand, sulfur is a macronutrient that has a key role against oxidative stress and improves plant growth in acidic soils; however, the implication of sulfate nutritional status in the modulation of short-term Al3+-toxicity tolerance mechanisms in plant leaves are barely reported. This study is focused on the role of sulfate on the leaf response of an Al3-sensitive perennial ryegrass (Lolium perenne cv. Jumbo) after 48 h of exposure. Lolium perenne cv. Jumbo seeds were cultivated in hydroponic conditions with modified Taylor Foy solutions supplemented with 120, 240, and 360 μM sulfate in the presence or absence of Al3+-toxicity. The L. perenne cv. Jumbo leaves were collected after 48 h of Al3+-toxicity exposure and processed to evaluate the effects of sulfate on Al3+ toxicity, measuring total proteins, mineral uptake, photosynthesis modulation, and ROS defense mechanism activation. The plants exposed to Al3+-toxicity and cultivated with a 240 µM sulfate amendment showed a recovery of total proteins and Ca2+ and Mg2+ concentration levels and a reduction in TBARS, along with no changes in the chlorophyll A/B ratio, gene expression of proteins related to photosynthesis (Rubisco, ChlAbp, and Fered), or ROS defense mechanism (SOD, APX, GR, and CAT) as compared with their respective controls and the other sulfate conditions (120 and 360 µM). The present study demonstrates that adequate sulfate amendments have a key role in regulating the physiological response against the stress caused by Al3+ toxicity.

Hexane defatting effects on rapeseed protein extractability and properties at neutral and mild pH 8.5

Rapeseed is a promising plant protein source yet to be fully explored. Traditionally, rapeseed proteins are extracted from hexane-defatted press cake at high alkaline conditions to maximise protein recovery. Instead of the traditional approach, this study explored the effects of extracting proteins from the whole-kernel seed, focusing on assessing the hexane defatting process followed by gentle protein extraction at neutral pH compared with pH 8.5. Extraction at neutral pH resulted in a napin-rich protein extract, while alkaline conditions favoured cruciferin extraction as expected. Hexane defatting destroyed the oil bodies's structure while detaching the cell membrane from the cell wall, causing dehydration, and enabling interactions between the seed constituents. Increasing cycles of hexane treatment greatly reduced the total protein recovery by 9 g/100 g under neutral pH and >60 g/100 g under mild alkaline conditions which affected the protein structure, leading to a more rigid structure with a slightly higher melting temperature.

Assessing protein and albumin recovery rates in different ascites filtration membrane washing methods for cell-free and concentrated ascites reinfusion therapy.

In cell-free and concentrated ascites reinfusion therapy (CART), the protein recovery rate decreases when the filtration membrane gets clogged. Employing a device with a filtration membrane washing feature prevents clogging, but it leads to the loss of ascites within the filter, resulting in reduced protein recovery. This study employed a device with a membrane washing function to investigate the relationship between protein recovery rate and the quantity of washing solution used, depending on the selected washing method. We analyzed cases of CART conducted at Fujita Health University Hospital between May 2021 and November 2022. The cases were divided and compared between two groups: one using flush and rinse as the washing method (flush+rinse group) and another using only flushing (flush group). We identified nine cases and 16 sessions. In the flush+rinse group, the median amount of washing solution used per membrane washing was 259mL per cycle, whereas it was 54mL per cycle in the flush group. This difference was statistically significant (p<0.0001). The median total protein recovery rate was 53.8% for the flush+rinse group and 78.8% for the flush group, with the latter showing a significantly higher value (p=0.0199). In CART using a membrane washing function, adopting a washing method that reduces the amount of washing solution leads to an increase in the total protein recovery rate.

Piperine, a phytochemical prevents the biofilm city of methicillin-resistant Staphylococcus aureus: A biochemical approach to understand the underlying mechanism

Methicillin-resistant Staphylococcus aureus (MRSA), a drug-resistant human pathogen causes several nosocomial as well as community-acquired infections involving biofilm machinery. Hence, it has gained a wide interest within the scientific community to impede biofilm-induced MRSA-associated health complications. The current study focuses on the utilization of a natural bioactive compound called piperine to control the biofilm development of MRSA. Quantitative assessments like crystal violet, total protein recovery, and fluorescein-di-acetate (FDA) hydrolysis assays, demonstrated that piperine (8 and 16 μg/mL) could effectively compromise the biofilm formation of MRSA. Light and scanning electron microscopic image analysis confirmed the same. Further investigation revealed that piperine could reduce extracellular polysaccharide production by down-regulating the expression of icaA gene. Besides, piperine could reduce the cell-surface hydrophobicity of MRSA, a crucial factor of biofilm formation. Moreover, the introduction of piperine could interfere with microbial motility indicating the interaction of piperine with the quorum-sensing components. A molecular dynamics study showed a stable binding between piperine and AgrA protein (regulator of quorum sensing) suggesting the possible meddling of piperine in quorum-sensing of MRSA. Additionally, the exposure to piperine led to the accumulation of intracellular reactive oxygen species (ROS) and potentially heightened cell membrane permeability in inhibiting microbial biofilm formation. Besides, piperine could reduce the secretion of diverse virulence factors from MRSA. Further exploration revealed that piperine interacted with extracellular DNA (e-DNA), causing disintegration by weakening the biofilm architecture. Conclusively, this study suggests that piperine could be a potential antibiofilm molecule against MRSA-associated biofilm infections.

Screening and validation of an alkaline-tolerant biomimetic affinity chromatography A5–87 resin for purification of discarded bovine serum Immunoglobulin G

It is important to recycle the bovine blood discarded at slaughter and develop it into high value-added bovine serum products. Biomimetic affinity chromatography (BiAC) resins have been developed to specifically purify bovine serum immunoglobulin G (Bs-IgG). The BiAC strategy was used to screen the resins with the best purification effect on Bs-IgG. Four resins with specificity for Bs-IgG adsorption were selected from 90 BiAC resins. Finally, BiAC-A5–87 was selected and used to purify Bs-IgG based on the results of SDS-PAGE and BCA protein quantification analysis. The adsorption capacity and purity of BiAC-A5–87 were 32.79 ± 3.57 mg/mL and 85.9 ± 1.21 % for Bs-IgG, respectively. The total protein recovery rate of Bs-IgG purified by BiAC-A5–87 was 89.78±3.52 %. The resin of BiAC-A5–87 column was recycled in 40 breakthrough cycles, and its Bs-IgG adsorption efficiency decreased by less than 10 %. After soaking BiAC-A5–87 in 1.0 moL NaOH solution for 64 h, its adsorption capacity for Bs-IgG was almost the same as that before soaking. The development of waste bovine serum not only realizes the utilization of blood resources and produces high economic benefits but also reduces the pollution of the environment.

Piperine, a Plant Alkaloid, Exhibits Efficient Disintegration of the Pre-existing Biofilm of Staphylococcus aureus: a Step Towards Effective Management of Biofilm Threats.

Staphylococcus aureus causes a range of chronic infections in humans by exploiting its biofilm machinery and drug-tolerance property. Although several strategies have been proposed to eradicate biofilm-linked issues, here, we have explored whether piperine, a bioactive plant alkaloid, can disintegrate an already existing Staphylococcal biofilm. Towards this direction, the cells of S. aureus were allowed to develop biofilm first followed by treatment with the test concentrations (8 and 16µg/mL) of piperine. In this connection, several assays such as total protein recovery assay, crystal violet assay, extracellular polymeric substances (EPS) measurement assay, fluorescein diacetate hydrolysis assay, and fluorescence microscopic image analysis confirmed the biofilm-disintegrating property of piperine against S. aureus. Piperine reduced the cellular auto-aggregation by decreasing the cell surface hydrophobicity. On further investigation, we observed that piperine could down regulate the dltA gene expression that might reduce the cell surface hydrophobicity of S. aureus. It was also observed that the piperine-induced accumulation of reactive oxygen species (ROS) could enhance biofilm disintegration by decreasing the cell surface hydrophobicity of the test organism. Together, all the observations suggested that piperine could be used as a potential molecule for the effective management of the pre-existing biofilm of S. aureus.

Obesity management for the pre–liver transplant and post–liver transplant patient

Obesity management for the pre–liver transplant and post–liver transplant patient

Effect of milk protein standardisation using buffalo milk protein co‐precipitates on the texture, composition and yield of paneer

Buffalo milk was utilised for preparing paneer samples after standardisation of protein:fat ratio to 0.68 with the addition of buffalo milk protein co‐precipitates (BMPC). These paneer samples along with control paneer (from buffalo milk) were analysed for yield, composition, microstructure, sensory, texture profile and protein loss in whey during manufacture. Paneer prepared using BMPC was considerably higher in yield and recovery of total solids, protein and calcium content than control paneer and could withstand the frying conditions in terms of retention of shape and integrity. Texture profile analysis and microstructure evaluation revealed the differences in textural properties of paneer samples prepared using BMPC with control paneer.

Honey powder produced by spray drying as an ingredient: A new perspective using a plant-based protein as a carrier

Honey is an excellent alternative to be used in different applications by the food, pharmaceutical, and cosmetics industries. However, the use of honey in its liquid form can present several disadvantages due to its high viscosity and density. In addition, honey can naturally crystallize during storage time, limiting its application in new products by industries. Thus, an alternative to using liquid honey in industry is the powder form. The objective of this study was to obtain honey powder by spray drying using a plant-based protein as a carrier. The honey used as raw material showed acceptable physicochemical parameters by the legislation of honey quality control, such as pH of (4.0 ± 0.1), the water content of (20.70 ± 0.3) g/100 g, ash of (0.48 ± 0.02) g/100 g, the fructose content of (35.42 ± 0.14) g/100 g, (43.05 ± 0.46) g/100 g for glucose, (0.56 ± 0.02) g/100 g for sucrose, and 5-hydroxymethylfurfural (HMF) value of (37.47 ± 1.04) mg/kg of honey, leading to the production of the honey powder. The mixture of fresh honey and plant-based protein was made with dispersions of 15, 16 and 18 g/100 g of honey using isolated rice protein as a carrier (total solid at 30 g/100 g d.b.). Finally, the honey powder produced was physicochemically characterized. The dispersion with 16 g/100 g of honey achieved an intermediary sugar/honey recovery of 35.93 ± 0.82% and 68.54 ± 2.16% of total protein recovery, and the spray drying yield was satisfactory > 50%. The overall results suggested that this dispersion is the ideal formulation when commercialization of the honey powder as an ingredient is intended.

629 Validation of a wound fluid collection method for subsequent identification of prognostic biomarkers for healing

Several matrix metalloproteinases (MMPs) are hypothesized to play key roles in the wound healing process due, to their ability to break down various extracellular matrix components. Expression levels of MMPs, or their inhibitors, may be useful for predicting the risk of wound healing or non-healing. However, methods used to collect human wound fluid are diverse with little evidence of rigorous method validation in the literature. Therefore, we aimed to develop a robust method for the collection of fluid from wound dressings. We have validated this method using both human serum and wound fluid samples. Serum was acquired commercially whilst wound dressings were collected from ten patients undergoing treatment of leg or foot ulcers. Total protein concentrations and relative protein abundances were measured for extracted and control samples. The developed method gave normalised total protein recoveries (arbitrary units, a.u.) of 0.64, 0.85, 0.60 and 0.56 a.u. when extracting serum from alginate (Kaltostat), foam (Mepilex), surgical (Softpore) and film (Tegaderm) dressings respectively in comparison to serum controls (1.00 a.u.). Fluid extracted from patient dressings had a mean total protein concentration of 21.5 mg/ml and gave consistent identification and quantification of several MMPs and their inhibitors via Western blot. The developed method therefore facilitated consistent recovery of serum with minimal protein loss, specifically when using foam dressings. However, these data show the variability in total protein recovery between dressing materials. This method’s suitability was further demonstrated with the successful extraction of wound fluid from patient dressings and quantification of proteins involved in wound healing. This method is therefore suitable for further wound fluid collection procedures and development of prognostic biomarker studies.

Polysorbates versus Hydroxypropyl Beta-Cyclodextrin (HPβCD): Comparative Study on Excipient Stability and Stabilization Benefits on Monoclonal Antibodies.

Polysorbates (PS 20 and PS 80) are the most widely used surfactants in biopharmaceutical formulations to protect proteins from denaturation, aggregation, and surface adsorption. To date, around 70% of marketed therapeutic antibodies contain either PS 20 or PS 80 in their formulations. However, polysorbates are chemically diverse mixtures, which are prone to degradation by oxidation and hydrolysis to produce peroxides and fatty acids, which, in turn, induce protein oxidation, aggregation, and insoluble particle formation. These will negatively impact protein quality and stability. Thus, polysorbate degradation has emerged as one of the major challenges in the development and commercialization of therapeutic protein products. KLEPTOSE® HPβCD (hydroxypropyl beta-cyclodextrin), a new multifunctional excipient, has been shown to provide protein stabilization functions in biopharmaceutical downstream processes and in their final formulations. This study aims to evaluate HPβCD, a new molecule of its class, against polysorbates as a stabilizer in biologics formulations. In this study, the chemical stability of KLEPTOSE® HPβCDs is compared with polysorbates (20 and 80) under various stress conditions. When subjected to heat stress, HPβCDs show little change in product recovery (90.7–100.7% recovery for different HPβCDs), while polysorbates 20 and 80 show significant degradation, with only 11.5% and 7.3% undegraded product remaining, respectively. When subjected to other chemical stressors, namely, autoclave, light, and oxidative stresses, HPβCD remains almost stable, while polysorbates show more severe degradation, with 95.5% to 98.8% remaining for polysorbate 20 and 85.5% to 97.4% remaining for polysorbate 80. Further, profiling characterization and degradation analysis reveal that chemical structures of HPβCDs remain intact, while polysorbates undergo significant hydrolytic degradation and oxidation. Lastly, the physicochemical stability of monoclonal antibodies in formulations is investigated. When subjected to light stress, adalimumab, as a model mAb, formulated in the presence of HPβCD, shows a significant decrease in protein aggregation, and superior monomer and total protein recovery compared to PS 80-containing formulations. HPβCD also reduces both agitation and thermal stress-induced protein aggregation and prevents subvisible particle formation compared to PS 80.

Longitudinal and time-to-event modeling for prognostic implications of radical surgery in retroperitoneal sarcoma.

Retroperitoneal sarcoma (RPS) is a rare malignancy which can be difficult to manage due to the variety of clinical behaviors. In this study, we aimed to develop a parametric modeling framework to quantify the relationship between postoperative dynamics of several biomarkers and overall/progression‐free survival of RPS. One hundred seventy‐four patients with RPS who received surgical resection with curative intent at the Peking University Cancer Hospital Sarcoma Center were retrospectively included. Potential prognostic factors were preliminarily identified. Longitudinal analyses of body mass index (BMI), serum total protein (TP), and white blood cells (WBCs) were performed using nonlinear mixed effects models. The impacts of time‐varying and time‐invariant predictors on survival were investigated by parametric time‐to‐event (TTE) models. The majority of patients experienced decline in BMI, recovery of TP, as well as transient elevation in WBC counts after surgery, which significantly correlated with survival. An indirect‐response model incorporating surgery effect described the fluctuation in percentage BMI. The recovery of TP was captured by a modified Gompertz model, and a semimechanistic model was selected for WBCs. TTE models estimated that the daily cumulative average of predicted BMI and WBC, the seventh‐day TP, as well as certain baseline variables, were significant predictors of survival. Model‐based simulations were performed to examine the clinical significance of prognostic factors. The current work quantified the individual trajectories of prognostic biomarkers in response to surgery and predicted clinical outcomes, which would constitute an additional strategy for disease monitoring and intervention in postoperative RPS.

Isolation and Characterization of Plasma-Derived Exosomes from the Marine Fish Rock Bream (Oplegnathus fasciatus) by Two Isolation Techniques

Exosomes are important mediators of intercellular communication and modulate many physiological and pathological processes. Knowledge of secretion, content, and biological functions of fish exosomes during pathological infection is still scarce due to lack of suitable standardized isolation techniques. In this study, we aimed to isolate exosomes from the plasma of marine fish, rock bream (Oplegnathus fasciatus), by two isolation methods: differential ultracentrifugation (UC) and a commercial membrane affinity spin column technique (kit). Morphological and physicochemical characteristics of the isolated exosomes were determined by these two methods, and the efficiencies of the two methods were compared. Exosomes isolated by both methods were in the expected size range (30–200 nm) and had a characteristic cup-shape in transmission electron microscopy observation. Moreover, more intact exosomes were identified using the kit-based method than UC. Nanoparticle tracking analysis demonstrated a heterogeneous population of exosomes with a mean particle diameter of 114.6 ± 4.6 and 111.2 ± 2.2 nm by UC and a kit-based method, respectively. The particle concentration obtained by the kit method (1.05 × 1011 ± 1.23 × 1010 particles/mL) was 10-fold higher than that obtained by UC (4.90 × 1010 ± 2.91 × 109 particles/mL). The kit method had a comparatively higher total protein yield (1.86 mg) and exosome protein recovery (0.55 mg/mL plasma). Immunoblotting analysis showed the presence of exosome marker proteins (CD81, CD63, and HSP90) in the exosomes isolated by both methods and suggests the existence of exosomes. However, the absence of cytotoxicity or adverse immune responses to fish and mammalian cells by the exosomes isolated by the UC procedure indicates its suitability for functional studies in vitro. Overall, our basic characterization results indicate that the kit-based method is more suitable for isolating high-purity exosomes from fish plasma, whereas UC has higher safety in terms of yielding exosomes with low toxicity. This study provides evidence for the existence of typical exosomes in rock beam plasma and facilitates the selection of an efficient exosome isolation procedure for future applications in disease diagnosis and exosome therapy as fish medicine.

Quantification of Soy-Derived Ingredients in Model Bread and Frankfurter Matrices with an Optimized Liquid Chromatography–Tandem Mass Spectrometry External Standard Calibration Workflow

The detection and quantification of soy protein is important for food allergen management and identifying the presence of undeclared soy proteins. Heat processing and matrix interactions can affect the accuracy of allergen detection methods. The sensitivity of enzyme-linked immunosorbent assay methods can be compromised if protein epitopes are modified during processing. Therefore, a mass spectrometry (MS)-based method was evaluated for the recovery of total soy protein in incurred matrices. MS-based quantification of total soy protein was assessed by using a combination of external and internal standards. The reproducibility of the standard curves was investigated by comparing within-day and among-day variation. Incurred samples were prepared using bread and frankfurters as model food matrices. Several soy-derived ingredients were used to prepare the matrices with varying levels of soy protein (1, 10, 50, or 100 ppm of total soy protein). A pooled standard curve was used to estimate the total soy protein concentration of the incurred food matrices and the percent total protein recovery. The variation of replicate standard curves between days and among all days was not significant. The differences in slopes obtained from replicate standards run on different days were minimal. The most influential factor on the quantitative protein recovery in incurred samples was the effect of the physical matrix structure on protein extraction. The lowest percent protein recoveries, less than 50%, were calculated for uncooked matrices. The cooked matrices had percentage recoveries between 50 and 150% for all total soy protein levels. Other factors, such as type of ingredient, were determined to be not as impactful on recovery. The MS method described in this study was able to provide sensitive detection and accurate quantification of total soy protein from various soy-derived ingredients present in processed food matrices.

Improved protein extraction from thermally processed shrimp (Litopenaeus vannamei) for reliable immunodetection via a synergistic effect of buffer additives

Extraction of targeted protein is a significant step for the quantitative detection of allergenic proteins, especially for detection in processed foods. To optimize the extraction step before immunodetection, six commonly used buffers were compared for their extraction efficiency and detectability of total proteins and tropomyosin (TM) from raw and thermally processed shrimp (Litopenaeus vannamei). Then the potential of three additives, including dithiothreitol (DTT), sodium dodecyl sulfate (SDS) and Tween 20, were applied to improve the extraction efficiency. The results showed the recovery of total proteins and TM extracted from processed shrimp was significantly reduced compared to unheated shrimps. Coca's buffer displayed higher efficacy in recovering total proteins and TM from raw and thermally processed shrimp. Moreover, principal component analysis (PCA) analysis revealed Coca's buffer with DTT, SDS, and Tween 20 additives displayed a synergistic effect in improving recovery of total protein and TM from raw and thermal processed shrimp. Indirect enzyme-linked immunosorbent assay (ELISA) and dot blotting analysis indicated the allergenicity of shrimp extracts could be highly suppressed by thermal treatment. Nevertheless, TM from various heated shrimps, except autoclaved samples, might have higher potential allergenicity, confirmed by western blotting analysis.

Influence of preservation methods on biochemical composition and downstream processing of cultivated Saccharina latissima biomass

Saccharina latissima biomass cultivated along the Swedish west coast was subjected to four different scalable preservation methods after harvest; freezing, sun-drying, oven-drying and ensiling. Freeze-drying and freezing at −80 °C were also included to provide dry and wet references. The effects of the different preservation methods on the composition of Saccharina biomass (on dry weight, DW, basis), and the recovery as well as properties of high-quality protein, alginate and biogas were evaluated. Sun-drying significantly reduced protein, alginate and fatty acid content of the seaweeds and thereby concentrated ash in the biomass compared to the other methods. Protein/amino acids and fatty acids were significantly concentrated in ensiled biomass, while mannitol and laminarin were reduced compared to the other biomasses. Oven-drying and −20 °C freezing affected the composition the least, with lower ash content and alterations in some specific amino and fatty acids. Sun-drying and ensiling resulted in significantly lower protein solubility at high pH compared to the other biomasses which translated into the lowest total seaweed protein recovery using the pH-shift process. Highest protein yield was obtained with the freeze-dried reference. Ensiling lead to a significant decrease in the molecular weight of alginate, while sun-drying caused a negative effect on alginate by inducing a shift in the guluronic and mannuronic acids composition of alginate. Sun-drying gave the lowest methane yield in the anaerobic digestion experiments while freezing at −80 °C gave the highest yield, closely followed by freezing at −20 °C and ensiling. To conclude, preservation methods must be carefully chosen to protect the valuable component in Saccharina latissima, and to achieve an efficient downstream processing ultimately yielding high quality products as part of a seaweed biorefinery.

Foam Fractionation Technology for Enrichment and Recovery of Cheese Whey Proteins

Background: Foam fractionation technology works on the adsorptive bubble separation principle. This technique involves adsorption of the surface-active substances on to a gas-liquid interface and separation of these components from the liquid along with bubbles as foam. The foam separation technology has been successfully utilized in the recovery of proteins from solutions containing either a single protein or binary mixtures. To develop a foam fractionation technology for selective enrichment and recovery of whey proteins, it is essential to investigate the effect of different feed and process variables that affect the foam fractionation process. The aim of the current study was to investigate the effect of two important feed variables, such as pH and initial protein concentration on recovery and enrichment of total whey proteins as well as α-lactalbumin and β-lactoglobulin. Methods: All the experiments were conducted in Agriculture and biosystems engineering lab and Alfred Dairy Science lab at South Dakota State University, Brookings, South Dakota during 2011-2013. The experiments used four levels of initial protein concentration and five levels of feed pH. Yield and enrichment ratios were determined for total whey proteins, α-Lactalbumin (α-La) and β-Lactoglobulin (β-Lg). Result: Whey protein yields ranged from 51.58 to 90.92%, while the enrichment ratios were between 1.2 to 5. The yield of α-La varied from 59 to 94% and the highest enrichment ratio of 8.45 was obtained with the treatment combination of initial protein concentration of 109 mg/L and pH of 5.1. Selective enrichment of α-La over β-Lg was observed at a pH of 4.65 with α-La to β-Lg ratio of 0.49. These findings will be helpful in selective enrichment and recovery of valuable proteins from Cheddar cheese whey using the foam fractionation process.

A comparison between pylorus-preserving and distal gastrectomy in surgical safety and functional benefit with gastric cancer: a systematic review and meta-analysis

BackgroundDue to better functional outcomes, pylorus-preserving gastrectomy (PPG) has been widely applied for early gastric cancer (EGC) patients as an alternative to distal gastrectomy (DG). However, controversies still persist regarding the surgical efficacy and oncological safety of PPG.MethodsOriginal studies comparing PPG and DG for EGC were searched in PubMed, Embase, and the Cochrane Register of Controlled Trials up to December 2019. The weight mean difference, standardized mean difference, or odds risk was used to calculate the short-term and long-term outcomes between the two groups.ResultsTwenty-one comparative studies comprising 4871 patients (1955 in the PPG group and 2916 in the DG group) were enrolled in this systematic review and meta-analysis. PPG showed longer hospital day, decreased harvested lymph nodes, and more delayed gastric emptying. However, PPG had the benefits of lower incidence of anastomosis leakage, early dumping syndrome, gastritis and bile reflux, and better recovery of total protein, albumin, hemoglobin, and weight. No difference was found in operative time, blood loss, and overall complications. Moreover, the long-term survival and recurrence rate were similar in two groups.ConclusionOwing to the non-inferiority of surgery and oncology outcomes and the superiority of function outcomes in PPG, we revealed that PPG can be clinically applicable instead of DG in EGC. However, more high-quality comparative studies and randomized clinical trials would be required for further confirmation.

A unified protocol for simultaneous extraction of DNA and proteins from archaeological dental calculus

Archaeological materials are a finite resource, and efforts should be made to minimize destructive analyses. This can be achieved by using protocols combining extraction of several types of biomolecules or microparticles, which decreases the material needed for analyses while maximizing the information yield. Archaeological dental calculus is a source of several different types of biomolecules, as well as microfossils, and can tell us about the human host, microbiome, diet, and even occupational activities. Here, we present a unified protocol allowing for simultaneous extraction of DNA and proteins from a single sample of archaeological dental calculus. We evaluate the protocol on dental calculus from six individuals from a range of time periods and estimated preservation states, and compare it against previously published DNA-only and protein-only protocols. We find that most aspects of downstream analyses are unaltered by the unified protocol, although minor shifts in the recovered proteome can be detected, such as a slight loss of hydrophilic proteins. Total protein recovery depends on both the amount of starting material and choice of extraction protocol, whereas total DNA recovery is significantly reduced using the unified protocol (mean 43%). Nevertheless, total DNA recovery from dental calculus is generally very high, and we found no differences in DNA fragment characteristics or taxonomic profile between the protocols. In conclusion, the unified protocol allows for simultaneous extraction of two complementary lines of biomolecular evidence from archaeological dental calculus without compromising downstream results, thereby minimizing the need for destructive analysis of this finite resource.

Analysis of multiple protein detection methods in human osteoporotic bone extracellular matrix: From literature to practice.

The punctual analysis of bone Extracellular Matrix (ECM) proteins represents a pivotal point for medical research in bone diseases like osteoporosis. Studies in this field, historically done to appreciate bone biology, were mainly conducted on animal samples and, up to today, only a few studies on protein detection in human bone are present. The challenges in bone ECM protein extraction and quantitation protocols are related to both the separation of proteins from the mineral content (i.e. hydroxyapatite) and the difficulty of avoiding protein denaturation during the extraction processes. The aim of the present work was to define appropriate protocol(s) for bone ECM protein extraction that could be applied to investigate both normal and pathological conditions. We compared and optimised some of the most used protocols present in the literature, modifying the protein precipitation method, the buffer used for resuspension and/or the volume of reagent used. Bradford and BCA assays and Western Blotting were used to evaluate the variations in the total protein recovery and the amount of selected proteins (Type I Collagen, TGF-β, IGF-1, Decorin, Osteopontin, Bone Sialoprotein-2 and Osteocalcin). Collectively, we were capable to draw-up two single-extract protocols with optimal recovery and ideal protein content, that can be used for a detailed analysis of ECM proteins in pathological bone samples. Time-consuming multi-extract procedures, optimised in their precipitation methods, are however crucial for a precise detection of specific proteins, like osteocalcin. As the matter of fact, also the demineralization processes, commonly suggested and performed in several protocols, could hinder an accurate protein detection, thus inherently affecting the study of a pathological bone ECM. This study represents a starting point for the definition of appropriate strategies in the study of bone extracellular matrix proteins involved in the onset and maintenance of bone diseases, as well as a tool for the development of customized scaffolds capable to modulate a proper feedback loop in bone remodelling, altered in case of diseases like osteoporosis.