Total Protein Assay Research Articles

A-136 Icteric Interference is not Resolved with Sample Dilution in Serum Creatinine and Total Protein Assays

Abstract Background Icterus is a common endogenous interference that can cause falsely low results for serum creatinine and total protein. It can be defined by elevated unconjugated (indirect) and conjugated (direct) bilirubin levels and is most commonly associated with liver dysfunction. Recent literature suggests that dilutions can be a reliable method to reduce bilirubin interference. The purpose of this study was to determine whether the vendor’s on-board sample dilution can eliminate icteric interference with creatinine and total protein. Methods Creatinine (enzymatic) and total protein interference studies were performed on an automated chemistry analyzer (Roche Diagnostics, cobas c702). Low, mid, and high sample pools for each analyte were divided into four equal volumes. To achieve an icterus index (II) of approximately 40 (approximately 40 mg/dL of bilirubin), one sample pool was spiked with unconjugated bilirubin, and one pool was spiked with conjugated bilirubin. An equal amount of diluent was added into each control pool and remained at an II of approximately 0. The icteric and control samples were measured for conjugated and unconjugated bilirubin, serum indices, and the target analyte. Using the on-board dilution function, the icteric creatinine and total protein pools were diluted using a 1:4 and 1:3 dilution, respectively. All samples were analyzed in triplicate. Percent variance was found by calculating the difference between the average dilution result and control result and then dividing by the average control result. Results Conclusions Only modest alleviation of icteric interference was noted with dilution. The average bias was 10% for creatinine and 24% with total protein, after dilution. The on-board sample dilution is not effective in eliminating icteric interference when measuring both creatinine and total protein.

Dachengqi decoction dispensing granule ameliorates LPS-induced acute lung injury by inhibiting PANoptosis in vivo and in vitro

Ethnopharmacological relevanceAcute lung injury (ALI) is a serious health-threatening syndrome of intense inflammatory response in the lungs, with progression leading to acute respiratory distress syndrome (ARDS). Dachengqi decoction dispensing granule (DDG) has a pulmonary protective role, but its potential modulatory mechanism to alleviate ALI needs further excavation. Aim of the studyThis study aims to investigate the effect and potential mechanism of DDG on lipopolysaccharide (LPS)-induced ALI models in vivo and in vitro. Materials and methodsLPS-treated Balb/c mice and BEAS-2B cells were used to construct in vivo and in vitro ALI models, respectively. Hematoxylin-eosin (HE), Wet weight/Dry weight (W/D) calculation of lung tissue, and total protein and Lactic dehydrogenase (LDH) assays in BALF were performed to assess the extent of lung tissue injury and pulmonary edema. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), and interleukin-18 (IL-18) in BALF, serum, and cell supernatant. The qRT-PCR was used to detect inflammatory factors, Z-DNA binding protein 1 (ZBP1), and receptor-interacting protein kinase 1 (RIPK1) expression in lung tissues and BEAS-2B cells. Double immunofluorescence staining and co-immunoprecipitation were used to detect the relative expression and co-localization of ZBP1 and RIPK1. The effects of LPS and DDG on BEAS-2B cell activity were detected by Cell Counting Kit-8 (CCK-8). Western blot (WB) was performed to analyze the expression of PANoptosis-related proteins in lung tissues and BEAS-2B cells. ResultsIn vivo, DDG pretreatment could dose-dependently improve the pathological changes of lung tissue in ALI mice, and reduce the W/D ratio of lung, total protein concentration, and LDH content in BALF. In vitro, DDG reversed the inhibitory effect of LPS on BEAS-2B cell viability. Meanwhile, DDG significantly reduced the levels of inflammatory factors in vitro and in vivo. In addition, DDG could inhibit the expression levels of PANoptosis-related proteins, especially the upstream key regulatory molecules ZBP1 and RIPK1. ConclusionDDG could inhibit excessive inflammation and PANoptosis to alleviate LPS-induced ALI, thus possessing good anti-inflammatory and lung-protective effects. This study establishes a theoretical basis for the further development of DDG and provides a new prospect for ALI treatment by targeting PANoptosis.

Detection of residual T7 RNA polymerase used in mRNA in vitro transcription by Simple Western.

Therapeutic messenger RNA (mRNA) has been demonstrated as a scalable and versatile vaccine platform for the rapid development and manufacture of new vaccine candidates. mRNA is synthesized enzymatically through in vitro transcription (IVT) using bacteriophage T7 RNA polymerase (T7 RNAP), a 99kDa protein with high binding affinity for the promoter sequence and a low error rate. Post-IVT, mRNA is purified to remove impurities, but if T7 RNAP is insufficiently cleared, undesirable clinical side effects may result. Therefore, it is important to quantitate T7 RNAP concentrations in IVT and process intermediates to understand clearance during downstream purification. A high-throughput T7 RNAP assay was developed using Simple Western (SW), a capillary immunoassay technology, to quantitate concentrations as low as 5.3ng/mL with good precision and accuracy. Compared to existing T7 RNAP immunoassays or total protein assays such as bicinchoninic acid assays or Bradford, the SW T7 RNAP assay is specific to T7 RNAP, requires <10µL of sample volume, and consists of minimal sample handling and hands-on time. This work highlights the development and optimization of a highly sensitive and robust T7 RNAP quantitation assay using the SW platform.

Quantitation of milk proteins in thermally treated milk samples and commercial food products by ELISA test kits

This study evaluated four ELISA kits for quantitation of milk proteins in thermally treated milk samples and food products. How reference materials may be used for comparison of kit performance was examined. Protein contents determined by Veratox Total Milk generally reflected those determined by the 660 nm total protein assay. BioKits BLG Kit was less affected by thermal treatment but resulted in overestimation of protein contents in samples that were boiled, autoclaved or dry-heated at ≤149 °C, while ELISA Systems Casein (ES Casein) and Beta-Lactoglobulin (ES BLG) assays underestimated protein levels in these samples. The four kits gave similar results for ice cream. Veratox registered higher concentrations in all products tested but its sensitivity was greatly lowered in retorted products. ES Casein underperformed Veratox for baked and retorted products. BioKits BLG maintained a better sensitivity towards fried, baked and retorted products while ES BLG exhibited reduced sensitivity for these products.

Resistive spontaneous breathing exacerbated lipopolysaccharide-induced lung injury in mice

BackgroundSpontaneous respiratory mechanical force interacted with the primary lung injury and aggravated the progression of ARDS clinically. But the exact role and involved mechanism of it in the pathogenesis of ARDS animal model remained obscure. AimThis study was to investigate the effect of spontaneous respiratory mechanical force on lung injury of ARDS in mice. MethodsFemale C57BL/6 mice were subjected to resistive spontaneous breathing (RSB) by tracheal banding after 4–6 h of intranasal inhalation of LPS. Pulmonary function was examined by Buxco system, partial pressures of oxygen and carbon dioxide (PO2 and PCO2) were measured by a blood gas analyzer, and lung pathological changes were analyzed with hematoxylin and eosin staining. The levels of inflammatory markers were quantified by ELISA, total protein assay, and FACS analysis. The expression levels of mechanosensitive ion channels were detected by qRT-PCR and immunohistochemistry. ResultsThe airway resistance (Raw) was increased and the tidal volume (TV) was decreased remarkedly in RSB group. RSB treatment did not affect PO2, PCO2, pathology and inflammation levels of lung in mice. The Raw increased and ventilatory indicators decreased in RSB + ARDS compared to ARDS significantly. Besides, RSB treatment deteriorated the changes of PO2, PCO2 and level of lactic acid induced by LPS. Meanwhile, RSB significantly promoted LPS-induced pulmonary histopathological injury, and elevated the levels of IL-1β, IL-6, TNF-α and total proteins, increased neutrophils infiltration. The expression level of Piezo1 in RSB + ARDS group was remarkably reduced compared to ARDS group and consistent with the severity of pulmonary damage. ConclusionRSB exacerbated LPS-induced ARDS hypoxemia and hypercapnia, inflammation and damage. The mechanosensitive protein Piezo1 expression decreased and may play an important role in the process.

Preparation and evaluation of ecofriendly nanocreams containing some plant essential oils and monoterpenes against adults of Culex pipiens L. with some biochemical and histological studies on albino rat

Plant essential oils (EOs) are considered a vital tool of novel natural mosquito repellents and botanical adulticides. Five plant EOs (cinnamon, cypress, lavender, lemon eucalyptus and tea tree) and their major constituents (cinnamaldehyde, citronellal, β-cymene, (R)-linalool, and α-terpinyl acetate) were investigated against adults of Culex pipiens. The efficacy of the tested compounds was manipulated as mortality and knockdown using a fumigation technique. After that, the most active compounds against adults (lemon eucalyptus oil and linalool) were investigated once more as repellents after incorporating them on a cream base against C. pipiens adults compared to their nano-cream using arm-in-cage technique. In addition, the biochemical and histological effects of dermal treatment of linalool, lemon eucalyptus,, and their nanoemulsions (NEs) were studied on male albino rats. Total protein assay, acetylcholinesterase (AChE), adenosine triphosphatase (ATPase), and liver and kidney functions were determined in blood serum. Complete blood count (CBC) was determined in whole blood. The results showed that lemon eucalyptus oil and (R)-linalool caused the highest knockdown activity against C. pipiens adults with Kt50 40.29 s and 12.73 s, respectively. The repellent effect (RC50) of nanocream formulations of lemon eucalyptus oil (10.03 mg/L) and (R)-linalool (68.11 mg/L) were higher than the original effects of these compounds with RC50 values = 100.82 mg/L and 998.54 mg/L, respectively. There are no obvious harmful side effects of the dermal topical treatments of (R)-linalool and lemon eucalyptus oil on the tested biochemical parameters of treated albino rats compared with the control. Furthermore, there are no obvious effects of the dermal topical treatments of (R)-linalool and lemon eucalyptus oil on the histological status of the treated skin of albino rats compared with untreated treatment. The tested oils and monoterpenes could be considered promising candidates for botanical adulticides against C. pipiens. Also, nano-cream of lemon eucalyptus oil and (R)-linalool could be considered promising ecofriendly repellents for C. pipiens adults.

Development of a smartphone enabled, paper-based quantitative diagnostic assay using the HueDx color correction system.

Color correction is an important methodology where a digital image's colors undergo a transformation to more accurately represent their appearance using a predefined set of illumination conditions. Colorimetric measurements in diagnostics are sensitive to very small changes in colors and therefore require consistent, reproducible illumination conditions to produce accurate results, making color correction a necessity. This paper presents an image color correction pipeline developed by HueDx, Inc., using transfer algorithms that improve upon existing methodologies and demonstrates real-world applications of this pipeline in colorimetric clinical chemistry using a smartphone enabled, paper-based total protein diagnostic assay. Our pipeline is able to compensate for a variety of illumination conditions to provide consistent imaging for quantitative colorimetric measurements using white-balancing, multivariate gaussian distributions and histogram regression via dynamic, non-linear interpolating lookup tables. We empirically demonstrate that each point in the color correction pipeline provides a theoretical basis for achieving consistent and precise color correction. To show this, we measure color difference with deltaE (ΔE00), alongside quantifying performance of the HueDx color correction system, including the phone hardware, color sticker manufacturing quality and software correction capabilities. The results show that the HueDx color correction system is capable of restoring images to near-imperceptible levels of difference independent of their original illumination conditions including brightness and color temperature. Comparisons drawn from the paper-based total protein assay calibrated and quantified with and without using the HueDx color correction pipeline show that the coefficient of variation in precision testing is almost twice as high without color-correcting. Limits of blank, detection and quantitation were also higher without color-correction. Overall, we were able to demonstrate the HueDx platform improves reading and outcome of the total protein diagnostic assay and is useful for the development of smartphone-based quantitative colorimetric diagnostic assays for point-of-care testing.

Evaluation of ELISA Test Kits for Detection of Milk Protein in Frying Oil Treated at Different Temperatures

Milk is a common ingredient in fried foods. Allergen cross-contact can occur through the reuse of frying oil. To enable assessment of the allergy risk of reused oil, methods for quantification of milk protein in oil are needed. This study evaluated four commercial ELISA test kits in comparison with the 660 nm total protein assay for the detection of milk protein in oil after frying. Corn oil spiked with nonfat or whole milk powder were fried at 150 °C or 180 °C for 3 min and were analyzed by ELISA kits either directly or after preextraction with phosphate-buffered saline containing 0.05% Tween (PBST). All four ELISA kits performed well in quantifying milk protein in unheated oil, achieving normalized recoveries of 72.1–115.9% compared with that determined in reference solutions (PBST spiked with nonfat or whole milk powder, 100%). Frying lowered the amount of protein detected, but the extent of reduction differed between test kits. In nonfat milk powder-spiked oil fried at 150 °C, normalized recoveries determined by Veratox Total Milk and BioKits BLG Assay (49.9% and 43.6%, respectively) were higher than that determined by the 660 nm assay (25.4%). Normalized recoveries determined by ELISA Systems Casein and Beta-Lactoglobulin (BLG) kits were substantially lower (9.7% and 2.4%, respectively). In samples fried under typical frying temperature (180 °C), very little protein (0.1–7.4%) was detected. Inclusion of PBST preextraction improved the detection of the two test kits targeting BLG but lowered the level of protein detected by Veratox and ELISA Systems Casein in fried samples. Overall, the ELISA kits evaluated could effectively quantify milk protein in unheated oil without the need to remove the oil phase prior to analysis. Heat treatment was the key factor negatively affecting protein quantitation. Such impact needs to be considered when ELISA test results are used for assessing the allergy risk of reused frying oil.

An aluminium adjuvant compound with ginseng stem leaf saponins enhances the potency of inactivated Pseudomonas plecoglossicida vaccine in large yellow croaker (Larimichthys crocea)

Large yellow croaker (Larimichthys crocea) farm industry in China suffered from huge economic loss caused by Pseudomonas plecoglossicida infection. Due to multi-antibiotic resistance, efficient vaccines are urgent to be developed to combat this pathogen. In this study, an inactivated vaccine was developed with an aluminium adjuvant (Alum) plus ginseng stem and leaf saponins (GSLS). As a result, the relative percentage survival (RPS) against P. plecoglossicida was up to 67.8 %. Comparatively, RPS of groups that vaccinated with only inactivated vaccine and vaccine containing Alum or Montanide™ 763A as adjuvant were 21.8 %, 32.2 % and 62.1 %, respectively. Assays for total serum protein and serum lysozyme activity in group vaccinated with inactivated vaccine plus Alum + GSLS adjuvant were significantly higher than that in control group. Moreover, specific antibody in serum elicited a rapid and persistent level. According to the expression of some immune related genes, inactivated vaccine plus Alum + GSLS adjuvant induced a stronger cellular immune response which was vital to defend against P. plecoglossicida. In conclusion, our study demonstrated that the compound Alum and GSLS adjuvant is a potential adjuvant system to develop LYC vaccine.

Prophylactic activities of Olax viridis in the liver of rats challenged with carbon tetrachloride

Olax viridis is a plant used for its ethnomedical properties. This experiment was focused at assessing the prophylactic activities of O. viridis against carbon tetrachloride (CCl4) - induced hepatotoxicity. Twenty-five male albino rats were randomly divided into five categories. Categories 1 and 2 received distilled water, category 3 received the extract at 100 mg/kg body weight (bw), 4 received the extract at 200 mg/kg while 5 received Silymarin at 100 mg/kg. The route of administration was per os, 12 hourly for five days. One hour after the last treatment, groups 2-5 were challenged orally with 0.15 ml/kg of CCl4 in olive oil. Eighteen hours later, blood samples were collected for serum assay of aspartate amino-transferase, alanine amino-transferase, alkaline phosphatase, total bilirubin and total protein. The effect of the extract on CCl4 alteration of pentobarbitone sleeping time was assessed using another 5 groups of five rats each. The animals were treated as described for the first experiment. There was no significant difference (P &lt; 0.05) between the serum biochemical markers of the animals given 100mg/kg bw of the extract, those given 100mg/kg bw of silymarin and those of the control group but they were significantly different (P &gt; 0.05) from those of the category treated with 200mg/kg of the extract and the category given only CCl4. Similar trend was observed in the pentobarbitone-induced sleeping time experiment. This study reveals that the methanolic root extract of O. viridis can protect the liver against CCl4 induced hepatotoxicity.

A-111 There is More to Diluting Icterus Interference than Meets the Eye

Abstract Background Total protein is measured in serum and body fluids as part of routine evaluations of general nutritional status and differentiating exudative and transudative effusions. Spectral interference thresholds for bilirubin (e.g., icterus) is set by assay manufacturers to prevent inaccurate results from being reported. The Roche Cobas serum total protein assay package insert states no significant interference from bilirubin up to an icterus (I) index of 20. Body fluids tend to have lower total protein concentrations; therefore, thresholds were derived during prior validation studies and set to I = 10, a threshold that is exceeded routinely. Laboratories can mitigate some interferences by recollecting (e.g., hemolysis) or ultracentrifuging (e.g., lipemia) specimens. Serial dilutions are another option used; however, it is imperative that the laboratory understands the mechanism of interference and can demonstrate that it is mitigated by sample dilution. The aim of this study was to investigate whether bilirubin interference can be mitigated by sample dilution in pleural fluid, peritoneal fluid, and serum specimens. Methods Residual clinically ordered pleural fluid (n = 3), peritoneal fluid (n = 3), and serum (n = 3) samples submitted for protein testing were split into two equal volume aliquots with concentrations ranging 2.7 to 4.3 g/dL in pleural fluids, 3.9 to 5.4 g/dL in peritoneal fluids, and 3.8 to 11.0 g/dL in serum. One aliquot was spiked (&amp;lt;10% by volume) with a bilirubin conjugate solution (∼1000 mg/dL) to surpass the icterus index threshold (spiked). The second aliquot was spiked with an equal volume of 0.9% saline (control). Total protein was measured on the Roche Cobas c701 instrument in both samples to calculate difference (Spiked-Control). The mean(SD) difference was calculated for each sample type. Each spiked sample was serially diluted (2-fold, 4-fold and 8-fold) with 0.9% saline. Total protein was measured for each diluted sample. Mean(SD) difference was calculated (DilutedX-Control), where X = 2-fold, 4-fold, 8-fold. Linear regression analysis was performed by plotting the measured (x-axis) vs expected (y-axis) total protein concentration. Slope, y-intercept, and R2 were determined from serially diluted samples where the expected concentrations were derived using the spiked concentration to replicate the practice of diluting an icteric sample. Results Bilirubin spiking (average icterus index = 33.5) into pleural fluid, peritoneal fluid, and serum, respectively demonstrated mean(SD) bias (g/dL) = −1.0(0.2), −0.9(0.0), and −0.7(0.0). Subsequent 2-fold dilution demonstrated mean(SD) bias (g/dL) = −1.0(0.2), −1.0(0.0), and −0.8(0.1), 4-fold dilution with mean(SD) bias (g/dL) = −1.1(0.2), −1.0(0.0), and -1.0(0.1), and 8-fold dilution with mean(SD) bias (g/dL) = −1.3(0.2), −1.0(0.0), and −1.4(0.1). Diluting pleural, peritoneal, and serum samples, respectively spiked with bilirubin resulted in slope = 0.99, 1.00, and 0.99; y-intercept = 0.04, 0.01, and 0.07; R2 = 0.99, 0.99, 0.99. Conclusion The dilutions exhibited a linear dilution response, however the dilution results never returned to baseline (non-spiked) concentration. Laboratories should exercise caution when conducting serial dilutions to diminish bilirubin interference in samples when measuring total protein as the mechanism of interference does not appear to be reversible by dilution. This behavior was demonstrated in both body fluids and serum.

Hepatoprotective effect of Vernonia amygdalina and Ocimum gratissimum on Wistar rats exposed to long-term administration of artemisinin-based combination therapies

In this study the protective effect of Vernonia amygdalina (VA) and Ocimum gratissimum (OG) on rats exposed to long term oral administration of artesunate-amodiaquine and arthemeter-lumefantrine were investigated. Forty-two albino rats were divided into seven groups. They were given the drugs, artesunate amodiaquine (AA) and artemether lumefantrine (AL) base on their body weight. Group 1: Control, received distilled water, group 2, received of 2.86 mg/7.7 mg AA, group 3, received of 1.14 mg/6.86 mg AL, group 4 received of 2.86 mg/7.7 mg AA + 200 mg VA, group 5 received of 1.14 mg/6.86 mg AL + 200 mg VA, group 6 received of 2.86 mg/7.7 mg AA + 200 mg OG and group 7 received of 1.14 mg/6.86 mg AL + 200 mg OG. The animals were sacrificed and blood samples obtained through cardiac puncture for biochemical investigations. Biochemical assay for total protein, albumin, total bilirubin, direct bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma glutamyltransferase (γ-GT) were done. Co-administration of artesunate-amodiaquine and arthemeter-lumefantrine significantly decreased (P&lt; 0.05) total protein and albumin while there was significant increase (P&lt; 0.05) in total bilirubin, direct bilirubin, AST, ALT, ALP and γ-GT when compared with control group. The administration of VA and OG significantly increased the total protein and albumin and significantly decreased (P&lt; 0.05) total bilirubin and liver enzymes when compared with control group. The results indicate hepatic injury in the rats exposed to long term administration of artemisinin-based combination therapies (ACTs) which was reversed by the administration of VA and OG. There is need for caution while taking ACTs as malaria chemotherapy.

Cola acuminata Mitigates Cognitive Deficit and Oxidative Stress in Mercury Chloride-induced Neurotoxicity in Male Wistar Rats

Cola acuminata is used in traditional medicine for the management of memory impairment and other neurodegenera-tive conditions. This study investigated the effects of Cola acuminata aqueous leaves extract (ALECA) on mercury chloride-induced neurotoxicity in Wistar rats. Twenty male Wistar rats weighing between 160 and 210 g were randomly assigned to four groups (n = 5). The control group received 0.5 mL of distilled water; the mercury chloride (HgCl2) group received HgCl2 (5 mg/kg b.w.); the ALECA100 and ALECA300 groups received ALECA (100 and 300 mg/kg b.w., respectively), followed by the administration of HgCl2 (5 mg/kg b.w.) for two weeks. The rats were subjected to behavioural tests in the Morris water maze and light and dark field box. The rats were then sacrificed to obtain their brains, which were homogenized for biochemical assays of acetylcholinesterase (AChE), malondialdehyde (MDA), total protein (TP), and glutathione (GSH) using standard methods. The results revealed a significant increase in escape latency, a significant decrease in probing frequency and brain GSH, and a significant (p&lt;0.05) increase in brain MDA and TP levels and AChE activity in the rats exposed to HgCl2. However, administration of either 100 or 300 mg/kg ALECA protected against memory impairment with a significantly reduced escape latency, increased probing frequency and brain GSH, and decreased (p&lt;0.05) MDA, TP and AChE. This study concludes that ALECA mitigated HgCl2-induced neurotoxicity via reduction of oxidative stress and enhanced cholinergic functions

Straightforward monitoring of honey with foreign diastase by leveraging the differentiation in LC-UV proteome profiles of authentic and fraudulent samples

Honey adulteration using sugar syrups is ubiquitous and adulterated honey may also be prepared by direct addition of α-amylases from different sources to match the legislation for honey trade. Colorimetric diastase assays may report false negatives in terms of foreign diastase (FD) detection owing to using substrates being prone to be cleaved by any α-amylases. The main objective of this study was to develop a reliable analytical method to determine FD contained honey. For this purpose, intrinsic honey proteins were first isolated, enriched, and cleaned up via experimentally designed serial ultrafiltration methodology. Next, diastase and invertase as abundant honey enzymes were purified simultaneously from obtained crude protein isolate by means of FPLC and novel purification protocol. Total protein and enzyme activity assays along with SDS-PAGE analysis were conducted for purity check. Subsequently, two purity-confirmed enzymes were used as analytical references in method development. With the hyphenation of UV detection, optimized chromatographic resolution, and practical dilute & shoot sample pretreatment, Foreign Amylase Monitoring (FAM) method has been introduced for routine analysis. Purified authentic enzymes were labeled in LC-UV chromatogram of honey proteome profile to distinguish any unnatural enzyme/protein signal. According to the validation of the FAM method, precise (RSDR; 1.27%), and linear (mean R2 >0.995) results were able to obtain. Commercial honey samples (n = 202) collected over 3 consecutive years were probed using the FAM method and industrial α-amylases were also analyzed for verification. The diastase activities were also measured prior to FAM application to elucidate if any positive correlation. The developed method rapidly and reliably detected the presence of FD qualitatively. A total of 74 samples were found to contain FD. It was observed that the presence of FD was correlated in samples with abnormally high diastase activities and an apparent FD peak was identified readily in all enzyme adulterated samples. The developed FAM method and performed trend analysis allowed us to decipher a dramatic increase in FD existence and this revealed the recently emerging problem of honey authenticity.

Volatile Components’ Variation Analysis on Ginseng Stems and Leaves at Different Growth Ages by HS-SPME-GC-MS

Panax ginseng is a famous valuable folk herb, the price of which particularly depends on its cultivation age. Since the classical chemical analysis usually needs to destroy the plants which is not suitable for the old wild ginseng due to the huge cost, in the current study, we employed the headspace solid-phase microextraction (HS-SPME) GC-MS methodology which has the advantage of time-saving, nondestructive, and green to analyze the growth age of ginseng (roots) via determining the volatile components of ginseng stem and leaves (GSLs) combined with metabolic profiles way. 100 mg of finely ground GSL samples (3–7 years) were extracted by a 65 μm PDMS-DVB fiber in the headspace and then analyzed within 20 mins. The content assay of total saponins, crude polysaccharides, and total protein was also included. As a result of GC-MS-based profiling, the orthogonal partial least-squares discriminant analysis’ (OPLS-DA) score plots showed excellent classification of four comparison groups (the 3- and 4-, 3-and 5-, 3- and 6-, and 3- and 7-year-old GSL) with 100% discrimination rate, respectively. 263 common differential variables were gained referring to the VIP and P values. 32 volatile metabolites were identified and showed good age discrimination capacities with an area under the curve (AUC) value of more than 0.8 by the receiver operating characteristic (ROC) analysis. Remarkably, there was one volatile component of sesquiterpenes, 1-cyclohexene-1-carboxaldehyde, 2, 6, 6-trimethyl-, which showed an excellent prediction with the AUC value of 1.0 among all the four compared groups, which could be the potential biomarker to distinguish all the four compared groups. The total contents of different growth ages of components of ginseng stems and leaves were distinct. The contents of total protein (4.80 ± 0.27%) and crude polysaccharide (24.15 ± 0.89%) in the 4th year of GSLs were the highest among the five cultivation years. The content of total saponins (4.66 ± 0.38%) in the 5th year of GSLs was the highest. These results could provide a novel reference for the GSLs’ harvest and application periods. Moreover, the current study could also supply a promising way to set up a nondestructive, in situ, and green approach to discriminate the growth age of Panax ginseng rapidly without digging the roots out instead of detecting the VOCs of GSLs.

Studies on Acute and Sub-Chronic Toxicities of N-Hexane Seed Extract of Ricom-1013-J in Wistar Rats

The study investigated the acute and sub-chronic effects of the n-hexane seed extract of RICOM-1013-J in biochemical and haematological parameters of mature adult female Wistar rats with an average weight of 110 g. The objective of the study was to determine the immediate, short-term and prolonged toxicological profile of the extract. Thirty-four (34) adult female rats were randomly divided into three groups (n=8) of rats. Two animals (group I) and coded A received 0.2 ml/100 g rat of vegetable oil, and served as control. Two other animals (group II) coded B received 5 mg/kg, subcut. of the extract, a third set of animals (group III) code C was administered 20 mg/kg, subcut. The last two animals (group IV) coded D were injected with 30 mg/kg, subcut. of the extract. The same dosage regimen was administered to a different set of animals of equal number in groups II and III in pairs, and coded accordingly. SGOT, SGPT, urea, total protein, alkaline phosphatase, bilirubin and haematological assays were carried out at weeks I, IV and VIII in groups I, II and III respectively. Findings from the experiment showed no significant (P&gt;0.05) differences in the mean values of the biochemical parameters and haematological indices test groups relative to control. The study concluded that the n-hexane seed extract of RICOM-1013-J is relatively safe in rats when administered subcutaneously.

Performance verification of a biochemical detection system for hydrothorax and ascites and clinical diagnostic accuracy evaluation of exudate and tuberculous effusion.

Lactate dehydrogenase (LDH), total protein (TP) and glucose (Glu) in pleural hydrothorax and ascites can be used in the diagnosis of exudate, and adenosine deaminase (ADA) can be used in the diagnosis of tuberculous effusion. However, the manufacturers do not claim that their biochemical reagents can be used to detect hydrothorax and ascites samples. Therefore, medical laboratories must conduct suitability studies on biochemical reagents for hydrothorax and ascites samples to comply with regulatory requirements for humor detection. This study aimed to verify the analytical performance and clinical diagnostic accuracy of the Mindray biochemical reagents, including LDH, TP, Glu and ADA, for hydrothorax and ascites. The repeatability, detection limits and reference intervals of Mindray biochemical reagents (LDH, TP, Glu, ADA) in detecting hydrothorax and ascites were determined. The comparison of different measurement procedures was performed. Meanwhile, the diagnostic accuracy of LDH, TP, Glu and ADA were assessed. The quality control results of LDH, TP, Glu, and ADA were all under control. The repeatability coefficient of variation (%) of LDH, TP, Glu, and ADA were all less than 1%. The limits of blank of LDH, TP, Glu, and ADA were 0.33 U/L, 0.45 g/L, 0.00 mmol/L, and 0.04 U/L, respectively; the limits of detection were 1.57 U/L, 1.85 g/L, 0.05 mmol/L, and 0.12 U/L, respectively. Compared with the reference measurement program, the correlation coefficients of LDH, TP, Glu and ADA were 0.9931, 0.9983, 0.9996 and 0.9966, respectively; the regression equations were y=1.0082x-10.06, y=0.9965x-0.4732, y=0.9903x+0.0522 and y=1.0051x-0.0232, respectively. The reference intervals of LDH, TP, Glu, and ADA in hydrothorax and ascites were ≤198.39 U/L, ≤32.97 g/L, ≥5.03 mmol/L. and ≤11.00 U/L respectively. For differentiating between exudates and transudates, the area under the curve (AUC) of LDH, TP, and Glu were 0.913, 0.875, and 0.767, respectively; the AUC of ADA for the differential diagnosis of tuberculous and nontuberculous effusions was 0.876. The LDH, TP, Glu, and ADA assays were validated for use with the Mindray BS-2800 analyzer for hydrothorax and ascites evaluation. LDH, TP, and Glu in hydrothorax and ascites are applicable to the differential diagnosis of exudates and transudates; ADA in hydrothorax and ascites can be employed to differentiate and diagnose tuberculous and nontuberculous effusions.

Analysis of influencing factors of serum total protein and serum calcium content in plasma donors.

The adverse effects of plasma donation on the body has lowered the odds of donation. The aim of this study was to investigate the prevalence of abnormal serum calcium and total serum protein related to plasma donation, identify the influencing factors, and come up with suggestions to make plasma donation safer. Donors from 10 plasmapheresis centers in five provinces of China participated in this study. Serum samples were collected before donation. Serum calcium was measured by arsenazo III colorimetry, and the biuret method was used for total serum protein assay. An automatic biochemical analyzer was used to conduct serum calcium and total serum protein tests. The mean serum calcium was 2.3±0.15 mmol/L and total serum protein was 67.75±6.02 g/L. The proportions of plasma donors whose serum calcium and total serum protein were lower than normal were 20.55% (815/3,966) and 27.99% (1,111/3,969), respectively. There were significant differences in mean serum calcium and total serum protein of plasma donors with different plasma donation frequencies, gender, age, regions, and body mass index (BMI), (all p<0.05). Logistic regression analysis revealed that donation frequencies, age, BMI and regions were significantly associated with a higher risk of low serum calcium level, and donation frequencies, gender, age and regions were significant determinants factors of odds of abnormal total serum protein. Donation frequencies, gender, age, regions, and BMI showed different effects on serum calcium and total serum protein. More attention should be paid to the age, donation frequency and region of plasma donors to reduce the probability of low serum calcium and low total serum protein.

Total protein assay by PCA-based RGB-spectrum conversion methods with smartphone-acquired digital images.

Total protein concentrations in the aqueous solutions were determined from the absorption spectra reproduced from smartphone-captured digital color images. We employed two different procedures for protein determination: the pyrogallol red molybdate method and Bradford's method. The principal-component-analysis-based reproduction process, which was previously reported by our research group, enabled the conversion of RGB values to score values for a linear combination of loading vectors to generate reproduced absorption spectra. The reproduced spectra were identical to those measured using a commercially available spectrophotometer. The total protein assays of commercial soymilk and human serum samples were carried out with both coloration reagents, and the obtained results were in good agreement with those attained using a conventional spectrophotometer. These results show that the proposed method enables smartphone-based ratiometric analysis of real samples without requiring any monochromating equipment.

Implementation of a Practical Teaching Course on Protein Engineering.

Simple SummaryProteins are the workhorses of the cell. With different combinations of the 20 common amino acids and some modifications of these amino acids, proteins have evolved with a staggering array of new functions and capabilities due to Protein Engineering techniques. The practical course presented was offered to undergraduate bioengineering and chemical students at the Faculty of Engineering of the University of Porto (Portugal) and consists of sequential laboratory sessions to learn the basic skills related to the expression and purification of recombinant proteins in bacterial hosts. These experiments were successfully applied by students as all working groups were able to isolate a model recombinant protein (the enhanced green fluorescent protein) from a cell lysate containing a mixture of proteins and other biomolecules produced by an Escherichia coli strain and evaluate the performance of the extraction and purification procedures they learned.Protein Engineering is a highly evolved field of engineering aimed at developing proteins for specific industrial, medical, and research applications. Here, we present a practical teaching course to demonstrate fundamental techniques used to express, purify and analyze a recombinant protein produced in Escherichia coli—the enhanced green fluorescent protein (eGFP). The methodologies used for eGFP production were introduced sequentially over six laboratory sessions and included (i) bacterial growth, (ii) sonication (for cell lysis), (iii) affinity chromatography and dialysis (for eGFP purification), (iv) bicinchoninic acid (BCA) and fluorometry assays for total protein and eGFP quantification, respectively, and (v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for qualitative analysis. All groups were able to isolate the eGFP from the cell lysate with purity levels up to 72%. Additionally, a mass balance analysis performed by the students showed that eGFP yields up to 46% were achieved at the end of the purification process following the adopted procedures. A sensitivity analysis was performed to pinpoint the most critical steps of the downstream processing.