Crude Serum Research Articles

Effects of feeding frequency on growth performance, muscle composition and serum biochemical indices of juvenile <italic>Siganus canaliculatus</italic>

The growth of fish is usually affected by the size and type of bait, feeding frequency, feeding rate and the ability of fish to absorb nutrients. Recent studies have found that feeding frequency is one of the key factors affecting the growth performance of fish. In this study, juvenile <italic>Siganus canaliculatus</italic> was used as the research object. The growth indices, muscle crude nutrients and serum biochemical indices were measured at different feeding frequencies. According to the physiological and biochemical indices of the fish body, the growth benefit under each feeding frequency was evaluated. The purpose is to find out the scientific feeding frequency for juvenile <italic>Siganus canaliculatus</italic>, and to provide the corresponding theoretical basis and scientific support for the actual production of <italic>Siganus canaliculatus</italic> and other fishes. To investigate the effects of feeding frequencies on the growth and physiology of juvenile <italic>Siganus canaliculatus</italic>, four feeding frequencies (1 meal·d^-1, 2 meals·d^-1, 3 meals·d^-1, 4 meals·d^-1) were set, marked as F1, F2, F3 and F4. Three parallel groups were set in each group for 16 weeks in a microfluidic breeding tank (effective volume 125 L). Results of the study showed: 1) Growth indicators: Body weight and body length in each group increased linearly with the breeding time, the growth trend was similar between F3 and F4. The feeding rate and feed conversion ratio were the highest in F3, significantly higher than those of F1 (<italic>P</italic><0.05). The hepatosomatic index of F4 was significantly higher than that of F1 (<italic>P</italic><0.05), and ratio of intestine weight to body weight and ratio of intestine length to body length of F1 were significantly higher than those of F4 (<italic>P</italic><0.05), condition factor of F3 and F4 was significantly higher than that of F1 and F2(<italic>P</italic><0.05). 2) Muscle crude nutrition: The muscle moisture content of F1 was significantly higher than that of the other three groups (<italic>P</italic><0.05), while the muscle crude protein content of F1 was significantly lower than that of the other three groups (<italic>P</italic><0.05). The muscle crude ash content of F2 and F3 was significantly higher than that of F1 and F4 (<italic>P</italic><0.05). 3) Serum biochemical indicators: The contents of glucose and total cholesterol were not significantly affected by feeding frequencies. The content of total protein (TP) of F2 and F3 was significantly higher than that of F1 and F4(<italic>P</italic><0.05), while the content of albumin (ALB) of F1 and F4 was significantly higher than that of F2 and F3 (<italic>P</italic><0.05). The content of triglyceride(TG) increased significantly with the breeding time (<italic>P</italic><0.05). The content of high-density lipoprotein (HDL) was the highest in F4, significantly higher than that of F1 and F2 (<italic>P</italic><0.05), and the content of low-density lipoprotein (LDL) of F4 was significantly lower than that of other three groups(<italic>P</italic><0.05). The activity of aspartate transaminase (AST) and alanine aminotransferase (ALT) was significantly lower than that of the other three groups (<italic>P</italic><0.05). Results showed that the optimal growth benefit could be gained at the feeding frequency of 3 meals·d^-1.

CLINICO-LABORATORY CHARACTERISTICS, RISK FACTORS AND OUTCOME OF ACUTE KIDNEY INJURY

OBJECTIVE: To determine the clinico-laboratory characteristics, risk factors and outcome related to acute kidney injury (AKI) in hospitalized patients. METHODS: This prospective study on 101 consecutive patients admitted to the Northwest General Hospital &amp; Research Centre, Peshawar, Pakistan was carried out from January-March 2019. Patients were staged according to Kidney Disease Improving Global Outcomes (KDIGO) guidelines and outcomes were measured in terms of in-hospital mortality and change in KDIGO staging. RESULTS: Majority (n=57/101; 56.43%) were males. Mean age of patients was 57.90±16.93 years. Twenty-seven (26.73%) patients were in AKI stage-I, 29 (28.71%) in stage-2 and 45 (44.55%) were in stage-3. Hypertension (n=74/101; 73.27%) was the commonest co-morbid condition. Sepsis (n=39/101; 38.61%) and hypovolemia (n=21/101; 20.8%) were most common risk factors for AKI. There was complete recovery in 43 (42.57%) patients. In-hospital mortality was 6.9% (n=7/101) and 57.1% (n=4/7) of these patients had KDIGO stage-3. Serum creatinine levels declined in 22 (21.78%) cases, remained static in 23 (22.8%) and worsened in 06 (5.9%) cases. Male gender and presence of hypovolumia (p&lt;0.05) significantly differed in survivors compared to non-survivors. Furthermore, factors associated with declined in serum creatinine included stage-II AKI and length of hospital stay, while stage-II and III AKI on admission, absence of oliguria crude odd ratio (cOR: 2.681, p=0.027, 95% CI: 1.12-6.44)&nbsp; and serum creatinine levels on admission (cOR: 0.668, p&lt;0.001, 95% CI: 0.54-0.83) were associated with complete recovery. CONCLUSION: Sepsis and hypovolemia constituted the major risk factors. Gender and hypovolemia were the significant factors between the survivors and non-survivors.

Serum IgM heavy chain sub-isotypes and light chain variants revealed in giant grouper (Epinephelus lanceolatus) via protein A affinity purification, mass spectrometry and genome sequencing

Two IgM heavy (H) chain sub-isotypes (80 and 40 kDa) and two light (L) chain variants (25 and 30 kDa) were detected in the serum of giant grouper (Epinephelus lanceolatus), purified by ammonium sulphate precipitation followed by protein A affinity chromatography. This method yielded 5.6 mg/mL high purity IgM from grouper serum, with efficiency estimated at 39.5% recovery from crude serum. The H and L chains were identified by SDS-PAGE and mass spectrometry (MS). Nanopore long-read sequencing was used to generate a genomic contig (MW768935), containing Cμ, Cδ loci, VH regions, and a H chain Joining segment. cDNA sequencing of Cμ transcripts (MW768933 and MW768934) were used to polish the genomic contig and determine the exons and introns of the corresponding locus. MS peptide mapping revealed that the 80 kDa H chain consisted of CH1-4 domains while peptides from the 40 kDa H chain only mapped to CH1-2 domains. Our genomic contig showed the Cμ locus has a Cμ1-Cμ2-Cμ3-Cμ4 arrangement on the same strand as the other Ig loci identified in this genomic sequence. Our study corrects the NCBI annotations of the opposing Cμ loci (LOC117268697 and LOC117268550) in chromosome 16 (NC_047006). Further, we identified both κ and λ L chain isotypes in serum IgM. The molecular weight differences observed may result from different combinations of CL and VL genes. Putative IgM sub-isotypes have also been reported in Epinephelus itajara and Epinephelus coioides. The presence of IgM sub-isotypes may be a conserved trait among Epinephelus species.

Molecularly imprinted spongy columns for Angiotensin(II) recognition from human serum.

Angiotensin II (AngII), the effector peptide of the renin angiotensin system and has an important role in regulating cardiovascular hemodynamics and structure. AngII is an important biomarker for certain diseases that are associated with cardiovascular disorders, i.e., influenza, SARS‐CoV‐2, tumors, hypertension, etc. However, AngII presents in blood in very low concentrations and they are not stable due to their reactivity, therefore spontaneous detection of AngII is a big challenge. In this study, AngII‐imprinted spongy columns (AngII‐misc) synthesized for AngII detection from human serum, and characterized by surface area measurements (BET), swelling tests, scanning electron microscopy (SEM), FTIR studies. AngII binding studies were achieved from aqueous environment and maximum binding capacity was found as 0.667 mg/g. It was calculated that the AngII‐miscs recognized AngII 8.27 and 14.25 times more selectively than competitor Angiotensin I and Vasopressin molecules. Newly produced AngII‐misc binds 60.5 pg/g AngII from crude human serum selectively. It has a great potential for spontaneous detection of AngII from human serum for direct and critical measurements in serious diseases, that is, heart attacks, SARS‐CoV‐2, etc.

Fluorescence excitation properties of bilirubin in solution and in serum

The bichromophore nature of bilirubin explains the presence of at least two partially overlaying bands in both absorption and fluorescence emission spectra, and accounts for interchromophore exciton transfer events responsible for the emission sensitivity to the molecular environment and excitation wavelength. These concepts were likely responsible for the previously reported good yield of the unexpected remarkable bilirubin fluorescence emission under excitation at 366 nm, at which bilirubin absorption is very low. In this connection, aim of this work is to further investigate bilirubin spectral excitation properties and their diagnostic potential, until now poorly considered. Fluorescence excitation spectra of pure bilirubin in solution with solubilizing agents observed at 520 and 570 nm showed a wide region in the 430–510 nm range, similar to the absorption profile. In addition, an excitation band centered at about 400 nm was detected. Comparable excitation features were detected in rat serum. The 430–510 nm excitation region was well separated from a main band at shorter wavelength, ascribable to other endogenous fluorophores, with a shoulder at about 400 nm which was also easily discriminated by spectral fitting analysis. The bands ascribable to bilirubin showed changes of their relative contribution to the overall spectral region after liver ischemia/reperfusion, comparable to bilirubin biochemical data. Excitation spectra proved to discriminate the fluorescence of serum bilirubin at levels much lower than emission spectra, opening promising perspectives to improve the real time fluorescence analysis of crude serum in the absence of any exogenous labelling agent, and advance the diagnostic application of optical-biopsy in experimental hepatology and biomedicine.

Performance, serum biochemical indices and crude protein utilisation by broiler chickens fed diets based on two varieties of cassava (Manihot esculenta Crantz) grits I. Starter Phase

Effect of dietary inclusion of cassava grits (Manihot esculenta Crantz) from two cassava varieties TME 419 and TMS 01/1371 as replacement for maize on performance, serum biochemical indices and crude protein utilization of broiler chicks was investigated in a three-week trial with Abor Acre plus broiler chicks (n=210). In a completely randomized design, birds were allotted to seven treatments, each replicated thrice with 10 birds per replicate. Diet 1 comprised 50 % maize and 0 % cassava grits, diets 2, 3 and 4 had their respective maize content replaced at 25, 50 and 75 % with cassava grits from TMS 01/1371 while diets 5, 6 and 7 was replaced correspondingly with cassava grits from TME 419 at 25, 50 and 75 %, respectively for a duration of 21-day. Increased dietary cassava grits significantly lowered (P&lt;0.05) weight gain and metabolizable crude protein of broiler chicks. Birds on diets 3, 4, 6 and 7 had similar weight gain, feed conversion ratio and metabolizable crude protein. Except for significantly increased (P&lt;0.05) total protein and alkaline phosphatase due to interactions of cassava varieties and inclusion levels, effects of cassava varieties and inclusion levels on other serum indices were similar (P&gt;0.05). Regression of metabolizable crude protein on graded cassava grit inclusion for both varieties were negative and significant (P&lt;0.05) for TME 419 (R2 =0.355) and highly significant (P&lt;0.01) for TMS 01/1371 (R2 =0.6639). Both graphs clearly depicted decreased metabolizable crude protein due to increased cassava grits in the diets. Increased dietary cassava grits of both varieties resulted in lowered metabolizable crude protein and serum indices with concomitant increased feed conversion ratio of broiler starter chicks.

Direct colorimetric LAMP assay for rapid detection of African swine fever virus: A validation study during an outbreak in Vietnam

African swine fever (ASF) is a highly infectious viral disease with high mortality. The most recent ASF outbreak in Vietnam began in 2019, posing a threat to spread to the neighbouring Asian countries. Without a commercial vaccine or efficient chemotherapeutics, rapid diagnosis and necessary biosecurity procedures are required to control the disease. While the diagnostic method of ASF recommended by the World Organization of Animal Health is real-time PCR, the ideal diagnosis procedure including master mix setup, template extraction and a high-cost qPCR equipment for many samples being tested simultaneously is not portable. In this study, a colorimetric loop-mediated isothermal amplification (LAMP) assay was modified and evaluated for ASF virus detection using crude serum samples collected from domestic pigs in Vietnam during the 2019 outbreak. The LAMP results can be readily visualized to the naked eye within 30min without the requirement of DNA extraction and sophisticated equipment. The sensitivity, specificity and limit of detection of direct colorimetric LAMP assay were comparable to a commercial diagnostic real-time PCR kit. Results strongly indicate that the adapted colorimetric LAMP assay has a remarkable potential for the in-field diagnosis of ASF.

(Invited) Plasmonic and Surface Plasmon Electrochemiluminescence Sensors for Detecting Biomarkers and Drugs

In this presentation, a series of different plasmonic sensors for the detection of cancer biomarkers, cancer therapeutic drugs and the response of patients to oncological biological drugs will be presented. The plasmonic sensors are based on a portable 4-channel SPR instrument and surface chemistry developed to present fouling of the sensors in crude serum or plasma. Sensors for detecting methotrexate and for the immune response induced by asparaginase in leukemia treatments directly in sera of patients during ongoing therapies will be presented. Our work towards the detection of HER-2 positive breast cancers will also be presented, with a comparison of commercial and point of care SPR instruments. Our initial work on the development of a SPR-electrochemiluminescence instrument will be presented. We recently revealed that performing SPR and electrochemiluminescence on a single instrument provides unprecedented information about the interfacial processes occurring on the electrode. In addition, studying the energy transfer between the ECL and the plasmon revealed that the optically excited plasmon reduced the ECL intensity in the far-field by about 40% due to a lower plasmon mediated luminescence process. Hence, the combination of SPR and ECL is highly advantageous to study electrochemical processes.

Spectrofluorometric Analysis of Autofluorescing Components of Crude Serum from a Rat Liver Model of Ischemia and Reperfusion.

Autofluorescence (AF) of crude serum was investigated with reference to the potential of its intrinsic AF biomarkers for the noninvasive diagnosis of liver injury. Spectral parameters of pure compounds representing retinol (vitamin A) and fluorescing free fatty acids were characterized by spectrofluorometry, to assess spectral parameters for the subsequent AF analysis of serum, collected from rats undergoing liver ischemia/reperfusion (I/R). Differences in AF spectral profiles detected between control and I/R were due to the increase in the AF components representing fatty acids in I/R serum samples. No significant changes occurred for retinol levels, consistently with the literature reporting that constant retinol levels are commonly observed in the blood, except for malnutrition or chronic severe liver disease. Conversely, fatty acids, in particular arachidonic and linoleic acid and their derivatives, act as modulating agents in inflammation, representing both a protective and damaging response to stress stimuli. The biometabolic and pathophysiological meaning of serum components and the possibility of their direct detection by AF spectrofluorometry open up interesting perspectives for the development of AF serum analysis, as a direct, cost effective, supportive tool to assess liver injury and related systemic metabolic alterations, for applications in experimental biomedicine and foreseen translation to the clinics.

Screening for Human Immunodeficiency Virus Infection by Use of a Fourth-Generation Antigen/Antibody Assay and Dried Blood Spots: In-Depth Analysis of Sensitivity and Performance Assessment in a Cross-Sectional Study.

We evaluated the performance of a fourth-generation antigen/antibody (Ag/Ab) assay for detecting HIV-1 infection on dried blood spots (DBS) both in a conventional laboratory environment and in an epidemiological survey corresponding to a real-life situation. Although a 2-log loss of sensitivity compared to that with plasma was observed when using DBS in an analytical analysis, the median delay of positivity between DBS and crude serum during the early phase postacute infection was 7 days. The performance of the fourth-generation assay on DBS was approximately similar to that of a third-generation (antibody only) assay using crude serum samples. Among 2,646 participants of a cross-sectional study in a population of men having sex with men, 428 DBS were found reactive, but negative results were obtained from 5 DBS collected from individuals who self-reported a positive HIV status, confirmed by detection of antiretroviral (ARV) drugs in their DBS. The data generated allowed us to estimate a sensitivity of 98.8% of the fourth-generation assay/DBS strategy in a high-risk population, even including a broad majority of individuals on ARV treatment among those HIV positive. Our study brings additional proofs that DBS testing using a fourth-generation immunoassay is a reliable strategy able to provide alternative approaches for both individual HIV testing and surveillance of various populations.

Use of Human Umbilical Cord Serum to Treat Animal Skin Burns

Objective: The objective of the study was to test the hypothesis, that umbilical cord blood crude serum applied topically with its high concentration of growth factors may promote an early healing for animal models with burn injury.&#x0D; Methods: Fresh human umbilical cord blood UCBS was collected and screened for transmitted diseases such as hepatitis B, hepatitis C and HIV. Mice and rabbits were scalded by boiling water and chemical burning by NaOH, then treated with UCBS. In comparison with a Cetrimide treated group and a control group (without treatment). The UCBS was subjected to microbial testing to demonstrate the presence or absence of extraneous viable contaminating microorganisms.&#x0D; Results: Topical application of UCBS promoted the healing process; complete healing was seen after 10 days for the mice group and in 7 days in the rabbit group. Cetrimide group applied to the rabbit showed slow healing and needed 10 days for hair regrowth. Control group in both mice and rabbits showed very slow response and the burn area diameter remained the same for over 10 days, and no hair regrowth was obvious after 10 days. &#x0D; Conclusion: The results of the current study indicated that UCBS is a promising therapy for the healing of burns by boiling water and/or chemical substance, more clinical trials are needed to further explore the long-term effects after UCBS use.

CRUDE POLYSACCHARIDES EFFECT OF Coriolus versicolor ON Mycobacterium fortuitum-INDUCED IMMUNE DYSFUNTION IN MICE

Coriolus versicolor contains crude polysaccharides which have I²-glucan active ingredients. It can activate granulocyte, monocyte, and macrophage. Administration of crude polysaccharide of C. versicolor against bacterial infection is hypothesized to increase immune response. This study assessed the crude polysaccharides activity of C. versicolor in improving immune response after induction of non-tuberculosis mycobacteria, Mycobacterium fortuitum. Animal model was female mice strain BALB/c aged 8-10 weeks. A crude polysaccharide of C. versicolor was administered before and/or after the bacteria infection for 10 days at a dose of 50 mg/kg body weight. Mice exposure to M. fortuitum was performed twice at a dose of 0.5 Mc. Farland. After crude polysaccharides treatment, both serum and peritoneal fluid were isolated. All data collected were analyzed statistically using ANOVA and Duncan test. Oral administration of crude polysaccharide was found to increase phagocyte number (P 0.05, TNF-I± levels after crude polysaccharides administration were relatively the same as controls). Administration of crude polysaccharides of C. versicolor could enhance non-specific immune response, specific immune response, and pro-inflammatory cytokine in mice infected by M. fortuitum. These results suggested that crude polysaccharides of C. versicolor may be applied as an effective immunostimulatory agent.

Effects of different carbohydrate levels in puffed feed on digestive tract morphological function and liver tissue structure of snakeheads (Channa argus)

The major objective of this study conducted for 8 weeks was to investigate the influence of different carbohydrate levels in the feed on the digestion and tissue structure of snakeheads (Channa argus) with prolonged feeding time. The control (CT) group was fed chilled fish, and the experimental groups were fed flour as a carbohydrate source to prepare puffed feed containing 13%, 16% and 19% flour (WS13, WS16 and WS19 respectively). The results demonstrated that carbohydrate levels in the feed had significant effects on amylase activity in the intestinal segment and Na+, K+-ATPase activity in the stomach (p < .05). In all the experimental groups, the pepsin activity in the gastrointestinal tract showed a decreasing trend over time in different feeding cycles. The alkaline phosphatase (AKP) activities in all groups showed a downward trend, and with increased feeding time, AKP in all experimental groups had an increasing trend. The liver index, liver crude fat content and serum triglyceride of snakeheads increased significantly with the increase in carbohydrate content in feed at 4 weeks (p < .05). There were no differences in the stomach structure among the groups as determined using light microscopy. The intestinal villi were injured, and the liver cells became larger and showed damage in a time and carbohydrate level-dependent manner. In conclusion, when the carbohydrate content is too high, it not only affects the morphological function of the snakeheads' digestive tract, but also affects the normal structure of liver cells and the metabolic function of the liver.

Clinical and Genome-Wide Analysis of Serum Platinum Levels after Cisplatin-Based Chemotherapy.

Serum platinum is measurable for years after completion of cisplatin-based chemotherapy (CBC). We report the largest investigation of serum platinum levels to date of 1,010 testicular cancer survivors (TCS) assessed 1-35 years after CBC and evaluate genetic contributions to these levels. Eligible TCS given 300 or 400 (±15) mg/m2 cisplatin underwent extensive audiometric testing, clinical examination, completed questionnaires, and had crude serum platinum levels measured. Associations between serum platinum and various risk factors and toxicities were assessed after fitting a biexponential model adjusted for follow-up time and cumulative cisplatin dose. A genome-wide association study (GWAS) was performed using the serum platinum residuals of the dose and time-adjusted model. Serum platinum levels exceeded the reference range for approximately 31 years, with a strong inverse relationship with creatinine clearance at follow-up (age-adjusted P = 2.13 × 10-3). We observed a significant, positive association between residual platinum values and luteinizing hormone (age-adjusted P = 6.58 × 10-3). Patients with high residual platinum levels experienced greater Raynaud phenomenon than those with medium or low levels (age-adjusted ORhigh/low = 1.46; P = 0.04), as well as a higher likelihood of developing tinnitus (age-adjusted ORhigh/low = 1.68, P = 0.07). GWAS identified one single-nucleotide polymorphism (SNP) meeting genome-wide significance, rs1377817 (P = 4.6 × 10-8, a SNP intronic to MYH14). This study indicates that residual platinum values are correlated with several cisplatin-related toxicities. One genetic variant is associated with these levels.

Evaluation of an emulsifier blend on growth performance, nutrient digestibility, blood lipid profiles, and fecal microbial in growing pigs fed low energy density diet

Energy is one of the major cost components in swine diets. Improving lipid digestibility is related with saving cost. Exogenous emulsifiers could help emulsification processing in gastrointestinal tract, thereby enhancing the digestion of lipids. The aim of this study was to evaluate the effect of dietary supplementation of an emulsifier [10.21% of glycerol polyethylene glycol ricinoleate (GPGR) and 2.2% of lysophospholipids (LPL) blend (GLB)] on growth performance, nutrient digestibility, blood lipid profiles, and fecal microbial in growing pigs fed a relatively low energy diet. In total, 135 female and castrated male crossbred growing pigs [(Yorkshire × Landrace) × Duroc] with an average initial body weight (BW) of 20.07 ± 1.76 kg were used in a 6-wk experiment. Pigs were blocked based on BW and sex and randomly assigned to 3 dietary treatments with 9 replication pens per treatment: CON, basal diet; RET, reduced energy treatment (50 kcal/kg); RET5, RET + 0.05% GLB. Overall, no significant result (P > 0.05) was observed between CON groups and RET groups on performance parameters, nutrient digestibility, blood lipid profiles of high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and cholesterol concentrations, and fecal microbial counts. At the end of the experiment, pigs fed RET5 diet had significantly (P < 0.05) increased BW, average daily gain (ADG), dry matter (DM) and crude fat (CF) digestibility, serum HDL-C and triglyceride concentrations, and fecal Lactobacillus counts compared with those fed RET diet. Moreover, pigs in RET5 group also showed significant (P < 0.05) increase in DM and serum triglyceride concentration compared with CON groups. These results indicated that supplementation with the GPGR and LPL blended emulsifier could improve the growth performance and nutrient digestion and absorption of growing pigs fed lower energy diet.

Occurrence of natural lectin with bacterial agglutination property in the serum of lepidopteran pest, Parasa lepida

AbstractInsects depend on lectins for non‐self recognition and clearance of invading pathogens. Naturally occurring lectin showing specificity for galactose was purified from the serum of lepidopteran pest Parasa lepida by affinity chromatography using Sepharose 6B coupled with galactose as a gel matrix. Preliminary studies on crude serum agglutinin revealed that the agglutinin molecule showed varying degrees of specificity to avian and mammalian red blood cells tested. Among them, the highest titer of 128 was recorded against rabbit red blood cell type. The agglutinin molecule in the crude serum was stable up to 60°C and at pH between 6 and 9. Also, the hemagglutinating activity was neither dependent on divalent cations nor sensitive to ethylenediaminetetraacetic acid treatment. Galactose inhibited the hemagglutinating activity at minimum inhibitory concentration of 12.5 mM and hence it was used as a ligand for affinity chromatography. Native polyacrylamide gel electrophoresis analysis revealed a single band and the molecular weight of the lectin was found to be approximately 90 kDa. Bacterial agglutination activity of the purified lectin with two significant toxin bacteria, namely Salmonella typhi and Bacillus thuringiensis, was observed.

Serum and tissue proteomic signatures of patients with hepatocellular carcinoma using 2‑D gel electrophoresis

Hepatocellular carcinoma (HCC) accounts for ~85% of primary liver cancer cases and is a leading cause of mortality worldwide. Effective early diagnosis is difficult for HCC; however, effective biomarkers may be beneficial for diagnosis. In the current study, serum samples, and HCC and adjacent tissue samples were obtained from patients with HCC for the detection of biomarkers using 2-D gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight (TOF)/TOF mass spectrometry. The crude serum samples did not need to be prepared for removal of high abundance proteins. The mRNA expression levels of HCC-associated proteins were detected in tissues using reverse transcription-quantitative PCR. Statistical analysis and database matching were used to identify the differentially expressed proteins detected in the serum and tissue groups. Immunohistochemistry (IHC) was performed to detect the expression of significant proteins in HCC and adjacent tissues. The results revealed ~800 protein spots on a 2-DE gel that were detected in serum samples, and 1,200 spots were identified in the tissue samples. The protein and mRNA expression levels of oxysterol binding protein-like 11 (OSBPL11) in HCC serum and tissue samples were consistent. Pathway analysis demonstrated that members of the apolipoprotein family, particularly apolipoprotein E (APOE), and RAS family members were closely associated in HCC, either directly or via ferratin heavy polypeptide 1. IHC results demonstrated that the APOE protein serves an important role in liver cancer development. The lysis buffer used in the current study was effective for serum protein separation in 2-DE sample preparation. In addition, the present study revealed that downregulated OSBPL11 may be a potential indicator for HCC, and the apolipoprotein family, particularly APOE, and the RAS family may cooperatively serve an important role.

Genotyping of Single Nucleotide Polymorphisms Using Allele-Specific qPCR Producing Amplicons of Small Sizes Directly from Crude Serum Isolated from Capillary Blood by a Hand-Powered Paper Centrifuge

The cell-free genomic DNA (gDNA) concentration in serum ranges from 1500 to 7500 copies/mL within 2 h after phlebotomy (6–24 times the concentration observed in plasma). Here, we aimed to evaluate the gDNA size distribution in serum with time after coagulation and to test if crude serum can be directly used as a source of gDNA for qPCR. Next, we investigated if single nucleotide polymorphisms (SNPs) could be genotyped directly from the crude serum isolated from capillary blood using a hand-powered paper centrifuge. All tested PCR targets (65, 100, 202 and 688 base pairs) could be successfully amplified from DNA extracted from serum, irrespective of their amplicon size. The observed qPCR quantitation cycles suggested that the genomic DNA yield increased in serum with incubation at room temperature. Additionally, only 65 and 101 base pair qPCR targets could be amplified from crude serum soon after the coagulation. Incubation for 4 days at room temperature was necessary for the amplification of PCR targets of 202 base pairs. The 688 base pair qPCR target could not be amplified from serum directly. Lastly, serum was successfully separated from capillary blood using the proposed paper centrifuge and the genotypes were assigned by testing the crude serum using allele-specific qPCR, producing small amplicon sizes in complete agreement with the genotypes assigned by testing the DNA extracted from whole blood. The serum can be used directly as the template in qPCR for SNP genotyping, especially if small amplicon sizes are applied. This shortcut in the SNP genotyping process could further molecular point-of-care diagnostics due to elimination of the DNA extraction step.

Purification of Plant ESCRT Proteins for Polyclonal Antibody Production.

Most endosomal sorting complex required for transport (ESCRT)-III proteins are not fully functional when expressed as fusion of fluorescent or epitope tags, frequently making the use of specific antibodies the only available method for their detection. Heterologous expression of ESCRT-III proteins in bacteria often results in the formation of insoluble aggregates or inclusion bodies that interfere with their purification. However, inclusion bodies are usually pure protein aggregates with high antigenicity. In addition, since proteins within inclusion bodies are presented in a range of folding states, immunization with inclusion bodies can potentially result in antibodies with specificity for different folding states of the protein under study. We describe here a protocol to isolate bacterial inclusion bodies of plant ESCRT-III proteins for production of polyclonal antibodies. We also describe a nitrocellulose-based immunoaffinity purification method that allows the immobilization of ESCRT-III proteins and the subsequent isolation of specific antibodies from a crude serum.

Effects of dietary yeast extract supplementation on growth, body composition, non-specific immunity, and antioxidant status of Chinese mitten crab (Eriocheir sinensis)

This study was conducted to evaluate the effects of dietary supplementation with yeast extract on growth, body composition, non-specific immunity, and antioxidant status of Chinese mitten crab (Eriocheir sinensis). A total of 432 crabs (initial average weight, 4.62 ± 0.11 g) were randomly distributed into four treatment groups with six replicates of 18 crabs. The crabs were fed a basal diet or the same diet supplemented with 2.5, 5, and 10 g/kg yeast extract for 8 weeks. The results showed that dietary yeast extract inclusion enhanced the edible viscera index (linear, P < 0.001), edible viscera crude protein (CP) content (linear, P = 0.025) and serum phenoloxidase (ProPO) activity (quadratic, P = 0.023) at 56 day, increased the total superoxide dismutase (T-SOD) activity at 28 day (quadratic, P = 0.037) and catelase (CAT) activity at 56 day (quadratic, P = 0.034) of edible viscera, and muscular T-SOD activity (quadratic, P = 0.020) at 56 day in Chinese mitten crab. Compared with the control group, the inclusion of 5 g/kg yeast extract in the diet increased the edible viscera index, enhanced the CAT activity of edible viscera at 56 day in Chinese mitten crab (P < 0.05). Dietary 10 g/kg yeast extract inclusion enhanced the edible viscera index at 56 day in Chinese mitten crab than that of the control group (P < 0.05). These results implied that dietary yeast extract inclusion improved the edible viscera index and crude protein content of edible viscera, enhanced serum immunity, and increased the antioxidant status of edible viscera and muscle in Chinese mitten crab, especially when it is supplemented at 5 g/kg yeast extract in the diet.