Short-term preservation of Colossoma macropomum oocytes by different methods and chilling temperatures.

A preservation protocol has not been established for Colossoma macropomum oocytes, and its development may improve the production and breeding programs of this South American fish species. Thus, this study aimed to determine the effect of different methods and protocols for the preservation of C. macropomum oocytes. Seven experiments were conducted throughout the breeding season of this species. The oocytes were collected and stored in sterile conditions. Preserved oocytes were subjected to storage in the following treatments: room temperature (RT, 27 °C), centrifugation followed by ovarian fluid removal (Cen), vacuum (Vac), chilled temperature (ChT), centrifugation and vacuum (CV), vacuum and chilled temperature (VChT), and centrifugation, vacuum, and chilled temperature (CVChT) in dry sterilized plastic containers and plastic bags. Chilled storage was tested at 4 and 13 °C. Fertilization and hatching rates were assessed at 0, 30, 60, 90, and 120 min after stripping (MAS) for preservation protocols. The larval malformation rate was analyzed at 0 and 30 MAS for RT and ChT. Quantitation and identification (by mean of MALDI-TOF MS) of bacteria were performed at 0, 60, 90, and 120 MAS, and scanning electron microscopy (SEM) analyses were carried out at 0, 60, and 90 MAS. The fertilization and hatching rates decreased over preservation time and breeding season. RT samples fertilized at 0, 30, and 60 MAS yielded similar fertilization rates at both the beginning and end of the season. By the end of the season, oocytes from treatment ChT at 13 °C 30 MAS yielded higher fertilization and hatching rates, and a lower percentage of larvae malformation than RT 30 MAS. The treatment ChT at 4 °C triggered low a fertilization rate. The treatments ChT (13 °C) and Cen provided good fertilization rate when used alone and with other approaches, i.e., treatments VChT, CV, and CVChT. The treatment Vac presented inconsistent results, so no conclusion could be made. Bacterial colony counts were low (10-1.6 × 105 CFU-mL-1), and a total of 18 bacteria species were identified in all batches analyzed; however, the treatments did not influence the number of bacteria. C. macropomum female breeders presented distinct bacteria species in their oocytes and the presence of bacteria did not impair oocyte quality until 120 MAS. Moreover, SEM analyses showed that the micropyle was not occluded during oocyte storage, and ovarian fluid was observed on the surface of chilled oocytes. Therefore, Colossoma macropomum oocytes could be preserved under chilled storage at 13 °C for 30 min throughout its breeding season.

Validation of a simple liquid chromatography coupled to tandem mass spectrometry method for the simultaneous determination of tacrolimus, sirolimus, everolimus and cyclosporin A in dried matrix on paper discs.

INTRODUCTION: Due to its high specificity and sensitivity, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is the gold standard method for immunosuppressant quantification in therapeutic drug monitoring. In this context, dried blood spots (DBS) have become a promising strategy as a sample collection procedure. Although the advantages of DBS over venipuncture are well known, this approach has limitations that strongly influence the acceptance of analytical results. Among them, the most important is hematocrit (Ht). The easiest way of overcoming this problem is by analyzing complete spots. In this strategy, called dried matrix on paper discs (DMPD), blood is volumetrically applied on pre-punched discs. OBJECTIVES: To validate an LC-MS/MS method for the quantification of tacrolimus, sirolimus, everolimus and cyclosporin A using DMPD. METHODS: The procedure was validated according to international guidelines using a commercial kit. The following performance parameters were evaluated: selectivity, carryover, linearity, accuracy, precision, lower limit of quantitation, relative recovery, commutability and stability. In addition, a method comparison study was performed to evaluate the clinical influence of Ht on the results. RESULTS: All performance parameters were within acceptance criteria and, hence, it was determined that the validated method is fit for the intended purpose. Likewise, calculated bias values on medical decision levels showed that there was no clinical influence of Ht on the results. CONCLUSION: Unlike other similar methodologies that have been published, here, a simple method has been fully validated. This is the first LC-MS/MS methodology adapting a commercial kit to use DMPD as a sampling strategy.

Evaluation of the Efficiency of Commercial Vaccines Against Infectious Bronchitis Virus (IBV) Belonging to the GI-16 Lineage Isolated in an Argentinean Outbreak.

In this study we evaluated the effectiveness of adding serotype 793B vaccine to an immunization program in order to control the infectious bronchitis virus (IBV) GI-16 lineage. Therefore, two different experiments were performed. First, a virus cross-neutralization test was carried out, which indicated that neither the Massachusetts (Mass) nor 793B serotypes are antigenically related to the field isolate A13 (GI-16). We also performed a challenge trial to evaluate if the Mass/793B combination is more efficient than Mass/Connecticut (Conn) to protect chickens against the Argentinian variant A13. Thus, 40 chickens were organized in four groups. Chickens in Group A were vaccinated at 1 day of age with Mass serotype and then at 14 days old with Mass plus Conn serotypes. Chickens in Group B received Mass and 793B serotypes at 1 and 14 days old, respectively. Groups C and D remained unvaccinated. At 28 days of age, Groups A, B, and C were challenged with the A13 isolate, while Group D remained as the negative control. The statistical analysis of the ciliostasis evaluation, performed at 7 days postchallenge (dpch), indicated that the difference between Mass/793B and Mass/Conn was not significant (p > 0.05). However, the comparison against the negative control showed that only Group A was significantly different, suggesting a slightly better performance on blocking ciliostasis for the Mass/793B combination. On the other hand, no significant differences were observed in the viral load, quantified by reverse-transcription quantitative real-time PCR (RT-qPCR) in tracheal swabs and kidneys (at 3 and 7 dpch, respectively) between vaccinated groups. Furthermore, some amounts of the viral genome were found in both vaccinated groups that could indicate that neither the Mass/793B nor the Mass/Conn combinations totally inhibited the viral replication. Such viral replication in vaccinated chickens should seriously be taken into consideration because it could promote the selection of new variants in the future.

Nota de investigación­Evaluación de la eficacia de vacunas comerciales contra el virus de la bronquitis infecciosa (IBV) perteneciente al linaje GI-16 aislado durante un brote argentino. En este estudio se evaluó la efectividad de agregar la vacuna del serotipo 793B a un programa de inmunización para controlar al virus de la bronquitis infecciosa (con las siglas en inglés IBV) linaje GI-16. Por tanto, se realizaron dos experimentos diferentes. Primeramente, se llevó a cabo una prueba de neutralización cruzada de virus, que indicó que ni los serotipos Massachusetts (Mass) ni 793B están antigénicamente relacionados con el aislado de campo A13 (GI-16). También se realizó una prueba de desafío para evaluar si la combinación Massachussets/793B era más eficiente que Massachussets/Connecticut (Conn) para proteger a los pollos contra la variante argentina A13. De esta forma, 40 pollos se organizaron en cuatro grupos. Los pollos del Grupo A se vacunaron al día de edad con el serotipo Massachussets y luego a los 14 días con los serotipos Massachussets más Connecticut. Los pollos del Grupo B recibieron los serotipos Massachussets y 793B a los 1 y 14 días de edad, respectivamente. Los grupos C y D permanecieron sin vacunar. A los 28 días de edad, los Grupos A, B y C fueron desafiados con el aislado A13, mientras que el Grupo D permaneció como control negativo. El análisis estadístico de la evaluación de la ciliostasis, realizada a los 7 días después del desafío (dpch), indicó que la diferencia entre el tratamiento Massachussets/793B y Massachussets/Connecticut no fue significativa (P> 0.05). Sin embargo, la comparación con el control negativo mostró que solo el Grupo A fue significativamente diferente, lo que sugiere un desempeño ligeramente mejor en el bloqueo de la ciliostasis para la combinación Massachussets/793B. Por otro lado, no se observaron diferencias significativas (P> 0.05) en la carga viral, cuantificada mediante transcripción reversa y PCR cuantitativa en tiempo real de hisopos traqueales y riñones (a 3 y 7 días después del desafío, respectivamente) entre los grupos vacunados. Además, se encontraron algunas cantidades del genoma viral en ambos grupos vacunados que podrían indicar que ni las combinaciones Massachussets/793B ni Massachussets/Connecticut inhibieron totalmente la replicación viral. Esta replicación viral en pollos vacunados debe tenerse muy en cuenta porque podría promover la selección de nuevas variantes en el futuro.

Detection, Isolation, and 1H NMR Quantitation of the Nitrile Glycoside Sarmentosin from a Bryophyllum pinnatum Hydro-Ethanolic Extract.

Bryophyllum pinnatum (Lam) Pers. (Crassulaceae) is widely used in folk medicine as leaf juice, aqueous, or hydro-ethanolic extracts. It is also listed as a medicinal plant in several countries such as France and Brazil. The main reported constituents are flavone glycosides, especially those with the rare 3-O-α-l-arabinopyranosyl-(1 → 2)-α-l-rhamnopyranoside moiety. Despite several phytochemical screenings indicating the presence of cyanide derivatives or alkaloids, there are no reports of nitrogenous metabolite characterization from this plant species. Nevertheless, the occurrence and the type of such compounds are of particular interest, as they may account for some of the numerous biological activities and ethnomedicinal uses described for B. pinnatum and could be regarded as chemical/taxonomic markers. Consequently, a hydro-ethanolic extract of B. pinnatum was investigated by using UHPLC-HRMS/MS and the nitrile glucoside sarmentosin was detected for the first time within the genus Bryophyllum/Kalanchoe. Considering the wide use of B. pinnatum and its closely related species for health purposes, the target metabolite was isolated by a combination of centrifugal partition chromatography in elution/extrusion mode and MPLC in order to confirm its structure. A linear, selective, precise, fast, and reliable 1H NMR quantitation method was then developed and validated and may become a tool for easy quality assessment of the plant species. The amount of sarmentosin was determined as 2.07% of the examined sample. Sarmentosin was also detected in Kalanchoe laciniata, confirming the occurrence of this compound within the genus.

Hevea brasiliensis latex proteomics: a review of analytical methods and the way forward.

Natural rubber or latex from the Hevea brasiliensis is an important commodity in various economic sectors in today's modern society. Proteins have been detected in latex since the early twentieth century, and they are known to regulate various biological pathways within the H. brasiliensis trees such as the natural rubber biosynthesis, defence against pathogens, wound healing, and stress tolerance. However, the exact mechanisms of the pathways are still not clear. Proteomic analyses on latex have found various proteins and revealed how they fit into the mechanisms of the biological pathways. In the past three decades, there has been rapid latex protein identification due to the improvement of latex protein extraction methods, as well as the emergence of two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). In this manuscript, we reviewed the methods of latex protein extraction that keeps on improving over the past three decades as well as the results of numerous latex protein identification and quantitation.

Using a toxicoproteomic approach to investigate the effects of thiamethoxam into the brain of Apis mellifera.

Neonicotinoids have been described as toxic to bees. In this context, the A. mellifera foragers were exposed to a sublethal concentration of thiamethoxam (LC50/100: 0,0227 ng de thiamethoxam/µL-1 diet), a neurotoxic insecticide, for 8 days; and it was decided to investigate the insecticide effect on the brain by a shotgun proteomic approach followed by label-free quantitative-based proteomics. A total of 401 proteins were identified in the control group (CG); and a total of 350 proteins in the thiamethoxam exposed group (TMX). Quantitative proteomics data showed up 251 proteins with significant quantitative values in the TMX group. These findings demonstrated the occurrence of shared and unique proteins with altered expression in the TMX group, such as ATP synthase subunit beta, heat shock protein cognate 4, spectrin beta chain-like, mushroom body large-type Kenyon cell-specific protein 1-like, tubulin alpha-1 chain-like, arginine kinase, epidermal growth factor receptor, odorant receptor, glutamine synthetase, glutamate receptor, and cytochrome P450 4c3. Meanwhile, the proteins that were expressed uniquely in the TMX group are involved mainly in the phosphorylation, cellular protein modification, and cell surface receptor signalling processes. Interaction network results showed that identified proteins are present in five different metabolic pathways - oxidative stress, cytoskeleton control, visual process, olfactory memory, and glutamate metabolism. Our scientific outcomes demonstrated that a sublethal concentration of thiamethoxam can impair biological processes and important metabolic pathways, causing damage to the nervous system of bees, and in the long term, can compromise the nutrition and physiology of individuals from the colony.

Determination of Chitin Content in Insects: An Alternate Method Based on Calcofluor Staining.

Chitin is an aminopolysaccharide present in yeast cells and arthropod cuticle and is one of the most abundant biopolymers. The conventional methods for the quantitation of chitin content in biological samples are based on its hydrolysis (acid or enzymatic), and the assessment of the byproduct, glucosamine. However, previously described methodologies are time-consuming, laborious, low throughput, and not applicable to insect samples in many cases. Here we describe a new approach to chitin content quantitation based on calcofluor fluorescent brightener staining of samples, followed by microplate fluorescence readings. Calcofluor is a specific chitin stain commonly used for topological localization of the polymer. The protocol was tested in three important disease vector species, namely Lutzomyia longipalpis, Aedes aegypti, and Rhodnius prolixus, and then compared to a classic colorimetric chitin assessment method. Results show that chitin content in the tested insects can vary largely in a range of 8-4600 micrograms of chitin per insect, depending on species, sex, and instar. Comparisons between measurements from the previous protocol and calcofluor method showed statistically significant differences in some samples. However, the difference might be due to interference in the classic method from non-chitin sources of glucosamine and reducing agents. Furthermore, chitinase hydrolysis reduces the total chitin mass estimated between 36 and 74%, consolidating the fluorescent measurements as actual stained chitin in the same extent that was observed with the standard protocol. Therefore, the calcofluor staining method revealed to be a fast and reliable technique for chitin quantitation in homogenized insect samples.

LC-MS bioanalysis of intact proteins and peptides.

Bioanalysis assays that reliably quantify biotherapeutics and biomarkers in biological samples play pivotal roles in drug discovery and development. Liquid chromatography coupled with mass spectrometry (LC-MS), owing to its superior specificity, faster method development and multiplex capability, has evolved as one of the most important platforms for bioanalysis of biotherapeutics, particularly new scaffolds such as half-life extension platforms for proteins and peptides, as well as antibody drug conjugates. Intact LC-MS analysis is orthogonal to bottom-up surrogate peptide approach by providing whole molecule quantitation and high-level sequence and structure information. Here we review the latest development in LC-MS bioanalysis of intact proteins and peptides by summarizing recent publications and discussing the important topics such as the comparison between top-down intact analysis and bottom-up surrogate peptide approach, as well as simultaneous quantitation and catabolite identification. Key bioanalytical issues around intact protein bioanalysis such as sensitivity, data processing strategies, specificity, sample preparation and LC condition are elaborated. For peptides, topics including quantitation of intact peptide vs. digested surrogate peptide, metabolites, sensitivity, LC condition, assay performance, internal standard and sample preparation are discussed.

48 weeks outcome after cessation of nucleos(t)ide analogue therapy in chronic hepatitis B patients.

INTRODUCTION AND OBJECTIVE: The aim of the present study was to investigate the significance of serum HBsAg levels in treatment cessation of nucleoside analogues (NAs) in patients with chronic hepatitis B (CHB) infection. METHODS: In 158 CHB patients with long-term NAs treatment, 74 patients were in HBeAg negative and had a HBsAg level <1500IU/mL, 36 of whom were informed and consented to cease NAs. HBsAg, HBV DNA and liver function were examined in the 1st, 3rd, 6th, 9th and 12th month after treatment cessation. RESULTS: The sustained response rate was 88.89% (32/36) within one year after NAs cessation. Sub-group analysis was based on HBsAg levels of patients with NAs cessation, there was no relapse case in 11 patients whose HBsAg <50IU/mL, and the negative predictive value (NPV) was 100%. Seroconversion of HBsAg occurred in 3 patients. 2 patients from 21 cases whose HBsAg was between 50IU/mL and 1000IU/mL relapsed. 2 of 4 patients whose in HBsAg >1000IU/mL relapsed. HBsAg of patients with a sustained response decreased slowly. In contrast, HBsAg levels increased gradually in relapsed patients, and the increase of HBsAg was precedent to relapses of HBV DNA and ALT. Multivariate analysis suggested that only HBsAg level showed a close correlation with HBV DNA relapses. ROC curve analysis suggested that the increase of HBsAg level in the 3rd and 6th month after NAs cessation had a great predictive value for relapses. CONCLUSION: Monitoring of base line HBsAg level can predict outcomes of NAs cessation in HBeAg-negative chronic hepatitis B. HBsAg <50IU/mL has higher predictive values of better sustained responses in HBeAg-negative CHB patients.

A qPCR Targeted Against the Viral Replication Origin Designed to Quantify Total Amount of Filamentous Phages and Phagemids.

Filamentous bacteriophages are widely used in phage display technology. The most common quantification method is lysis plaque formation test (PFT). This technique has several disadvantages, and only quantifies infective phages and is not effective when phagemids are used. We developed a qPCR method directed against the M13 replication origin, which detects between 3.3 × 103 and 3.3 × 108 viral genome copies with a linearity of R 2 = 0.9998. Using this method we were able to observe a difference of approximately ten more phages than with the PFT. This difference was not due to the presence of a free genome, which suggests the presence of non-infective particles. Using a DNaseI treatment, we observed the presence of 30% to 40% of unpackaged genome in recombinant phage modified in PIII or PVIII. The qPCR method with a DNase I treatment is an efficient method to quantify the total amount of filamentous phages.

Identification and Determination of Seven Phenolic Acids in Brazilian Green Propolis by UPLC-ESI-QTOF-MS and HPLC.

Brazilian green propolis is a complex mixture of natural compounds that is difficult to analyze and standardize; as a result, controlling its quality is challenging. In this study, we used the positive and negative modes of ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time of flight mass spectrometry in conjunction with high-performance liquid chromatography for the identification and characterization of seven phenolic acid compounds in Brazilian green propolis. The optimal operating conditions for the electrospray ionization source were capillary voltage of 3500 V and drying and sheath gas temperatures of 320 °C and 350 °C, respectively. Drying and sheath gas flows were set to 8 L/min and 11 L/min, respectively. Brazilian green propolis was separated using the HPLC method, with chromatograms for samples and standards measured at 310 nm. UPLC-ESI-QTOF-MS was used to identify the following phenolic compounds: Chlorogenic acid, caffeic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, caffeic acid phenethyl ester (CAPE), and artepillin C. Using a methodologically validated HPLC method, the seven identified phenolic acids were then quantified among different Brazilian green propolis. Results indicated that there were no significant differences in the content of a given phenolic acid across different Brazilian green propolis samples, owing to the same plant resin sources for each sample. Isochlorogenic acid B had the lowest content (0.08 ± 0.04) across all tested Brazilian green propolis samples, while the artepillin C levels were the highest (2.48 ± 0.94). The total phenolic acid content across Brazilian green propolis samples ranged from 2.14-9.32%. Notably, artepillin C quantification is an important factor in determining the quality index of Brazilian green propolis; importantly, it has potential as a chemical marker for the development of better quality control methods for Brazilian green propolis.

Monoclonal Antibodies Against the Capsular Polysaccharides A, C, Y, W, and X of Neisseria meningitidis: A Platform for the Quality Control of Meningococcal Vaccines.

Vaccination has reduced morbidity and mortality of many diseases that previously caused devastating epidemics and deaths globally. Vaccines as a biological product may contain microorganisms or their derivatives. This aspect together with the fact that they are administered to healthy individuals (mainly children) means that approximately 70% of vaccines development time is dedicated to quality control. Monoclonal antibodies (MAbs) have become essential analytical tools for application in ELISAs, Western and Dot blotting, immunoprecipitation, and flow cytometric assays that ensure the quality control of vaccines. The aim of this work is to present a review of the methods used to obtain a platform of MAbs against Neisseria meningitidis polysaccharide antigens to use as an analytical tool for quality control of anti-meningococcal polysaccharide (Ps) vaccines. The MAbs obtained are used in five sandwich ELISAs developed for Ps quantification. The assays showed good reproducibility and repeatability, with quantitation and detection limits below 1 ng/mL. Dot Blot, as the Identity test of the Ps vaccine, was carried out to positively identify licensed and experimental vaccines. All assays described are suitable for the screening of multiple vaccine samples and could be useful for monitoring lot-to-lot consistency and stability.

A sensitive and robust UPLC-MS/MS method for quantitation of estrogens and progestogens in human serum.

OBJECTIVE: With the widespread use of sex-steroid hormones in contraceptives and hormone replacement therapy, there is an increasing need for reliable analytical methods. We report the development of a sensitive and robust UPLC-MS/MS method for quantitation of both endogenous and synthetic sex-steroid hormones in human serum. STUDY DESIGN: We developed and validated a UPLC-MS/MS method to quantify progestogens (etonogestrel, levonorgestrel, medroxyprogesterone acetate, norethindrone, progesterone) and estrogens (estradiol and ethinyl estradiol) with good accuracy, high sensitivity, and excellent robustness. We then applied the method to the analysis of sex-steroid hormones in serum from 451 clinical research participants. RESULTS: Each UPLC-MS/MS analysis was 6.5 min. The lower limits of quantitation (LLOQs) were 25 pg/ml for the progestogens, and 2.5 and 5.0 pg/ml for estradiol and ethinyl estradiol, respectively. When estradiol was analyzed without assessment of progestogens, the LLOQ was reduced to 1 pg/ml. The calibration curves were linear from 25-50,000, 2.5-2000 (1-2000 for estrogens-only analysis) and 5-2000 pg/ml, respectively. Both the accuracy and precision were below±15% not only for routine validation (intraday and interday), but for long-term (>2 years) assay robustness with external controls, thereby, demonstrating the utility of this method for multi-year clinical trial assessments of progestogens and estrogens. We applied the method to quantify sex-steroid levels in 1804 clinical samples. CONCLUSIONS: We successfully developed a UPLC-MS/MS method, and overcame the matrix suppression to allow sensitive quantitation of both synthetic and endogenous sex-steroid hormones in human serum. IMPLICATIONS: We developed a sensitive and robust UPLC-MS/MS method to accurately measure the levels of sex-steroid hormones in serum. The method overcame matrix interference barriers and achieved excellent long-term stability and reproducibility (≥96.9% accuracy; ≤13.0% relative variability measured with external controls over 2 years), demonstrating its utility in clinical sample analysis.

Gene mapping strategy for Alu elements rearrangements: Detection of new large deletions in the SERPING1 gene causing hereditary angioedema in Brazilian families.

BACKGROUND: Hereditary angioedema (HAE) is a rare genetic disorder mainly caused by mutations in the SERPING1 gene, determining a deficit of C1 inhibitor (C1-INH). In approximately 10% of the cases, HAE with C1-INH deficiency (C1-INH-HAE) is caused by large gene rearrangements, which are not detected by Sanger sequencing. Here we present the exon quantification technique (EQT), a molecular diagnostic test for the detection of large genetic rearrangements in SERPING1, mapping the exact size and location of the deletion caused by the recombination of Alu elements. EQT analysis was performed on total DNA extracted from blood of patients belonging to two Brazilian families with a medical history of HAE, low plasma levels of C4 and C1-INH and no pathogenic alteration in SERPING1 analyzed by Sanger sequencing. RESULTS: Two large deletions were found, one of 1356 pb and one of 1804 pb, which resulted from recombination of two Alu elements present in introns 3 and 4 of the gene. CONCLUSION: These results showed that the EQT could be used as a simple, rapid, and efficient diagnosis test for analysis of large deletions and insertions involving SERPING1, otherwise not detected by Sanger sequencing, serving as a support technique for molecular diagnosis of HAE.

Cytokine gene transcription in the trachea, Harderian gland, and trigeminal ganglia of chickens inoculated with virulent infectious laryngotracheitis virus (ILTV) strain.

The objective of this study was to determine how cytokine transcription profiles correlate with patterns of infectious laryngotracheitis virus (ILTV) replication in the trachea, Harderian gland, and trigeminal ganglia during the early and late stages of infection after intratracheal inoculation. Viral genomes and transcripts were detected in the trachea and Harderian gland but not in trigeminal ganglia. The onset of viral replication in the trachea was detected at day one post-infection and peaked by day three post-infection. The peak of pro-inflammatory (CXCLi2, IL-1ß, IFN-γ) and anti-inflammatory (IL-13, IL-10) cytokine gene transcription, 5 days post-infection, coincided with the increased recruitment of inflammatory cells, extensive tissue damage, and limiting of virus replication in the trachea. In contrast, transcription of the IFN-ß gene in the trachea remained unaffected suggesting that ILTV infection blocks type I interferon responses. In the Harderian gland, the most evident transcription change was the early and transient upregulation of the IFN-γ gene at 1 day post-infection, which suggests that the Harderian gland is prepared to rapidly respond to ILTV infection. Overall, results from this study suggest that regulation of Th1 effector cells and macrophage activity by Th1/2 cytokines was pertinent to maintain a balanced immune response capable of providing an adequate Th1-mediated protective immunity, while sustaining some immune homeostasis in preparation for the regeneration of the tracheal mucosa.

Development of a droplet digital RT-PCR for the quantification of foot-and-mouth virus RNA.

Foot-and-mouth-disease (FMD) is a highly contagious disease of domestic animals which can result in substantial economic losses, caused by the FMD virus (FMDV). The aim of this study was to develop and standardize a novel reverse transcriptase droplet digital PCR (RT-ddPCR) assay for the quantification of FMDV RNA. This assay was based upon an OIE-recognized real-time RT-PCR that detects the 3D-encoding region of FMDV. The limit of detection at 101.4 TCID50/mL and 26.5 copies was determined using FMDV-A24-Cruzeiro-virus and a plasmid containing the 3D-FMDV sequences, respectively. FMDV O, A and C serotypes and 11 species of non-FMDV were used to confirm the sensitivity and specificity of the assay. The RT-ddPCR was standardized using 60 bovine samples (representing negative and positive samples of epithelium and/or oesophageal-pharyngeal [OP] fluid) from animals suspected of vesicular diseases and previously tested by RT-qPCR. The RT-ddPCR showed robustness, sensitivity, specificity and accuracy, with similar results to the RT-qPCR. Moreover, the new RT-ddPCR diagnostic tool allowed the absolute quantification of FMDV RNA from epithelium and OP-fluid samples, as well as having the advantages of direct quantification by endpoint, eliminating the need for a calibration standard curve required in quantitative real-time RT-PCR.

A Rapid Extraction Method for mammalian cell cultures, suitable for quantitative immunoblotting analysis of proteins, including phosphorylated GCN2 and eIF2α.

Many studies require the detection and relative quantitation of proteins from cell culture samples using immunoblotting. Limiting factors are the cost of protease inhibitors, the time required to break cells and generate samples, as well as the high risk of protein loss during cell breakage procedures. In addition, a common problem is the viscosity of lysed samples due to the released genomic DNA. As a consequence, the DNA needs to be broken down prior to denaturing polyacrylamide protein gel electrophoresis (SDS-PAGE), e.g. by passing the sample through a syringe gauge needle, sonication, or DNase treatment. In a quest to find a more cost-effective, fast, and yet robust procedure, we found that cell lysis, protein denaturation, and DNA fragmentation can be done in only two steps: harvesting followed by a simple non-laborious 2nd step. Similarly to many pre-existing cell breakage procedures, in our Rapid Protein Extraction (RPE) method, proteins liberated from cells are immediately exposed to a denaturing environment. However, advantages of our method are: •No breaking buffer is needed, instead proteins are liberated directly into the denaturing protein loading buffer used for SDS-PAGE. Consequently, our RPE method does not require any expensive inhibitors.•The RPE method does not involve post-lysis centrifugation steps; instead all cell material is dissolved during the 2nd step, the mixing-heat-treatment step which is new to this method. This prevents potential protein loss that may occur during centrifugation. In addition, this 2nd step simultaneously shears the genomic DNA, making an additional step for DNA fragmentation unnecessary.•The generated samples are suitable for high-quality quantitative immunoblotting. With our RPE method we successfully quantified the phosphorylated forms of protein kinase GCN2 and its substrate eIF2α. In fact, the western signals were stronger and with less background, as compared to samples generated with a pre-existing method.

Selective Reaction Monitoring Mass Spectrometry for Quantitation of Glycolytic Enzymes in Postmortem Brain Samples.

Patients with psychiatric disorders exhibit dysfunctions in peripheral and central metabolism. This may be a root cause of impaired neuronal function, manifested as changes in mood, behavior, and cognitive capabilities in patients suffering with these conditions. Here we describe a selective reaction monitoring mass spectrometry (SRM-MS)-based targeted proteomic protocol for precise simultaneous quantitation of three glycolytic enzymes in postmortem brain tissue extracts. The SRM-MS approach has several advantages in terms of sensitivity, reproducibility, and reduced sample consumption, compared to traditional MS methods.

Selective Reaction Monitoring for Quantitation of Cellular Proteins.

Proteins and proteomes are dynamic and complex. The accurate identification and measurement of their properties such as abundance, location, and turnover are challenging tasks. Even though high-throughput proteomics has significantly evolved, the technique still lacks fully quantitative and reproducible qualities. A mass spectrometry-based targeted proteomic strategy called selective reaction monitoring (SRM) has emerged in recent years as an important multiplex platform to precisely quantify sets of proteins in multiple samples. This has several advantages in terms of sensitivity, reproducibility, and sample consumption compared to classical methods including those based on antibodies. Here, we present a detailed protocol for quantitation of panels of proteins from cell line extracts using the SRM targeted proteomics approach.