Equine synovial fluid protein equalization via combinatorial peptide ligand libraries

To gain further knowledge of the equine synovial fluid (SF) proteome, we propose a protocol based on the equalization of the relative concentrations of its proteins, which leads to the modification of the standard electrophoretic pattern revealing low-abundance proteins that otherwise remain undetected. Fresh SF samples were collected from ten macroscopically normal metacarpophalangeal joints of crossbred horses. The samples were processed using standard procedures as the control and via combinatorial peptide ligand libraries (CPLL) using low ionic forces (NaH2PO4 10 mM) at different pHs (4.0, 7.0, and 9.3) with 10% sodium dodecyl sulfate (SDS) and 25 mM DTT for protein resolubilization. Proteins were then separated by conventional 8% SDS-PAGE and stained with coomassie blue. After separation of the equalized proteins, there was a significant reduction in the albumin (the most abundant protein in the SF) and, at the same time, other protein bands arise that were not visible without CPLL processing. In addition, there was variation in the protein profiles at different pHs. The results suggest that protein equalization of the equine SF by CPLL could be a useful tool to better understand the articular homeostasis and/or for the detection of new biomarkers of joint pathology.

Leishmania major cause of a cutaneous leishmaniasis in a car driver from east Azerbaijan province: A case report

Background: Leishmaniasis is a zoonotic disease and its the known as a health problem in all of the world. Case presentation: The patient, a 47-year-old man car driver from city of Bostan abad, with a history of traveling to cutaneous leishmaniasis endemic provinces last year, was referred to city health center with a 17 mm, no discharge and pus with necrotic appearance skin lesion. Biochemical, hematological parameters and urine culture and analysis were normal. Three smears was prepared with sterile vaccinostyle from the margins of wound and transferred to specific medium in sterile conditions. After staining the prepared smears from the wound, amastigotes with large nucleus and small kinetoplast in macrophages and active promastigotes in the liquid phase of medium were observed. Polymerase chain reaction (PCR) was performed using ITS-1 by specific primers. In the PCR product electrophoresis were compared with the marker bands and 350 bp band weight determined. With electrophoretic pattern and comparison with positive control band, isolates parasites belonged to Leishmania genus. In order to definitively diagnose the parasite species, sequencing method was used. The results showed 99% homology with Leishmania major. Finally, the patient with a diagnosis of rural cutaneous leishmaniasis was induction with Glucantime drug. Conclusion: Diagnosis of suspected cases with cutaneous leishmaniasis is the major importance. If an infected patient is diagnosed in the early stages of the disease, can be prevented the extent of the scar after recovery, as well as the possible complications of rural infection.

Serum protein electrophoretic pattern in piglets during the early postnatal period

The pattern of serum proteins, the typical features of the electrophoretogram in newborn piglets and during their postnatal development is not completely described. Therefore, the aim of this study was to characterize the changes in serum protein electrophoretic pattern and features of the electrophoretograms during the early postnatal period. Significant changes during the monitored period were found in all evaluated parameters (P < 0.001). The most marked changes were observed mainly in the period before weaning. The concentrations of total proteins, albumin and γ-globulins were before colostrum intake low, γ-globulins represented the smallest proportion of protein fractions. The proportion of α1-globulins was after birth a dominant protein fraction. Significant increase of total proteins, α2-, β- and γ-globulins and decrease of α1-globulins was found 2 days after colostrum intake. The albumin and A/G values increased after birth gradually until weaning. After weaning a significant changes were found in absolute concentrations of total protein and albumin, and in relative values of β-globulin fractions. Presented results showed marked developmental alterations in the serum protein pattern in piglets along with the age. The study also brings new knowledge in the field of description of typical features of electrophoretograms in the observed period of piglet’s life.

Antithrombin glycoforms from type II antithrombin deficient patients selectively adsorb onto the surface of plasma extracellular vesicles

AT is a glycoprotein produced by the liver and acts as the most important antagonist of clotting factors. A deficit in AT production or function leads to coagulation disorders. Two kinds of AT deficiencies are reported, named quantitative (or type I) and qualitative (or type II) defects. The first is characterized by low levels of AT in the bloodstream, the latter by impaired AT activity related to dysfunctional domains of AT and it is challenging to diagnose. Although being a soluble protein, evidence of AT transported by plasma EVs has been found but the physicochemical features of the association of AT to EVs are missing. We separated and characterized EVs from the plasma of healthy subjects, focusing on AT association. We found AT is localized on the external leaflet of the EV membrane. Furthermore, 2D-electrophoresis conducted on plasma and EVs of healthy subjects highlighted that specific AT glycoforms are selectively enriched onto the EVs with respect to whole plasma, suggesting that glycosylation plays a role in the partitioning of AT between the EV surface and liquid plasma phase, and ultimately on the EV exofacial topology. Finally, we separated EVs from the plasma of 8 patients affected by type II AT defect. The comparison of the AT 2D-electrophoretic pattern of patients and healthy subjects highlighted a difference in AT adsorption onto EV surface, supporting the role of EVs in coagulation and suggesting a promising approach to improve diagnosis and management of type II AT deficiencies.

Bisalbuminemia: A Pathologist’s Insight of an Uncommon Phenomenon

Background The incidence of a bifid electrophoretic pattern in the albumin region on serum protein electrophoresis is an infrequent phenomenon. The availability of literature from India is scarce and is limited to case reports. Objective The aim of the study is to analyze the frequency of bisalbuminemia in an Indian referral facility. The study delved into their clinical associations. Material and Methods The retrospective case records of the patient from the departmental database were scrutinized. The study subjects were for an 8-year study period. Results There were about 39,900 serum electrophoresis performed in an 8-year study period. A total of 40 cases of bisalbuminemia were detected. The incidence in our cohort was 0.01%. Conclusion Bisalbuminemia, an overtly benign condition, is infrequent in Indian population although not rare. It is associated with several clinical disorders; however, the association seems to be plausibly coincidental.

The proteolytic inactivation of protein Z-dependent protease inhibitor by neutrophil elastase might promote the procoagulant activity of NETs

Septic shock is the archetypal clinical setting in which extensive cross talk between inflammation and coagulation dysregulates the latter. The main anticoagulant systems are systematically impaired, depleted and/or downregulated. Protein Z-dependent protease inhibitor (ZPI) is an anticoagulant serpin that not only targets coagulation factors Xa and XIa but also acts as an acute phase reactant whose plasma concentration rises in inflammatory settings. The objective of the present study was to assess the plasma ZPI antigen level in a cohort of patients suffering from septic shock with or without overt-disseminated intravascular coagulation (DIC). The plasma ZPI antigen level was approximately 2.5-fold higher in the patient group (n=100; 38 with DIC and 62 without) than in healthy controls (n=31). The elevation’s magnitude did not appear to depend on the presence/absence of DIC. Furthermore, Western blots revealed the presence of cleaved ZPI in plasma from patients with severe sepsis, independently of the DIC status. In vitro, ZPI was proteolytically inactivated by purified neutrophil elastase (NE) and by NE on the surface of neutrophil extracellular traps (NETs). The electrophoretic pattern of ZPI after NE-catalyzed proteolysis was very similar to that resulting from the clotting process - suggesting that the cleaved ZPI observed in severe sepsis plasma is devoid of anticoagulant activity. Taken as a whole, our results (i) suggest that NE is involved in ZPI inactivation during sepsis, and (ii) reveal a novel putative mechanism for the procoagulant activity of NETs in immunothrombosis.

Impact of Extraction Method on the Detection of Quality Biomarkers in Normal vs. DFD Meat

The objective of this work was to demonstrate how the extraction method affects the reliability of biomarker detection and how this detection depends on the biomarker location within the cell compartment. Different extraction methods were used to study the sarcoplasmic and myofibrillar fractions of the Longissimus thoracis et lumborum muscle of young bulls of the Asturiana de los Valles breed in two quality grades, standard (Control) or dark, firm, and dry (DFD) meat. Protein extractability and the expression of some of the main meat quality biomarkers—oxidative status (lipoperoxidation (LPO) and catalase activity (CAT)), proteome (SDS-PAGE electrophoretic pattern), and cell stress protein (Hsp70)—were analyzed. In the sarcoplasmic fraction, buffers containing Triton X-100 showed significantly higher protein extractability, LPO, and higher intensity of high-molecular-weight protein bands, whereas the TES buffer was more sensitive to distinguishing differences in the protein pattern between the Control and DFD meat. In the myofibrillar fraction, samples extracted with the lysis buffer showed significantly higher protein extractability, whereas samples extracted with the non-denaturing buffer showed higher results for LPO, CAT, and Hsp70, and higher-intensity bands in the electrophoretic pattern. These findings highlight the need for the careful selection of the extraction method used to analyze the different biomarkers considering their cellular location to adapt the extractive process.

Identification, Virulence, and Molecular Characterization of a Recombinant Isolate of Grass Carp Reovirus Genotype I

The hemorrhagic disease of grass carp (HDGC) caused by grass carp reovirus (GCRV) still poses a great threat to the grass carp industry. Isolation and identification of the GCRV genotype I (GCRV-I) has been rarely reported in the past decade. In this study, a new GCRV was isolated from diseased fish with severe symptoms of enteritis and mild hemorrhages on the body surface. The isolate was further identified by cell culture, transmission electron, indirect immunofluorescence, and SDS-PAGE electrophoretic pattern analysis of genomic RNA. The results were consistent with the new isolate as a GCRV-I member and tentatively named GCRV-GZ1208. Both grass carp and rare minnow infected by the GCRV-GZ1208 have no obvious hemorrhagic symptoms, and the final mortality rate was ≤10%, indicating that it may be a low virulent isolate. GZ1208 possessed highest genomic homology to 873/GCHV (GCRV-I) and golden shiner reovirus (GSRV). Additionally, it was found a 90.7–98.3% nucleotide identity, a 96.4–100% amino acid identity, and <50% identity with GCRV-II and III genotypes. Interestingly, the sequences of some segments of GZ1208 were similar to GCRV-8733/GCHV, whereas the remaining segments were more closely related to GSRV, suggesting that a recombination event had occurred. Bootscan analysis of the complete genomic sequence confirmed this hypothesis, and recombination events between 873/GCHV and other GSRV-like viruses were also accompanied by gene mutations.

Salt-Cured Atlantic Cod Skin: A Sustainable Source of Acid-Soluble Type I Collagen

Collagen is the most abundant protein in the animal kingdom. Industrial collagen is mainly bovine and porcine origin. However, due to religious beliefs, allergic issues, and infectious diseases, alternative sources of collagen as marine are gaining increasing interest. In this work, the acid-soluble collagen (ASC) were extracted from salt-cured Atlantic cod (Gadus morhua) skin and characterized. The extraction yield was about 2.0%, equivalent to the extraction yield reported for other fish skins. The electrophoretic pattern showed the typical type I structure (&alpha;, &beta; and &gamma; chains). UV-VIS and FTIR absorbance spectra suggested a very pure ASC with an intact triple helical structure. The integrity and the adequate porosity required for different applications were then confirmed by electron micrograph. Our findings allow us to say that, for the first time, we extracted acid-soluble type I collagen from salt-cured Atlantic cod skin, with characteristics suitable for application in various fields, such as biomedical.

Modulation of Serum Protein Electrophoretic Pattern and Leukocyte Population in Horses Vaccinated against West Nile Virus

This study aimed to evaluate the hematological and serum protein electrophoretic profiles of horses after inactivated West Nile virus (WNV) vaccine administration. Blood samples were collected from 10 horses before (T0), after 24 h, 48 h, 72 h, 1 week, 2 weeks and 3 weeks (T1I, T2I, T3I, T4I, T5I and T6I) from the first WNV vaccine-dose administration, before the vaccine-booster (TPREII), and after 24 h, 48 h, 72 h, 1 week, 2 weeks and 3 weeks (T1I I, T2II, T3II, T4II, T5II, T6II) from the WNV vaccine-booster. There was a significant increase in lymphocytes and a decrease in neutrophils after both the first vaccine-dose and vaccine-booster administration compared to the baseline values (p < 0.01). Monocytes showed higher values after 72 h, 1 week and 2 weeks from the vaccine-booster (p < 0.01). Higher serum total protein values were found in horses after both the first vaccine-dose and booster administration (p < 0.05). α1-lobulins increased after the vaccine-booster with the highest levels measured at T4II (p < 0.05); α-2- and β-globulin fractions increased throughout the post-vaccine period compared to the baseline values (p < 0.05); and higher γ-globulin values were found before the vaccine-booster (TPREII) and after 24 h, 72 h and 3 weeks from the vaccine-booster (T1II, T3II and T6II). The findings allow us to conclude that the WNV vaccine used in the current study does not alter the overall hemogram picture of horses although it is associated with modulation of leukocyte populations and the serum protein electrophoretic pattern.