Homologous Serum Research Articles

Different Neutralizing Antibody Responses of Heterologous Sera on Sheeppox and Lumpy Skin Disease Viruses.

Sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV) are the three members of the genus Capripoxvirus within the Poxviridae family and are the etiologic agents of sheeppox (SPP), goatpox (GTP), and lumpy skin disease (LSD), respectively. LSD, GTP, and SPP are endemic in Africa and Asia, causing severe disease outbreaks with significant economic losses in livestock. Incursions of SPP and LSD have occurred in Europe. Vaccination with live attenuated homologous and heterologous viruses are routinely implemented to control these diseases. Using the gold standard virus neutralization test, we studied the ability of homologous and heterologous sera to neutralize the SPPV and LSDV. We found that LSD and SPP sera effectively neutralize their homologous viruses, and GTP sera can neutralize SPPV. However, while LSD sera effectively neutralizes SPPV, SPP and GTP sera cannot neutralize the LSDV to the same extent. We discuss the implications of these observations in disease assay methodology and heterologous vaccine efficacy.

Development and validation of enzyme-linked immunosorbent assay for anti-mouse pertussis immunoglobulin G using international reference anti-Bordetella pertussis mouse serum NIBSC 97/642.

In this study, an in-house enzyme-linked immunosorbent assay (ELISA) was developed and validated. The titer of ELISA was calculated using the reference line (RFL) method based on the standard curve drawn using the international reference anti-mouse serum NIBSC (National Institute for Biological Standards and Control) 97/642. In the development step, signal to noise was depicted to select the buffers that showed the most appropriate ratio. In the validation step, standard range, precision, dilution linearity, and specificity were confirmed, and RFL and parallel line (PLL) methods were compared in precision and dilution linearity. Coating concentration for plate was achieved at 0.1 µg/mL for pertussis toxin (PT), 0.15 µg/mL for filamentous hemagglutinin antigen (FHA), and 0.25 µg/mL for pertactin (PRN). The signal to noise ratio was 22.02 for PT, 14.93 for FHA, and 8.02 for PRN with 0.25% goat serum in phosphate-buffered saline (PBS) as a dilution buffer, and 2% skim milk in PBS as a blocking buffer. Based on the precision results, we assessed the lower limit of quantification by 1, 0.2, and 1.5 EU/mL concentration for PT, FHA, and PRN which met the ICH (International Council for Harmonization) M10 criteria of a 25% accuracy and total error of 40%. In specificity, homologous serum was spiked into heterologous serum and the accuracy met the criteria. There was no difference in the results between RFL and PLL calculations (p-value=0.3207 for PT, 0.7394 for FHA, 0.2109 for PRN). ELISA validated with RFL calculation method in this study is a relatively accurate assay for mouse humoral immunogenicity test.

Insight into the Effect of Submicellar Concentrations of Sodium Deoxycholate on the Structure, Stability, and Activity of Bovine and Human Serum Albumin: An Interesting Comparison between Single and Double Tryptophan Proteins.

The progressive escalation in the applications of bile salts in diverse fields has triggered research on their interaction with various biological macromolecules, especially with proteins. A proper understanding of the interaction process of bile salts, particularly in the lower concentrations range, with the serum albumin seems important since the normal serum concentration of bile salts is approximately in the micromolar range. The current study deals with a comprehensive and comparative analysis of the interaction of submicellar concentrations of sodium deoxycholate (NaDC) with two homologous transport proteins: bovine serum albumin (BSA) and human serum albumin (HSA). HSA and BSA with one and two tryptophans, respectively, provide the opportunity for an interesting comparison of tryptophan fluorescence behavior on interaction with NaDC. The study suggests a sequential interaction of NaDC in three discrete stages with the two proteins. A detailed study using warfarin and ibuprofen as site markers provides information about the sites of interaction, which is further confirmed by inclusive molecular dynamics simulation analysis. Moreover, the comparison of the thermodynamics and stability of the NaDC-serum albumin complexes confirms the stronger interaction of NaDC with BSA as compared to that with HSA. The differential interaction between the bile salt and the two serum albumins is further established from the difference in the extent of decrease in the esterase-like activity assay of the proteins in the presence of NaDC. Therefore, the present study provides important insight into the effect of submicellar concentrations of NaDC on the structure, stability, and activity of the two homologous serum albumins and thus can contribute not only to the general understanding of the complex nature of serum albumin-bile salt interactions but also to the design of more effective pharmaceutical formulations in the field of drug delivery and biomedical research.

Assessment of the antigenic evolution of a clade 6B.1 human H1N1pdm influenza virus revealed differences between ferret and human convalescent sera

Influenza viruses continually acquire mutations in the antigenic epitopes of their major viral antigen, the surface glycoprotein haemagglutinin (HA), allowing evasion from immunity in humans induced upon prior influenza virus infections or vaccinations. Consequently, the influenza strains used for vaccine production must be updated frequently. To better understand the antigenic evolution of influenza viruses, we introduced random mutations into the HA head region (where the immunodominant epitopes are located) of a pandemic H1N1 (H1N1pdm) virus from 2015 and incubated it with various human sera collected in 2015-2016. Mutants not neutralized by the human sera were sequenced and further characterized for their haemagglutination inhibition (HI) titers with human sera and with ferret sera raised to H1N1pdm viruses from 2009 to2015. The largest antigenic changes were conferred by mutations at HA amino acid position 187; interestingly, these antigenic changes were recognized by human, but not by ferret serum. H1N1pdm viruses with amino acid changes at position 187 were very rare until the end of 2018, but have become more frequent since; in fact, the D187A amino acid change is one of the defining changes of clade 6B.1A.5a.1 viruses, which emerged in2019. Our findings indicate that amino acid substitutions in H1N1pdm epitopes may be recognized by human sera, but not by homologous ferret sera. This project was supported by funding from the NIAID-funded Center for Research on Influenza Pathogenesis (CRIP, HHSN272201400008C).

Interactions between hydroxylated polycyclic aromatic hydrocarbons and serum albumins: multispectral and molecular docking analyses.

Hydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) can bind to serum albumin and influence their distribution and elimination in organisms. Here, multispectral analysis and molecular docking methods were used to investigate the binding mechanism of two OH-PAHs, 1-hydroxyphenanthrene (1-OHPhe) and 9-hydroxyphenanthrene (9-OHPhe), with two homologous serum albumins, human serum albumin (HSA) and bovine serum albumin (BSA). The quenching constants of HSA with 1-OHPhe and 9-OHPhe were much larger than those for BSA. Energy transfer from the tryptophan (Trp) residues in HSA to 1-OHPhe and 9-OHPhe was more probable than from Trp in BSA. The interactions of 1-OHPhe and 9-OHPhe with Trp in HSA and BSA altered the microenvironment of Trp. Molecular docking results revealed that the binding modes and binding forces of 1-OHPhe and 9-OHPhe with HSA and BSA were different. The two OH-PAHs were used as fluorescent probes to analyze the microenvironmental hydrophobicities of HSA and BSA, which were distinctly different. The structural difference between HSA and BSA induced significant variations in their binding behaviour with 1-OHPhe and 9-OHPhe. Moreover, HSA was more susceptible to 1-OHPhe and 9-OHPhe than BSA. This work suggests that the differences between the two serum albumins should be considered in related studies.

Abstract 11 Outcome of Patients Treated with Umbilical Cord Blood and Homologous Serum Eye Drops

IntroductionSeveral authors have demonstrated the safety and efficacy of CBS eye drops in the treatment of severe DE with or without Sjogren’s syndrome (SS-I), ocular graft versus host disease (GVHD), persistent corneal epithelial defects, recurrent corneal erosions, chemical burns, and neurotrophic keratitis.ObjectiveTo compare the efficacy of cord blood serum (CBS) and adult donor peripheral blood serum (PBS) eye drops (prepared, standardized, and controlled in the Emilia Romagna Cord Blood Bank) in the treatment of severe dry eye disease (DED), a multicenter randomized, double masked, crossover clinical trial was designed.MethodsSixty patients diagnosed with severe DED associated with persistent corneal epithelial defects were randomized and equally assigned to group A (treated with CBS) or group B (treated with PBS) , eye drops administered 8 times/day for 1 month. Primary outcome was the pre-treatment and post-treatment change in corneal fluorescein staining. Secondary outcomes included the pretreatment and post-treatment change in ocular surface disease index (OSDI), questionnaire and Visual Analogue Score (VAS) of subjective symptoms, Schirmer I test, tear break-up time, and conjunctival staining. Patients with relapse in signs or symptoms after further 2 months switched to the remaining group for one additional month.ResultsCorneal staining was more significantly reduced after CBS treatment; both VAS and OSDI score reduction was observed in both groups, but group A reported significantly less grittiness and pain. Nineteen patients shifted in the crossover period. The within individual comparison confirmed a better recovery in the CBS treatment period. Epidermal growth factor, transforming growth factor, and platelet-derived growth factor content was associated with reduction of epithelial damage. Levels of interleukins were positively associated with symptoms decrease.DiscussionDED signs improved after both CBS and PBS treatments with potential advantages of CBS for subjective symptoms and corneal damage reduction. In addition to the very important therapeutic value of EDHO products, considering the CB units not suitable for transplantation, we have identified novel non-wasteful clinical applications of those allogeneic biological products, resulting in an increase of ethical motivation of families generously donating CB for public benefit.Figure 1.Slit-lamp photographs with fluorescein staining in patient running both period 1 and period 2 of the study. Before PBS treatment, the corneal epithelium was stained diffusely (top left). One month after treatment, the severity of the damage decreased, especially in the central area (top right). Two months after the end of the treatment, the level of epithelial damage worsened and patient entered period 2 (bottom left). After 1 month of CBS treatment, a diffuse decrease in staining was observed in all areas. In this patient, a better improvement was observed after the CBS treatment compared with the PBS treatment.

African Swine Fever Virus Hemadsorption Inhibition Assay.

Adsorption of red blood cells (hemadsorption) on the surface of macrophages infected with African swine fever virus (ASFV) is a unique phenomenon allowing to determine virus infectious titer in hemadsorption unit (HAU) and differentiate virus strains phenotypically. In the meantime, hemadsorption of particular ASFV strain can by inhibited by homologous anti-ASFV serum containing antibody to the serogroup-specific virus protein (CD2v). Here, we describe a hemadsorption inhibition assay (HADIA) to phenotype ASFV strains to one of the known nine serogroups using blood-derived swine macrophages. The HADIA is a powerful method in the ASFV immunopathology and vaccine research since it provides additional antigenic and phenotypic characteristics of virus strains that can't be defined by other assays.

A Comparison of Peripheral Blood Smears, Autologous Cell Cultures, and Reverse Line Blot Hybridisation in Screening for Anaplasma/Ehrlichia in Roaming Dogs and Symptomatic Dogs in Trinidad.

This study compared two methods to detect cases of canine ehrlichiosis in a field setting. One method was a polymerase chain reaction for the 16S rRNA gene followed by reverse line blot hybridisation with genera and species-specific probes for Anaplasma/Ehrlichia. The second method was an autologous cell culture of peripheral leucocytes isolated from heparinised blood and maintained in a homologous canine serum in Dulbecco’s Modified Eagle medium without antibiotics. The cultures were examined under light microscopy for inclusion bodies after 48 h. Leucocytes were successfully propagated for 20 of the 34 samples submitted for autologous cell culture. Inclusion bodies were observed after cell culture in leucocytes of eight dogs. Two dogs were positive to the Anaplasma/Ehrlichia genera probe and six dogs were positive to the E. canis probe after reverse line blot hybridisation. There was acceptable agreement between reverse line blot hybridisation and cell culture results. Both reverse line blot hybridisation and autologous cell cultures can be used to detect E. canis in subclinical and clinical cases of disease. A definitive diagnosis of E. canis is best achieved by a combination of clinical signs, positive autologous cell culture, and reverse line blot hybridisation results.

Difference in an intermolecular disulfide-bond between two highly homologous serum proteins Leg1a and Leg1b implicates their functional differentiation

Zebrafish Liver-enriched gene 1a (Leg1a) and Leg1b are liver-produced serum proteins encoded by two adjacently linked homologous genes leg1a and leg1b, respectively. We previously showed that maternal-zygotic (MZ) leg1a null mutant developed a small liver at 3.5 days post-fertilization (dpf) during winter-time or under UV-treatment and displayed an abnormal stature at its adulthood. It is puzzling why Leg1b, which shares 89.3% identity with Leg1a and co-expressed with Leg1a, cannot fully compensate for the loss-of-function of Leg1a in the leg1azju1 MZ mutant. Here we report that Leg1a and Leg1b share eight cysteine residues but differ in amino acid residue 358, which is a serine in Leg1a but cysteine (C358) in Leg1b. We find that Leg1b forms an intermolecular disulfide bond through C358. Mutating C358 to Methionine (M358) does not affect Leg1b secretion whereas mutating other conserved cysteine residues do. We propose that the intermolecular disulfide bond in Leg1b might establish a rigid structure that makes it functionally different from Leg1a under certain oxidative conditions.

Preparation of serum capped silver nanoparticles for selective killing of microbial cells sparing host cells

Following access into the cell, colloidal silver nanoparticles exhibit generalized cytotoxic properties, thus appear as omnipotent microbicidal, but not suitable for systemic use unless are free of toxic effects on host cells. The AgNP-Serum-18 when prepared from silver nitrate, using dextrose as reducing and group-matched homologous serum as a stabilizing agent, selective endocytosis, and oxidative stress-dependent bio-functional damages to the host are mostly eliminated. For their bio-mimicking outer coat, there is the least possibility of internalization into host cells or liberation of excess oxidants in circulation following interaction with erythrocytes or vascular endothelial cells. The presence of infection-specific antibodies in the serum can make such nano-conjugates more selective. A potent antimicrobial action and a wide margin of safety for mammalian cells in comparison with very similar PVA-capped silver nanoparticles have been demonstrated by the in-vitro challenge of such nanoparticles on different microbes, human liver cell-line, and in-vivo study on mice model. This may open up wide-range therapeutic prospects of colloidal nanoparticles.

Pathology, tissue tropism and antibody response of nephropathogenic infectious bronchitis virus (IBV) Indian isolate in experimentally infected chicken.

The online version contains supplementary material available at 10.1007/s13337-021-00693-4.

Comparison of the protective antigen variabilities of prevalent Newcastle disease viruses in response to homologous/heterologous genotype vaccines

The genotype VII Newcastle disease virus (NDV) vaccine has begun to replace the traditional genotype II NDV vaccine and is widely used in the commercial poultry of China. However, the effect of homologous and heterogeneous anti-NDV serum on the evolution of prevalent NDV is unknown. To understand the effect of genotype II and VII anti-NDV serum on the evolution of genotype VII NDV strains, ZJ1 (waterfowl origin) and CH/SD/2008/128 (ND128; chicken origin) were used for serial passage of 30 generations in DF-1 cells without anti-NDV serum or with genotype II and VII anti-NDV serum independently. The F and HN genes of the 2 viruses were amplified for the 10th, 20th, and 30th generations of each serial passage group and compared with their respective original viruses. We found that there was only one mutation at position 248 in the F gene of ZJ1 due to the serum pressure of genotype VII anti-NDV. Similarly, mutations at residue 527 of the F gene, and position 9 and 319 of the HN gene of ND128 were noted in both anti-NDV serum groups. The results show that the nonsynonymous (NS)-to-synonymous (S) ratio of the F gene of ZJ1 virus was 1.6, and for the HN gene, it was 2.5 in the anti-II serum group. In the anti-VII serum group, the NS/S ratio for the F gene was 2.1, and for the HN gene, it was 2.5. The NS/S ratio of the F gene of the ND128 virus was 0.8, and for the HN gene, it was 3 in the anti-II serum group. Furthermore, the NS/S ratio of the F gene was 0.8, and the HN gene was 2.3 in the anti-VII group. Taken together, our findings highlight that there was no significant difference in the variation of protective antigens in genotype VII NDV under the selection pressure of homologous and heterogeneous genotype NDV inactivated vaccines.

Aggregation of Mouse Serum Amyloid A Protein Was Promoted by Amyloid-Enhancing Factors with the More Genetically Homologous Serum Amyloid A

Amyloid A (AA) amyloidosis is a condition in which amyloid fibrils characterized by a linear morphology and a cross-β structure accumulate and are deposited extracellularly in organs, resulting in chronic inflammatory diseases and infections. The incidence of AA amyloidosis is high in humans and several animal species. Serum amyloid A (SAA) is one of the most important precursor amyloid proteins and plays a vital step in AA amyloidosis. Amyloid enhancing factor (AEF) serves as a seed for fibril formation and shortens the onset of AA amyloidosis sharply. In this study, we examined whether AEFs extracted and purified from five animal species (camel, cat, cattle, goat, and mouse) could promote mouse SAA (mSAA) protein aggregation in vitro using quantum-dot (QD) nanoprobes to visualize the aggregation. The results showed that AEFs shortened and promoted mSAA aggregation. In addition, mouse and cat AEFs showed higher mSAA aggregation-promoting activity than the camel, cattle, and goat AEFs. Interestingly, homology analysis of SAA in these five animal species revealed a more similar amino acid sequence homology between mouse and cat than between other animal species. Furthermore, a detailed comparison of amino acid sequences suggested that it was important to mSAA aggregation-promoting activity that the 48th amino acid was a basic residue (Lys) and the 125th amino acid was an acidic residue (Asp or Glu). These data imply that AA amyloidosis exhibits higher transmission activity among animals carrying genetically homologous SAA gene, and may provide a new understanding of the pathogenesis of amyloidosis.

Comparison of AA Amyloid Fibril Morphology and Serum Amyloid A Gene Sequence in 5 Animal Species.

Amyloid fibrils are characterized by a linear morphology and a cross-β structure. Polymorphic and multiple fibril morphologies can be found when amyloid fibrils are extracted from amyloid-laden tissue. In this study, we report on the purification and transmission electron microscopic analysis of amyloid fibrils from 5 different animal species (mouse, cow, goat, dog, and camel) with AA amyloidosis. The results show that amyloid fibrils had a linear morphology with a cross-structure and irregular length in vivo. Although the fibrils from these different species showed highly similar conformations, there were significant differences in fibril width and crossover distance. We analyzed the sequences of homologous amyloid proteins and serum amyloid A, an evolutionarily conserved protein and a major amyloid precursor. We found 78.23% homology between the most distant amyloid proteins. The findings suggested similar fibril width and crossover distance in different animal species that displayed high homology of amyloid protein sequences. Dog and camel, as well as goat and cow, showed high genetic homology and similar fibril morphology. These data indicate that the fibrils from different animal species have similar genetic homology and morphology, which may provide a better understanding of the pathogenesis of amyloidosis.

Selection and antigenic characterization of immune-escape mutants of H7N2 low pathogenic avian influenza virus using homologous polyclonal sera

Understanding the dynamics of the selection of influenza A immune escape variants by serum antibody is critical for designing effective vaccination programs for animals, especially poultry where large populations have a short generation time and may be vaccinated with high frequency. In this report, immune-escape mutants of A/turkey/New York/4450/1994 H7N2 low pathogenic avian influenza virus, were selected by serially passaging the virus in the presence of continuously increasing concentrations of homologous chicken polyclonal sera. Amino acid mutations were identified by sequencing the parental hemagglutinin (HA) gene and every 10 passages by both Sanger and deep sequencing, and the antigenic distance of the mutants to the parent strain was determined. Progressively, a total of five amino acid mutations were observed over the course of 30 passages. Based on their absence from the parental virus with deep sequencing, the mutations appear to have developed de novo. The antigenic distance between the selected mutants and the parent strain increased as the number of amino acid mutations accumulated and the concentration of antibodies had to be periodically increased to maintain the same reduction in virus titer during selection. This selection system demonstrates how H7 avian influenza viruses behave under selection with homologous sera, and provides a glimpse of their evolutionary dynamics, which can be applied to developing vaccination programs that maximize the effectiveness of a vaccine over time.

Serological relationship between a novel ovine pestivirus and classical swine fever virus.

The genus Pestivirus comprises globally distributed members of the family Flaviviridae, which cause severe losses in livestock. The most common species of the genus are bovine viral diarrhoea virus type 1 (BVDV-1) and type 2 (BVDV-2), classical swine fever virus (CSFV) and border disease virus (BDV). Recently, a novel ovine pestivirus was repeatedly detected in aborted lamb foetuses on a farm located in the Brescia Province (Italy). Complete genome characterization of this isolate showed that it was highly divergent from known pestivirus species and that it was genetically closely related to CSFV. The aim of this study was to determine the serological relatedness between the identified novel pestivirus and BVDV, BDV and CSFV selected strains for which homologous serum was available, by antigenic characterization performed using cross-neutralization assays. The serological relatedness was expressed as the coefficient of antigenic similarity (R). Both field and specific antisera raised against the ovine pestivirus neutralized the CSFV reference strain Diepholz with titres significantly higher than those specific for the BDV and BVDV strains. Furthermore, the calculated R values clearly indicated that the novel ovine pestivirus is antigenically more related to CSFV than to ruminant pestiviruses, in agreement with the results of the genomic analysis. This would have severe consequences on CSFV serology in the event of a switch to porcine hosts with implications for CSFV surveillance and porcine health management.

In Vivo Confocal Microscopy Automated Morphometric Analysis of Corneal Subbasal Nerve Plexus in Patients With Dry Eye Treated With Different Sources of Homologous Serum Eye Drops.

To investigate the comparative effect of allogeneic peripheral blood serum (allo-PBS) and cord blood serum (CBS) eye drops on the status of the corneal subbasal nerve plexus in patients with dry eye disease by using an automated analysis system of in vivo confocal microscopy images. This prospective, randomized, double-blind study included 30 patients with severe dry eye disease assigned to receive allo-PBS (group 1) or CBS (group 2) eye drops 8 times a day for 30 days. The following in vivo confocal microscopy parameters were calculated with ACCMetrics before (visit 1 [V1]) and after treatment (visit 2 [V2]): corneal nerve fiber density, corneal nerve branch density, corneal nerve fiber length, corneal nerve total branch density, corneal nerve fiber area, corneal nerve fiber width, and corneal nerve fractal dimension (CNFrD). In overall patients, the values of corneal nerve fiber density, corneal nerve fiber length, and CNFrD significantly increased, whereas the value of corneal nerve fiber width significantly decreased at V2 compared with V1 (respectively, 20.4 ± 7.9 vs. 17.4 ± 10.1 n/mm; 13.5 ± 4.0 vs. 12.0 ± 5.1 mm/mm; 1.466 ± 0.046 vs. 1.475 ± 0.033; and 0.022 ± 0.002 vs. 0.023 ± 0.002; all P < 0.05). In the subanalysis according to the treatment type, the increase of CNFrD value from V1 to V2 was higher in group 2 compared with group 1 (respectively, from 1.455 ± 0.041 to 1.471 ± 0.030 and from 1.479 ± 0.050 to 1.481 ± 0.035; P = 0.030). Overall, both treatments significantly improved corneal subbasal nerve plexus parameters, and in particular, nerve density, length, width, and fractal dimension. Treatment with CBS eye drops was associated with a higher increase of CNFrD compared with allo-PBS.

Profiling of patient-specific myocytes identifies altered gene expression in the ophthalmoplegic subphenotype of myasthenia gravis

BackgroundWhile extraocular muscles are affected early in myasthenia gravis (MG), but respond to treatment, we observe a high incidence of treatment-resistant ophthalmoplegia (OP-MG) among MG subjects with African genetic ancestry. Previously, using whole exome sequencing, we reported potentially functional variants which associated with OP-MG. The aim of this study was to profile the expression of genes harbouring the OP-MG associated variants using patient-derived subphenotype-specific ‘myocyte’ cultures.MethodsFrom well-characterised MG patients we developed the ‘myocyte’ culture models by transdifferentiating dermal fibroblasts using an adenovirus expressing MyoD. These myocyte cultures were treated with homologous acetylcholine receptor antibody-positive myasthenic sera to induce muscle transcripts in response to an MG stimulus. Gene expression in myocytes derived from OP-MG (n = 10) and control MG subjects (MG without ophthalmoplegia; n = 6) was quantified using a custom qPCR array profiling 93 potentially relevant genes which included the putative OP-MG susceptibility genes and other previously reported genes of interest in MG and experimental autoimmune myasthenia gravis (EAMG).ResultsOP-MG myocytes compared to control MG myocytes showed altered expression of four OP-MG susceptibility genes (PPP6R2, CANX, FAM136A and FAM69A) as well as several MG and EAMG genes (p < 0.05). A correlation matrix of gene pair expression levels revealed that 15% of gene pairs were strongly correlated in OP-MG samples (r > 0.78, p < 0.01), but not in control MG samples. OP-MG susceptibility genes and MG-associated genes accounted for the top three significantly correlated gene pairs (r ≥ 0.98, p < 1 × 10− 6) reflecting crosstalk between OP-MG and myasthenia pathways, which was not evident in control MG cells. The genes with altered expression dynamics between the two subphenotypes included those with a known role in gangliosphingolipid biosynthesis, mitochondrial metabolism and the IGF1-signalling pathway.ConclusionUsing a surrogate cell culture model our findings suggest that muscle gene expression and co-expression differ between OP-MG and control MG individuals. These findings implicate pathways not previously considered in extraocular muscle involvement in myasthenia gravis and will inform future studies.

Fast protocol for the production of Histoplasma capsulatum antigens for antibody detection in the immunodiagnosis of histoplasmosis

BackgroundCurrent methods for the production of Histoplasma capsulatum antigens are problematic in terms of standardization, specificity, stability, repeatability and reproducibility. AimsIn this study, we sought to optimize the methodology for producing H. capsulatum antigens, and to evaluate its applicability. MethodsAntigenic preparations obtained from 12 H. capsulatum isolates were evaluated by double immunodiffusion and immunoblotting assays against homologous and heterologous sera. ResultsThe evaluated and optimized protocol allowed a more stable production, as well as repeatable, reproducible, with shorter culture time and less costly. By double immunodiffusion and immunoblotting assays, the best pattern of reactivity was observed for antigens obtained with 33 days of culture from the isolates 200 and 406 against the M antigen and for the isolate 200 with 15 days against H antigen. The SDS-PAGE presented antigenic components of molecular masses between 17 and 119kDa. The immunoblotting sensitivity was 95.5% and 100% with histoplasmosis sera from ill patients and sera from H. capsulatum infected but otherwise healthy patients, respectively, to the antigen derived from isolates 200 and 406. ConclusionsWe suggest the employment of the antigen from isolate 200, with 15 or 30 days of culture, in the double immunodiffusion and immunoblotting assays due to its good ability to discriminate both sera from patients with histoplasmosis illness and histoplasmosis infection, in addition to its high specificity against heterologous sera.

Is the Sudlow site I of human serum albumin more generous to adopt prospective anti-cancer bioorganic compound than that of bovine: A combined spectroscopic and docking simulation approach

A comparative biophysical study on the individual conformational adaptation embraced by two homologous serum albumins (SA) (bovine and human) towards a potential anticancer bioorganic compound 2-(6-chlorobenzo[d] thiazol-2-yl)-1H-benzo[de] isoquinoline-1,3(2H)- dione (CBIQD) is apparent from the discrimination in binding behavior and the ensuing consequences accomplished by combined in vitro optical spectroscopy, in silico molecular docking and molecular dynamics (MD) simulation. The Sudlow site I of HSA although anion receptive, harbors neutral CBIQD in Sudlow site I (subdomain IIA, close to Trp) of HSA, while in BSA its prefers to snugly fit into Sudlow site II (subdomain IIIA, close to Tyr). Based on discernable diminution of HSA mean fluorescence lifetime as a function of biluminophore concentration, facile occurrence of fluorescence resonance energy transfer (FRET) is substantiated as the probable quenching mechanism accompanied by structural deformations in the protein ensemble. CBIQD establishes itself within HSA close to Trp214, and consequently reduces the micropolarity of the cybotactic environment that is predominantly constituted by hydrophobic amino acid residues. The stronger association of CBIQD with HSA encourages an allosteric modulation leading to slight deformation in its secondary structure whereas for BSA the association is comparatively weaker. Sudlow site I of HSA is capable to embrace a favorable conformation like malleable gold to provide room for incoming CBIQD, whereas for BSA it behaves more like rigid cast-iron which does not admit any change thus forcing CBIQD to occupy an altogether different binding location i.e. the Sudlow site II. The anticancer CBIQD is found to be stable within the HSA scaffold as vindicated by root mean square deviation (RMSD) and root mean square fluctuation (RMSF) obtained by MD simulation. A competitively inhibited esterase-like activity of HSA upon CBIQD binding to Lys199 and Arg257 residues, plausibly envisions that similar naphthalimide based prodrugs, bearing ester functionality, can be particularly activated by Sudlow site I of HSA. The consolidated spectroscopic research described herein may encourage design of naphthalimide based pro-drugs for effective in vivo biodistribution using HSA-based drug delivery systems.