Protein Bands Research Articles

Overexpression of miR-155 Modulates Interferon Response and Inhibits Viral Replication of IHNV and IPNV in EPC Cells.

Infections caused by RNA viruses, including infectious haematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV), result in substantial economic losses in the aquaculture industry. MicroRNAs (miRNAs), particularly miR-155, play crucial roles in regulating host immune responses and viral infections. In this study, we investigated the overexpression effect of miR-155 in Epithelioma papulosum cyprini (EPC) cells infected with IHNV and IPNV and analysed the mechanisms underlying these antiviral activities. EPC cells were transfected with miR-155 at 40 pmol and then infected with IHNV or IPNV at an MOI of 0.01. The cytopathogenic effect (CPE) was observed for 5 days post-infection. The cells transfected with miR-155 did not show any signs of CPE or exhibit any viral growth over time after infection with both viruses. Additionally, real-time qPCR of type I interferon-related immune genes (ISG15 and Mx1) showed upregulation at 0, 24, and 48 h.p.i in cells transfected with miR-155. At 48 h post-infection, the cells transfected with miR-155 did not show any bands of viral protein by western blot. Furthermore, the overexpression of miR-155 in EPC cells significantly enhanced the expression of interferon response genes by targeting BCL2 and CYLD and suppressed the viral replication through directly targeting viral genes, including the L gene of IHNV and the VP2 gene of IPNV. These findings elucidate the dual mechanism of miR-155's antiviral effect through immune modulation and direct viral gene targeting, offering insights for developing novel antiviral strategies in aquaculture.

Theranostic Near-Infrared Monoamine Oxidase Inhibitor (NMI) Protein Binding Interactions with MAOA and Albumin.

The protein binding interactions of near-infrared monoamine oxidase inhibitor (NMI) are reported here. NMI-bound proteins were examined by fluorescent SDS-PAGE and mass spectrometry using tumor tissues from brain and colon cancer mouse models. This study shows protein interactions with NMI, a chemical conjugate of MAOA inhibitor clorgyline and tumor-seeking dye, MHI-148. NMI fluorescence in MAOA knock-out (KO) mice was significantly lower compared to WT mice, including whole animal, organs, and tissue lysates which indicated that NMI binds to MAOA. Pure recombinant MAOA protein was detectable as a single fluorescent band that migrated at ~ 65kD. NMI inhibited MAOA activity (IC50 1-5µM). In a glioma mouse model, NMI targeted specifically to tumor with high contrast to adjacent normal brain, shown by a 65 kD protein band. Recent studies demonstrated heptamethine cyanine dyes (e.g., MHI-148) interact with serum albumin, contributing to tumor uptake and cancer cell internalization. Our study shows NMI binds to albumin but highly prefers MAOA, providing a plausible mechanism for systemic drug delivery via serum albumin to the tumor target and subsequent MAOA inhibition. Further studies in a colon cancer mouse model found the ~ 65 kD SDS-PAGE band, bound to NMI, contained both MAOA and albumin proteins by mass spectrometry. NMI was shown to interact with MAOA and the blood carrier protein, albumin. This study provides insights for drug delivery and protein target specificity of NMI to image and treat cancer.

Comparative Study of Electrophoretic Patterns of Protein Sub Units in Various Tissues of Two Fresh Water Fishes (Heteropneustes fossilis and Labeo rohita) Through Gel Electrophoresis

The present investigation is determine the comparative study of electrophoretic patterns of protein sub units in two different fishes i.e. Heteropneustes fossilis and Labeo rohita. All the tissues were examined on 7.5% SDS-PAGE. In this investigation we observed gill, liver, intestine, muscle and brain tissues of both fishes. In this Gill and Muscle tissues of two fishes exhibits highest protein bands (10 and 7 respectively). The lowest bands are observed in intestine in both fishes(7,5 respectively).In this investigation we observed homology in protein bands with minor variations.

Corosolic acid and its derivatives targeting MCCC1 against insulin resistance and their hypoglycemic effect on type 2 diabetic mice.

Corosolic acid (CA), a natural triterpenoid, exhibits various biological activities and is often called as plant-derived insulin due to its significant hypoglycemic effects, making it especially beneficial for individuals with diabetes or high blood glucose levels. However, CA has notable in vitro toxicity, low water solubility, and poor pharmacokinetic properties. To address these limitations, a series of CA derivatives were synthesized, resulting in the identification of derivative H26, which demonstrates a significantly enhanced hypoglycemic effect, reduced toxicity, and improved pharmacokinetic characteristics compared to CA. To identify the target protein of CA and investigate its therapeutic potential, a chemical probe derived from natural products, called CA-biotin, was designed and synthesized. By employing an avidin-biotin affinity binding system, we distinguished the differential protein bands between CA-biotin and biotin. This quantitative proteomic analysis revealed, for the first time, that the biotin-containing enzyme methylcrotonoyl-CoA carboxylase 1 (MCCC1) directly binds to CA. The interaction between H26 and MCCC1 was examined in vitro. The research on the mechanisms by which CA and H26 address Type 2 diabetes mellitus (T2DM) focused on the insulin resistance signaling pathway, specifically targeting MCCC1. The results indicated that H26 shows significant promise as a potential hypoglycemic agent, while MCCC1 may serve as a valuable target for addressing insulin resistance. This presents a promising opportunity for developing new medications aimed at improving the health of patients with type 2 diabetes mellitus (T2DM) or hyperglycemia.

Evaluating serum proteinogram methodologies for the diagnosis of inflammation in fish: Acute and chronic patterns in gilthead seabream (Sparus aurata) injected with λ-carrageenan.

Proteinogram is a semiquantitative method specially used in clinic to separate the serum proteins from patients for use in the diagnosis of diseases. However, this methodology has only been applied very recently with this approach in farmed fish. Thus, the aim of this study was to explore the changes in the serum proteinogram of gilthead seabream (Sparus aurata), after triggering an acute or chronic sterile inflammation. For this, two experiments were carried out: i) Acute inflammation experiment: seabream specimens were injected intramuscularly with 50μL of λ-carrageenan (0.5mg fish-1) or buffer (control) and blood samples were collected at 3, 6 and 24h post-injection; ii) Chronic inflammation experiment: specimens were injected at 0, 7 and 14 days with 500, 250 and 250μL of λ-carrageenan, respectively (20mg fish-1) or buffer, and blood samples were collected at 15 days post-injection. In both cases, serum was obtained and processed by electropherograms and HPLC-mass spectrometry. Results of electropherograms of control fish revealed four major proteins of 19.5, 76.3, 104.4, and 156.7kDa in the serum. These four proteins were correlated with apolipoprotein A-II (II (the counterpart of mammalian albumin, Apo fraction), serotransferrin (β fraction), inter-α-trypsin inhibitor heavy chain H3-like (α1 fraction) and α-2-macroglobulin-like (α2 fraction) according to the results obtained with HPLC-mass spectrometry. In a statistical view (p<0.05), no variations were detected in the four major serum protein bands between the control and the acutely inflamed groups. However, in chronically inflamed fish, the Apo fraction decreased statistically compared to the control group. In contrast, the α1 and α2 fractions were statistically increased in the serum of fish sampled 15 days after λ-carrageenan injection, compared to those found in the control fish. α1 and α2 protein fractions are recognized indicators of inflammation in mammals. Consequently, our study presents a novel method for assessing both acute and chronic λ-carrageenan-induced sterile inflammation in gilthead seabream, which could be applicable to other marine species for diagnostic purposes.

Phosphonylated tyrosine and cysteine disulfide adducts both generated from immunoglobulin G and human serum albumin indicate exposure to the nerve agent VX in vitro.

Pronase-catalyzed proteolysis is shown to produce single amino acid adducts of tyrosine (Tyr) and cysteine (Cys) obtained from both human serum albumin (HSA) and immunoglobulin G (IgG) after in vitro exposure of plasma to the nerve agent VX. Total plasma as well as isolated HSA and IgG yielded the Tyr residue phosphonylated with the ethyl methylphosphonic acid moiety, Tyr(-EMP). Furthermore, a Cys residue adducted with the diisopropylaminoethane thiol leaving group of the agent bound via a disulfide bridge, Cys(-DPAET), was also obtained from both proteins. Even though Tyr(-EMP) represents an internationally well-accepted biomarker of a VX-like agent its origin from plasma IgG has never been shown before. In addition, this is the first time that Cys(-DPAET) is presented as a biomarker of VX exposure clearly identifying the chemical nature of the V-type nerve agent's leaving group. Both biomarkers were detected after selective affinity-based solid-phase extraction (SPE) from plasma that yielded highly purified HSA and IgG as documented by sodium dodecyl polyamide gel electrophoresis (SDS-PAGE). Both biomarkers were found in the corresponding protein bands of HSA and IgG each after in-gel proteolysis with pronase. A micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry method (LC-ESI HR-MS/MS) was developed for the simultaneous detection of Tyr(-EMP) and Cys(-DPAET). The time for proteolysis was optimized for maximum biomarker yield. The method showed excellent selectivity and sensitivity, and the adducted proteins and biomarkers were found to be highly stable during storage. Accordingly, the presented method sheds more light on the molecular toxicology of VX and broadens the spectrum of methods suited for biomedical verification.

Structural and Functional Analysis of Hemoglobin Binding to the Peritrophic Matrix During Blood Digestion in Aedes aegypti

The Aedes aegypti mosquito is responsible for transmitting pathogens such as the Dengue, Zika, and Chikungunya viruses. The peritrophic matrix (PM) is an extracellular chitin-rich structure that lines the midgut of arthropods, providing a crucial protective barrier for the gut epithelium against mechanical damage, ingested pathogens, and toxic substances. During blood digestion, hemoglobin is lysed, releasing free heme into the midgut lumen. Part of this heme binds strongly to the PM, mitigating its harmful effects on the mosquito epithelial cells. Our study focused on investigating the interaction dynamic between hemoglobin and the PM during blood digestion in A. aegypti. Optical microscopy was employed to observe the temporal progression of blood digestion in the A. aegypti midgut, highlighting significant morphological changes in the blood bolus. An electrophoresis analysis revealed distinct protein bands in the PM extract, some of which were associated with hemoglobin and its subunits. The presence of PM-associated hemoglobin was confirmed by amino-terminal sequencing and an immunoblot analysis using anti-hemoglobin antibodies. Furthermore, fluorescence microscopy revealed overlapping labeling between hemoglobin and chitin, suggesting an interaction between hemoglobin and PM chitin. Corroborating these results, hemoglobin showed an affinity with chitin in the chromatography and molecular docking assays, in which the hemoglobin subunits interacted with the oligosaccharide (NAG)4. Thus, hemoglobin may perform a function similar to that of peritrophins. Further experiments demonstrated the protective role of the PM against hemoglobin proteolysis during blood digestion. Overall, this study provides valuable insights into the intricate interactions between hemoglobin and the PM, enhancing our understanding of mosquito digestive physiology and potentially contributing to the development of vector control strategies.

Blastocystis ST1: Protein Profile and Specific Serum Immunoglobulin in Irritable Bowel Syndrome (IBS) Patients.

Blastocystis sp. is a common enteric human parasite, which recently has been linked to gastrointestinal disorders i.e. Irritable Bowel Syndrome (IBS) and symptomatic patients (non IBS). Analyzing antibodies level in these patients could help in differential diagnosis. The current study aimed to identify the protein profile of the Blastocystis ST1 (Syrian strain: OR537347) lysates and to investigate the differences in IgG serum immunoglobulin between patients with IBS and non IBS. Twenty two IBS (Rome III) and nineteen patients suffering from different gastrointestinal disorders (non IBS), positive for Blastocystis were enrolled in this study. SDS-PAGE was used to identify the protein profile of the Blastocystis ST1 lysates and immunoblotting using sera from patients was used for reactivity compared to known Blastocystis protein targets. The crude protein profile of Blastocystis ST1 showed 24 protein bands ranged between 10 and 130kDa. Western blot demonstrated that the proteins (27-29);32;(39-42);(50-51) kDa had similar immunogenicity characteristic in IBS and non IBS patients while the proteins (60-95kDa) only interacted with IBS patients' sera. Our results highlighted the importance of Blastocystis proteins 60-95kDa (probably a metalloproteases) in IBS patients compared to non IBS, suggesting that these metalloproteases may be important Blastocystis antigens and can be used as a serologic test tool or as a biomarker for differential diagnosis.

DOP119 Predicting response to anti-tumour necrosis factor-alpha therapy-α in fibrostenotic Crohn’s disease using infra-red microspectroscopy

Abstract Background Fibrostenotic strictures are common in Crohn’s disease, have limited treatment options and lack validated biomarkers to predict treatment response. Fourier-transform infra-red (FTIR) spectroscopy provides reproducible biochemical information from biospecimens using a rapid, label-free, non-destructive technique via molecular vibrations. We previously completed a proof-of-concept study that identified spectral biomarkers for inflammation in a model of colitis.1 The aim of this study was to investigate whether the tissue biochemical "fingerprint" using FTIR spectroscopy can predict patient clinical response to anti-tumour necrosis-factor-α (anti-TNFα) therapy, and identify spectral biomarkers for treatment response. Methods Hyperspectral images were acquired using the Agilent Cary 670 FPA-IR coupled with the Agilent Cary 620 microscope in transflection mode in the wavenumber region 1800 – 800 cm-1 from fixed ileo-colonoscopic biopsies from 62 patients with Crohn’s disease and 12 healthy controls. A subset of patients (n = 24) were included from STRIDENT2, a randomised study assessing adalimumab therapy for intestinal Crohn’s strictures. Thousands of spectra were obtained per sample, pre-processed via transformation to the second derivative, normalised and reduced to 25 spectra per sample prior to analysis using partial least squares discriminant analysis (PLSDA) with internal cross-validation. Spectral biomarkers were identified by the most contributive infra-red bands from the latent variables (LV) and significant variable importance in projection (VIP) scores of &amp;gt;1. Adjacent histological sections were stained with H&amp;E, Masson’s trichrome and α-smooth muscle actin with inflammation and fibrosis scored by a clinical pathologist. Results Spectra from baseline biopsies from the 24 patients in the STRIDENT study demonstrated excellent performance using PLSDA in discriminating between responders and non-responders in relation to the 12 month clinical outcomes, defined by area under the receiver operating characteristic curve (AUC) &amp;gt;0.9 for MRI and IUS responses, normalisation of faecal calprotectin and CRP, CDAI and pain responses (Table 1). Spectral biomarkers of interest for stricture response to anti-TNFα are shown in Figure 1 and include the amide I and II protein bands (peaks at 1651 and 1543 cm-1, respectively), the carboxylate group of proteins (1454 cm-1), lipid bands (1750 and 1395 cm-1), collagen band (1236 cm-1), glycoprotein bands (1081 and 1030 cm-1), and nucleic acid bands (1236, 1081 and 966 cm-1). Conclusion The novel technique of FTIR spectroscopy detects tissue biochemical "fingerprints" in fibrostenotic Crohn’s disease that demonstrate excellent discrimination for predicting clinical response to adalimumab therapy.

Two Novel Peptides from Buthotus saulcyi Scorpion Venom: Proteomic Analysis and Approaches

Background and Objectives: Venomous scorpions play a crucial role in medicine and public health. Buthotus saulcyi scorpion is known as one of the most populous species in East Asia and Iran, while its venom proteome has still not been fully determined. background: In the present study, we report for the first time a proteomic profile of Buthotus saulcyi scorpion venom with the aim of looking ahead and determining the structural and functional characteristics of these compounds for use as medical tools. Aims: In the current research, the proteomic profile of Buthotus saulcyi scorpion venom to determine the structural and functional characteristics of its compounds used for treatment will be examined for the first time. objective: In the present study, we report for the first time a proteomic profile of Buthotus saulcyi scorpion venom with the aim of looking ahead and determining the structural and functional characteristics of these compounds for use as medical tools. Method:: 2D-PAGE, HPLC, SDS-PAGE, sequencing, and MALDI-TOF MS techniques were used to investigate the properties of these peptides. Result: The 2D-PAGE analysis of crude toxin from B. saulcyi revealed a minimum of 96 protein spots, with isoelectric points ranging from 4 to 9 and molecular weights spanning from 3.6 to 205 kDa. Following this, HPLC was used to isolate 14 fractions of crude toxins, and the protein content of these fractions was measured. SDS-PAGE analysis identified 7 protein bands within the B. saulcyi crude toxin fractions, with molecular weights ranging from 13 to 217 kDa. Further examination of fraction 7 through amino acid sequencing resulted in the identification of two protein bands labeled peptide 3 and peptide 4. Ultimately, these protein bands were extracted, and their molecular mass and amino acid sequences were analyzed using MALDI-TOF MS. result: 2D-PAGE data from the B. saulcyi crude toxin showed at least 96 protein spots in isoelectric point between 4 and 9 and a molecular weight between 3.6 and 205 kDa. Then 14 fractions of crude toxins were isolated using HPLC and their protein content was estimated. SDS-PAGE analysis of fractions of B. saulcyi crude toxin showed 7 protein bands with a molecular weight between 13 and 217 kDa. Subsequently, the amino acid sequencing of Fraction 7 led us to two protein bands designated as Peptide 3 and Peptide 4 peptides. Finally, these protein bands were removed, and molecular mass and amino acid sequence analysis was performed using MALDI-TOF MS. Conclusion:: According to our results, the alignment of P3 and P4 protein sequences revealed the highest similarity to chrysophsin 2 and pheromone-bound protein 2, respectively.

Histone H2B lysine lactylation modulates the NF-κB response via KPNA2 during CSFV infection.

Histone lysine lactylation (Kla) has recently been reported to participate in various biological processes, regulating transcription, inflammation, and immune-related diseases. However, the mechanism of histone Kla in innate immunity and viral infection remains largely unknown. Here, we observed fluorescent Kla signals in all four histones (H2A, H2B, H3, and H4) in PK-15 cells. Immunoprecipitation analysis showed prominent histone Kla protein bands, with H2B being the most abundant. We generated the H2B K16R mutant plasmid and identified K16 as one of the Kla modification sites in H2B. Further exploration revealed increased global H2B Kla and H2BK16la levels upon classical swine fever virus (CSFV) infection. By employing the Kla agonist (L-lactate), inhibitor (oxamate), or siLDHA, we demonstrated that H2BK16la and pan Kla in PK-15 cells rely on the LDHA-lactate axis, which is also crucial for CSFV-induced H2BK16la and pan Kla. Moreover, our data proved the interaction between H2B and CSFV NS4A protein. Notably, H2B Kla can modulate CSFV proliferation. Mechanistically, H2BK16la and pan Kla activate the nuclear factor kappa-B (NF-κB) pathway by mediating p65 nuclear translocation via karyopherin α2 (KPNA2), thereby inducing type III interferon (IFN-λ) expression and inhibiting CSFV replication. In conclusion, our study unveils the role of H2B Kla in regulating the NF-κB pathway during viral infection, presenting a novel mechanism. These findings significantly contribute to understanding the pathogenic mechanisms during viral infection and hold promise for the development of viral therapeutic strategies.

T4 Phage Displaying Dual Antigen Clusters Against H3N2 Influenza Virus Infection.

The current H3N2 influenza subunit vaccine exhibits weak immunogenicity, which limits its effectiveness in preventing and controlling influenza virus infections. In this study, we aimed to develop a T4 phage-based nanovaccine designed to enhance the immunogenicity of two antigens by displaying the HA1 and M2e antigens of the H3N2 influenza virus on each phage nanoparticle. Specifically, we fused the Soc protein with the HA1 antigen and the Hoc protein with the M2e antigen, assembling them onto a T4 phage that lacks Soc and Hoc proteins (Soc-Hoc-T4), thereby constructing a nanovaccine that concurrently presents both HA1 and M2e antigens. The analysis of the optical density of the target protein bands indicated that each particle could display approximately 179 HA1 and 68 M2e antigen molecules. Additionally, animal experiments demonstrated that this nanoparticle vaccine displaying dual antigen clusters induced a stronger specific immune response, higher antibody titers, a more balanced Th1/Th2 immune response, and enhanced CD4+ and CD8+ T cell effects compared to immunization with HA1 and M2e antigen molecules alone. Importantly, mice immunized with the T4 phage displaying dual antigen clusters achieved full protection (100% protection) against the H3N2 influenza virus, highlighting its robust protective efficacy. In summary, our findings indicate that particles based on a T4 phage displaying antigen clusters exhibit ideal immunogenicity and protective effects, providing a promising strategy for the development of subunit vaccines against various viruses beyond influenza.

CXCL13 levels in cerebrospinal fluid in relapsing-remitting multiple sclerosis: The role of Borrelia in neuroinfections.

This study was compared the Borrelia antibodies and chemokine ligand 13 (CXCL13) levels in cerebrospinal fluid (CSF) samples from cases diagnosed with relapsing-remitting multiple sclerosis (RRMS), radiologically isolated syndrome (RIS), and pseudotumour cerebri (PTC). A total of 43 CSF samples were collected from patients diagnosed with RRMS, RIS and PTC. We prospectively investigated Borrelia IgG and IgM antibodies in the CSF samples of the cases by enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) method, and CXCL13 levels by ELISA. Data were statistically analysed using the the Spearman rank correlation test. Five antigens (protein 19, 20, 21, 58, and outer surface protein C (OspC)) were positive due to confirmation of the positive samples for Borrelia antibodies by the WB method. There were no significant differences in CSF CXCL13 levels between the three groups. The CXCL13 level was found to be statistically higher in the demyelinating group compared to the PTC group (p=0.001). The Borrelia antibodies were found positive in CSF samples of RRMS patients. The coexistence of high CXCL13 (may be a potential biomarker) suggests that LNB may also play a role in the etiopathogenesis of RRMS. In addition, the positive detection of OspC and p58 WB bands in most cases suggests that these protein bands can be used as in the differential diagnosis of neurodegenerative diseases and Lyme disease.

Retracted] Differential effects of PXD101 (belinostat) on androgen-dependent and androgen-independent prostate cancer models.

Following the publication of the above article, a concerned reader drew to the Editor's attention that certain of the western blot assay data shown in Fig. 4G on p. 717 were strikingly similar to data that had appeared in a paper published previously in the journal Molecular Cancer Therapeutics, which had been written by different authors at different research institutes. Moreover, there appeared to be as many as five sets of duplicated protein bands in Fig. 4B and C, including the appearance of apparently the same protein band in different orientations in Fig. 4C in one case, and certain data shown in Fig. 4B and G had apparently been re-used in these figure parts. After having performed an independent assessment of these issues in the Editorial Office, these concerns of the reader were found to have been validated; therefore, the Editor of International Journal of Oncology has decided that this paper should be retracted from the publication. After having been in contact with the authors, they accepted the decision to retract the paper. The corresponding author, Claudio Festuccia, takes full responsibility for the raised concerns and clarifies that the other co-authors were not directly involved in the preparation of the submitted figures. The Editor apologizes to the readership for any inconvenience caused. [International Journal of Oncology 40: 711-720, 2012; DOI: 10.3892/ijo.2011.1270].

Immunoproteomic analysis and identification of possible allergenic proteins in Artemisia annua pollen.

Artemisia annua (A. annua) is a wind-pollinated weed and a major allergen responsible for allergic respiratory diseases in Northern China. This study involved the separation of pollen proteins from A. annua utilizing SDS-PAGE and Coomassie Blue staining techniques. The effectiveness of extracting allergens from Artemisia annua pollen (AAP) were confirmed both in vivo and in vitro. A mouse model was established using A. annua pollen (AAP) extracts. In vitro, the interaction between allergenic proteins in the AAP extract and specific IgE antibodies present in patients' sera was analyzed, using IgE-immunoblotting and ELISA methods. Protein bands were subsequently analyzed by mass spectrometry. The recombinant Art an 3 (rArt an 3) protein was obtained via recombination, expression, and purification. Finally, the binding activity of rArt an 3 specific IgE was subsequently examined using Western blot and inhibition ELISA (iELISA). The electrophoretic profiles of AAP showed band patterns ranging from 10 to 70kDa, with the most prominet IgE-binding pollen proteins detected at approximately 12kDa. After stimulation of AAP, the sensitized group of mice exhibited significant allergic symptoms compared to the control group. Mass spectrometry analysis identified 7 proteins, including putative aldehyde oxidase Art an 7, Art v 1-like protein, Art an 3.0101 allergen precursor, Art an 3.0102 allergen precursor, Art an 3.0201 allergen precursor, lipid transfer protein 3, and non-specific lipid-transfer protein. The results of immunoblotting and iELISA showed that rArt an 3 could bind to IgE antibodies in the patient sera, and co-incubation of rArt an 3 with serum, the binding serum significantly inhibited IgE binding to the crude AAP. The Art an 3 is a key allergenic protein in AAP with a high IgE sensitization rate in the studied population sample. These findings enhance our understanding of the sensitization components of AAP and its sensitization characteristics within the Chinese population, thereby promoting the development of precise molecular diagnostics and therapeutic interventions.

Preparation and Purification of β-1,3-glucan-Linked Candida glabrata Cell Wall Proteases by Ion-Exchange Chromatography, Gel Filtration, and MDPF-Gelatin-Zymography Assay.

Candida glabrata is an opportunistic pathogen that may cause serious infections in an immunocompromised host. C. glabrata cell wall proteases directly interact with host cells and affect yeast virulence and host immune responses. This protocol describes methods to purify β-1,3-glucan-bonded cell wall proteases from C. glabrata. These cell wall proteases are detached from the cell wall glucan network by lyticase treatment, which hydrolyzes β-1,3-glucan bonds specifically without rupturing cells. The cell wall supernatant is further fractioned by centrifugal devices with cut-offs of 10 and 50 kDa, ion-exchange filtration (charge), and gel filtration (size exclusion). The enzymatic activity of C. glabrata proteases is verified with MDPF-gelatin zymography and the degradation of gelatin is visualized by loss of gelatin fluorescence. With this procedure, the enzymatic activities of the fractions are kept intact, differing from methods used in previous studies with trypsin digestion of the yeast cell wall. The protein bands may be eventually located from a parallel silver-stained gel and identified with LC-MS/MS spectrometry. The advantage of this methodology is that it allows further host protein degradation assays; the protocol is also suitable for studying other Candida yeast species. Key features • Uses basic materials and laboratory equipment, enabling low-cost studies. • Facilitates the selection and identification of proteases with certain molecular weights. • Enables further functional studies with host proteins, such as structural or immune response-related, or enzymes and candidate protease inhibitors (e.g., from natural substances). • This protocol has been optimized for C. glabrata but may be applied with modifications to other Candida species.

Ventricular septal defects in cats – a retrospective study

Significant deterioration in semen quality is associated with increased male infertility. Alterations in the sperm proteome resulting from infertility are not well understood. The aim of this study was to examine the connection between the proteome and the quality of spermatozoa in fertile and infertile dogs. The research was conducted on 11 male dogs, including 3 German shepherds, 3 Turkish Kangal, 2 Cane Corso, and 3 Golden Retrievers, aged 4 and 6 years (4.81 ± 0.75). Four of the 11 dogs constituted the infertile study group because of conception failure observed in at least 3 (3-5) matings with different fertile females within 1 year, and seven dogs from different owners constituted the fertile control group. Semen was manually manipulated and collected from all dogs in the presence of a female dog in heat. Ejaculates were used for spermatological examination and protein analysis. Computer-assisted semen analysis (CASA) was used to evaluate semen samples. In spermatological parameters, motility, kinematic parameters, morphology and plasma membrane integrity were evaluated. Gel electrophoresis (SDS-PAGE) was used to analyze proteins in sperm. The semen quality of infertile dogs was significantly lower. Motility, progressive motility, viability and plasma membrane integrity were statistically significantly lower in the infertile group than in the fertile group (P &lt; 0.05). In morphological parameters, acrosome, head, middle part, tail and total defects were found to be statistically significantly higher in sterile dogs than in fertile dogs (P &lt; 0.05). In the study, a total of 8 protein bands with molecular weights (MW) ranging from 11.0 to 245.0 kDa were identified in the protein analysis of semen. Variations in the sperm proteome composition were shown to be dependent on fertility. These proteins were associated with important metabolite pathways. Additionally, correlations of these bands with various spermatological parameters were revealed. The result of the study suggests that seminal plasma proteins play a role in semen quality and may be potential fertility biomarkers.

Molluscicidal assessment of certain toxicants: Impact on biochemical alterations and electrophoretic protein patterns in Massylaea vermiculata (O. F. Müller, 1774) snails.

Massylaea vermiculata snails are a significant gastropod pest in Egypt, threatening agriculture. Due to increasing concerns about conventional pesticides, it is imperative to find effective alternatives that are less harmful. We assessed the molluscicidal activity of abamectin, methoxyfenozide, and spinosad using the leaf-dipping method in vitro and the effect of LC50 of these compounds on biochemical aspects and protein electrophoresis. Results showed that these compounds exhibited molluscicidal activity, with LC50 values of 0.21, 0.63, and 0.65 mg/l for abamectin, methoxyfenozide and spinosad, respectively. Biochemical assays on treated snails showed increased aspartate and alanine aminotransferase and alkaline phosphatase activities and reduced total protein compared to controls. For the most effective compound (abamectin), these values were 195.36, 105.82, 276.76, and 2.49, compared to control values of 88.00, 47.67, 124.67, and 5.52, after 10 days post-treatment. Protein electrophoresis revealed variations in protein bands. Thus, these compounds can be effective within integrated control programs.

Characterization of Fertile and Infertile Dog Seminal Plasma Proteins and Their Correlation with Semen Quality: Preliminary Study

Significant deterioration in semen quality is associated with increased male infertility. Alterations in the sperm proteome resulting from infertility are not well understood. The aim of this study was to examine the connection between the proteome and the quality of spermatozoa in fertile and infertile dogs. The research was conducted on 11 male dogs, including 3 German shepherds, 3 Turkish Kangal, 2 Cane Corso, and 3 Golden Retrievers, aged 4 and 6 years (4.81 ± 0.75). Four of the 11 dogs constituted the infertile study group because of conception failure observed in at least 3 (3-5) matings with different fertile females within 1 year, and seven dogs from different owners constituted the fertile control group. Semen was manually manipulated and collected from all dogs in the presence of a female dog in heat. Ejaculates were used for spermatological examination and protein analysis. Computer-assisted semen analysis (CASA) was used to evaluate semen samples. In spermatological parameters, motility, kinematic parameters, morphology and plasma membrane integrity were evaluated. Gel electrophoresis (SDS-PAGE) was used to analyze proteins in sperm. The semen quality of infertile dogs was significantly lower. Motility, progressive motility, viability and plasma membrane integrity were statistically significantly lower in the infertile group than in the fertile group (P &lt; 0.05). In morphological parameters, acrosome, head, middle part, tail and total defects were found to be statistically significantly higher in sterile dogs than in fertile dogs (P &lt; 0.05). In the study, a total of 8 protein bands with molecular weights (MW) ranging from 11.0 to 245.0 kDa were identified in the protein analysis of semen. Variations in the sperm proteome composition were shown to be dependent on fertility. These proteins were associated with important metabolite pathways. Additionally, correlations of these bands with various spermatological parameters were revealed. The result of the study suggests that seminal plasma proteins play a role in semen quality and may be potential fertility biomarkers.

Efeito do ultrassom aplicado a baixa temperatura na extração e modificação de proteinas de sub-produtos da indústria pesqueira

ABSTRACT: This study explored the use only of ultrasound (US) for extracting proteins from grass carp (Ctenopharyngodon idella) backbones at low temperature and examined its impact on hydrophobicity, solubility, electrophoretic profile, and sulfhydryl levels. Backbones were treated with 35 and 130 kHz for 20, 30, and 40 minutes at 14 ºC, resulting in two protein fractions: solid (TPS) and liquid (TPL). US increased yield compared to the non-sonicated fraction. TPL (35 kHz) exhibited a 16% reduction in total and free sulfhydryl levels and a 25% increase in hydrophobicity. US induced protein conformation and band intensity alterations, particularly between 25 to 100 kDa for TPL at 130 kHz and below 30 kDa for TPS at 35 kHz. This study demonstrated the efficacy of US for protein extraction from fish by-products and its capacity to modify protein characteristics, facilitating the development of innovative food products.