Concentration Of Recombinant Protein Research Articles

OPTIMIZATION OF THE MAIN PARAMETERS OF A LATERAL FLOW ASSAY BASED ON THE RECOMBINANT Р32 ANTIGEN OF LUMPY SKIN DISEASE VIRUS

Lumpy skin disease is a highly contagious infectious transboundary disease of cattle, resulting in significant economic losses. The wide and rapid spread of lumpy skin disease to new territories emphasizes the objective need to improve diagnostic methods. The article outlines the findings of lateral flow assay parameter optimization for lumpy skin disease detection using recombinant P32 antigen. It was found that the optimal amount of protein for immobilization on 20 nm colloidal gold particles was 20 µg/mL. Bovine serum albumin at a final concentration of 0.25% was used to stabilize the conjugate. The optimal dilution of the conjugate was 1:2, and the concentration of recombinant protein and anti-species antibodies for application to the membrane was 500 μg/ml and 250 μg/ml, respectively. The colloidal gold conjugate interacted with antibodies and specifically reacted with the recombinant protein. No cross-reaction with other proteins was noticed at the same time. The created express test allowed for the detection of antibodies from immunized animals when the serum was diluted up to 1:200, however, 1:50 was the ideal ratio. The conjugate remained active after 90 days of storage at room temperature. The findings demonstrate the express method's potential for use in serological research for the prevention and control of the spread of lumpy skin disease.

Effects of Recombinant SARS-CoV-2 Spike Protein Variants on Platelet Morphology and Activation.

Platelets are central elements of hemostasis and also play a pivotal role in the pathogenesis of thrombosis in coronavirus disease 2019. This study was planned to investigate the effects of different severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) recombinant spike protein variants on platelet morphology and activation. Citrated whole blood collected from ostensibly healthy subjects was challenged with saline (control sample) and with 2 and 20 ng/mL final concentration of SARS-CoV-2 recombinant spike protein of Ancestral, Alpha, Delta, and Omicron variants. Platelet count was found to be decreased with all SARS-CoV-2 recombinant spike protein variants and concentrations tested, achieving the lowest values with 20 ng/mL Delta recombinant spike protein. The mean platelet volume increased in all samples irrespective of SARS-CoV-2 recombinant spike protein variants and concentrations tested, but especially using Delta and Alpha recombinant spike proteins. The values of both platelet function analyzer-200 collagen-adenosine diphosphate and collagen-epinephrine increased in all samples irrespective of SARS-CoV-2 recombinant spike protein variants and concentrations tested, and thus reflecting platelet exhaustion, and displaying again higher increases with Delta and Alpha recombinant spike proteins. Most samples where SARS-CoV-2 recombinant spike proteins were added were flagged as containing platelet clumps. Morphological analysis revealed the presence of a considerable number of activated platelets, platelet clumps, platelet-monocyte, and platelet-neutrophils aggregates, especially in samples spiked with Alpha and Delta recombinant spike proteins at 20 ng/mL. These results provide support to the evidence that SARS-CoV-2 is capable of activating platelets through its spike protein, though such effect varies depending on different spike protein variants.

Effects of Different Types of Recombinant SARS-CoV-2 Spike Protein on Circulating Monocytes' Structure.

This study investigated the biological effects on circulating monocytes after challenge with SARS-CoV-2 recombinant spike protein. Whole blood collected from seven ostensibly healthy healthcare workers was incubated for 15 min with 2 and 20 ng/mL final concentration of recombinant spike protein of Ancestral, Alpha, Delta, and Omicron variants. Samples were analyzed with Sysmex XN and DI-60 analyzers. Cellular complexity (i.e., the presence of granules, vacuoles and other cytoplasmic inclusions) increased in all samples challenged with the recombinant spike protein of the Ancestral, Alpha, and Delta variants, but not in those containing Omicron. The cellular content of nucleic acids was constantly decreased in most samples, achieving statistical significance in those containing 20 ng/mL of Alpha and Delta recombinant spike proteins. The heterogeneity of monocyte volumes significantly increased in all samples, achieving statistical significance in those containing 20 ng/mL of recombinant spike protein of the Ancestral, Alpha and Delta variants. The monocyte morphological abnormalities after spike protein challenge included dysmorphia, granulation, intense vacuolization, platelet phagocytosis, development of aberrant nuclei, and cytoplasmic extrusions. The SARS-CoV-2 spike protein triggers important monocyte morphological abnormalities, more evident in cells challenged with recombinant spike protein of the more clinically severe Alpha and Delta variants.

Development of a novel capillary electrophoresis method for quantitative measurements of intracellular recombinant protein titer

The measurement of volumetric titer is an integral step in the assessment and selection of a production cell line and cell culture process. The production of monoclonal antibodies (mAbs), a major class of therapeutic proteins, in Chinese Hamster Ovary (CHO) cell lines is challenging due to the clone-to-clone variations in the intrinsic capability to secrete a biologically complex protein. The measurement of intracellular mAb concentration could be a valuable tool to determine the ratio of intracellular to secreted product and be part of the evaluation of potential mAb productive cell lines. High throughput automation is a valuable tool that is used in bioprocess development to reduce work intensive steps. When coupled with the Simple Western (Wes) platform, automated capillary electrophoresis is an efficient method to measure recombinant protein concentration. In this study, we demonstrate the utility of using the automated Wes to rapidly measure intracellular titer and then compare the intracellular titer, volumetric titer and specific productivity between high and low production CHO clones expressing a model human IgG1 mAb.

Macrophage migration inhibitory factor facilitates astrocytic production of the CCL2 chemokine following spinal cord injury.

Spinal cord injury causes accumulation of a large number of leukocytes at the lesion site where they contribute to excessive inflammation. Overproduced chemokines are responsible for the migratory process of the leukocytes, but the regulatory mechanism underlying the production of chemokines from resident cells of the spinal cord has not been fully elucidated. We examined the protein levels of macrophage migration inhibitory factor and chemokine C-C motif chemokine ligand 2 in a spinal cord contusion model at different time points following spinal cord injury. The elevation of macrophage migration inhibitory factor at the lesion site coincided with the increase of chemokine C-C motif chemokine ligand 2 abundance in astrocytes. Stimulation of primary cultured astrocytes with different concentrations of macrophage migration inhibitory factor recombinant protein induced chemokine C-C motif chemokine ligand 2 production from the cells, and the macrophage migration inhibitory factor inhibitor 4-iodo-6-phenylpyrimidine attenuated the stimulatory effect. Further investigation into the underlying mechanism on macrophage migration inhibitory factor-mediated astrocytic production of chemokine C-C motif chemokine ligand 2 revealed that macrophage migration inhibitory factor activated intracellular JNK signaling through binding with CD74 receptor. Administration of the macrophage migration inhibitory factor inhibitor 4-iodo-6-phenylpyrimidine following spinal cord injury resulted in the reduction of chemokine C-C motif chemokine ligand 2-recruited microglia/macrophages at the lesion site and remarkably improved the hindlimb locomotor function of rats. Our results have provided insights into the functions of astrocyte-activated chemokines in the recruitment of leukocytes and may be beneficial to develop interventions targeting chemokine C-C motif chemokine ligand 2 for neuroinflammation after spinal cord injury.

A novel aptamer beacon for rapid screening of recombinant cells and in vivo monitoring of recombinant proteins

Recombinant protein drugs, which are typically produced by mammalian host cells, have been approved for the treatment of a range of diseases. Accordingly, systems for selecting recombinant cell lines with efficient protein expression and for testing the content of recombinant proteins in vivo are crucial to the large-scale production and application of protein-based therapeutic drugs. In this study, we designed three aptamer beacons to detect His-tag, a common label of recombinant proteins. We found that all three beacons could specifically and quantitatively measure the His-tagged recombinant proteins with a short reaction time. Among these three beacons, the 6H5-MU beacon had the highest sensitivity for His polypeptides with a detection limit of 250ng/mL and the shortest detection time within 1min. Furthermore, we established a rapid and highly effective recombinant cell line construction system, which could obtain monoclonal cell lines with high yields of target proteins within 21days, by combining 6H5-MU with pSB, a novel plasmid composed of a Sleeping Beauty transposase and a transposon. Finally, 6H5-MU also discriminately tested the serum concentration of His-tagged recombinant proteins in vivo, with consistent results compared to enzyme-linked immunosorbent assay (ELISA). We thus established a rapid and high-throughput method for generating recombinant cell lines and in vivo monitoring of recombinant protein levels, thereby providing a new platform for the development and preparation of recombinant protein drugs. KEY POINTS: • The 6H5-MU aptamer beacon rapidly and accurately binds to His-tagged recombinant proteins. • A system for rapid and high-throughput generation of recombinant cell lines is established using 6H5-MU and pSB. • 6H5-MU allows in vivo monitoring of recombinant protein levels.

Development and Optimization of an Immunoassay for the Detection of Antibodies Against SARS-CoV-2 with In-house Recombinant RBD Protein

COVID-19 caused by SARS-CoV-2 poses a major threat to the global community, particularly in Indonesia. Countermeasures to prevent the spread of this disease have also been implemented, including the implementation of a vaccination program. An immunoassay technique that can be used to analyze antibodies that might develop following vaccination is the indirect enzyme-linked immunosorbent assay (ELISA). We produced the recombinant spike protein used in this study. The optimization comprised adjusted concentrations of spike recombinant protein (5 and 10 ng/mL), blocking agent (2.5% and 5%), and conjugate (1:1000 and 1:5000). The optimal conditions in this study included a spiked concentration of 10 ng/mL, a blocking agent concentration of 5%, sample dilution of 1:33, and a conjugate concentration of 1:1000. The intra-assay value of this optimized indirect ELISA was 7.3, and the inter-assay value was 5.3. The commercial MyBioSource kit and immunodiagnostic were utilized as a reference in the T-test, with P-values of 0 and 0.313, indicating that the recombinant protein in-house ELISA kit in this study demonstrated the same ability as the commercial immunodiagnostic kit in detecting SARS-CoV-2 antibodies, allowing it to be used for post-vaccination efficacy evaluation.

Quantification of pharmacokinetic profiles of a recombinant canine PD-1 fusion protein by validated sandwich ELISA method.

Tumors are becoming a serious threat to the quality of life of human and dogs. Studies have shown that tumors have caused more than half of the deaths in older dogs. Similar to human, dogs will develop various and highly heterogeneous tumors, but there are currently no viable therapies for them. In human, immunotherapy has been used widely and considered as an effective treatment for tumors by immune checkpoint targets, which are also expressed on canine tumors, suggesting that immunotherapy may be a potential treatment for canine tumors. In this work, we developed a sandwich ELISA method to detect the concentration of recombinant canine PD-1 fusion protein in canine serum and investigated pharmacokinetics in canines after intravenous infusion administration. After being validated, the ELISA method showed an excellent linear relationship in 25.00–3,200.00 ng/ml in serum, and the R2 was more than 0.99 with four-parameter fitting. The precision and accuracy of intra-assay and inter-assay at the five different concentrations met the requirements of quantitative analysis. At the same time, no hook effect was observed at the concentration above ULOQ, and the stability was good under different predicted conditions with accuracy > 80%. The pharmacokinetic study in dogs has shown that the recombinant canine PD-1 fusion protein exhibited a typical biphasic PK profile after intravenous infusion administration, and the linear pharmacokinetic properties were observed between 1.00 and 12.00 mg/kg. Meanwhile, the T1/2 after intravenous infusion administration with non-compartmental analysis was about 5.79 days.

Optimized Purification of CIDRα-PfEMP1 Plasmodium falciparum Recombinant Protein with Affinity Chromatography

Interaction of Cysteine-rich Interdomain Region (CIDR)α-Plasmodium falciparum Erythrocyte Membrane Protein (PfEMP1) and Endothelial Cell Receptors especially CD36 on host cells is main malaria pathogenesis, makes this domain as a malaria vaccine candidate. Recently, the development of the malaria vaccine is conducted by recombinant technology, and the purification of the CIDRα-PfEMP1 recombinant protein is a pivotal step. This study aimed to determine an optimal condition to purify the CIDRα-PfEMP1 recombinant protein by affinity chromatography through imidazole and NaCl concentration. The purified recombinant protein was visualized using SDS-PAGE and its concentration was measured using Image J software and Bradford Assay. The data were analyzed using SPSS 26 software, and the Paired T-Test analysis was conducted to compare the concentration of purified recombinant protein from two different methods. The result showed that thetarget band of purified recombinant protein was 27 kDa. The thickest target protein band was observed in purified recombinant protein using 140 mM imidazole and 300 mM NaCl. The recombinant protein concentration using Image J software was 0.025 µg/µL, while the Bradford Assay was 0.56 µg/µL. The Paired T-Test analysis has a significance value of 0.010 (p<0.05), meaning there was a significant difference between the concentration measurement using Image J software and Bradford Assay. In conclusion, the optimized condition to purify the CIDRα-PfEMP1 recombinant protein by affinity chromatography was using 140 mM imidazole and 300 mM NaCl. It is suggested to measure the purified CIDRα-PfEMP1 recombinant protein concentration using the Bradford Assay method due to its convenience and sensitivity.

대장균에서 발현된 넙치 PKCβ1의 활성도 조사

Oliver flounder (Paralichthys olivaceus) is the second most farmed fish species in Korea in 2020. Thus, oliver flounder was studied on aquaculture methods, pathogenic viruses and bacteria, and gene duplication of teleostei fish have been actively conducted. Protein kinase C (PKC) is an enzyme that phosphorylates serine/threonine residues of proteins. PKC is divided to several subfamilies (conventional, novel, atypical) depending on how it is activated. PKCβ is activated by Ca2+ and diacylglycerol (DAG) in the presence of phosphatidylserine. The recombinant protein of oliver flounder PKCβ1 (PoPKCβ1) was expressed and purified, and the activity was measured in several reaction conditions such as temperature, pH, and recombinant protein concentration. As a result, it exhibited a wide range of activity from 10°C to 50°C by reaction temperature, and sufficient activity from 5 to 10 by pH. And 50 ng/μl showed the highest activity in the recombinant protein concentration. Therefore this study suggests that PoPKCβ1 can perform phosphorylation of proteins under a wide range of conditions.

Spike protein of SARS-CoV-2 suppresses gonadotrophin secretion from bovine anterior pituitaries.

Coronavirus disease (COVID-19), the ongoing global pandemic, is caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Recent evidence shows that the virus utilizes angiotensin-converting enzyme 2 (ACE2) as a spike protein receptor for entry into target host cells. The bovine ACE2 contains key residues for binding to the spike protein receptor-binding domain. This study evaluated the hypothesis that bovine gonadotroph expresses ACE2, and spike protein suppresses luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion from cultured bovine anterior pituitary (AP) cells. ACE2 mRNA expression and ACE2 protein expression were detected in the bovine AP cells using reverse transcription PCR and western blot analysis. Immunofluorescence microscopy analysis with the anti-ACE2 antibody revealed the co-localization of ACE2 and gonadotropin-releasing hormone (GnRH) receptor on the gonadotroph plasma membrane. Approximately 90% of GnRH receptor-positive cells expressed ACE2, and approximately 46% of ACE2-positive cells expressed the GnRH receptor. We cultured bovine AP cells for 3.5 days and treated them with increasing concentrations (0, 0.07, 0.7, or 7 pM) of recombinant spike protein having both S1 and S2 regions. The spike protein (0.07–7 pM) suppressed both basal and GnRH-induced LH secretion (P < 0.05). Spike protein (0.7–7 pM) suppressed GnRH-induced (P < 0.05), but not basal FSH secretion. In contrast, pre-treatment with ERK 1/2/5 inhibitor (U0126) partially restored the GnRH-induced LH and FSH secretion from the spike protein suppression. Collectively, the results indicate that gonadotrophs express ACE2, a receptor for coronavirus 2 spike protein, which in turn suppresses LH and FSH secretion from AP cells.

Enhanced Bone Regeneration in Variable-Type Biphasic Ceramic Phosphate Scaffolds Using rhBMP-2.

Our aim was to investigate the bone regeneration capacity of powder-type biphasic ceramic scaffold (BCP powder), block-type BCP (BCP block), and collagen-added block-type BCP (BCP collagen) with different concentrations of recombinant human bone morphogenetic protein 2 (rhBMP-2) in an animal model. Four rabbits were assigned to each of the following groups: no graft + rhBMP-2 (0.1/0.2 mg/mL), BCP powder + rhBMP-2 (0.1/0.2 mg/mL), BCP block + rhBMP-2 (0.1/0.2 mg/mL), and BCP collagen + rhBMP-2 (0.1/0.2 mg/mL), i.e., a total of 32 rabbits. Polycarbonate tubes (Φ 7 mm × 5 mm) for supporting scaffolds were fixed into a 7 mm round border. Subsequently, 0.1 mL of rhBMP-2 solutions with different concentrations was injected into the tubes. Both radiological and histomorphometric analyses showed that osteogenesis was not enhanced by increasing the concentration of rhBMP-2 in all groups at both 3 and 6 weeks. Radiological analysis showed that bone formation was higher in the BCP collagen group than in the BCP powder and BCP block groups at both rhBMP-2 concentrations at 3 weeks. rhBMP-2 enhanced bone formation; however, as the concentration increased, bone formation could not be enhanced infinitely. Collagen-added alloplastic graft material may be useful for mediating rapid bone formation in initial stages.

Molecular characterization, immune responses, and functional activities of manganese superoxide dismutase in disk abalone (Haliotis discus discus)

Superoxide dismutases (SODs) are metalloenzymes that convert superoxide radicals to H2O2 and O2. Although SODs have been extensively studied in mammals and other species, comparative studies in invertebrates, such as abalones, are lacking. Here, we aimed to characterize manganese superoxide dismutase in disk abalone (Haliotis discus discus) (AbMnSOD) by assessing its transcriptional levels at different embryonic developmental stages. Additionally, the temporal expression of AbMnSOD in different abalone tissues in response to bacterial, viral, and pathogen-associated molecular pattern (PAMP) stimuli was investigated. SOD activity was measured at various recombinant protein concentrations via the xanthine oxidase/WST-1 system. Cell viability upon exposure to H2O2, wound healing ability, and subcellular localization were determined in AbMnSOD-transfected cells. AbMnSOD was 681 bp long and contained the SOD-A domain. AbMnSOD expression was higher at the trochophore stage than at the other stages. When challenged with immune stimulants, AbMnSOD showed the highest expression at 6 h post-injection (p.i.) for all stimulants except lipopolysaccharides. In the gills, the highest AbMnSOD expression was observed at 6 h p.i., except for the Vibrio parahaemolyticus challenge. Recombinant AbMnSOD showed concentration-dependent xanthine oxidase activity. Furthermore, AbMnSOD-transfected cells survived H2O2-induced apoptosis and exhibited significant wound gap closure. As expected, AbMnSOD was localized in the mitochondria of the cells. Our findings suggest that AbMnSOD is an essential antioxidant enzyme that participates in regulating developmental processes and defense mechanisms against oxidative stress in hosts.

Myostatin suppresses adipogenic differentiation and lipid accumulation by activating crosstalk between ERK1/2 and PKA signaling pathways in porcine subcutaneous preadipocytes.

The current study was undertaken to determine the effect of myostatin (MSTN) on lipid accumulation in porcine subcutaneous preadipocytes (PSPAs) and to further explore the potential molecular mechanisms. PSPAs isolated from Meishan weaned piglets were added with various concentrations of MSTN recombinant protein during the entire period of adipogenic differentiation process. Results showed that MSTN treatment significantly reduced the lipid accumulation, intracellular triglyceride (TG) content, glucose consumption, and glycerol phosphate dehydrogenase activity, while increased glycerol and free fatty acid release. Consistent with above results, the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway was obviously activated and thus key adipogenic transcription factors peroxisome proliferator-activated receptor-gamma (PPAR-γ), CCAAT/enhancer-binding protein-alpha (C/EBP-α), and their downstream enzymes fatty acid synthase and acetyl-CoA carboxylase were all inhibited. However, chemical inhibition of ERK1/2 signaling pathway by PD98059 markedly reversed the decreased TG content by increasing PPAR-γ expression. In addition, MSTN activated the cyclic AMP/protein kinase A (cAMP/PKA) pathway and stimulated lipolysis by reducing the expression of antilipolytic gene perilipin, thus elevated key lipolytic enzymes adipose triglyceride lipase and hormone-sensitive lipase (HSL) expression and enzyme activity. On the contrary, pretreatment with PKA inhibitor H89 significantly reversed TG accumulation by increasing PPAR-γ expression and thus inhibiting ERK1/2, perilipin, and HSL phosphorylation, supporting the crosstalk between PKA and ERK1/2 pathways in both the anti-adipogenic and pro-lipolytic effects. In summary, our results suggested that MSTN suppressed adipogenesis and stimulated lipolysis, which was mainly mediated by activating crosstalk of ERK1/2 and PKA signaling pathways, and consequently decreased lipid accumulation in PSPAs, our findings may provide novel insights for further exploring MSTN as a potent inhibitor of porcine subcutaneous lipid accumulation.

Effects of HMGB1 on Proliferation and Secretion of Human Bone Marrow Mesenchymal Stem Cells

To investigate the effect of high mobility group protein 1(HMGB1) on the proliferation and cytokine expression of human bone marrow mesenchymal stem cells (MSC). Different concentrations of recombinant human HMGB1 protein (100, 200, 400, 800 and 1000 ng/ml) were incubated with MSC for 24, 48, 72 h and the proliferation of MSC were detected respectively by using the CCK-8 method and flow cytometry. The best concentrations of HMGB1 incubated with MSC was determined (200 ng/ml, 1000 ng/ml), and the flow cytomerty was used to determine the effect of HMGB1 on the proliferation of MSC. The mRNA expression levels of IL-10, TGF- β1, TSG-6 and IFN-γ in MSC incubated with HMGB1 protein were detected by real-time quantitative PCR and ELISA. The result of MSC identification and flow cytometry showed that the CD105, CD73 and CD90 were expressed, but did not expression CD45, CD34, CD11b, CD19 and HLA-DR; CCK-8 showed that HMGB1 at the concentrations of 100 ng/ml, 200 ng/ml and 400 ng/ml could promote the proliferation of MSC incubated for 24, 48 and 72 h as compared with the control group (P<0.05), and the most effective concentration was 200 ng/ml; flow cytometry showed that the compared with the control group, HMGB1 200 ng/ml could induce MSC from G1 phase to S phase to promote the proliferation of MSC; QPCR showed that the mRNA expression of MSC cytokines IL-10, TGF-β1, TSG-6 increased while IFN-γ decreased at the concentration of 200 ng/ml HMGB1 as compared with the control group. ELISA experiments showed that the HMGB1 200 ng/ml acting on MSC for 48 h could significantly promoted the secretion of IL-10, TGF-β 1 and TSG-6(P<0．05), while IFN-γ showed no significant difference as compared with control group. Recombinant human HMGB1 can promote the proliferation and secretion of MSC in healthy people.

Expression and biological functions of Ancylostoma ceylanicum saposin-like protein.

Ancylostoma ceylanicum is a common zoonotic nematode that inhabits the small intestine of humans, dogs, and cats. Saposin-like proteins (SLPs) have hemolytic and antibacterial activities and could be used as diagnostic or vaccine candidates. To explore the biological functions of Ancylostoma ceylanicum SLP (Ace-SLP-1), cDNA-encoding Ace-SLP-1 mature peptide was cloned into prokaryotic expression vector pET-28a and transformed into Escherichia coli BL21 (DE3) to induce expression. After incubation of canine red blood cell suspension with different concentrations of recombinant Ace-SLP-1, the supernatant was separated to measure OD value and calculate the hemolysis rate. The different concentrations of recombinant protein were co-cultured with E. coli and Enterococcus faecalis, and colony-forming units (CFU) were determined by the plate counting method. Peripheral blood mononuclear cells (PBMCs) from healthy dogs were incubated with different concentrations of recombinant Ace-SLP-1, and the cytokine expression was evaluated by relative quantitative PCR. Our results showed that the hemolytic activity of Ace-SLP-1 increased with the increase in protein concentration from 25 to 100μg/mL. The recombinant protein had no antibacterial activity against the two kinds of bacteria but could stimulate the secretion of cytokines (IL-4, IL-10, IL-12, and IL-13) in canine PBMCs. These data suggest that Ace-SLP-1 is involved in hookworm blood-feeding and survival and has good immunogenicity, supporting its potential as a diagnostic and vaccine target molecule.

Recombinant TSG-6 protein inhibits the growth of capsule fibroblasts in frozen shoulder via suppressing the TGF-\u03b2/Smad2 signal pathway

BackgroundThe tumor necrosis factor-stimulated gene-6 (TSG-6) has been confirmed to inhibit inflammation. It is now generally accepted that local inflammatory stimulation around shoulder capsule causes proliferative fibrosis. This study aims to investigate the mechanism of recombinant TSG-6 protein inhibiting the growth of capsule fibroblasts in frozen shoulder via the TGF-β/Smad2 signal pathway.MethodsHuman frozen shoulder capsule tissue was taken for primary and passage culture, and the 3rd generation fibroblasts from pathological frozen shoulder capsule were treated with different concentrations of recombinant TSG-6 protein, or with TGF-β1 agonist SRI-011381. Immunoconfocal analysis was used to identify the isolated fibroblasts, and MTT assay, colony formation assay, and flow cytometry were used to detect the viability, proliferation, and apoptosis rate of fibroblast. The contents of fibrosis and inflammation indexes COL1A1, TNF-α, IL-6, and IL-1β in the cell supernatant were detected using ELISA and then further examined by qRT-PCR. The expression of Bax, Bcl-2, and proteins related to TGF-β/Smad2 pathway were detected by Western Blot.ResultsCompared with the blank control group, fibroblasts intervened with TSG-6 (2 μg and 5 μg) showed significantly decreased viability and proliferation ability and enhanced cell apoptosis, concurrent with the reductions in Bcl-2 expression; COL1A1, TNF-α, IL-6, and IL-1β levels; and the expression of TGF-β1 and phosphorylated Smad22, and an increase in Bax expression, while SRI-011381 treatment would reverse the effect of recombinant TSG-6 protein.ConclusionRecombinant TSG-6 protein inhibited the growth of primary fibroblasts from human frozen shoulder capsule by suppressing the TGF-β/Smad2 signaling pathway.

Measurement over 1 Year of Neutralizing Antibodies in Cattle Immunized with Trivalent Vaccines Recombinant Alpha, Beta and Epsilon of Clostridium perfringens.

The alpha (CPA), beta (CPB) and epsilon (ETX) toxins of Clostridium perfringens are responsible for causing diseases that are difficult to eradicate and have lethal potential in production animals. Vaccination of herds is still the best control strategy. Recombinant clostridial vaccines have shown good success at inducing neutralizing antibody titers and appear to be a viable alternative to the conventional production of commercial clostridial toxoids. Research is still needed on the longevity of the humoral immune response induced by recombinant proteins in immunized animals, preferably in target species. The objective of this study was to measure the humoral immune response of cattle immunized with trivalent vaccines containing the recombinant proteins alpha (rCPA), beta (rCPB) and epsilon (rETX) of C. perfringens produced in Escherichia coli at three different concentrations (100, 200, and 400 µg) of each protein for 12 months. The recombinant vaccines containing 200 (RV2) and 400 µg (RV3) yielded statistically similar results at 56 days. They performed better throughout the study period because they induced higher neutralizing antibody titers and were detectable for up to 150 and 180 days, respectively. Regarding industrial-scale production, RV2 would be the most economical and viable formulation as it achieved results similar to RV3 at half the concentration of recombinant proteins in its formulation. However, none of the vaccines tested induced the production of detectable antibody titers on day 365 of the experiment, the time of revaccination typically recommended in vaccination protocols. Thus, reiterating the need for research in the field of vaccinology to achieve greater longevity of the humoral immune response against these clostridial toxins in animals, in addition to the need to discuss the vaccine schedules and protocols adopted in cattle production.

Bridging Offline Functional Model Carrying Aging-Specific Growth Rate Information and Recombinant Protein Expression: Entropic Extension of Akaike Information Criterion.

This study presents a mathematical model of recombinant protein expression, including its development, selection, and fitting results based on seventy fed-batch cultivation experiments from two independent biopharmaceutical sites. To resolve the overfitting feature of the Akaike information criterion, we proposed an entropic extension, which behaves asymptotically like the classical criteria. Estimation of recombinant protein concentration was performed with pseudo-global optimization processes while processing offline recombinant protein concentration samples. We show that functional models including the average age of the cells and the specific growth at induction or the start of product biosynthesis are the best descriptors for datasets. We also proposed introducing a tuning coefficient that would force the modified Akaike information criterion to avoid overfitting when the designer requires fewer model parameters. We expect that a lower number of coefficients would allow the efficient maximization of target microbial products in the upstream section of contract development and manufacturing organization services in the future. Experimental model fitting was accomplished simultaneously for 46 experiments at the first site and 24 fed-batch experiments at the second site. Both locations contained 196 and 131 protein samples, thus giving a total of 327 target product concentration samples derived from the bioreactor medium.

Next Generation Immunotherapies – Emerging Strategies for Immune Modulation against Cancer, Infections, and Beyond

Next Generation Immunotherapies – Emerging Strategies for Immune Modulation against Cancer, Infections, and Beyond