Tris-HCl Buffer pH Research Articles

Optimizing PES microfiltration membranes for sterile filtration: A comparative study of polydopamine coating in acidic and basic conditions to minimize protein adsorption

The increasing need for effective sterile filtration in the pharmaceutical industry calls for advancements in microfiltration (MF) membrane technology that can effectively reduce fouling and preserve essential protein products. Our study highlights the crucial role of improving the hydrophilicity throughout the entire membrane, not just on the surface, to decrease both protein adsorption and fouling. We have developed a facile but effective method for producing hydrophilic porous polyethersulfone (PES) MF membranes using vapor-induced phase separation (VIPS) followed by a hydrophilic coating. By meticulously adjusting the vapor exposure time, we fabricated two specific types of symmetric membranes: a standard one with a mean pore size of 0.22 μm and another designed with slightly larger pores for the subsequent polydopamine (PDA) coating. All membranes developed in this study showed an outstanding sterilization performance (over 99.99999 % bacteria rejection) due to the absence of defects. Our comparative studies of PDA coating in both acidic and basic environments revealed that the PDA-modified membranes in a Tris-HCl buffer (pH 8.0) (PDA-b-PES2M) outperform others in performance of protein microfiltration. While the membrane modified under acidic conditions underwent a gradual degradation of PDA leading to a poor filtration performance, the PDA-b-PES2M membranes showcased remarkable anti-fouling properties, greatly minimizing the adsorption of bovine serum albumin (BSA) and simplifying the removal of adsorbed materials. Our optimized membrane has a great potential for sterile filtration applications where minimizing protein loss is crucial.

Automated Standardization of Melanin Bleaching Procedures of Heavily Pigmented Melanocytic Lesions With Low-Concentration Hydrogen Peroxide on an Automated Platform.

Melanin is a natural pigment in the human body that is primarily found in the skin and hair. It protects the skin from damage by ultraviolet radiation. Although this pigment plays a crucial role in protecting the human body, it represents a challenge for pathologists to evaluate highly pigmented tissue samples from melanoma or pigmented skin lesions. Abundant melanin may obscure tissue morphology, which makes it very difficult for pathologists to make a differential diagnosis. Melanin pigment is brown-to-black and granular, and its distribution is often uneven in tissues. The presence of these pigments can complicate the analysis of immunohistochemistry (IHC) for 2 reasons. First, they have a direct physical masking effect on antigen-antibody interactions. Second, 3,3-diaminobenzidine, the most commonly used chromogen, has a brown color that is difficult to distinguish from melanin pigment. Therefore, melanin bleaching has become a crucial step in handling pigmented melanocytic lesions. Bleaching techniques aid pathologists in histopathologic examination of melanin-rich tissue. In this study, we integrated melanin bleaching and IHC on an automated IHC platform to set up a rapid and fully automated procedure. Bleaching steps were performed before antigen retrieval. Samples were treated with 1% hydrogen peroxide solution in Tris-HCl buffer (pH 10) at 80°C for 8 minutes, achieving optimal conditions for melanin bleaching while preserving tissue morphology and antigenicity. This rapid, effective, fully automated, and standardized workflow can be applied to routine staining procedures in clinical laboratories, thereby improving the quality of pathological diagnosis.

Inhibition of some multidrug-resistant bacteria using prepared essential oil Nanoemulsion formulas and their mode of action

Inhibition of some multidrug-resistant bacteria using prepared essential oil Nanoemulsion formulas and their mode of action

Development of a fluorescence-based excipient screening for improved stability and shelf-life of recombinant chitin deacetylase

Chitin deacetylase (CDA) modifies chitin into chitosan by removing acetyl groups, but its inherent instability poses a challenge for successful crystallisation. Despite limited successes in crystallizing CDAs, prior attempts with recombinant chitin deacetylase (BaCDA) failed due to poor stability. To address this, we propose an enzyme buffer formulation as a cost-effective strategy to enhance stability, prolong shelf life, and increase the likelihood of crystallisation. Utilizing the high-throughput screening technique FTSA, we developed a screening method correlating BaCDA stability with its activity. The optimised formulation comprises 50 mM Tris-HCl buffer pH 7, 1 M NaCl, 20 % glycerol, and 1 mM Mg2+ as excipients. This formulation significantly improves BaCDA's thermostability (140.47 % increase) and enzyme activity (2.9-fold enhancement). BaCDA remains stable in the formulated buffer at −20 °C and −80 °C for 30 days and at 4 °C for 15 days. The current study has designed a high-throughput screening method approach to assess the stability of CDA enzyme formulations. The results of this study could contribute to the exploration of formulation elements that enhance the structural stability of CDA, thereby facilitating investigations into the enzyme's structure-function relationships.

Identification and characterization of a PL35 GAGs lyase with 4-O-sulfated N-acetylgalactosamine (A-type)-rich structures producing property

Glycosaminoglycan (GAG) lyases are important tools for investigating the structure of GAGs and preparing low-molecular-weight GAGs. The PL35 family, a recently established polysaccharide lyase family, should be further investigated. In this study, we discovered a new GAG lyase, CHa1, which belongs to the PL35 family. When expressed heterologously in Escherichia coli (BL21), CHa1 exhibited high expression levels and solubility. The optimal activity was observed in Tris-HCl buffer (pH 7.0) or sodium phosphate buffer (pH 8.0) at 30 °C. The specific activities towards HA, CSA, CSC, CSD, CSE, and HS were 3.81, 13.03, 36.47, 18.46, 6.46, and 0.50 U/mg protein, respectively. CHa1 digests substrate chains randomly that acting as an endolytic lyase and shows a significant preference for GlcA-containing structures, prefers larger oligosaccharides (≥UDP8) and can generate a series of oligosaccharides composed mainly of the A unit when digesting CSA. These oligosaccharides include ΔC-A, ΔC-A-A, ΔC-A-A-A, ΔC-A-A-A-A, and ΔC-A-A-A-A-A. The residues Tyr257 and His421 play crucial roles in the catalytic process, and Ser211, Asn212, Asn213, Trp214, Gln216, Lys360, Arg460 and Gln462 may participate in the binding process of CHa1. This study on CHa1 contributes to our understanding of the PL35 family and provides valuable tools for investigating the structure of GAGs.

Hemostatic activity <i>in vitro</i> of lectin-containing substances of bryathypes on the example of some Antarctic and Belarus representatives

BACKGROUND: The modern market of medical devices in Belarus and Russia is represented by a wide range of hemostatic agents, of which the most popular are local hemostatics of plant origin possessing the significant technological potential for renewal and improvement. A promising reserve for this may be biologically active compounds of mosses, which are characterized by anti-inflammatory, antibacterial and antifungal effects. However, their hemostatic effect is almost not studied, which determines the relevance of this work.
 AIM: The aim of this work is to study the effect of lectin-containing substances from mosses of three species collected in East Antarctica and Belarus on the parameters of human blood hemostasis in vitro.
 MATERIALS AND METHODS: We studied mosses of the genera Bryum, Ceratodon, and Coscinodon, collected in the area of the Belarusian Antarctic station Gora Vechernyaya in East Antarctica and in Belarus. Lectin-containing substances of mosses were obtained by extracting shoots in 0.05 M tris-HCl buffer (pH 8.0), centrifugation, filtration. The assessment of the biological activity of lectin-containing substances in mosses was carried out by the agglutination reaction of rabbit erythrocytes, as well as the effect on human platelet aggregation and in the test for activated partial thromboplastin time.
 RESULTS: It was established that lectin-containing substances of the studied moss species had agglutinating activity against erythrocytes in the range from 11708.28 (Belarusian samples) to 1333979.59 U/mg of protein (Antarctic samples) depending on the species and localization; initiated the aggregation of human platelets (25–80% of the effect of thrombin) regardless of blood group, Rh and gender of donors; influenced the plasma link of hemostasis, reducing activated partial thromboplastin time (by 15–18%).
 CONCLUSIONS: It was found that some species of mosses of the genera Bryum, Ceratodon and Coscinodon of Antarctica and Belarus had an agglutinating and hemostatic effect on erythrocytes and platelets, with the greatest activity noted for Antarctic species. A hypothesis has been put forward that the observed phenomenon is due to the structural features of proteins, including lectins. It is assumed that lectins are possible inducers of erythrocyte agglutination and platelet aggregation in mosses. It is shown that the moss species Bryum pseudotriquetrum and Ceratodon purpureus have a certain resource potential in Belarus for their annual harvest. The results obtained expand the list of moss species with hemostatic activity, and can be used to develop new hemostatics of plant origin for local use from Belarusian plant materials.

HPLC purification of antioxidant and antibacterial peptides from a lichen "Parmotrema perlatum (Huds.) M. Choisy": Identification by LC-MS/MS peptide mass fingerprinting.

Parmotrema perlatum, a lichen belonging to the family Parmeliaceae, is well known for its culinary benefits and aroma used as a condiment in Indian homes is also known as the "black stone flower" or "kalpasi" in India. This research intends to analyze the antioxidant power of the crude extracts using four pH-based buffers solubilized proteins/peptides and RP-HPLC fractions of P. perlatum obtained by purification. The proteins that were extracted from the four different buffers were examined using LC-MS/MS-based peptide mass fingerprinting. When compared to the other buffers, the 0.1M of Tris-HCl buffer pH 8.0 solubilized proteins/peptides had the strongest antioxidant capacity. The sequential purification of the peptide was carried out by using a 3-kDa cut-off membrane filter and semipreparative RP-HPLC. Additionally, the purified fractions of the peptide's antioxidant activity were assessed, and effects were compared with those of the crude and 3kDa cut--off membrane filtrates. The peptide fractions were sequenced by LC-MS/MS, which reveals that fraction 2 from RP-HPLC with the sequence LSWFMVVAP has shown the highest antioxidant potential in comparison with other fractions which can serve as the potential natural antioxidant drug. Further, fraction 2 also showed antibacterial activity against the selected microorganisms.

Purification and characterization of human glycerol 3-phosphate dehydrogenases (mitochondrial and cytosolic) by NAD+/NADH redox method

Glycerol 3-phosphate (G3P) shuttle is composed of mGPDH and cGPDH and serves as the interface between carbohydrate- and lipid-metabolism. Recently, these metabolic enzymes have been implicated in type II diabetes mellitus but the detailed kinetic parameters and crystal structure of human mGPDH is unknown, though fewer studies on cGPDH are available. To characterize these enzymes, the human mGPDH and cGPDH genes were optimized and cloned into the pET-SUMO vector and pET-24a(+) vector, respectively, and over-expressed in Escherichia coli BL21 (DE3). However, SUMO-mGPDH was expressed as inclusion bodies. Hence, various culture parameters, solubilizing agents and expression vectors were used to solubilize the protein but they did not produce functional SUMO-mGPDH. Over-expression of SUMO-mGPDH along with molecular chaperone (pG-KJE8) produced a functional SUMO-mGPDH. The functional SUMO-mGPDH was purified and characterized using NAD+/NADH redox method. cGPDH was also over-expressed and purified for its characterization. DLS analysis and CD spectra of the purified proteins were performed. The mGPDH was a monomeric enzyme with MW of ∼74 kDa and displayed optimal activity in the Tris-HCl buffer (pH 7.4); while, cGPDH was a homodimer with a monomeric MW of ∼37 kDa and showed optimal activity in imidazole buffer (pH 8.0). The Kmapp was 0.475 mM for G3P, and 0.734 mM for DHAP. These methods may be used to characterize these enzymes to understand their role in metabolic disorders.

Biocatalysts based on complexes of carbon nanomaterials with cysteine proteases

The purpose of the research is to develop and study biocatalysts based on complexes of cysteine proteases with fullerenes and carbon nanotubes. During the formation of ficin complexes with fullerenes and carbon nanotubes, the activity of hybrid preparations was 70 and 45%, respectively. During the formation of papain complexes with fullerenes and carbon nanotubes, the proteolytic ability of the enzyme remained at the same level for the samples with fullerene and decreased by 27% for the preparations with carbon nanotubes. The formation of bromelain complexes with fullerenes and carbon nanotubes contributed to a decrease in the proteolytic activity of the biocatalyst by 18 and 48% as compared to the free enzyme. While determining the stability of complexes of nanomaterials and cysteine proteases during a 7-day incubation in 0.05 M tris-HCl buffer (pH 7.5) at 37 °C, we noticed a decrease in the proteolytic activity of the samples. Complexation with carbon nanoparticles and fullerenes increased the stability of ficin and bromelain, while the stability of papain in the complexes remained unchanged

Peroxidase activity as a biochemical marker of insecticide use in vegetables

The insecticides use is important for crop improvement and protection, but in excessive amounts, they would induce a dysfunction of metabolic enzymatic systems in plant tissues, leading to undesirable qualitative changes. In this context, we are interested in peroxidase (POD), an important enzyme in plant physiology but whose activity seems to be conditioned by the presence of insecticides in the soil. This work aims to study the impact of locally used insecticides (chlorpyrifos and dimethoate) on the activity of POD in parsley, onion, celery and garlic grown in soils treated or not. POD extraction was performed using Tris-HCl buffer (pH 7.3); its activity was measured using the substrate o-dianisidine in the presence of H2O2. Our result showed that POD activity for insecticide treated parsley, celery and onions increased by 30 % 127 % and 341 % respectively, however did not change significantly for garlic. Thus, the action of these chemicals is not trivial because they may alter non-target pathways, especially when doses are not adjusted accordingly. We found that insecticide stress would increase POD activity in all vegetables except garlic, which showed tolerance to insecticides. Our findings suggest that organic farming conditions could minimize peroxidase activity in parsley, celery and onion. We add that overproduction of POD negatively affects the quality and reduces the shelf life of vegetables, thus would be a very interesting biomarker of insecticide stress.

Biocatalysts based on papain associates with chitosan nanoparticles

The research purpose was to develop and study biocatalysts based on papain associates with chitosan nanoparticles. We obtained medium and high molecular weight chitosan nanoparticles, both with and without ascorbic acid . When the papainna-noparticles complexes with ascorbic acid were formed, the catalytic activity of the enzyme increased by 3 % for medium molecular weight chitosan and by 16 % for high molecular weight chitosan. After 168 hours of incubation in 0.05 M of Tris-HCl buffer (pH 7.5) at 37 °C, the free enzyme retained 15 % of its catalytic activity, whereas its associates with chitosan nanoparticles exhibited ~ 30 %. The papain complex with chitosan nanoparticles and ascorbic acid exhibited 40 % of the enzyme catalytic activity. We simulated the bonds and interactions within the chitosan-ascorbic acid-papain complex. The proposed biocatalysts have high prospects for effective use in cosmetology, biomedicine, and pharmacy.

FIBRINOGEN-SPECIFIC PROTEASE IN THE Vipera renardi SNAKE VENOM

Aim. To search fibrinogenolytic enzymes among protein components of Vipera renardi snake venom. Methods. Venom of V. renardi as the lyophilized powder was supplied by Trypillia serpentarium. It was dissolved in 0.05 M Tris-HCl buffer pH 8.3 and fractionated on Superdex G-75 using FPLC system Acta Prime. Peaks were tested for their ability to directly cleave fibrinogen. Hydrolytic products were analyzed by SDS-PAGE. Enzyme-electrophoresis with fibrinogen co-polymerized in 12% polyacrylamide gel was used for the identification of protein that can cleave fibrinogen.. Results. Venom of V. renardi was fractionated on 4 fractions using size-exclusion chromatography. SDS-PAGE of fibrinogen hydrolysis products showed the presence of fibrinogen-specific protease in the 1st and 2nd fractions of venom. 2nd fraction was much more active and according to the data of enzyme electrophoresis contained protease with molecular mass 25 kDa. Conclusions. Fractionation of V. renardi snake venom allowed to detect a protease with apparent molecular mass 25 kDa that can cleave fibrinogen molecule.

Potential of a methanolic extract of Lawsonia inermis (L.) leaf as an alternative sanitiser in the time of COVID-19 Pandemic and beyond

To harness the antimicrobial properties of a crude methanolic extract of Henna (Lawsonia inermis) leaf as a potential alternative sanitiser, there is the need to test its performance in different solutions. In this work, the effects of distilled water (dH20), Acetate-HCL (AH) Buffer (pH 4.6), Phosphate Buffer Saline (PBS) (pH 7.2) and Tris-HCL (TBH) Buffer (pH 8.6) on the antibacterial and antiviral activity of the extract were assessed. Through standard phytochemical screening and HPLC-MS (LCMS STANDARD 7.M), it was found that the extract consisted of about 30 different compounds including flavonoids. The extent of the antimicrobial activity of the extract in solutions was in the increasing order of AH > dH2O >>>> TBH > PBS. Under the same conditions, reduced antibacterial activity and complete cessation of the antiviral activity of the extract in TBH and PBS was observed. However, in AH and dH20, within 1–5 min, 1 mg ml−1, 0.125 mg ml−1 and 0.0625 mg ml−1 of the extract caused complete inactivation of E.coli (reductions of 8.2 log CFU ml−1), B. subtilis (reductions of 8.2 log CFU ml−1) and MS2 (reductions of 9.7 log PFU ml−1) respectively. The fluorescence microscopy images of the live/dead staining of the inactivated bacterial samples validated the extent of the inactivation. The broad spectrum and high antimicrobial activity of the extract, coupled with the plant not a staple food, has long history of safe use by humans as a medicine and cosmetic, cheaply available in abundance in many regions of the world, thus making the extract a potential candidate as an alternative sanitiser in the time of COVID-19 Pandemic and beyond.

A novel method for silkworm cocoons self-degumming and its effect on silk fibers

IntroductionConventional hot-alkaline cocoon degumming techniques greatly weaken the physicochemical and mechanical properties of silk fibroin fiber, thus affecting the quality of silk fabric. Moreover, it causes massive energy waste and serious environmental pollution. ObjectiveThis study aims to establish a novel cocoon self-degumming method by genetic modification of silkworm varieties and silk fibers. MethodsThe self-degummed cocoon material was generated by specifically overexpressing trypsinogen protein in the sericin layer of silk thread; the effect of cocoon self-degumming method was evaluated by the degumming rate of sericin protein, the cleanliness and equivalent diameter of silk fibroin fiber; the basic characteristics of silk fibroin fiber degummed by cocoon self-degumming method and conventional hot-alkaline degumming technique were determined by electron microscopy, Fourier infrared spectroscopy, X-ray diffraction and tensile tests; the composition and biological activity of degummed sericin protein was respectively analyzed by liquid chromatograph-mass spectrometry and cytological experiments. ResultsThe genetically engineered self-degumming cocoon containing trypsinogen protein was successfully created, and the content of trypsinogen protein in silk was 47.14 ± 0.90 mg/g. The sericin protein in the self-degumming cocoon was removed out in water or 1 mM Tris-HCl buffer (pH = 8.0). Compared to alkaline-degummed silk fibroin, self-degummed silk fibroin had better cleanliness, thicker equivalent diameter, more complete silk structure and better mechanical property. In addition, sericin protein degummed from self-degumming cocoons significantly promoted cell proliferation and caused no obvious cytotoxicity. ConclusionCompared to conventional hot-alkaline degumming technique, the cocoon self-degumming method by genetically overexpressing trypsinogen protein in sericin layer of silk thread can self-degummed in a mild degumming condition, and gain silk fiber with better quality and more biologically active sericin protein products. This strategy can not only reduce the environmental impact, but also generate greater economic value, which will accelerate its application in the silk and pharmaceutical industries.

Molecular Characterization of a Stable and Robust L-Asparaginase from Pseudomonas sp. PCH199: Evaluation of Cytotoxicity and Acrylamide Mitigation Potential

L-asparaginase is an important industrial enzyme widely used to treat acute lymphoblastic leukemia (ALL) and to reduce acrylamide formation in food products. In the current study, a stable and robust L-asparaginase from Pseudomonas sp. PCH199, with a high affinity for L-asparagine, was cloned and expressed in Escherichia coli BL21(DE3). Recombinant L-asparaginase (Pg-ASNase II) was purified with a monomer size of 37.0 kDa and a native size of 148.0 kDa. During characterization, Pg-ASNase II exhibited 75.8 ± 3.84 U/mg specific activities in 50.0 mM Tris-HCl buffer (pH 8.5) at 50 °C. However, it retained 80 and 70% enzyme activity at 37 °C and 50 °C after 60 min, respectively. The half-life and kd values were 625.15 min and 1.10 × 10−3 min−1 at 37 °C. The kinetic constant Km, Vmax, kcat, and kcat/Km values were 0.57 mM, 71.42 U/mg, 43.34 s−1, and 77.90 ± 9.81 s−1 mM−1 for L-asparagine, respectively. In addition, the enzyme has shown stability in the presence of most metal ions and protein-modifying agents. Pg-ASNase II was cytotoxic towards the MCF-7 cell line (breast cancer) with an estimated IC50 value of 0.169 U/mL in 24 h. Further, Pg-ASNase II treatment led to a 70% acrylamide reduction in baked foods. These findings suggest the potential of Pg-ASNase II in therapeutics and the food industry.

Dual Crosslinked Ion-Based Bacterial Cellulose Composite Hydrogel Containing Polyhexamethylene Biguanide.

Composite bacterial cellulose (BC) based hydrogel with alginate (A) or pectin (P) or alginate and pectin was fabricated via a physical crosslinking technique using calcium chloride (CaCl2) solution and incorporated with polyhexamethylene biguanide (PHMB) as an effective antimicrobial drug by immersion method. After that, the physicochemical properties of all hydrogel formulations were characterized. The result showed that the formulations with PHMB performed better physicochemical properties than the hydrogel without PHMB. Fourier transform infrared spectroscopy (FT-IR) showed the interaction between PHMB and the carboxylic group of alginate and pectin. BC/A-PHMB hydrogel performed suitable mechanical strength, fluid uptake ability, water retention property, drug content, high integrity value, and maximum swelling degree. Moreover, in vitro cell viability of BC/A-PHMB hydrogel revealed high biocompatibility with human keratinocyte cell line (HaCaT) and demonstrated prolong released of PHMB in Tris-HCl buffer pH 7.4, while rapid release in phosphate buffer saline pH 7.4. BC/A-PHMB hydrogel demonstrated good anti-bacterial activity against S. aureus and P. aeruginosa. In conclusion, BC/A-PHMB hydrogel could be a potential dual crosslinked ion-based hydrogel for wound dressing with anti-bacterial activity.

Intercalation of diafenthiuron insecticide with calf thymus DNA: spectroscopic and molecular dynamics analysis

A series of biophysical experiments like UV-Vis, fluorescence, circular dichroism (CD), competitive displacement assays, voltammetric studies, viscosity measurements and denaturation effect and metadynamics simulation studies were performed to establish the mode of binding of diafenthiuron (DF) insecticide with calf thymus DNA (CT-DNA). Analysis of absorption and fluorescence spectra in Tris-HCl buffer of pH 7.4 indicates the formation of a complex between DF and CT-DNA and the binding constant of which is in the order of 104 M−1. Competitive displacement assay with ethidium bromide (EB) and Hoechst 33258 suggests that the most probable mode of binding of DF with CT-DNA may be via intercalation mode. The results of other experiments such as CD spectral studies, viscosity measurements and the effect of denaturation agent urea support the intercalation of DF with CT-DNA. Thermodynamic parameters (ΔH o, ΔS o and ΔG o) reveal that hydrogen bonds (H-bonds) or van der Waals (vdW) force is the main binding force in the spontaneous interaction between DF and CT-DNA. Molecular dynamics (MD) simulation studies confirmed the intercalation of DF into the base pairs of CT-DNA. Communicated by Ramaswamy H. Sarma

Species differences in liver microsomal hydrolysis of acyl glucuronide in humans and rats

Acyl glucuronides (AGs) are known as one of the causes of idiosyncratic drug toxicity (IDT). Although AGs can be enzymatically hydrolysed by β-glucuronidase and esterase, much information on their characteristics and species differences is lacking. This study was aimed to clarify species differences in AG hydrolysis between human and rat liver microsomes (HLM and RLM). To evaluate the AG hydrolysis profile, and the contribution of β-glucuronidase and esterase towards AG hydrolysis in HLM and RLM, nonsteroidal anti-inflammatory drugs (NSAIDs) were used. AGs were incubated with 0.1 M Tris-HCl buffer (pH 7.4) and 0.3 mg/mL HLM or RLM in the absence or presence of β-glucuronidase inhibitor, D-saccharic acid 1,4-lactone (D-SL) and esterase inhibitor, phenylmethylsulfonyl fluoride (PMSF). AGs of mefenamic acid (MEF-AG) and etodolac (ETO-AG) showed significantly higher AG hydrolysis rates in RLM than in HLM. Esterases were found to serve as AG hydrolases dominantly in HLM, whereas both esterases and β-glucuronidase equally contribute to AG hydrolysis in RLM. However, MEF-AG and ETO-AG were hydrolysed only by β-glucuronidase. We demonstrated for the first time that the activity of AG hydrolases towards NSAID-AGs differs between humans and rats.

Chitosan-based formulation of hemagglutinin antigens for oculo-nasal booster vaccination of chickens against influenza viruses

Avian influenza viruses (AIVs) and especially highly pathogenic (HP) AIVs of the H5 and H7 subtypes are of both veterinary and public health concern worldwide. In response to the demand for effective vaccines against H5N1 HPAIVs, we produced recombinant protein based on hemagglutinin (HA), a protective viral antigen. A fragment of the HA ectodomain, with a multibasic cleavage site deletion, was expressed in Escherichia coli, refolded, and chromatographically purified from inclusion bodies. Finally, the protein was formulated in Tris-HCl buffer of pH 8.0 or PBS of pH 7.4 to obtain antigens denoted rH5–1 and rH5–2, respectively. The systemic prime and boost immunizations proved that rH5–1 adsorbed to aluminum hydroxide induces anti-H5 HA neutralizing antibodies and protective immune responses against H5N1 HPAIVs in chickens. The present studies were aimed at stimulating immune responses via the mucosal routes using the systemic prime-mucosal boost strategy. Efficacy trials were performed in commercial layer chickens. For systemic and mucosal immunizations, H5 HA antigens were adjuvanted with aluminum hydroxide and chitosan glutamate, respectively. The first dose of rH5–2 was administered subcutaneously, while its second dose was administered subcutaneously, intraocularly, oculo-nasally, or intranasally. rH5–1 was delivered to the subcutaneously primed chickens by the intranasal route. Post-vaccination sera were analyzed for anti-H5 HA antibodies, using homologous ELISA and heterologous FluAC H5 and hemagglutination inhibition tests. Intraocularly and oculo-nasally delivered rH5–2 mixed with chitosan glutamate was capable of stimulating anti-H5 HA IgY antibody responses in the subcutaneously primed chickens; however, it was ineffective when administered by the intranasal route. Efficient intranasal boosting was achieved using rH5–1. The enhanced production of antigen-specific antibodies was reflected in the development of H5-subtype specific and hemagglutination inhibiting antibodies. Conclusively, the subcutaneous prime and oculo-nasal boost vaccination is proposed as the target strategy for future optimization.

A dual-signal sensing strategy based on ratiometric fluorescence and colorimetry for determination of Cu2+ and glyphosate.

Herein, a dual-signal sensing strategy based on ratiometric fluorescence and colorimetry for Cu2+ and glyphosate determination was constructed. Fluorescence silicon nanoparticles (SiNPs) were prepared by hydrothermal reaction, which has maximum fluorescence intensity under the excitation of 355nm. o-Phenylenediamine (OPD) was oxidized through Cu2+ to generate 2,3-diaminophenazine (oxOPD). The obtained oxOPD showed a strong absorption peak at 417nm and quenched the fluorescence of SiNPs at 446nm due to fluorescence resonance energy transfer (FRET). Meanwhile, oxOPD produced a new fluorescence emission at 556nm forming a ratiometric state. With increasing Cu2+, the original solution changed from colorless to yellow. When glyphosate was present, the interaction between Cu2+ and the functional groups of glyphosate could reduce the oxidation of oxOPD, resulting in the enhancement of fluorescence at 446nm and the decrease of fluorescence at 556nm. Furthermore, the addition of glyphosate changed yellow solution to colorless. Under the optimal conditions of OPD (1mM), 20mM Tris-HCl buffer (pH 7.5), and incubation time (4h), the ratiometric fluorescence sensor had good selectivity and showed a wide linear range of 0.025-20μM with the LOD of 0.008μM for Cu2+ and 0.15-1.5μg/mL with the LOD of 0.003μg/mL for glyphosate, respectively. Besides, it is worth mentioning that this developed sensing system showed good performance in real samples, providing a simple and reliable dual-signal detection strategy.