Avidin Biotin Research Articles

Probing the interaction between biomolecules under sub-zero temperature conditions by electrophoresis in ice grain boundaries

BackgroundPsychrophiles can survive under cryogenic conditions because of various biomolecules. These molecules interact with cells, ice crystals, and lipid bilayers to enhance their functionality. Previous studies typically measured these interactions by thawing frozen samples and conducting biological assays at room temperature; however, studying these interactions under cryogenic conditions is crucial. This is because these biomolecules can function at lower temperatures. Therefore, a platform for measuring chemical interactions under sub-zero temperature conditions must be established. ResultsThe chemical interactions between biomolecules under sub-zero temperature conditions were evaluated within ice grain boundaries with a channel-like structure, which circumvents the need for thawing. An aqueous solution of sucrose was frozen within a microfluidic channel, facilitating the formation of freeze-concentrated solutions (FCSs) that functioned as size-tunable electrophoretic fields. Avidin proteins or single-stranded DNA (ssDNA) were introduced into the FCS in advance. Probe micro/nanospheres whose surfaces were modified with molecules complementary to the target analytes were introduced into the FCS. If the targets have functionalities under sub-zero temperature conditions, they interact with complementary molecules. The chemical interactions between the target molecules and nanospheres led to the aggregation of the particles. The size tunability of the diameter of the FCS channels enabled the recognition of aggregation levels, which is indicative of interaction reactivity. The avidin–biotin interaction and ssDNA hybridization served as models for chemical interactions, demonstrating interactivity under sub-zero temperature conditions. The results presented herein suggest the potential for in situ measurement of biochemical assays in the frozen state, elucidating the functionality of bio-related macromolecules at or slightly below 0 °C. SignificanceThis is the first methodology to evaluate chemical interactions under sub-zero temperature conditions without employing the freeze-and-thaw process. This method has the advantage of revealing the chemical interactions only at low temperatures. Therefore, it can be used to screen and evaluate the functionality of cryo-related biomolecules, including cold-shock and antifreeze proteins.

Development of Biotinylated Liposomes Encapsulating Metformin for Therapeutic Targeting of Inflammation-Based Diseases.

Inflammation is a physiological response to a damaging stimulus but sometimes can be the cause of the onset of neurodegenerative diseases, atherosclerosis, and cancer. These pathologies are characterized by the overexpression of inflammatory markers like endothelial adhesion molecules, such as Vascular Cell Adhesion Molecule-1 (VCAM-1). In the present work, the development of liposomes for therapeutic targeted delivery to inflamed endothelia is described. The idea is to exploit a three-step pretargeting system based on the biotin-avidin high-affinity interaction: the first step involves a previously described biotin derivative bearing a VCAM-1 binding peptide; in the second step, the avidin derivative NeutrAvidinTM, which strongly binds to the biotin moiety, is injected; the final step is the administration of biotinylated liposomes that would bind to NeutravidinTM immobilized onto VCAM-1 overexpressing endothelium. Stealth biotinylated liposomes, prepared via the thin film hydration method followed by extrusion and purification via size exclusion chromatography, have been thoroughly characterized for their chemico-physical and morphological features and loaded with metformin hydrochloride, a potential anti-inflammatory agent. The three-step system, tested in vitro on different cell lines via confocal microscopy, FACS analysis and metformin uptake, has proved its suitability for therapeutic applications.

The immunohistochemical expression Ki-67 In Polymorphous Low Grade Adenocarcinoma of the Salivary Glands

Background: Polymorphous Low Grade Adenocarcinoma is a rare malignant tumor of minor salivary glands. It has an unique histopathological, clinical and behavioural features. Most of its incidence has been presented in the palate. It shows a variety of growth patterns within the same one lesion, including cribriform, tubular, and solid areas that may mimic adenoid cystic carcinoma. The tumor cells histologically are characterized by lacking of nuclear atypical and hyperchromatism with rare mitoses. However, the prognosis of this tumor is good because of its low grade, although it has an unpredictable potential to metastasize.
 Objectives: To evaluate the histopathological expression of Ki-67 proliferation antigen in Polymorphous low grade adenocarcinoma and compare the results with the clinicopathological parameters and grading of the tumors.
 Materials and Methods: This study includes 20 cases with Polymorphous Low Grade Adenocarcinoma of minor salivary glands diagnosed histopathologically and treated surgically, were investigated for the immunohistochemical expression of Ki-67 antigen. Immunohistochemical staining was performed using a Labeled Strept- Avidin Biotin method (LSAB) and 0.05 (two-sided) was the level of significance.
 Results: Twenty cases of Polymorphous Low Grade Adenocarcinoma of salivary glands were histologically diagnosed in this retrospective study to evaluate the expression of Ki-67. The mean age for patients with PLGA was nearly (50.6). (12) (60%) cases were males and (8) (40%) cases were females and also male to female ratio was (1.5:1).
 In regarding to site distribution, the most common site was the palate followed by the submandibular gland and floor of the mouth. 
 Conclusions: it was observed that there is a higher rate of cellular proliferation in malignant tumors with high grade, indicating their role in the malignancy and aggressive behavior of these tumors.

The Immunohistochemical expressions and diagnostic values of E-cadherin and β-Catenin in suspected cervical lesion or cervical cancer

Background: Cervical lesions are the cellular changes of a pathologic process with a bacterial and viral etiology ranging from cervicitis, cervical intraepithelial neoplasm and invasive cancer. The integrity of the cervical epithelium is maintained by complex cell adhesion molecules. This work is aimed at determining the expression pattern diagnostic value of the adhesion molecules to enhance early detection of cancer. Methods: The study population was from 10 years retrospective archival tissue blocks of cervical cancer among women in Abuja metropolis from 2005 to 2015 Histopathological diagnosis made, from the 80 cases examined 20(25%) cases were cervicitis (CC), 15(18.8%) were cervical intraepithelial neoplasia (CINI), 15(18.8%) were cervical intraepithelial neoplasia (CIN2) while 30(37.3%) cases were squamous cell carcinoma SCC. They were examined immunohistochemically by Avidin Biotin Complex (ABC) method. E-Cadherin and Beta-Catenin, (cell adhesion molecules). Results: Statistically significant rate of positivity were found in the expression of β-catenin 100% among all the categories of the studied cases. β-catenin expression was significantly higher in CINI than in SCC, while in E-cadherin, therewere no statistically significant difference (p<0.05) between the distribution of high expression and low expression of E-cadherin in both CINI and SCC. In expression of E-cadherin among the cases studied, there was a significant difference (p<0.05) between rate of positivity and negativity for the expression of E-cadherin. A statistically significant rate of positivity was found in the expression of β-catenin among all the categories of the studied cases. In the expression E-cadherin, there was a gradual decrease in high expression from cervicitis to CINI, CIN2 and SCC. There was Significant (p<0.05) high degree of expression of E-cadherin in cervicitis and CINI against CIN2 and SCC, while a significant (p<0.05) low degree expression of E-cadherin in CIN2 and SCC against Cervicitis and CINI. In β-catenin expression, there as a gradual decrease in high expression from cervicitis to CIN2 and SCC expect in CINI were all had high expression. Conclusion: This study showed that most of the immunological markers examined can be used for identification of different stages involved in cancer progression, also for diagnosis of cancer, predictive prognosis and can be explore as therapy targets for cervicitis and Squamous cell carcinoma.

Biotinylated Quinone as a Chemiluminescence Sensor for Biotin-Avidin Interaction and Biotin Detection Application

Biotin, or vitamin B7, is essential for metabolic reactions. It must be obtained from external sources such as food and biotin/vitamin supplements because it is not biosynthesized by mammals. Therefore, there is a need to monitor its levels in supplements. However, biotin detection methods, which include chromatographic, immune, enzymatic, and microbial assays, are tedious, time-consuming, and expensive. Thus, we synthesized a product called biotin-naphthoquinone, which produces chemiluminescence upon its redox cycle reaction with dithiothreitol and luminol; then it was used as a chemiluminescence sensor for biotin–avidin interaction. When a quinone biotinylated compound binds avidin, the chemiluminescence decreases noticeably due to the proximity between quinone and avidin, and when free biotin is added in a competitive assay, the chemiluminescence returns. The chemiluminescence is regained as the free biotin displaces biotinylated quinone in its complex with avidin, freeing biotin-naphthoquinone. Many experiments, including the use of a biotin-free quinone, proved the competitive nature of the assay. The competitive assay method used in this study was linear in the range of 1.0–100 µM with a detection limit of 0.58 µM. The competitive chemiluminescence assay could detect biotin in vitamin B7 tablets with good recovery of 91.3 to 110% and respectable precision (RSD < 8.7%).

Spinal afferent innervation in flat-mounts of the rat stomach: anterograde tracing

The dorsal root ganglia (DRG) project spinal afferent axons to the stomach. However, the distribution and morphology of spinal afferent axons in the stomach have not been well characterized. In this study, we used a combination of state-of-the-art techniques, including anterograde tracer injection into the left DRG T7-T11, avidin–biotin and Cuprolinic Blue labeling, Zeiss M2 Imager, and Neurolucida to characterize spinal afferent axons in flat-mounts of the whole rat stomach muscular wall. We found that spinal afferent axons innervated all regions with a variety of distinct terminal structures innervating different gastric targets: (1) The ganglionic type: some axons formed varicose contacts with individual neurons within myenteric ganglia. (2) The muscle type: most axons ran in parallel with the longitudinal and circular muscles and expressed spherical varicosities. Complex terminal structures were observed within the circular muscle layer. (3) The ganglia-muscle mixed type: some individual varicose axons innervated both myenteric neurons and the circular muscle, exhibiting polymorphic terminal structures. (4) The vascular type: individual varicose axons ran along the blood vessels and occasionally traversed the vessel wall. This work provides a foundation for future topographical anatomical and functional mapping of spinal afferent axon innervation of the stomach under normal and pathophysiological conditions.

A novel visual biosensor for mercury detection based on a cleavable phosphorothioate-RNA probe and microfluidic bead trap

Mercury ion (Hg2+) detection plays a crucial role in safeguarding the environment and protecting human health. In this study, we developed a microfluidic biosensing platform that utilizes a phosphorothioate-RNA (PS-RNA) probe and gold-coated magnetic nanoparticles to quantify Hg2+ visually. The PS-RNA probe was linked to gold-coated magnetic nanoparticles via Au–S interaction and biotin–avidin interaction and then combined with polystyrene microspheres to form a self-assembled structure called a magnetic bead-AuNP-probe-polystyrene (MAPP) bead. We designed and fabricated a microfluidic chip that contained only a magnetic separator and a particle trap. The presence of Hg2+ cut the PS-RNA probe, leading to the separation of magnetic beads and polystyrene microspheres. The MAPP was removed by the magnetic separator, while the cut polystyrene microspheres flowed along the channel and accumulated in the particle trap, forming a long visible strip. The length of the polystyrene microsphere strip was proportional to the Hg2+ concentration. The microfluidic biosensing platform exhibited high sensitivity and specificity for Hg2+ detection, with a detection limit of 21.9 nM. By integrating the microfluidic magnetic manipulation system with a sensitive nucleic acid probe, we developed an automated and visual biosensor that is a valuable reference for the on-site visual detection of heavy metal ions.

Thermal/ultrasound-triggered release of liposomes loaded with Ganoderma applanatum polysaccharide from microbubbles for enhanced tumour ablation

The effectiveness of thermal ablation for the treatment of liver tumours is limited by the risk of incomplete ablation, which can result in residual tumours. Herein, an enhancement strategy is proposed based on the controlled release of Ganoderma applanatum polysaccharide (GAP) liposome–microbubble complexes (GLMCs) via ultrasound (US)-targeted microbubble destruction (UTMD) and sublethal hyperthermic (SH) field. GLMCs were prepared by conjugating GAP liposomes onto the surface of microbubbles via biotin–avidin linkage. In vitro, UTMD promotes the cellular uptake of liposomes and leads to apoptosis of M2-like macrophages. Secretion of arginase-1 (Arg-1) and transforming growth factor-beta (TGF-β) by M2-like macrophages decreased. In vivo, restriction of tumour volume was observed in rabbit VX2 liver tumours after treatment with GLMCs via UTMD in GLMCs + SH + US group. The expression levels of CD68 and CD163, as markers of tumour-associated macrophages (TAMs) in the GLMCs + SH + US group were reduced in liver tumour tissue. Decreased Arg-1, TGF-β, Ki67, and CD31 factors related to tumour cell proliferation and angiogenesis was evident on histological analysis. In conclusion, thermal/US-triggered drug release from GLMCs suppressed rabbit VX2 liver tumour growth in the SH field by inhibiting TAMs, which represents a potential approach to improve the effectiveness of thermal ablation.

Efficient delivery of mesenchymal stem/stromal cells to injured liver by surface PEGylation

BackgroundMesenchymal stem/stromal cells (MSCs) have been used in clinical trials for various diseases. These have certain notable functions such as homing to inflammation sites, tissue repair, and immune regulation. In many pre-clinical studies, MSCs administered into peripheral veins demonstrated effective therapeutic outcomes. However, most of the intravenously administered MSCs were entrapped in the lung, and homing to target sites was less than 1%. This occurred mainly because of the adhesion of MSCs to vascular endothelial cells in the lung. To prevent this adhesion, we modified the surface of MSCs with polyethylene glycol (PEG; a biocompatible polymer) using the avidin–biotin complex (ABC) method.MethodsThe surface of MSCs was modified with PEG using the ABC method. Then, the cell adhesion to mouse aortic endothelial cells and the tissue distribution of PEG-modified MSCs were evaluated. Moreover, the homing to the injured liver and therapeutic effect of PEG-modified MSCs were evaluated using carbon tetrachloride-induced acute liver failure model mice.ResultsThe PEG modification significantly suppressed the adhesion of MSCs to cultured mouse aortic endothelial cells as well as the entrapment of MSCs in the lungs after intravenous injection in mice. PEG-modified MSCs efficiently homed to the injured liver of carbon tetrachloride-induced acute liver failure model mice. More importantly, the cells significantly suppressed serum transaminase levels and leukocyte infiltration into the injured liver.ConclusionThese results indicate that PEG modification to the surface of MSCs can suppress the lung entrapment of intravenously administered MSCs and improve their homing to the injured liver.Graphical

Diagnostic Implications of Enhanced CD34 Expression in Hepatocellular Carcinoma: A Cross-Sectional Study

Background: Hepatocellular carcinoma is the most prevalent primary cancer of the liver globally and is responsible for the majority of cancer-related fatalities. The purpose of the research was to establish the frequency of enhanced cd34 expression in sinusoid-like vascular endothelial cells in Hepatocellular carcinoma. Methods: CMH Multan's Department of Histopathology carried out a cross-sectional study. Histochemical staining was performed. Before beginning the staining technique, sections were washed in phosphate buffer saline for 5 minutes. The usual immunohistochemistry staining method - Avidin Biotin Complex Method - was employed following hospital protocols. Results: 85 (67.5%) male and 41 (32.5%) female patients were among the 126 studied cases. 54 (42.9%) were from rural areas, while 72 (57.1%) were from urban areas. Diabetes was prevalent in 43 (34.1%), and smoking was found in 10 (7.9%). Obesity was found in 18 (14.3%), and hypertension was identified in 74 (58.7%). Only 5 (4.0%) of our research's cases had a family history of HCC. Enhanced cd34 expression was noted in 106 (84.1%). Conclusion: There found a very high frequency of enhanced cd34 expression of sinusoid-like vascular endothelial cells in HCC. The enhanced cd34 presentation can aid in the early recognition of HCC and the proper care of these patients to improve their quality of life. Keywords: Enhanced Expression, Hepatocellular carcinoma, cd34, Risk factors.

Prognostic role of CD74, CD10 and Ki-67 immunohistochemical expression in patients with diffuse malignant peritoneal mesothelioma: a retrospective study

BackgroundDiagnosis and treatment of diffuse malignant peritoneal mesothelioma (DMPM) are still challenging. The aim of the present study was to explore the correlation between CD74, CD10, Ki-67 and clinicopathological parameters, and identify independent prognostic factors of DMPM.MethodsSeventy patients with pathologically proven DMPM were retrospectively reviewed. The expression of CD74, CD10 and Ki-67 in peritoneal tissues was detected by immunohistochemical analysis using standard avidin biotin complex (ABC) immunostaining technique. Kaplan-Meier survival analysis and multivariate Cox regression analyses were performed to assess prognostic factors. The nomogram based on the Cox hazards regression model was established. C-index and calibration curve were performed to evaluate the accuracy of nomogram models.ResultsThe median age of DMPM was 62.34 years, and the male-to-female ratio was 1: 1.80. CD74 expression was identified in 52 (74.29%) of 70 specimens, CD10 in 34 (48.57%) specimens, and higher Ki-67 in 33(47.14%) specimens. CD74 was negatively associated with asbestos exposure(r = -0.278), Ki-67(r = -0.251) and TNM stage(r = -0.313). All patients were effectively followed up in the survival analysis. Univariate analysis revealed that PCI, TNM stage, treatment, Ki-67, CD74 and ECOG PS were associated with DMPM prognosis. CD74 (HR = 0.65, 95%Cl:0.46–0.91, P = 0.014), Ki-67(HR = 2.09, 95%Cl:1.18–3.73, P = 0.012),TNM stage (HR = 1.89, 95%Cl:1.16–3.09, P = 0.011), ECOG PS(HR = 2.12, 95%Cl:1.06–4.25, P = 0.034), systemic chemotherapy (HR = 0.41, 95%Cl:0.21–0.82, P = 0.011) and intraperitoneal chemotherapy (HR = 0.34, 95%Cl:0.16–0.71, P = 0.004) were independent predictors by multivariate Cox analysis. The C‑index of the nomogram for predicting overall survival (OS) was 0.81. The OS calibration curve showed good agreement between nomogram-predicted and observed survival.ConclusionsCD74, Ki-67, TNM stage, ECOG PS and treatment were independent factors affecting prognosis of DMPM. Reasonable chemotherapy treatment might improve the prognosis of patients. The proposed nomogram was a visual tool to effectively predict the OS of DMPM patients.

Seroprevalence of Infectious Bovine Rhinotracheitis (IBR) in Indian Buffaloes

Background: Infectious bovine rhinotracheitis (IBR) is an infectious disease of bovines causing respiratory problem, abortions and reduced milk yield leading to huge economic losses. IBR is endemic in India. India ranks first in buffalo population. Reports on seroprevalence of IBR in cattle in India are available but not in buffaloes. The study was aimed at conducting a seroprevalence of IBR in Indian buffaloes. Methods: A total of 1000 buffalo serum samples collected randomly from top 10 milk producing states of India and were tested for IBR antibodies using Avidin-Biotin (AB) ELISA. Result: Cumulative seropositivity was found to be 43.30%. Punjab and Andhra Pradesh states showed highest (58%) and lowest (27%) seropositivity respectively and were part of north and south zone of the country. India has highest number of buffalo population. Presently, India do not vaccinate bovines against IBR and should take call on vaccination of dairy buffalo after having witnessed a high seroprevalence of IBR. This is the first report of such kind of number of buffalo samples tested for IBR antibodies.

Biotin-Avidin System-Based Delivery Enhances the Therapeutic Performance of MSC-Derived Exosomes.

Exosomes (EXs) shed by mesenchymal stem cells (MSCs) are potent therapeutic agents that promote wound healing and regeneration, but when used alone in vivo, their therapeutic potency is diminished by rapid clearance and bioactivity loss. Inspired by the biotin-avidin interaction, we developed a simple yet versatile method for the immobilization of MSC-derived EXs (MSC-EXs) into hydrogels and achieved sustained release for regenerative purposes. First, biotin-modified gelatin methacryloyl (Bio-GelMA) was fabricated by grafting NHS-PEG12-biotin onto the amino groups of GelMA. Biotin-modified MSC-EXs (Bio-EXs) were then synthesized using an in situ self-assembling biotinylation strategy, which provided sufficient binding sites for MSC-EX delivery with little effect on their cargo composition. Thereafter, Bio-EXs were immobilized in Bio-GelMA via streptavidin to generate Bio-GelMA@Bio-EX hydrogels. An in vitro analysis demonstrated that Bio-EXs could be taken up by macrophages and exerted immunomodulatory effects similar to those of MSC-EXs, and Bio-GelMA@Bio-EX hydrogels provided sustained release of MSC-EXs for 7 days. After subcutaneous transplantation, a more constant retention of MSC-EXs in Bio-GelMA@Bio-EX hydrogels was observed for up to 28 days. When placed in an artificial periodontal multitissue defect, the functionalized hydrogels exhibited an optimized therapeutic performance to regrow complex periodontal tissues, including acellular cementum, periodontal ligaments (PDLs), and alveolar bone. In this context, Bio-GelMA@Bio-EX hydrogels exerted a robust immunomodulatory effect that promoted macrophage polarization toward an M2 phenotype. Our findings demonstrate that MSC-EXs delivered with the aid of the biotin-avidin system exhibit robust macrophage-modulating and repair-promoting functions and suggest a universal approach for the development of MSC-EX-functionalized biomaterials for advanced therapies.

Tissue Tropism of H9N2 Low-Pathogenic Avian Influenza Virus in Broiler Chickens by Immunohistochemistry

The H9N2 subtype of low-pathogenic avian influenza viruses (LPAIV) is a widespread pathogen of poultry that can also infect humans. The characterization of viral infections is a complex process, involving clinical, pathological, and virological investigations. The aim of this study was to adapt and optimize an immunohistochemical (IHC) technique developed for LPAIVs specifically for the detection of H9N2 virus antigens in infected tissues. Twenty-one-day-old broiler chickens were inoculated with three different strains of H9N2 virus by different infection routes (i.e., intranasal-intratracheal and intravenous) or co-infected with infectious bronchitis virus (IBV) and observed for 11 days post infection. The suggested IHC protocol was modified: (i) DAB (diamino-benzidine) was substituted with AEC (3-amino-9-ethyl carbazole) as chromogen; and (ii) indirect two-step immune reactions of monoclonal primary and peroxidase-labeled anti-mouse secondary antibodies were used instead of avidin-biotin complexes. Avian influenza virus antigen appears as a red precipitate in the nuclei of affected cells but can also be identified in the cytoplasm. Mild hyperemia and congestion were observed in the trachea, air sac, and lungs of the challenged birds, and fibrinous exudate was found at the bifurcation in a few cases. Neither gross pathological nor IHC lesions were found in the control group. Using the optimized protocol and an associated scoring scheme, it was demonstrated that the H9N2 strains tested exhibited respiratory and urinary tract tropism irrespective of the route of inoculation. On day 5, viral antigen was detected in the respiratory tract and kidney in 30-50% of the samples. On day 11, no IHC signal was observed, indicating the lack of viral replication. Slight differences in viral antigen expression were found between the different H9N2 virus strains, but, in contrast to highly pathogenic avian influenza (HPAI), no viral antigen was detected in the brain and pancreas. Thus, IHC can be considered as an informative, visual addition to the toolkit for the characterization of H9N2 LPAIV infections.

Moderate Constraint Facilitates Association and Force-Dependent Dissociation of HA-CD44 Complex.

Binding of cell surface glycoprotein CD44 to hyaluronic acid (HA) is a key event for mediating cell adhesion, motility, metastasis, inflammatory responses and tumor development, but the regulation mechanism and its molecular basis under diverse mechanical constraints remain unclear. We herein investigated interaction of CD44 HABD (HA binding site domain) to HA through free and steered molecular dynamics (MD) simulations as well as atomic force microscope (AFM) measurement using different constraints on HA. The middle, two ends or both of the constrained HA chains were fixed for MD simulations, while one and two biotin-avidin linkage or physical absorption were used to immobilize HA on substrates for AFM experiments, to model HA chains with low, moderate and high HA flexibilities, respectively. We found that binding of CD44 to moderate fixed HA was possessed of a better thermo-stability, a lower mechanical strength and a higher dissociation probability, while higher adhesive frequency, smaller rupture force and shorter lifetime were assigned to CD44 on the two biotin-immobilized HA rather than one biotin-immobilized or physically absorbed HA on substrates, suggesting a moderate HA flexibility requirement in favor of association and force-induced dissociation of CD44-HA complex. Tensile-induced convex conformation of HA chain was responsible for reduction of complex mechano-stability and did inversely a shrunken CD44 HABD under stretching; transition from catch bond to slip bond governed CD44-HA interaction. This study uncovered the regulation mechanism and its molecular basis for CD44-HA affinity under diverse mechano-microenvironments and provided a new insight into CD44-HA interaction-mediated cell inflammatory responses and tumor development.

Sortase A transpeptidation produces seamless, unbranched biotinylated nanobodies for multivalent and multifunctional applications.

Exploitation of the biotin-streptavidin interaction for advanced protein engineering is used in many bio-nanotechnology applications. As such, researchers have used diverse techniques involving chemical and enzyme reactions to conjugate biotin to biomolecules of interest for subsequent docking onto streptavidin-associated molecules. Unfortunately, the biotin-streptavidin interaction is susceptible to steric hindrance and conformational malformation, leading to random orientations that ultimately impair the function of the displayed biomolecule. To minimize steric conflicts, we employ sortase A transpeptidation to produce quantitative, seamless, and unbranched nanobody-biotin conjugates for efficient display on streptavidin-associated nanoparticles. We further characterize the protein-nanoparticle complex and demonstrate its usefulness in optical microscopy and multivalent severe acute respiratory syndrome coronavirus (SARS-CoV-2) antigen interaction. The approach reported here provides a template for making novel multivalent and multifunctional protein complexes for avidity-inspired technologies.

Seroprevalence of Infectious bovine rhinotracheitis (IBR) in India: A 5-year study.

Infectious bovine rhinotracheitis (IBR) is a highly contagious disease of bovines causing respiratory symptoms, abortions, and reduced milk yield, leading to huge economic losses. Reports on seroprevalence in bovines in India are available and restricted to districts/states. In the present study, a nationwide seroprevalence of IBR in bovines was conducted to provide a national IBR seroprevalence to the Chief Veterinarian who in turn can design the control strategies. A total of 15,592 cattle and buffalo serum samples from 25 states and 3 Union Territories viz., Jammu and Kashmir, Puducherry, and Andaman and Nicobar Islands were tested for IBR antibodies using Avidin‑Biotin (AB) ELISA. Cumulative seropositivity was found to be 31.37%. Maharashtra and Rajasthan states, part of the west zone of the country, showed the highest and lowest seroprevalence, respectively. A total of 11,423 cattle and 4,169 buffalo serum samples were tested, which showed 33.91% and 24.39% seropositivity, respectively. India has the highest buffalo population. Presently, India no IBR vaccination programs are implemented in India. Considering the high seroprevalence, the authorities should plan control strategies for vaccinating dairy cows and buffaloes in India.

Microbubble-Nanoparticle Complexes for Ultrasound-Enhanced Cargo Delivery.

Targeted delivery of therapeutics to specific tissues is critically important for reducing systemic toxicity and optimizing therapeutic efficacy, especially in the case of cytotoxic drugs. Many strategies currently exist for targeting systemically administered drugs, and ultrasound-controlled targeting is a rapidly advancing strategy for externally-stimulated drug delivery. In this non-invasive method, ultrasound waves penetrate through tissue and stimulate gas-filled microbubbles, resulting in bubble rupture and biophysical effects that power delivery of attached cargo to surrounding cells. Drug delivery capabilities from ultrasound-sensitive microbubbles are greatly expanded when nanocarrier particles are attached to the bubble surface, and cargo loading is determined by the physicochemical properties of the nanoparticles. This review serves to highlight and discuss current microbubble-nanoparticle complex component materials and designs for ultrasound-mediated drug delivery. Nanocarriers that have been complexed with microbubbles for drug delivery include lipid-based, polymeric, lipid-polymer hybrid, protein, and inorganic nanoparticles. Several schemes exist for linking nanoparticles to microbubbles for efficient nanoparticle delivery, including biotin-avidin bridging, electrostatic bonding, and covalent linkages. When compared to unstimulated delivery, ultrasound-mediated cargo delivery enables enhanced cell uptake and accumulation of cargo in target organs and can result in improved therapeutic outcomes. These ultrasound-responsive delivery complexes can also be designed to facilitate other methods of targeting, including bioactive targeting ligands and responsivity to light or magnetic fields, and multi-level targeting can enhance therapeutic efficacy. Microbubble-nanoparticle complexes present a versatile platform for controlled drug delivery via ultrasound, allowing for enhanced tissue penetration and minimally invasive therapy. Future perspectives for application of this platform are also discussed in this review.

Spontaneous symmetry breaking propulsion of chemically coated magnetic microparticles

Chemically coated micro/nanoparticles are often used in medicine to enhance drug delivery and increase drug up-take into specific areas of the body. Using a recently discovered spontaneous symmetry breaking propulsion mechanism, we demonstrate that chemically coated microparticles can swim through mucus solution under precise navigation and that certain functionalizations can dynamically change propulsion behavior. For this investigation biotin, Bitotin-PEG3-amine, and biotin chitosan were chemically functionalized onto the surfaces of magnetic microparticles using an avidin–biotin complex. These chemicals were chosen because they are used prolifically in drug delivery applications, with PEG and chitosan having well known mucoadhesive effects. Coated microparticles were then suspended in mucus synthesized from porcine stomach mucins and propelled using rotating magnetic fields. The relationship between different chemical coatings, microparticle velocity, and controllability were thoroughly explored and discussed. Results indicate that the biotinylated surface coatings altered the propulsion behavior of microparticles, with performance differences interlinked to both magnetic field properties and localized mucus properties. Precisely controlled drug carrying microparticles are envisioned to help supplant traditional drug delivery methods and enhance existing medical techniques utilizing micro/nanoparticles.

Gold-Nanoparticle-Coated Magnetic Beads for ALP-Enzyme-Based Electrochemical Immunosensing in Human Plasma.

A simple and sensitive AuNP-coated magnetic beads (AMB)-based electrochemical biosensor platform was fabricated for bioassay. In this study, AuNP-conjugated magnetic particles were successfully prepared using biotin–streptavidin conjugation. The morphology and structure of the nanocomplex were characterized by scanning electron microscopy (SEM) with energy-dispersive X-ray analysis (EDX) and UV–visible spectroscopy. Moreover, cyclic voltammetry (CV) was used to investigate the effect of AuNP-MB on alkaline phosphatase (ALP) for electrochemical signal enhancement. An ALP-based electrochemical (EC) immunoassay was performed on the developed AuNP-MB complex with indium tin oxide (ITO) electrodes. Subsequently, the concentration of capture antibodies was well-optimized on the AMB complex via biotin–avidin conjugation. Lastly, the developed AuNP-MB immunoassay platform was verified with extracellular vesicle (EV) detection via immune response by showing the existence of EGFR proteins on glioblastoma multiforme (GBM)-derived EVs (108 particle/mL) spiked in human plasma. Therefore, the signal-enhanced ALP-based EC biosensor on AuNP-MB was favorably utilized as an immunoassay platform, revealing the potential application of biosensors in immunoassays in biological environments.