HBcAg (hepatitis B core antigen) is a nanoplex bioproduct that has a great potential in the development of therapeutic drugs and vaccines. In the present study, a continuous-flow bead milling for the disruption of Escherichia coli was optimized and a direct recovery protocol to isolate the recombinant HBcAg from the unclarified E. coli disruptate was developed. The optimal condition for continuous-flow bead milling for the release of HBcAg from E. coli was achieved at a feed flow rate of 15 litres/h, biomass concentration of 10% [ww/v (wet weight/vol.)] and impeller tip speed of 14 m/s. The sucrose-density-gradient analysis showed that the particulate form of the HBcAg released by this optimal condition is still preserved. In the direct purification of HBcAg from the unclarified disruptate, the AE-EBAC (anion-exchange expanded-bed adsorption chromatography) technique was employed. A 54% adsorption and 50.7% recovery of HBcAg were achieved in this direct recovery process. The purity of HBcAg recovered was 49.8%, which corresponds to a purification factor of 2.0. ELISA showed that the HBcAg recovered is functionally active.
Non-small cell lung cancer (NSCLC) incited by epidermal growth factor receptor (EGFR) mutation makes up ∼85% of lung cancer diagnosed and death cases worldwide. The presented study introduced an alternative approach in detecting EGFR mutation using nano-silica integrated with polydimethylsiloxane (PDMS) polymer on interdigitated electrode (IDE) sensor. A 400 μm gap-sized aluminum IDE was modified with nano-polymer layer, which was made up of silica nanoparticles and PDMS polymer. IDE and PDMS-coated IDE (PDMS/IDE) were imaged using electron microscopes that reveals its smooth and ideal sensor morphology. The nano-silica-integrated PDMS/IDE surface was immobilized with EGFR probe and target to specify the lung cancer detection. The sensor specificity was justified through the insignificant current readouts with one-base mismatch and noncomplementary targets. The sensitivity of nano-silica-integrated PDMS/IDE was examined with mutant target spiked in human serum, where the resulting current affirms the detection of EGFR mutation. Based on the slope of the calibration curve, the sensitivity of nano-silica-integrated PDMS/IDE was 2.24E-9 A M-1 . The sensor recognizes EGFR mutation lowest at 1 aM complementary mutant target; however, the detection limit obtained based on 3σ calculation is 10 aM with regression value of 0.97.
A microbial fuel cell is a sustainable and environmental-friendly device that combines electricity generation and wastewater treatment through metabolic activities of microorganisms. However, low power output from inadequate electron transfer to the anode electrode hampers its practical implementation. Nanocomposites of oxidized carbon nanotubes and medium-chain-length polyhydroxyalkanoates (mcl-PHA) grafted with methyl acrylate monomers enhance the electrochemical function of electrodes in microbial fuel cell. Extensive polymerization of methyl acrylate monomers within mcl-PHA matrix, and homogenous dispersion of carbon nanotubes within the graft matrix are responsible for the enhancement. Modified electrodes exhibit high conductivities, better redox peak and reduction of cell internal resistance up to 76%. A stable voltage output at almost 700 mV running for 225 H generates maximum power and current density of 351 mW/m2 and 765 mA/m2 , respectively. Superior biofilm growth on modified surface is responsible for improved electron transfer to the anode hence stable and elevated power output generation.
Artificial intelligence of things (AIoT) has become a potential tool for use in a wide range of fields, and its use is expanding in interdisciplinary sciences. On the other hand, in a clinical scenario, human blood-clotting disease (Royal disease) detection has been considered an urgent issue that has to be solved. This study uses AIoT with deep long short-term memory networks for biosensing application and analyzes the potent clinical target, human blood clotting factor IX, by its aptamer/antibody as the probe on the microscaled fingers and gaps of the interdigitated electrode. The earlier results by the current-volt measurements have shown the changes in the surface modification. The limit of detection (LOD) was noticed as 1 pM with the antibody as the probe, whereas the aptamer behaved better with the LOD at 100 fM. The time-series predictions from the AIoT application supported the obtained results with the laboratory analyses using both probes. This application clearly supports the results obtained from the interdigitated electrode sensor as aptamer to be the better option for analyzing the blood clotting defects. The current study supports a great implementation of AIoT in sensing application and can be followed for other clinical biomarkers.
Biosynthesis and in vivo depolymerization of intracellular medium-chain-length poly-3-hydroxyalkanoates (mcl-PHA) in Pseudomonas putida Bet001 grown on lauric acid were studied. Highest mcl-PHA fraction (>50 % of total biomass) and cell concentration (8 g L-1 ) were obtained at carbon-to-nitrogen (C/N) ratio 20, starting cell concentration 1 g L-1 , and 48 H fermentation. The mcl-PHA comprised of 3-hydroxyhexanoate (C6 ), 3-hydroxyoctanote (C8 ), 3-hydroxydecanoate (C10 ), and 3-hydroxydodecanoate (C12 ) monomers. In vivo action was studied in a mineral liquid medium without carbon source, and in different buffer solutions with varied pH, molarity, ionic strength, and temperature. The monomer liberation rate reflected the mol percentage distribution of the initial polymer subunit composition. Rate and percentage of in vivo depolymerization were highest in 0.2 M Tris-HCl buffer (pH 9, strength = 0.2 M, 30 °C) at 0.21 g L-1 H-1 and 98.6 ± 1.3 wt%, respectively. There is a congruity vis-à-vis to specific buffer type, molarity, pH, ionic strength, and temperature values for superior in vivo depolymerization activities. Direct products from in vivo depolymerization matched the individual monomeric composition of native mcl-PHA. It points to exo-type reaction for the in vivo process, and potential biological route to chiral molecules.
The biosynthesis of medium-chain-length poly-3-hydroxyalkanoates by Pseudomonas putida Bet001 cultivated on mixed carbon sources was investigated. The mixed carbon sources consisted of heptanoic acid (HA) and oleic acid (OA). A relatively low PHA content at 1.2% (w/w) and 11.4% (w/w) was obtained when HA or OA was used as the sole carbon source. When these fatty acids were supplied as a mixture, PHA content increased threefold. Interestingly, the mixture-derived PHA composed of both odd and even monomer units, namely. 3-hydroxyheptanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate and no unsaturated monomer was detected. It is hypothesized that the even-numbered monomers were derived primarily from OA, whereas the odd-numbered monomer was derived from HA. This also points out to an efficient and yet distinct fatty acids metabolism that fed the PHA biosynthesis machinery of this particular microorganism. PHA obtained was elastomeric because melting temperature (Tm ) and crystallinity were absent. It showed good thermal stability with degradation temperature (Td ) ranging from 275.96 to 283.05 °C.
The current world condition is dire due to epidemics and pandemics as a result of novel viruses, such as influenza and the coronavirus, causing acute respiratory syndrome. To overcome these critical situations, the current research seeks to generate a common surveillance system with the assistance of a controlled Internet of Things operated under a Gaussian noise channel. To create the model system, a study with an analysis of H1N1 influenza virus determination on an interdigitated electrode (IDE) sensor was validated by current-volt measurements. The preliminary data were generated using hemagglutinin as the target against gold-conjugated aptamer/antibody as the probe, with the transmission pattern showing consistency with the Gaussian noise channel algorithm. A good fit with the algorithmic values was found, displaying a similar pattern to that output from the IDE, indicating reliability. This study can be a model for the surveillance of varied pathogens, including the emergence and reemergence of novel strains.
Limit of detection (LOD), limit of quantification, and the dynamic range of detection of hepatitis B surface antigen antibody (anti-HBs) using a surface plasmon resonance (SPR) chip-based approach with Pichia pastoris-derived recombinant hepatitis B surface antigen (HBsAg) as recognition element were established through the scouting for optimal conditions for the improvement of immobilization efficiency and in the use of optimal regeneration buffer. Recombinant HBsAg was immobilized onto the sensor surface of a CM5 chip at a concentration of 150 mg/L in sodium acetate buffer at pH 4 with added 0.6% Triton X-100. A regeneration solution of 20 mM HCl was optimally found to effectively unbind analytes from the ligand, thus allowing for multiple screening cycles. A dynamic range of detection of ∼0.00098-0.25 mg/L was obtained, and a sevenfold higher LOD, as well as a twofold increase in coefficient of variance of the replicated results, was shown as compared with enzyme-linked immunosorbent assay (ELISA). Evaluation of the assay for specificity showed no cross-reactivity with other antibodies tested. The ability of SPR chip-based assay and ELISA to detect anti-HBs in human serum was comparable, indicating that the SPR chip-based assay with its multiple screening capacity has greater advantage over ELISA.
Proteomic analysis of plants relies on high yields of pure protein. In plants, protein extraction and purification present a great challenge due to accumulation of a large amount of interfering substances, including polysaccharides, polyphenols, and secondary metabolites. Therefore, it is necessary to modify the extraction protocols. A study was conducted to compare four protein extraction and precipitation methods for proteomic analysis. The results showed significant differences in protein content among the four methods. The chloroform-trichloroacetic acid-acetone method using 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer provided the best results in terms of protein content, pellets, spot resolution, and intensity of unique spots detected. An overall of 83 qualitative or quantitative significant differential spots were found among the four methods. Based on the 2-DE gel map, the method is expected to benefit the development of high-level proteomic and biochemical studies of Andrographis paniculata, which may also be applied to other recalcitrant medicinal plant tissues.
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 0.1 M 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 3 kDa 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.
A white-rot fungus of Polyporus sp. S133 was isolated from an oil-polluted soil. The metabolism of pyrene by this fungus was investigated in liquid medium with 5 mg of the compound. Depletion of pyrene was evident during the 30-day growth period and was 21% and 90%, respectively, in cometabolism and metabolism of pyrene alone. Pyrene was absorbed to fungal cells or biodegraded to form simpler structural compounds. Seventy-one percent of eliminated pyrene was transformed by Polyporus sp. S133 into other compounds, whereas only 18% was absorbed in the fungal cell. The effects of pH and temperature on biomass production of Polyporus sp. S133 for pyrene were examined; the properties of laccase and 1,2-dioxygenase produced by Polyporus sp. S133 during pyrene degradation were investigated. The optimal values of pH were 3, 5, and 4 for laccase, 1,2-dioxygenase, and biomass production, respectively, whereas the optimal values of temperature were 25 °C for laccase and 50 °C for 1,2-dioxygenase and biomass production. Under optimal conditions, pyrene was mainly metabolized to 1-hydroxypyrene and gentisic acid. The structure of 1-hydroxypyrene and gentisic acid was determined by gas chromatography-mass spectrometry after identification using thin-layer chromatography.
Due to its thermostability and high pH compatibility, subtilisin is most known for its role as an additive for detergents in which it is categorized as a serine protease according to MEROPS database. Subtilisin is typically isolated from various bacterial species of the Bacillus genus such as Bacillus subtilis, B. amyloliquefaciens, B. licheniformis, and various other organisms. It is composed of 268-275 amino acid residues and is initially secreted in the precursor form, preprosubtilisin, which is composed of 29-residues signal peptide, 77-residues propeptide, and 275-residues active subtilisin. Subtilisin is known for the presence of high and low affinity calcium binding sites in its structure. Native subtilisin has general properties of thermostability, tolerance to neutral to high pH, broad specificity, and calcium-dependent stability, which contribute to the versatility of subtilisin applicability. Through protein engineering and immobilization technologies, many variants of subtilisin have been generated, which increase the applicability of subtilisin in various industries including detergent, food processing and packaging, synthesis of inhibitory peptides, therapeutic, and waste management applications.
One-pot synthesis of sugar-functionalized oligomeric caprolactone was carried out by lipase-catalyzed esterification of ε-caprolactone (ECL) with methyl-d-glucopyranoside (MGP) followed by the elongation of functionalized oligomer chain. Functionalization was performed in a custom-fabricated glass reactor equipped with Rushton turbine impeller and controlled temperature at 60 °C using tert-butanol as reaction medium. The overall reaction steps include MGP esterification of ECL monomer and its subsequent elongation by free 6-hydroxyhexanoate monomer units. A ping-pong bi-bi mechanism without ternary complex was proposed for esterification of ECL and MGP with apparent values of kinetic constant, namely maximal velocity (Vmax ), Michaelis constant for MGP (KmMGP ), and Michaelis constant for ECL (KmECL ) at 3.848 × 10-3 M H-1 , 8.189 × 10-2 M, and 6.050 M, respectively. Chain propagation step of MGP-functionalized ECL oligomer exhibits the properties of living polymerization mechanism. Linear relationship between conversion (%) and number average molecular weight, Mn (g mol-1 ), of functionalized oligomer was observed. Synthesized functionalized oligomer showed narrow range of molecular weight from 1,400 to 1,600 g mol-1 with more than 90% conversion achieved. Structural analysis confirmed the presence of covalent bond between the hydroxyl group in MGP with carboxyl end group of ECL oligomer.
Luteinizing hormone (LH)/Lutropin is an interstitial cell stimulating hormone playing a predominant role in the reproductive system, and highly correlated with the infertility treatment in both men and women. This research was concentrated to quantify LH level by using interdigitated electrode sensor. To improve the electric current flow, sensing electrode was modified with graphene oxide (GO) and the aptamer probe was attached on GO through biotin-streptavidin linker. Current responses were measured with aptamer-LH interaction at the target concentrations between 7.5 nM and 1 μM and the detection limit of LH was calculated as 60 nM with the determination co-efficient (R2 ) value, 0.9229 [y = 1.296x - 2.8435] on a linear range from 30 nM until 1 μM. Further, biofouling effect on sensing electrode surface was analysed with complementary aptamer sequence, control proteins (Albumin, and globulin). The above GO-aptamer modified interdigitated electrode sensor helps to quantify LH level and diagnose gynaecological endocrinology related complications. This article is protected by copyright. All rights reserved.
The traditional approach of fermentation by a free cell system has limitations of low productivity and product separation that need to be addressed for production enhancement and cost effectiveness. One of potential methods to solve the problems is cell immobilization. Microbial cell immobilization allows more efficient up-scaling by reducing the nonproductive growth phase, improving product yield and simplifying product separation. Furthermore, the emergence of nanomaterials such as carbon nanotubes, graphene, and metal-based nanomaterials with excellent functional properties provides novel supports for cell immobilization. Nanomaterials have catalytic properties that can provide specific binding site with targeted cells. However, the toxicity of nanomaterials towards cells has hampered its application as it affects the biological system of the cells, which cannot be neglected in any way. This gray area in immobilization is an important concern that needs to be addressed and understood by researchers. This review paper discusses an overview of nanomaterials used for cell immobilization with special focus on its toxicological challenges and how by understanding physicochemical properties of nanomaterials could influence the toxicity and biocompatibility of the cells.
Despite a lot of intensive research on cells-scaffolds interaction, focused are mainly on the capacity of construct scaffolds to regulate cell mobility, migration and cytotoxicity. The effect of the scaffold's topographical and material properties on the expression of biologically active compounds from stem cells is not well understood. In this study, the influence of cellulose acetate (CA) on the electrospinnability of gelatin and the roles of gelatin-cellulose acetate (Ge-CA) on modulating the release of biologically active compounds from amniotic fluid stem cells (AFSCs) is emphasized. It was found that the presence of a small amount of CA could provide a better microenvironment that mimics AFSCs' niche. However, a large amount of CA exhibited no significant effect on AFSCs migration and infiltration. Further study on the effect of surface topography and mechanical properties on AFSCs showed that the tailored microenvironment provided by the Ge-CA scaffolds had transduced physical cues to biomolecules released into the culture media. It was found that the AFSCs seeded on electrospun scaffolds with less CA proportions has profound effects on the secretion of metabolic compounds compared to those with higher CA contained and gelatin coating. The enhanced secretion of biologically active molecules by the AFSCs on the electrospun scaffolds was proven by the accelerated wound closure on the injured human dermal fibroblast (HDF) model. The rapid HDF cell migration could be anticipated due to a higher level of paracrine factors in AFSCs media. Our study demonstrates that the fibrous topography and mechanical properties of the scaffold is a key material property that modulates the high expression of biologically active compounds from the AFSCs. The discovery elucidates a new aspect of material functions and scaffolds material-AFSCs interaction for regulating biomolecules release to promote tissue regeneration/repair. To the best of our knowledge, this is the first report describing the scaffolds material-AFSCs interaction and the efficacy of scratch assays on quantifying the cell migration in response to the AFSCs metabolic products. This article is protected by copyright. All rights reserved.
Ovarian cancer starts in the ovaries in its earlier stages and then spreads to the pelvis, uterus, and abdominal region. The success of an ovarian cancer treatment depends on the stage of the cancer and the diagnostic system. Squamous cell carcinoma antigen (SCC-Ag) is one of the most efficient cancer biomarkers, and elevated levels of SCC-Ag in ovarian cancer cells have been used to identify ovarian cancer. Carbon is a potential material for biosensing applications due to its thermal, electrical, and physical properties. Multiwalled carbon nanotubes (MWCNTs) are carbon-based materials that can be used here to detect SCC-Ag. Anti-SCC-Ag antibody was immobilized on the amine-modified MWCNT dielectric sensing surface to detect SCC-Ag. The uniformity of the surface structure was measured with a 3D nanoprofiler, and the results confirmed the detection of SCC-Ag at ∼80 pM. The specific detection of SCC-Ag was confirmed with two control proteins (factor IX and human serum albumin), and the system did not show biofouling. This experimental set-up with MWCNTs a dielectric sensing surface can lead to the detection of ovarian cancer in its initial stages.
Phage display has been applied successfully as a tool for the generation of monoclonal antibodies (mAbs). Naive antibody libraries are unique as they are able to overcome several limitations associated with conventional mAb generation methods like the hybridoma technology. Here, we performed an in vitro selection and generation of Fab antibodies against Brugia malayi SXP protein (BmSXP), a recombinant antigen for the detection of lymphatic filariasis. We developed a naïve multi ethnic Fab antibody library with an estimated diversity of 2.99 × 109 . The antibody library was used to screen for mAbs against BmSXP recombinant antigen. Soluble monoclonal Fab antibodies against BmSXP were successfully isolated from the naïve library. The Fab antibodies obtained were expressed and analyzed to show its binding capability. The diversity obtained from a pool of donors from various ethnic groups allowed for a diverse antibody library to be generated. The mAbs obtained were also functional in soluble form, which makes it useful for further downstream applications. We believe that the Fab mAbs are valuable for further studies and could also contribute to improvements in the diagnosis of filariasis.
Gestational diabetes and jaundice are the correlated diseases predominantly found in mother and newborn child. Jaundice is a neonatal complication with an increased risk when mother has gestational diabetes. Mothers with diabetes at an early stage of gestational age are at higher risk for hyperbilirubinemia (jaundice) and hypoglycemia. So, it is mandatory to monitor the condition of diabetes and jaundice during the pregnancy period for a healthy child and safest delivery. On the other hand, nanotechnology has displayed a rapid advancement that can be implemented to overcome these issues. The development of high-performance diagnosis using appropriate biomarkers provides their efficacy in the detection gestational diabetes and jaundice. This review covers the aspects from a fast-developing field to generate nanosensors in the nanosized dimensions for the applications to overcome these complications by coupling diagnostics with biomarkers. Further, the serum-based biomarkers have been discussed for these inborn complications and also the diagnosis with the current trend.
Differentially expressed aqueous soluble proteins between Mycobacterium tuberculosis H37Ra and H37Rv were identified. The protein extracts were separated by two-dimensional gel electrophoresis followed by tandem mass spectrometric analysis. Twelve proteins were detected to be differentially expressed significantly between virulent strain H37Rv and attenuated strain H37Ra. The differentially expression of these proteins was validated by a recently isolated clinical virulent strains of M. tuberculosis, TB138. Out of the 12 proteins identified, which consisted of ten upregulated and two downregulated proteins, nine were belonged to intermediate metabolism and respiration protein group, two were in lipid metabolism, and one protein was involved in information pathways and virulence. Among these proteins, two of the upregulated proteins, namely, mmsA and pntAa, showed a consistent expression pattern in both virulent mycobacterium strains. These proteins can serve as potential biomarkers for the intervention treatment of TB.