Maximum Activity Research Articles

A Novel Cold‐Adapted Catechol 1,2‐Dioxygenase From Antarctic Sea‐Ice Bacterium Halomonas sp. ANT108: Characterization and Immobilization

ABSTRACTThe enzyme catechol 1,2‐dioxygenase (CAT) plays a critical role in the biosynthesis pathway of cis, cis‐muconic acid (CCMA), which serves as an indispensable raw material for various industrial applications. In this research, we cloned a novel cold‐adapted CAT (HaCAT) from the Antarctic sea ice bacterium Halomonas sp. ANT108. Homology modeling analysis revealed that HaCAT possessed the characteristic Fe3+ binding site and catalytic active site of typical CATs, and it exhibited unique structural adaptations to cold environments. The optimal temperature and pH for recombinant HaCAT (rHaCAT) were found to be 25°C and 6.5, respectively. At 0°C, the enzyme retained a maximum activity of 43.6%, and in the presence of 1.0 M NaCl, its activity reached 173.9%, demonstrating significant salt tolerance. Additionally, the Vmax and Km of rHaCAT were 6.68 μmol/min/mg and 128.90 μM at 25°C, respectively. Furthermore, rHaCAT was successfully immobilized in the metal‐organic framework ZIF‐8 and retained almost 50% of its activity after five reuse cycles, demonstrating excellent reusability. Overall, these results provided a new resource and theoretical foundation for the industrial biocatalytic production and modification of CAT.

The Relationship Between Length and Active Force for Submaximal Skeletal Muscle Contractions: a Review.

The force-length relationship is usually obtained for isometric contractions with maximal activation, but less is known about how sarcomere length affects force during submaximal activation. During submaximal activation, length-dependent alterations in calcium sensitivity, owing to changes in cross-bridge kinetics (rate of attachment and/or detachment), result in an activation-dependent shift in optimal length to longer sarcomere lengths. It is known that sarcomere length, as well as temperature and phosphorylation of the regulatory light chains of myosin, can modify Ca2⁺ sensitivity by altering the probability of cross-bridge interaction. This altered calcium sensitivity is particularly important for submaximal force levels, as it can change the shape of the length dependence of force, with peak force occurring at sarcomere lengths longer than those associated with maximal filament overlap. In athletic contexts, contractions typically do not reach maximal intensity. Therefore, understanding that the ability to produce force under both maximal and submaximal conditions can differ, and that peak force can be generated at different lengths, could influence the development of targeted training regimens optimal for each sport.

Enhancing crack self-healing properties of low-carbon LC3 cement using microbial induced calcite precipitation technique

Limestone Calcined Clay Cement (LC3) is a promising low-carbon alternative to traditional cement, but its reduced clinker content limits its self-healing ability for microcracks, affecting durability. This study explores the application of Microbial Induced Calcite Precipitation (MICP) technique to enhance the crack self-healing capacity of LC3-based materials. Bacillus pasteurii was utilized to induce calcium carbonate precipitation to improve the crack self-healing capacity of LC3, thereby addressing its limited durability due to reduced clinker content. Experimental tests focused on optimizing the growth conditions for B. pasteurii, evaluating the compressive strength, capillary water absorption, and crack self-healing rates of the modified LC3 material. Results showed that under optimal conditions (pH of 9, inoculation volume of 10%, incubation temperature of 30°C, and shaking speed of 150 rpm), the bacterial strain exhibited maximum metabolic activity. The Microbe-LC3 mortar demonstrated a self-healing rate of up to 97% for cracks narrower than 100 μm, significantly higher than unmodified LC3. Additionally, the compressive strength of Microbe-LC3 was enhanced by approximately 15% compared to standard LC3 mortar after 28 days. The capillary water absorption was reduced, indicating improved durability due to the microbial-induced calcium carbonate filling the pores. This study confirms that MICP technology is a viable approach to significantly enhance the performance of LC3, contributing to the development of more durable and sustainable cementitious materials for construction applications.

Evaluation of 210Po, 210Pb, and potentially toxic element concentrations in aquatic environments and polychaetes along the southeast coast of Chennai, Tamil Nadu: a baseline report

ABSTRACT The activity concentrations of radionuclides (210Po and 210Pb) and the concentrations of potentially toxic elements (PTEs) in water, sediment, and two polychaete species (Marphysa madrasi and Namalycastis jaya) collected from 12 stations, denoted as S1 to S12, along the coast of Tamil Nadu, Chennai, in southeast India has been investigated. The average activity concentrations of 210Po and 210Pb were 0.39 ± 0.01 mBq/L and 3.30 ± 0.06 mBq/L in water, and 0.80 ± 0.02 Bq/kg and 4.21 ± 0.19 Bq/kg in sediment, respectively. Marphysa madrasi exhibited higher 210Po and 210Pb activity concentrations (37.78 ± 0.43 Bq/kg and 17.25 ± 0.22 Bq/kg, respectively) compared to Namalycastis jaya (34.40 ± 0.42 Bq/kg and 15.91 ± 0.26 Bq/kg, respectively). Maximum activity concentrations for both radionuclides in water were observed at Kalpaakam (S11) and Nemmeli (S7), while maximum sediment activity concentrations were found at Nemmeli (S7). The elevated activity concentrations in polychaetes from Nemmeli (S7) likely reflect the significantly higher 210Po and 210Pb activity concentrations observed in sediments from this site when compared to other sites. Bioaccumulation values for 210Po and 210Pb averaged 6.89 × 101 and 4.20 for M. madrasi, and 6.17 × 101and 3.84 for N. jaya , respectively. Sediment PTE concentrations followed the order: Fe > Zn > Mn > Ni > Co > Cr > Pb > Cd > Cu. Risk assessment indicated alarming levels of PTE contamination in sediments, suggesting severe anthropogenic impacts. In contrast, average PTE concentrations in polychaetes followed the order: Fe > Cu > Zn > Mn> Cr > Ni > Co > Cd > Pb. Statistical analysis revealed strong and significant correlations (p < 0.05) between PTE concentrations in sediments and polychaetes. The present study offers baseline data for evaluatingthe levels of 210Po and 210Pb in water, sediment, and polychaetes in selected locations along Chennai, Tamil Nadu, India’s southeast coast. These findings lay the groundwork for further studies of polychaetes, specifically the ecological effects of PTEs and radionuclides on coastal creatures and biomagnification.

The whole-body and skeletal muscle metabolic response to 14 days of highly controlled low energy availability in endurance-trained females.

This study investigated the effects of 14 days low energy availability (LEA) versus optimal energy availability (OEA) in endurance-trained females on substrate utilization, insulin sensitivity, and skeletal muscle mitochondrial oxidative capacity; and the impact of metabolic changes on exercise performance. Twelve endurance-trained females (V̇O2max 55.2 ± 5.1 mL × min-1 × kg-1) completed two 14-day randomized, blinded, cross-over, controlled dietary interventions: (1) OEA (51.9 ± 2.0 kcal × kg fat-free mass (FFM)-1 × day-1) and (2) LEA (22.3 ± 1.5 kcal × kg FFM-1 × day-1), followed by 3 days OEA. Participants maintained their exercise training volume during both interventions (approx. 8 h × week-1 at 79% heart rate max). Skeletal muscle mitochondrial respiratory capacity, glycogen, and maximal activity of CS, HAD, and PFK were unaltered with LEA. 20-min time trial endurance performance was impaired by 7.8% (Δ -16.8 W, 95% CI: -23.3 to -10.4, p < .001) which persisted following 3 days refueling post-LEA (p < .001). Fat utilization was increased post-LEA as evidenced by: (1) 99.4% (p < .001) increase in resting plasma free fatty acids (FFA); (2) 270% (p = .007) larger reduction in FFA in response to acute exercise; and (3) 28.2% (p = .015) increase in resting fat oxidation which persisted during submaximal exercise (p < .001). These responses were reversed with 3 days refueling. Daily glucose control (via CGM), HOMA-IR, HOMA-β, were unaffected by LEA. Skeletal muscle O2 utilization and carbohydrate availability were not limiting factors for aerobic exercise capacity and performance; therefore, whether LEA per se affects aspects of training quality/recovery requires investigation.

Aerobic and anaerobic poise of white swimming muscles of the deep-diving scalloped hammerhead shark: comparison to sympatric coastal and deep-water species

Scalloped hammerhead sharks (Sphyrna lewini) routinely perform rapid dives to forage on mesopelagic prey. These deep dives consist of intensive swimming followed by recovery periods in the surface mixed layer. Swimming muscle temperature profiles suggest that S. lewini suppresses gill function as a means to reduce convective heat loss during dives into cool water. Such intensive swimming behavior coupled with reduced respiration prompted us to test whether the aerobic and anaerobic metabolic capacities of the white swimming muscle tissue of this species are greater than those of other shark species from the same region. The activities of key enzymes used in aerobic and anaerobic metabolism provide an indirect indicator of the metabolic potential (“poise”) of a tissue. Here we measured the maximal activities [international units (µmol substrate converted to product per min, U) per gram of wet tissue mass at 10°C] of the citric acid cycle enzymes citrate synthase (CS) and malate dehydrogenase (MDH) and glycolytic enzymes pyruvate kinase (PK) and lactate dehydrogenase (LDH) from white swimming muscle of S. lewini. Enzyme activities, and ratios of these enzyme activities that indicate relative indexes of aerobic to anaerobic capacity, were compared to those measured in three sympatric coastal carcharhinid sharks and two deep-dwelling species, Echinorhinus cookei and Hexanchus griseus. This is the first report of swimming-muscle enzyme activity for these deep-dwelling species. In comparison to the other species, S. lewini had significantly higher activities of both LDH and MDH in the white muscle, and a higher MDH/CS ratio. The high LDH activities suggest that the white muscle of S. lewini relies on relatively high rates of anaerobic ATP production, with would result in build up of high lactate levels, during deep foraging dives. High MDH activity in S. lewini white muscle suggests the potential for lactate levels to be rapidly reduced when aerobic conditions are restored while in the surface mixed layer between dives. These biochemical characteristics may enable S. lewini to swim rapidly while suppressing gill function during deep dives and thereby exploit a very different ecological niche from sympatric shark species (e.g., coastal carcharhinids) and hunt more rapidly via faster swimming for deep-water prey compared to species that permanently inhabit deep depths.

Microbial enzymatic indices for predicting composting quality of recalcitrant lignocellulosic substrates

Three different enzymes alkaline phosphatase, Urease and Dehydrogenase were measured during this study to monitor the organic matter dynamics during semi-industrial composting of mixture A with 1/3 sludge+2/3 palm waste and mixture B with ½ sludge+1/2 palm waste. The phosphatase activity was higher for Mix-A (398.7 µg PNP g−1 h−1) than Mix-B (265.3 µg PNP g−1 h−1), while Mix-B (103.3 µg TPF g−1d−1) exhibited greater dehydrogenase content than Mix-A (72.3 µg TPF g−1 d−1). That could contribute to the dynamic change of microbial activity together with high amounts of carbonaceous substrates incorporated with the lignocellulosic. The gradual increase in the dehydrogenase from the compost Mix-A implies that high lignocellulosic substrate requires gradual buildup of dehydrogenase activity to turn the waste into mature compost. A higher pick of urease with a maximum activity of 151.5 and 122.4 µg NH4-N g−1 h−1 were reported, respectively for Mix-A and B. Temperature and pH could also influence the expression of enzyme activity during composting. The machine learning well predicted the compost quality based on NH3/NO3, C/N ratio, decomposition rate and, humification index (HI). The root mean square error (RMSE) values were 1.98, 1.95, 4.61%, and 4.1 for NH+3/NO−3, C/N ratio, decomposition rate, and HI, respectively. The coefficient of determination between observed and predicted values were 0.87, 0.93, 0.89, and 0.94, for the r NH3/NO3, C/N ratio, decomposition rate, and HI. Urease activity significantly predicted the C/N ratio and HI only. The profile of enzymatic activity is tightly linked to the physico-chemical properties, proportion of lignocellulosic-composted substrates. Enzymatic activity assessment provides a simple and rapid measurement of the biological activity adding understunding of organic matter transformation during sludge-lignocellulosic composting.

Membrane-Targeted Quantum Dot-Based BACE1 Activity Sensors for In Vitro and In Cellulo Assays.

The need for the development of specific and robust methodologies to elucidate the intricate pathological mechanisms of neurodegenerative diseases and discover effective treatments for prevention and remediation is evident. Alzheimer's disease, in particular, has become more prevalent as the global population has aged. β-Secretase, the β-site amyloid precursor protein cleaving enzyme (BACE1), is the protease that produces the β-amyloid peptide, which is considered one of the driving factors of Alzheimer's disease and an important target for treatment development. However, an understanding of its activity, modulation, and regulation is far from complete. This is in large part due to the complex nature of following its activity. Beyond the common requirements for all biosensors (ease of preparation and use), BACE1 probes also demand both stability at acidic pH and membrane localization. To overcome these hurdles, we exploit the modular self-assembly provided by fluorescent quantum dot (QD) sensors. As compared to other fluorophores, QDs provide enhanced fluorescence brightness and photostability, and their large surface area enables functionalization with peptide substrates together with targeting elements that localize the sensor to the areas of maximal BACE1 activity, all achieved through His-tag self-assembly. In vitro, the sensor demonstrated stability under acidic conditions, and using high-throughput plate reader assays, we determined BACE1 activity in-line with literature values and enabled the obtainment of the inhibitor constant of verubecestat, a small molecule inhibitor. The sensor was also transitioned to cellular experiments, where it demonstrated sensitivity to BACE1 activity and its modulation upon inhibitor treatment in a neuroblastoma cell line.

Optimization of waste biomass demineralization through response surface methodology and enhancement of thermochemical and fusion properties

This study examines the impact of leaching with dilute hydrochloric acid solution on the reduction of ash content and the thermal degradation behavior of sugarcane bagasse. Response surface methodology (RSM) was used to statistically design the experiments and investigate the effect of three independent variables: treatment time, solid-to-liquid ratio, and reagent concentration. The leaching conditions were further optimized and experimentally validated for maximum ash reduction for suitability of treated biomass as feedstock for thermochemical conversion technologies. Reagent concentration and treatment time directly affected ash reduction, while the solid-to-liquid ratio inversely influenced it. Concentration had the highest impact, and treatment duration had the least. The maximum 78.2% ash reduction was achieved by treating the biomass with 1 M HCl for 80 min at a solid-to-liquid ratio of 50:1 (wt/vol). This ash reduction also resulted in a 9.82% increase in higher heating value (HHV). Hemicellulose hydrolysis during leaching was observed through chemical composition and Fourier-transform infrared spectroscopy (FTIR). Ash fusion temperatures increased, indicating more thermally stable biomass. Thermogravimetric analysis (TGA) showed elevated maximum degradation temperature and activation energy.

Efficient Biotransformation of Icariin to Baohuoside I Using Two Novel GH1 β-Glucosidases

Epimedium Folium (EF) is a traditional Chinese herbal medicine, and its primary bioactive ingredients, such as icariin, are flavonoid glycosides. A rare EF flavonoid, baohuoside I, exhibits superior bioactivities and enhanced bioavailability compared to its metabolic precursor icariin. The biotransformation of icariin to baohuoside I can be effectively and specifically achieved by β-glucosidases. In this study, 33 candidate full-length β-glucosidase genes were screened from a previously built carbohydrate active enzyme (CAZyme) gene dataset derived from cow fecal microbiota. Thirteen of them exhibited β-glucosidase activity, with DCF-bgl-26 and DCF-bgl-27 showing relatively high expression levels and β-glucosidase activity. The maximum β-glucosidase activity of DCF-bgl-26 and DCF-bgl-27 was achieved at 45 °C and pH 6.0, with DCF-bgl-26 demonstrating better thermostability and pH tolerance compared to DCF-bgl-27. The activities of DCF-bgl-26 and DCF-bgl-27 were 123.2 U/mg protein and 157.9 U/mg protein, respectively, both of which are higher than those of many bacterial β-glucosidases. Structure analysis suggested that both β-glucosidases possess canonical (β/α)8-TIM barrel fold structure of GH1 family β-glucosidases. Thin-layer chromatography results showed that both enzymes could efficiently convert icariin to baohuoside I in 30 min, indicating they have potential application in the production of high value rare baohuoside I.

Valorization of coconut (Cocos nucifera L.) testa as a biocolourant

IntroductionCoconut testa, a by-product of the coconut processing industry, is currently underutilised. This study aimed to extract a coconut testa-based food colourant using various organic solvents and physical methods, and to utilise this colourant in food product preparation.MethodsDifferent organic solvents, along with various time and temperature combinations, were employed for colourant extraction using both a laboratory-scale water bath and ultrasonication. The colour coordinate values (CIELab) of the testa-derived colourants were measured, and the colourants were screened for various phytochemicals. The in vitro antioxidant potential of the testa colourant was assessed by quantifying total phenolics, and the phytochemical composition, including monomeric anthocyanins, was evaluated.ResultsThe study determined the optimal combinations of organic solvents, temperature and time to obtain extracts with maximum antioxidant activity and total phenolic content (TPC). Acidified ethanol-based extracts of testa colourants yielded highest polyphenol content (154.39 ± 2.63 mg GAE/g) and flavonoids content (53.65 ± 0.62 mg QE/g). Similarly, ethanol-based extractants of coconut testa produced high anthocyanin content [823.02 ± 1.81 mg Cy-3-glc equivalents (C3GE)/100 g]. Acidified (0.3 M HCl) solvents at relatively high temperature and time combinations exhibited high antioxidant potential of testa colourant, as measured by CUPRAC, FRAP, and DPPH assays. Following the foam mat drying process of the colourant, a mature coconut water-based jelly was prepared by incorporating the testa colourant extracted with acidified ethanol.DiscussionThis study highlights the biochemical and antioxidant potential of the food colorant derived from coconut testa and explores its suitability for functional food applications. Therefore, coconut testa extract serves a dual purpose: it enhances the aesthetic appeal of food as a colourant and provides significant health-promoting properties due to its high anthocyanin content. Insights from this study could help in promoting the valorization of one of the beneficial by products of coconut industry.

Biological analysis of shoulder joint muscle strength during volleyball spike specific physical training

Volleyball spike requires extremely high shoulder joint muscle strength. Traditional research methods are single, with insufficient sample size, lack multidimensional analysis and long-term training effect evaluation, and have not fully combined biomechanical and physiological theories. This article combined biomechanical and physiological theories to collect field data and used a three-dimensional motion capture system and surface electromyography technology to analyze the performance of shoulder joint muscle strength in spiking. The experimental results show that Integrated Biomechanics and Physiology (IBP) is significantly superior to other training methods in improving the maximum strength, endurance, and muscle activation of the shoulder joint. Specific data show that after IBP training, the maximum strength of the shoulder joint increased by 50 N, and the endurance increased by 19.6%, and the muscle activation increased by 13.5%. Studies have shown that the IBP method has great potential for application in optimizing special physical training, providing a scientific basis for athletes’ physical training.

Structural determinants of protein kinase A essential for CFTR channel activation

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), the anion channel mutated in cystic fibrosis (CF) patients, is activated by the catalytic subunit of protein kinase A (PKA-C). PKA-C activates CFTR both noncatalytically, through binding, and catalytically, through phosphorylation of multiple serines in CFTR's regulatory (R) domain. Here, we identify key molecular determinants of the CFTR/PKA-C interaction essential for these processes. By comparing CFTR current activation in the presence of ATP or an ATP analog unsuitable for phosphotransfer, as well as pseudosubstrate peptides of various lengths, we identify two distinct specific regions of the PKA-C surface which interact with CFTR to cause noncatalytic and catalytic CFTR stimulation, respectively. Whereas the "substrate site" mediates CFTR phosphorylation, a distinct hydrophobic patch (the "docking site") is responsible for noncatalytic CFTR activation, achieved by stabilizing the R domain in a "released" conformation permissive to channel gating. Furthermore, by comparing PKA-C variants with different posttranslational modification patterns, we find that direct membrane tethering of the kinase through its N-terminal myristoyl group is an unappreciated fundamental requirement for CFTR activation: PKA-C demyristoylation abolishes noncatalytic, and profoundly slows catalytic, CFTR stimulation. For the F508del CFTR mutant, present in ~90% of CF patients, maximal activation by demyristoylated PKA-C is reduced by ~10-fold compared to that by myristoylated PKA-C. Finally, in bacterial genera that contain common CF pathogens, we identify virulence factors that demyristoylate PKA-C in vitro, raising the possibility that during recurrent bacterial infections in CF patients, PKA-C demyristoylation may contribute to the exacerbation of lung disease.

Isolation, identification and optimization for tyrosinase production by banana peel waste for industrial application

The study aimed to isolate, purify and optimise tyrosinase from banana peel waste for industrial use. Tyrosinase was extracted from banana peel using phosphate buffer. Purification initiated with centrifugation followed by ammonium sulphate precipitation. Further purification and characterisation was done through gel filtration. Bradford assay was used to determine the protein concentration. Enzyme activity of each sample was measured using tyrosinase activity assay. The pH and temperature optimisation of tyrosinase were also obtained. Results indicated that fractions obtained through ammonium sulphate precipitation at 70% saturation showed more activity than that of crude extract and 35% saturated fractions. Gel filtration column results showed that fraction number 17 &18 have maximum activity. Tyrosinase showed maximum activity at pH 7.0; 37 °C. Comparison between industrially purified and lab-isolated enzyme showed that both have similar optimum pH and temperature. It can be concluded that banana peel can be a source for synthesis of tyrosinase.

Molecular characterization and computational analysis of a highly specific L-glutaminase from a marine bacterium Bacillus australimaris NIOT30

An alkaline active L-glutaminase (BALG) producing bacterium was screened and identified from seamount sediment samples of the Arabian Sea. The isolate was confirmed to be Bacillus australimaris NIOT30 based on morphological characteristics and 16 S rRNA gene sequencing. The glutaminase gene, balg was PCR amplified, cloned and expressed in E. coli BL21 (DE3) host. The molecular weight of purified BALG was estimated to be 36 kDa and the enzyme showed a specific activity of 507 ± 27 Umg−1 against L-glutamine under optimal assay conditions of pH 7.0 and temperature at 37 °C for 15 min. The enzyme showed maximum activity at pH 7 and retained 95% activity at pH 10. BALG retained a relative activity of about 82% and 45% at 45 °C and 60 °C respectively. The kinetic parameters of BALG, Km and Kcat/Km were determined to be of 210 ± 11 mM and 4.4 × 102 M s−1 respectively. Homology modeling and substrate ligand interaction studies revealed the stability of the enzyme-substrate complex. The present study highlights the characterization of a highly active L-glutaminase from B. australimaris NIOT30. Further, mutational analyses of ligand binding residues would show insights into the affinity of L-Glutaminase.

Odd-Numbered Agaro-Oligosaccharides Produced by α-Neoagaro-Oligosaccharide Hydrolase Exert Antioxidant Activity in Human Dermal Fibroblasts

Agarases produce agar oligosaccharides with various structures exhibiting diverse physiological activities. α-Neoagaro-oligosaccharide hydrolase (α-NAOSH) specifically cleaves even-numbered neoagaro-oligosaccharides, producing 3,6-anhydro-l-galactose (l-AHG) and odd-numbered agaro-oligosaccharides (OAOSs). In this study, α-NAOSH from the agar-degrading marine bacterium Gilvimarinus agarilyticus JEA5 (Gaa117) was purified and characterized using an E. coli expression system to produce OAOSs and determine their bioactivity. Recombinant Gaa117 (rGaa117) showed maximum activity at pH 6.0 and 35 °C. rGaa117 retained &gt;80% of its initial activity after 120 min at 30 °C. The activity was enhanced in the presence of Mn2+. Km, Vmax, and Kcat/Km values of the enzyme were 22.64 mM, 246.3 U/mg, and 15 s−1/mM, respectively. rGaa117 hydrolyzed neoagarobiose, neoagarotetraose, and neoagarohexaose, producing OAOSs that commonly contained l-AHG. Neoagarobiose and neoagarotetraose mixtures, designated NAO24, and mixtures of l-AHG and agarotriose, designated AO13, were obtained using recombinant rGaa16B (β-agarase) and rGaa117, respectively, and their antioxidant activities were compared. AO13 showed higher hydrogen peroxide-scavenging activity than NAO24 in human dermal fibroblasts in vitro because of structural differences: AOSs have d-galactose at the non-reducing end, whereas NAOSs have l-AHG. In conclusion, OAOSs exhibited high ROS-scavenging activity in H2O2-induced human dermal fibroblasts. They may be applicable in cosmetics and pharmaceuticals for prevention of skin aging.

In vitro and in vivo study of 221Fr and 213Bi progeny release from the 225Ac-labelled TiO2 nanoparticles

BackgroundTargeted alpha therapy (TAT) is an effective option for cancer treatment. To maximize its efficacy and minimize side effects, carriers must deliver radionuclides to target tissues. Most of the nuclides used in TAT decay via the alpha cascade, producing several radioactive daughter nuclei with sufficient energy to escape from the original carrier. Therefore, studying these daughter atoms is crucial in the search for new carriers. Nanoparticles have potential as carriers due to their structure, which can prevent the escape of daughter atoms and reduce radiation exposure to non-target tissues. This work focuses on determining the released activity of 221Fr and 213Bi resulting from the decay of 225Ac labelled TiO2 nanoparticles. ResultsLabelling of TiO2 nanoparticles has shown high sorption rates of 225Ac and its progeny, 221Fr and 213Bi, with over 92 % of activities sorbed on the nanoparticle surface for all measured radionuclides. However, in the quasi-dynamic in vitro system, the released activity of 221Fr and 213Bi is strongly dependent on the nanoparticles concentration, ranging from 15 % for a concentration of 1 mg/mL to approximately 50 % for a nanoparticle concentration of 10 μg/mL in saline solution. The released activities of 213Bi were lower, with a maximum value of around 20 % for concentrations of 0.05, 0.025, and 0.01 mg/mL. The leakage of 225Ac and its progeny was tested in various biological matrices. Minimal released activity was measured in saline at around 10 % after 48 h, while the maximum activity was measured in blood serum and plasma at 20 %. The amount of 225Ac released into the media was minimal (<3 %). The in vitro results were confirmed in a healthy mouse model. The difference in %ID/g was clearly visible immediately after dissection and again after 6 h when 213Bi reached equilibrium with 225Ac. ConclusionThe study verified the potential release of 225Ac progeny from the labelled TiO2 nanoparticles. Experiments were performed to determine the dependence of released activity on nanoparticle concentration and the biological environment. The results demonstrated the high stability of the prepared 225Ac@TiO2 NPs and the potential release of progeny over time. In vivo studies confirmed our hypothesis. The data obtained suggest that the daughter atoms can escape from the original carrier and follow their own biological pathways in the organism.

Structural and catalytic properties of histidyl-tRNA synthetase: A potential drug target against leishmaniasis

Visceral leishmaniasis is caused by Leishmania donovani which affects the poorer sections of society, and despite the global spread, effective treatment is unavailable. The current study investigates the potential of leishmanial histidyl-tRNA synthetase (LdHisRS) as a drug target. LdHisRS delineated more closeness to other protozoan parasites than its mammalian counterparts and contained relevant differences in the active site residues. The important ATP-binding residues were mutated to alanine and all the proteins, including human HisRS, were purified to homogeneity. LdHisRS exhibited a dimeric state in solution and showed maximal amino acid activation activity in physiological conditions. It also demonstrated a greater affinity for substrate over cofactor, while magnesium and potassium enhanced its activity better than other tested metal ions. Comp-7 m, a benzothiazolo-coumarin derivative, proved to be specific inhibitor of LdHisRS with competitive mode of inhibition for ATP whereas it displayed lower binding affinity towards mutants. LdHisRS majorly contained α-helices and most of the aromatic residues were present in its hydrophobic core. Additionally, Comp-7 m superimposed on ATP adenine ring during docking analysis and LdHisRS-ligand complexes had comparable stability as well as rigidity in molecular dynamics simulation. We thus provide structural and functional insights of LdHisRS which can be useful for devising antileishmanials.

Inhibition of dipeptidyl peptidase-IV by hydrolysates of beta-lactoglobulin isolated from Gir cow milk

The current study attempts to assess the dipeptidyl peptidase – IV (DPP-IV) inhibitory potential of β-lactoglobulin (β-Lg) isolated from milk of Gir cow. Whey was separated from skim milk by precipitation of casein followed by ultrafiltration (UF) to remove lactose and salts as permeate. β-Lg was separated from the UF retentate using salting out technique. Its purity was confirmed by SDS-PAGE and RP-HPLC. Hydrolysates of β-Lg were prepared using three different enzymes viz., pepsin, trypsin and Flavourzyme at different enzyme to substrate (E:S) ratios (1:25, 1:50, 1:100) and treated for 2-12 h durations. Pepsin-treated hydrolysate showed maximum DPP-IV inhibition of 77.62 ± 0.98 % (IC50 =3.78 mg/mL). Hydrolysate was passed serially through UF filters of 10 and 3 kDa. The permeate of 3 kDa, which exhibited 52.10 ± 0.72 % DPP-IV inhibition was fractionated through RP-HPLC and time based fractions were collected. All 41 fractions were again analysed for DPP-IV inhibition activity and 3 fractions which showed maximum DPP-IV inhibition activity were chosen for LC-MS/MS analysis. One novel peptide was identified through LC-MS/MS, which showed adequate DPP-IV inhibition (IC50 = 6.64 mmolL-1).

Enhanced activity of split trehalase biosensors by interspecies domain combineering.

The split trehalase biosensor has potential as a versatile diagnostic technology. Split enzymes are engineered proteins, divided into inactive fragments, which can reassemble and regain activity when brought together by an analyte. The split TreA biosensor requires no sample processing and produces stable signals (in the form of glucose). Split trehalase reagents can function in blood, but periplasmic trehalase of E.coli requires blood acidification for maximal activity. The objective of this study was to obtain split trehalase with near physiological pH optimum. For this purpose, periplasmic trehalases of Cellvibrio spp. with higher activity at neutral pH, were split in analogy with the E. coli TreA into hood and catalytic domains. However, these split trehalases displayed self-complementation due to spontaneous reassembly. In contrast, when catalytic domains of Cellvibrio trehalases were combined with E.coli hood domains, these hybrids displayed conditional complementation capacity when split trehalase fragments fused to immunoglobulin-binding protein G (STIGA) were used to quantify immunoglobulin concentrations. Other hybrid combinations of Cellvibrio spp. had increased activity compared to the cognate pairs, albeit with strong self-complementation. A mutagenesis analysis of residues responsible for self-complementation led to uncoupling of self-complementation from allostery. The Michaelis-Menten kinetics of Cellvibrio enzymes and fragment pairs confirmed improved activity of a mutated hybrid pair of Cellvibrio hood and catalytic domains at physiological pH. In conclusion, the improvements in pH optimum and activity, demonstrated with STIGA, can now be leveraged to enhance other variations of the split trehalase biosensor platform, broadening its utility for testing clinical samples.