Expression, Purification, and Partial In Vitro Characterization of Biologically Active Human Coagulation Factor VIII Light Chain (A3-C1-C2) in Pichia pastoris

Recombinant coagulation factor VIII (FVIII) expressed in mammalian expression systems is used extensively in the treatment of hemophilia A. It is reported that the heavy (A1–A2) and light chains (A3–C1–C2) of factor VIII purified from plasma regained the coagulation activity by dimerization in vitro. In this work, cDNA coding for the light chain of human coagulation factor VIII (FVIII-LC) was cloned into pPICZα-A expression vector downstream of alcohol oxidase promoter and α-mating signal sequence from Saccharomyces cerevisiae in order to express the protein with a native N-terminus. The methylotrophic yeast, Pichia pastoris X-33, was transformed with this cassette, and transformants were selected for production of human factor VIII light chain into culture media. SDS-PAGE and Western blot analysis confirmed the expression of factor VIII light chain protein. The expressed protein was purified to near homogeneity using histidine ligand affinity chromatography (2.342 mg/L). The biological activity of FVIII-LC was confirmed by analyzing the interaction between FVIII-LC and phospholipid vesicles. The data presented here indicate the possibilities of exploring cost-effective systems to express complex proteins of therapeutic value.     

Simultaneous Delamination and Rutile Formation on the Surface of Ti3C2Tx MXene for Copper Adsorption

In this work, we studied the formation of the rutile phase of titanium dioxide (TiO₂) on delaminated MXene (d‐Ti₃C₂T_x) flakes by the reaction of Ti₃C₂T_x with amino acids in water. Three types of amino acids with varied side‐chain polarity were used to delaminate Ti₃C₂T_x. d‐Ti₃C₂T_x flakes formed stable colloidal solutions due to the negative surface charges of chemisorbed amino acids on the d‐Ti₃C₂T_x. Rutile formed on d‐Ti₃C₂T_x at room temperature upon the intercalation of aromatic amino acids and subsequent sonication of the solution, while flakes intercalated with aliphatic amino acids did not oxidize. X‐Ray diffraction (XRD), transmission electron microscopy (TEM) and Raman spectroscopy revealed the nanosize rutile formation on the surface of Ti₃C₂T_x flakes. The XPS results indicated the surface functionalization of histidine on d‐Ti₃C₂T_x flakes. As‐synthesized histidine functionalized rutile TiO₂@d‐Ti₃C₂T_x hybrid was used for adsorption of Cu²⁺ ions from aqueous solution with a maximum uptake of 95 mg g^?1.     

Solvent-free synthesis of dibenzo[b,j][1,10]phenanthroline derivatives using Eaton’s reagent as catalyst

A new class of dibenzo[b,j][1,10]phenanthrolines has been prepared. The synthon acridones were achieved in very good yield by a one-pot reaction of 2-amino-5-chloro or 2′-chloro/flouro-substituted benzophenones with 1,2-cyclohexanedione in the presence of freshly prepared Eaton’s reagent (phosphorus pentoxide–methanesulfonic acid) without solvent, through Friedländer synthesis. Then these intermediates were reacted with 2-amino-3,5-dibromobenzaldehyde to afford 1,3-dibromo-10-chloro-8-aryl-6,7-dihydrodibenzo[b,j][1,10]phenanthroline. Also an one-pot reaction between 2?mol of 2-amino-5-chloro aryl benzophenones with 1?mol of 1,2-cyclohexanedione to get 3,10-dichloro-5,8-diaryl-6,7-dihydrodibenzo[b,j][1,10]phenanthroline has also been reported. The newly synthesized structures of the compounds were deduced by spectroscopic techniques.     

Inhibition of peroxynitrite- and peroxidase-mediated protein tyrosine nitration by imidazole-based thiourea and selenourea derivatives

In the present study, the synthesis and characterization of a series of N-methylimidazole-based thiourea and selenourea derivatives are described. The new compounds were also studied for their ability to inhibit peroxynitrite (PN)- and peroxidase-mediated nitration of protein tyrosine residues. It has been observed that the selenourea derivatives are more efficient than the thiourea-based compounds in the inhibition of protein nitration. The higher activity of selenoureas as compared to that of the corresponding thioureas can be ascribed to the zwitterionic nature of the selenourea moiety. Single crystal X-ray diffraction studies on some of the thiourea and selenourea derivatives reveal that the C=S bonds in thioureas possess more of double bond character than the C=Se bonds in the corresponding selenoureas. Therefore, the selenium compounds can react with PN or hydrogen peroxide much faster than their sulfur analogues. The reactions of thiourea and selenourea derivatives with PN or hydrogen peroxide produce the corresponding sulfinic or seleninic acid derivatives, which upon elimination of sulfurous/selenous acids produce the corresponding N-methylimdazole derivatives.     

Study of Forced Degradation Behaviour of Brinzolamide Using LC–ESI–Q-TOF and In Silico Toxicity Prediction

Brinzolamide (BZA) is a topical ophthalmic drug which is generally used to lower the intraocular pressure during glaucoma. It was subjected to forced degradation studies under hydrolytic (acidic, basic), oxidative, photolytic and thermal stress conditions; the drug degraded significantly in hydrolytic and oxidative conditions, leading to the formation of seven degradation products in total. It was stable on exposure to light and dry heat in the solid state. An ultra-performance liquid chromatography (UPLC) method was developed on a Waters CSH phenyl hexyl column (100 × 2.1 mm, 1.7 µm), using gradient elution of 0.1 % formic acid and methanol as mobile phase. The method was extended to quadrupole time-of-flight mass spectrometry (Q-TOF–MS) for the structural characterisation. All degradation products were comprehensively characterised by UHPLC–ESI/MS/MS experiments. The most probable mechanisms for the formation of degradation products were also proposed. In silico toxicity of the drug and its degradation products was determined using TOPKAT toxicity prediction software.

     

Development of Optical Biosensor for the Detection of Glutamine in Human Biofluids Using Merocyanine Dye

Merocyanine dye based fluorescent organic compound has been synthesized for the detection of glutamine. The probe showed remarkable fluorescent intensity with glutamine through ICT (Intermolecular Charge Transfer Mechanism). Hence, it is tested for the detection of glutamine using colorimetric and fluorimetric techniques in physiological and neutral pH (7.2). Under optimized experimental conditions, the probe detects glutamine selectively among other interfering biomolecules. The probe has showed a LOD (lower limit of detection) of 9.6?×?10^–8 mol/L at the linear range 0–180 µM towards glutamine. The practical application of the probe is successfully tested in human biofluids.

Graphical abstract

     

Efficient Protocol for Synthesis of Pyrazolo[3,4-a]acridines

A new class of pyrazolo[3,4-a]acridines have been prepared. The synthon acridones were obtained in very good yield by a one-pot reaction of 2-amino-5-chloro or nitro substituted benzophenones with 1,3-cyclic diketones in the presence of freshly prepared Eaton’s reagent without solvent, using Friedländer synthesis. The intermediates were reacted with ethylformate followed by hydrazine hydrate to afford pyrazolo[3,4-a]acridines. All of the compounds were purified by recrystallization only, and no chromatographic workup was required. The structures of the synthesized compounds were deduced by spectroscopic techniques, including single-crystal x-ray diffraction.     

Measurement of gamma radiation from rocks used as building material in Tiruchirappalli district,Tamil Nadu,India

Tiruchirappalli district is naturally endowed with rich building material resources which are also used in neighboring districts. Hence, measurements of activity concentrations and absorbed dose rate of primordial radionuclides (²³⁸U, ²³²Th and ⁴⁰K) in the rock samples collected from 14 sedimentary rocks and 9 igneous rocks were carried out employing gamma ray spectrometry. In sedimentary rocks the geometric mean activity concentrations of ²³⁸U, ²³²Th and ⁴⁰K were found to be 7.4, 29.5 and 233.6 Bq kg^?1, respectively. On the other hand in igneous rocks geometric mean activity concentrations of ²³⁸U, ²³²Th and ⁴⁰K were distinctly higher and found to be 13.1, 105.7 and 888.8 Bq kg^?1, respectively. The mean radium equivalent activity (Ra_eq) recorded in both sedimentary (20.8 Bq kg^?1) and igneous rocks (245.4 Bq kg^?1) were well within the limit prescribed for dwellings (370 Bq kg^?1) except Vilathupatti (689.3 Bq kg^?1) and Narthamalai (371.6 Bq kg^?1). The mean absorbed dose rate from igneous rock (115 nGy h^?1) exceeded the prescribed limit of 55 nGy h^?1. The mean indoor annual effective dose from the sedimentary rock (0.056 mSv y^?1) and igneous rock (0.580 mSv y^?1) did not exceed the prescribed limit (1 mSv y^?1) except the igneous rock from Vilathupatti (1.51 mSv y^?1). The study concludes that sedimentary and igneous rocks analyzed were radiologically safe when used as building materials except igneous rock from Vilathupatti and Narthamalai.     

Compatibility study between sitagliptin and pharmaceutical excipients used in solid dosage forms

Differential scanning calorimetry (DSC) is a primary technique for measuring the thermal properties of materials, which reflects the physico-chemical properties of drug substances. In the present study, it is used as a screening technique for assessing the compatibility of sitagliptin with some currently employed pharmaceutical excipients. The influence of processing conditions and their effects (simple blending, co-grinding or kneading) on drug stability was evaluated. Sitagliptin showed a sharp endothermic peak at 212.1 °C with an enthalpy change of 131.5 J g^?1 indicating melting of drug. Facile transformation of dehydrated sitagliptin to monohydrate form was observed in some mixtures, disappearance of sharp melting endothermic peak of sitagliptin was observed in some mixtures. On the basis of DSC results, sitagliptin was found to be compatible with micro crystalline cellulose, croscarmellose, and pregelatinized starch. Some excipient interaction was observed with magnesium stearate, ascorbic acid, and citric acid. X-ray diffractometry and FT-IR were used as supportive tools in interpreting the DSC results. Overall, the excipients selected were compatible with the API and the mixtures are stable within the tested conditions. These results would be useful for formulation development of the film coated tablets of sitaglitptin.     

Toward the synthesis of macrolide aspergillide D

Stereoselective synthesis of 16-membered macrocyclic core of marine natural product, Aspergillide D is described using linear strategy. The salient features of this synthetic study include the Sharpless asymmetric kinetic resolution followed by regioselective ring-opening reaction of corresponding epoxide to establish the stereo centers and ring-closing metathesis (RCM) by Grubbs'-II catalyst for the construction of macrocyclic ring.