An efficient process of generating bispecific antibodies via controlled Fab-arm exchange using culture supernatants.

Bispecific antibody generation is actively pursued for therapeutic and research antibody development. Although there are multiple strategies for generating bispecific antibodies (bsAbs); the common challenge is to develop a scalable method to prepare bsAbs with high purity and yield. The controlled Fab-arm exchange (cFAE) method combines two parental monoclonal antibodies (mAbs), each with a matched point mutation, F405L and K409R in the respective CH3 domains. The conventional process employs two steps: the purification of two parental mAbs from culture supernatants followed by cFAE. Following a reduction/oxidation reaction, the bispecific mAb is formed with greater than 95% heterodimerization efficiency. In this study, cFAE was initiated in culture supernatants expressing the two parental mAbs, thereby eliminating the need to first purify the parental mAbs. The bsAbs formed in culture supernatant was then purified using a Protein A affinity chromatography. The BsAbs generated in this manner had efficiency comparable to the conventional method using purified parental mAbs. BsAbs prepared by two different routes showed indistinguishable characteristics by SDS capillary electrophoresis, analytical size exclusion, and cation exchange chromatography. This alternative method significantly shortened timelines and reduced resources required for bsAb generation, providing an improved process with potential benefits in large-scale bsAb preparation, as well as for HTP small-scale bsAb matrix selection.

AIEE activated Pyrene-Dansyl coupled FRET probe for discriminating detection of lethal Cu2+ and CN-: Bio-Imaging, DNA binding studies and prompt prognosis of Menke's disease.

In present work a pyrene-dansyl dyad functionalized chemoreceptor, DPNS is unveiled towards ultrasensitive chromo-fluorogenic detection of heavy and transition metal ions (HTMs) like Cu2+ and pernicious CN-. It demonstrated distinct chromogenic responses; colorless to faint yellow (Cu2+), intense yellow (CN-) from contaminant aqueous sources. Cu2+ instigated alteration in DPNS fluorescence from feeble emission to sparkling green with LOD: 37.75 × 10-9 M, cyan emission for CN- having LOD 61.51 × 10-8M. In particular, chemical scaffold of DPNS consists of -C = N, O = S = O donor entitities that escalates overall polarity thereby providing an excellent binding pocket for simultaneous Cu2+ and CN- recognition with distinct photophysical signaling. Impressively, presence of two fluorophoric moieties triggers FRET, CHEF phenomenon. The conceivable host:guest interactive pathway is manifested by LMCT- FRET-PET-CHEF, C = N isomerization for Cu2+ and ICT-H-bonding for CN-. An exquisite experimental and theoretical corroboration further strengthened the recognition phenomenon. In addition owing to pyrene excimer formation, DPNS exhibits AIEE with increasing water fraction. Notably, DPNS could successfully undergo intracellular tracking of Cu2+ in Tecoma Stans, Peperomia Pellucida. DPNS•••Cu2+ adduct displayed significant intercalative DNA binding activity rationalized by spectral investigation, competitive EB binding, viscosity study. The overall findings, excellent properties endows DPNS a potential contender towards discriminative detection of Cu2+ and CN- like toxic industrial contaminants.

Unveiling of a smartphone-mediated ratiometric chemosensor towards the nanomolar level detection of lethal CN-: combined experimental and theoretical validation with the proposition of a molecular logic circuitry.

A promising naphthalene-functionalized ratiometric chemosensor (E)-1-((naphthalen-5-yl) methylene)-2-(2,4-dinitrophenyl) hydrazine (DNMH) is unveiled in the present work. DNMH demonstrates brisk discernible colorimetric response from yellow to red in the presence of CN-, a lethal environmental contaminant, in a near-perfect aqueous medium with a LOD of 278 nM. The "key role marker" controlling the electrochemical and non-covalent H-bonding interaction between DNMH and CN- is through the commendable role of acidic -NH functionalities. Kinetic studies reveal a pseudo second order reaction rate and the formation of an unprecedented photostable adduct. The negative value of ΔG as evaluated from ITC substantiates the spontaneity of the DNMH⋯CN- interaction. The sensing mechanism was further reinforced with state-of-the-art theoretical investigations, namely DFT, TDDFT and Fukui indices (FIs). Moreover, the proposition of a reversible multi-component logic circuitry implementing Boolean functions in molecular electronics has also been triggered by the turn-over spectrophotometric response of the ditopic ions CN- and Cd2+. The cytotoxicity of DNMH towards Bacillus thuringiensis and Escherichia coli is successfully investigated via the MTT assay. Impressively, "dip stick" and "easy to prepare" test paper device and silica gel-based solid-phase CN- recognition validate the on-site analytical application of DNMH. Furthermore, the involvement of a synergistic approach between 'chemistry beyond the molecule' and 'engineering' via an exquisitely implemented smartphone-assisted colorimetric sensory prototype makes this work unprecedented among its congeners and introduces a new frontier in multitudinous material-based functional product development.

Design of a potent D-peptide HIV-1 entry inhibitor with a strong barrier to resistance.

The HIV gp41 N-trimer pocket region is an ideal viral target because it is extracellular, highly conserved, and essential for viral entry. Here, we report on the design of a pocket-specific D-peptide, PIE12-trimer, that is extraordinarily elusive to resistance and characterize its inhibitory and structural properties. D-peptides (peptides composed of D-amino acids) are promising therapeutic agents due to their insensitivity to protease degradation. PIE12-trimer was designed using structure-guided mirror-image phage display and linker optimization and is the first D-peptide HIV entry inhibitor with the breadth and potency required for clinical use. PIE12-trimer has an ultrahigh affinity for the gp41 pocket, providing it with a reserve of binding energy (resistance capacitor) that yields a dramatically improved resistance profile compared to those of other fusion inhibitors. These results demonstrate that the gp41 pocket is an ideal drug target and establish PIE12-trimer as a leading anti-HIV antiviral candidate.

Calcium ion incorporated hydrous iron(III) oxide: synthesis, characterization, and property exploitation towards water remediation from arsenite and fluoride.

Calcium ion-incorporated hydrous iron(III) oxide (CIHIO) samples have been prepared aiming investigation of efficiency enhancement on arsenic and fluoride adsorption of hydrous iron(III) oxide (HIO). Characterization of the optimized product with various analytical tools confirms that CIHIO is microcrystalline and mesoporous (pore width, 26.97 Å; pore diameter, 27.742 Å with pore volume 0.18 cm3 g-1) material. Increase of the BET surface area (> 60%) of CIHIO (269.61 m2 g-1) relative to HIO (165.6 m2 g-1) is noticeable. CIHIO particles are estimated to be ~ 50 nm from AFM and TEM analyses. Although the pH optimized for arsenite and fluoride adsorptions are different, the efficiencies of CIHIO towards their adsorption are very good at pH 6.5 (pHzpc). The adsorption kinetics and equilibrium data of either tested species agree well, respectively, with pseudo-second order model and Langmuir monolayer adsorption phenomenon. Langmuir capacities (mg g-1at 303 K) estimated are 29.07 and 25.57, respectively, for arsenite and fluoride. The spontaneity of adsorption reactions (ΔG0 = - 18.02 to - 20.12 kJ mol-1 for arsenite; - 0.2523 to - 3.352 kJ mol-1 for fluoride) are the consequence of entropy parameter. The phosphate ion (1 mM) compared to others influenced adversely the arsenite and/or fluoride adsorption reactions. CIHIO (2.0 g L-1) is capable to abstract arsenite or fluoride above 90% from their solution (0 to 5.0 mg L-1). Mechanism assessment revealed that the adsorption of arsenite occurs via chelation, while of fluoride occurs with ion-exchange.

Nanomolar-level selective dual channel sensing of Cu2+ and CN- from an aqueous medium by an opto-electronic chemoreceptor.

A dual-channel chromogenic and fluorogenic fused-aromatic-system-based chemoreceptor (2-(benzo[d]thiazol-2-yl)-1-((pyren-8-yl)methylene)hydrazine) (TyM) was designed for ditopic sensitivity towards heavy and transition metal ions (HTMs), such as Cu2+, in an aqueous medium and lethal CN- in a semi-aqueous medium. The chemoreceptor displayed proclivity towards the targeted analytes with a distinct optical response (yellow to colourless in the case of Cu2+ and yellow to bright red for CN-). TyM formed a 2 : 1 adduct with Cu2+ with a detection limit of 40 nM. A 1 : 1 binding stoichiometry was confirmed with the chemoreceptor TyM with CN- in sub-nano molar limit of detection. In addition to sophisticated spectroscopic analysis, such as UV-vis, fluorescence, FTIR, 1H-NMR, 13C-NMR, ESI-MS, and HRMS, the plausible mechanistic course of sensing was also established from a theoretical perspective. The reversible UV-vis response of the chemoreceptor TyM towards CN- and H+ can mimic different molecular logic functions and therefore can be exploited for designing several complex electronic circuits principally based on Boolean Algebra. In vitro fluorescence imaging in male microspores of seed plants (Bohonia Nigalandra) and Monilia Albicans (diploid fungus) with TyM and Cu2+ confirmed the permeability of the chemoreceptor TyM at the cellular level as well as its ability to investigate transition metals, such as Cu2+, in biological samples.

Trace Level Recognition of Zn2+ and Cd2+ by Biocompatible Chemosensor inside Androecium, Diagnosis of Pick's Disease from Urine and Biomimetic ß-Cell Exocytosis.

Two chemosensors with varying substitution have been synthesized for selective detection of d10 metal ion analyte Zn2+ and Cd2+ by fluorometric method from aqueous medium at very low limit of detection. Density functional theory (DFT)-based Loewdin spin population analysis reveals that methoxy-substituted chemosensor is much stronger donor than bromo-substituted chemosensor. Eventually, bromo and methoxy substituted chemosensors are moderate and strong donor, respectively, toward selective detection of Cd2+ and Zn2+ by luminescence induced phenomenon (blue for Cd2+ and cyan for Zn2+). The mechanism of sensing could be explained by PET-CHEF-C = N isomerization-ILCT pathway. 1H NMR, ESI-MS and FT-IR has been carried out in order to explore the selective ion sensing mechanism. Intracellular detection of Zn2+ and Cd2+ has been carried out inside androecium (filament and pollens) of Tecoma Stans. Extracellular detection of Zn2+ for yeast cells represents the bio mimetic model experiments toward ß-cells exocytosis as a marker of diabetes mellitus. The unprecedented and novel feature of the present biocompatible chemosensor is its application as biosensor to detect in vivo Zn2+ from human urine specimen which could be a next generation diagnostic tool for Pick's disease.