Monitoring effects of heavy metal stress on biochemical and spectral parameters of cotton using hyperspectral reflectance.

Monitoring heavy metal pollution in agricultural ecosystems is crucial to ensure environmental safety. Heavy metals interfere with plants' biochemical characteristics, such as chlorophyll content and photosynthesis, and also influence leaves' spectral properties. Spectral changes caused by heavy metal stress can easily be measured using proximal sensing or in-field spectroscopy. This research utilizes a combined approach of biochemical and spectral characteristics to evaluate cotton crops' performance under different heavy metal (Pb & Cd) stress after artificial contamination with the metal under study. A detailed study of spectroscopy and lab-based measurements for chlorophyll and heavy metal content during the crop's growth cycle revealed some significant findings. Results indicated that the chlorophyll pigments decreased significantly with increased heavy metal levels. Pb accumulation is high in cotton as compared to Cd. The most sensitive stage for the accumulation of Pb is the initial vegetative stage of cotton. The transfer factor from soil to plant was higher for Pb, indicating the feasibility of growing cotton in Pb-contaminated soil. The spectral measurement showed no characteristic changes in standard reflectance spectra due to heavy metal stress. Wavelet decomposition of reflectance spectra amplified the changes indicating Pb stress in cotton during the initial vegetative stage. The significant correlation of greater than - 0.70 between the reconstructed detail wavelet coefficients at the third level of the decomposition in the wavelength range of 651-742 nm suggested that Pb stress caused spectral changes in near-infrared and visible ranges in cotton plants. The effects of Cd stress on the cotton plant were negligible due to less absorption. Thus, detailed wavelet coefficients at the third level of decomposition in the range of 651-742 nm are a potential indicator of Pb stress. The results of this study can provide a basis for quantifying heavy metal stress in a particular region.

Evaluation of physicochemical and biological properties of nonreconstituted MYL-1401O vials, reconstituted MYL-1401O suspension in vial, and diluted MYL-1401O suspension in infusion bags (0.9% saline) for extended duration.

BACKGROUND: MYL-1401O; trastuzumab-dkst (Ogivri™; Mylan Inc.) is a biosimilar to the trastuzumab reference product (Herceptin®; Genentech, USA). Assessment of physicochemical stability and biological activity for the non-reconstituted, reconstituted, and infused solution over an extended, clinically relevant duration is critical for ensuring optimal patient outcomes and health resource utilization. METHODS: The physicochemical and biological stability of MYL-1401O was assessed in non-reconstituted vials stored at 25 °C ± 2 °C/60% ± 5% relative humidity (RH) for 6 months, reconstituted 21 mg/mL solution in vials stored at 2 °C to 8 °C for 10 days, and diluted in 0.9% saline-containing infusion bags at 0.3 mg/mL and 4.0 mg/mL stored for 77 days at 2 °C to 8 °C, plus an additional 2 days at 25 °C ± 2 °C/60% ± 5% RH. RESULTS: At all storage conditions tested, MYL-1401O was physicochemically and biologically stable for extended duration and under various temperature and humidity conditions. CONCLUSIONS: MYL-1401O retained its physicochemical and biological stability under different storage conditions, which supports advanced preparation of MYL-1401O, better efficiency, less wastage, and cost-savings for better patient management.

Effect of arginine on protein aggregation studied by fluorescence correlation spectroscopy and other biophysical methods.

Arginine has been used extensively as an excipient in the formulation development of protein-based biopharmaceuticals. We investigate the role of arginine in suppressing protein aggregation and its mechanism by using bovine serum albumin as a model system. By using sedimentation velocity and other analytical techniques, we show that the use of arginine inhibits temperature-induced aggregation of the protein. We use fluorescence correlation spectroscopy and other spectroscopic techniques to show that arginine inhibits accumulation of partially folded intermediates, potentially involved in the aggregation process. The hydrodynamic radii of the protein in its native, unfolded, and intermediate states have been determined using fluorescence correlation spectroscopy at single-molecule resolution. A possible mechanism of the effects of arginine and its role as an aggregation suppressor has been discussed.

Predicting Protein-Protein Interactions of Concentrated Antibody Solutions Using Dilute Solution Data and Coarse-Grained Molecular Models.

Protein-protein interactions for solutions of an IgG1 molecule were quantified using static light scattering (SLS) measurements from low to high protein concentrations (c2). SLS was used to determine second osmotic virial coefficients (B22) at low c2, and excess Rayleigh profiles (Rex/K vs. c2) and zero-q structure factors (Sq=0) as a function of c2 at higher c2 for a series of conditions (pH, sucrose concentration, and total ionic strength [TIS]). Repulsive (attractive) interactions were observed at low TIS (high TIS) for pH 5 and 6.5, with increasing repulsions when 5% w/w sucrose was also present. Previously developed and refined coarse-grained antibody models were used to fit model parameters from B22 versus TIS data. The resulting parameters from low-c2 conditions were used as the sole input to multiprotein Monte Carlo simulations to predict high-c2Rex/K and Sq=0 behavior up to 150 g/L. Experimental results at high-c2 conditions were quantitatively predicted by the simulations for the coarse-grained models that treated antibody molecules as either 6 or 12 (sub) domains, which preserved the basic shape of a monoclonal antibody. Finally, preferential accumulation of sucrose around the protein surface was identified via high-precision density measurements, which self-consistently explained the simulation and experimental SLS results.

Relating Protein-Protein Interactions and Aggregation Rates From Low to High Concentrations.

At low protein concentrations (c2), non-native protein aggregation rates are known to be sensitive to changes in conformational stability and "weak" or "colloidal" protein-protein interactions. Protein-protein interactions are also known to be strong functions of c2. In the present work, protein-protein interactions and rates of aggregation were quantified systematically for a monoclonal antibody (MAb) across a broad range of c2 at pH 5.1 and 6.5, with or without 5 wt/wt % sucrose or 100 mM NaCl present. Aggregation rates were determined from initial-rate analysis with size-exclusion chromatography, and interactions were quantified with static and dynamic laser light scattering. A number of hypotheses were tested regarding whether changes in protein-protein interactions can be predictive of changes in aggregation rates versus c2. Hypotheses were based on (i) changes in thermodynamic activity; (ii) statistical mechanical fluctuation theory; and (iii) surface-contact probabilities. Arguments based on (i) and (ii) were qualitatively inconsistent with experimental rates and scattering. Hypothesis (iii) was reasonably successful and resulted in a semiquantitative correlation between rates and protein-protein interactions across almost 2 orders of magnitude in c2. However, (iii) requires one to assume that the concentration-dependent protein-protein Kirkwood-Buff integral is a reasonable surrogate for contact probabilities.

pH-induced structural change of a multi-tryptophan protein MPT63 with immunoglobulin-like fold: identification of perturbed tryptophan residue/residues.

The structural change of M. tuberculosis MPT63, which is predominantly a ß-sheet protein having an immunoglobulin like fold, has been investigated in the pH range 7.5-1.5 using various biophysical techniques along with low-temperature phosphorescence (LTP) spectroscopy. MPT63 contains four Tryptophan (Trp) residues at 26, 48, 82, and 129. Although circular dichroism, steady-state and time-resolved fluorescence, time-resolved anisotropy, 1-aniline-8-naphthalene sulfonic (ANS) acid binding, and analytical ultracentrifuge depict more open largely unfolded structure of MPT63 at pH 1.5 and also more accessible nature of Trp residues to neutral quencher at pH 1.5, it is, however, not possible to assign the specific Trp residue/residues being perturbed. This problem has been resolved using LTP of MPT63, which shows optically resolved four distinct (0, 0) bands corresponding to four Trp residues in the pH range 7.5-3.0. LTP at pH 1.5 clearly reveals that the solvent-exposed Trp 82 and the almost buried Trp 129 are specifically affected compared with Trp 48 and Trp 26. Lys 8 and Lys 27 are predicted to affect Trp 129. Tyrosine residues are found to be silent even at pH 1.5. This type of specific perturbation in a multi-Trp protein has not been addressed before. LTP further indicates that partially exposed Trp 48 is preferentially quenched by acrylamide compared with other Trp residues at both pH 7.5 and 1.5. The solvent-exposed Trp 82 is surprisingly found to be not quenched by acrylamide at pH 7.5. All the results are obtained using micromolar concentration of protein and without using any Trp-substituted mutant.

Unusual optical resolution of all four tryptophan residues in MPT63 protein by phosphorescence spectroscopy: assignment and significance.

MPT63, a secreted protein of unknown function that is specific to Mycobacterium tuberculosis and a potential drug target, contains four Tryptophan (Trp/W) residues located at positions 26, 48, 82, and 129 in the amino acid sequence. All of the four Trp residues have been optically resolved by simple inexpensive phosphorescence spectroscopy at 77 K. The protein architecture provides a delicate micro-environment and location of Trp residues giving rise to four different (0,0) bands in the phosphorescence spectra. Calculation of intra Trp energy transfer (ET) efficiency, accessible surface area (ASA) of Trp residues, and environment of Trp in the wild-type (WT) and the mutant W26F [where, Trp 26 is replaced by phenyl alanine (Phe/F)] reveal: E(T1) (W82) > E(T1) (W48) > E(T1) (W129) > E(T1) (W26), where E(T1) is the lowest (π-π*) triplet state energy of Trp. The (0,0) band observed at 421.6 nm assigned for Trp 26 is found to be the longest wavelength (0,0) band so far reported in the literature. Fluorescence in WT and W26F is dominated by buried or partially exposed Trp residues indicated by time-resolved spectra. Circular Dichroism (CD) studies and the time-resolved anisotropy measurement confirm the unaltered secondary and tertiary structure of the mutant compared to that of the WT. Excitation energy dependent phosphorescence spectra suggest that the intensity of the different (0,0) bands could be tuned and Tyrosine (Tyr/Y) residue is silent in emission. Optical resolution of all the Trp residues will help understand the role of each Trp residue in the folding/unfolding mechanism and in the interaction with other systems.