Cadmium exposure induces inflammation through the canonical NF-κΒ pathway in monocytes/macrophages of Channa punctatus Bloch.

A vast range of research related to the toxicity of the heavy metal cadmium (Cd) has been carried out in a wide variety of fish species. However, Cd induced immunomodulation in monocytes/macrophages of Channa punctatus Bloch. has rarely been explored. The present study was designed to determine Cd induced immune response, role of NF-κB (nuclear factor kappa B) pathway and the subsequent downstream molecular responses in monocytes/macrophages of C. punctatus. Fish were sampled and acclimatized, with one group treated with cadmium chloride (CdCl2) (1.96 mg/L) and another kept as untreated control group, both under observation for 7 days. Exposure to CdCl2 was found to alter hematological profile of C. punctatus in addition to incurring histo-architectural damages in the HK (head kidney) and ultrastructural changes in the monocytes/macrophages. The innate immune potential was found to be significantly compromised as evident from decreased phagocytosis, intracellular killing, cell adhesion and reduced release of nitric oxide (NO) and myeloperoxidase (MPO) in Cd intoxicated group. Also Cd triggered ROS generation, reduced cellular NO levels by forming peroxynitrite along with the upregulated expression of the inflammatory marker iNOS (inducible nitric oxide synthase) in monocytes/macrophages, both at mRNA and protein levels, indicating inflammation. Inflammation is further verified from the upregulated expression of proinflammatory cytokines viz. TNF-α, IL-1ß, IL-6, IL-12 along with a central inflammatory mediator NF-κΒ and downregulation of the anti-inflammatory cytokine IL-10, both at mRNA and protein levels. It can be concluded that, a sub-lethal exposure of Cd in C. punctatus for 7 days caused significant alterations in the hematological, histological and ultrastructural profile in monocytes/macrophages; impaired innate immune parameters, triggers ROS generation and inflammation as validated from the upregulated expression of NF-κΒ, iNOS, TNF-α, IL-1ß, IL-6, IL-12 and IL-10 downregulation.

Theoretical spectroscopy of isotopically dilute water and hydrophobicity.

Hydrophobic solutes significantly alter the water hydrogen bond network. The local alteration of solvation structures gets reflected in the vibrational spectroscopic signal. Although it is possible to detect this microscopic feature by modern infrared spectroscopy, bulk phase spectra often come with a formidable challenge of establishing the connection of experimental spectra to molecular structures. Theoretical spectroscopy can serve as a more powerful tool where spectroscopic data cannot provide the microscopic picture. In the present work, we build a theoretical spectroscopic map based on a hybrid quantum-classical molecular simulation approach using a methane-water system. The single oscillator O-H stretch frequency is well correlated with a collective variable solvation energy. We construct the spectroscopic maps for fundamental transition frequencies and also the transition dipoles. A bimodal frequency distribution with a blue-shifted population of transition frequency illustrates the presence of gas like water molecules in the hydration shell of methane. This observation is further complemented by a shell-wise decomposition of the O-H stretch frequencies. We observe a significant increase in the ordering of the first solvation water molecules, except those which are directly facing the methane molecule. This is manifested in the redshift of the observed transition frequencies. Temperature dependent simulations depict that the water molecules facing the methane molecule behave similarly to the high temperature water, and a few of the first shell water molecules behave more like cold water.

Sugar Production from Hybrid Poplar Sawdust: Optimization of Enzymatic Hydrolysis and Wet Explosion Pretreatment.

Wet explosion pretreatment of hybrid poplar sawdust (PSD) for the production of fermentable sugar was carried out in the pilot-scale. The effects of pretreatment conditions, such as temperature (170-190 °C), oxygen dosage (0.5-7.5% of dry matter (DM), w/w), residence time (10-30 min), on cellulose and hemicellulose digestibility after enzymatic hydrolysis were ascertained with a central composite design of the experiment. Further, enzymatic hydrolysis was optimized in terms of temperature, pH, and a mixture of CTec2 and HTec2 enzymes (Novozymes). Predictive modeling showed that cellulose and hemicellulose digestibility of 75.1% and 83.1%, respectively, could be achieved with a pretreatment at 177 °C with 7.5% O2 and a retention time of 30 min. An increased cellulose digestibility of 87.1% ± 0.1 could be achieved by pretreating at 190 °C; however, the hemicellulose yield would be significantly reduced. It was evident that more severe conditions were required for maximal cellulose digestibility than that of hemicellulose digestibility and that an optimal sugar yield demanded a set of conditions, which overall resulted in the maximum sugar yield.

Enhancement of reaction rate in small-sized droplets: A combined analytical and simulation study.

Several recent mass spectrometry experiments reveal a marked enhancement of the reaction rate of organic reactions in microdroplets. This enhancement has been tentatively attributed to the accumulation of excess charge on a surface, which in turn can give rise to a lowering of activation energy of the reaction. Here we model the reactions in droplets as a three-step process: (i) diffusion of a reactant from the core of the droplet to the surface, (ii) search by diffusion of the reactant on the surface to find a reactive partner, and finally (iii) the intrinsic reaction leading to bond breaking and product formation. We obtain analytic expressions for the mean search time (MST) to find a target located on the surface by a reactant in both two- and three-dimensional droplets. Analytical results show quantitative agreement with Brownian dynamics simulations. We find, as also reported earlier, that the MST varies as R2/D, where R is the radius of the droplet and D is the diffusion constant of the molecules in the droplet medium. We also find that a hydronium ion in the vicinity can substantially weaken the bond and hence lowers the activation barrier. We observe a similar facilitation of bond breaking in the presence of a static dipolar electric field along any of the three Cartesian axes. If the intrinsic reaction is faster compared to the mean search time involved, it becomes primarily a diffusion-controlled process; otherwise the reaction cannot be accelerated in the droplet medium. The air-droplet interface provides a different environment compared to the interior of the droplet. Hence, we might also expect a completely different mechanism and products in the case of droplet reactions.

Study of distance dependence of hydrophobic force between two graphene-like walls and a signature of pressure induced structure formation in the confined water.

We examine the separation distance dependence of the hydrophobic force by systematically varying the distance (d) between two walls. The hydrophobic force exhibits a distance mediated crossover from a liquid-like to a gas-like behavior at around d ∼ 12 Å for 1 atm pressure. The distance dependence can be fitted to a bi-exponential form, with the longer distance part displaying a correlation length of 20 Å. In addition, the crossover is found to be accompanied by a divergent-like growth of the local relative number fluctuation of the water molecules confined between the two surfaces. Furthermore, at a fixed separation (d = 20 Å), we observe a pressure induced structural modification of confined water at high pressure. The confined water is found to form an ordered structure at high pressure (10 000 atm) and room temperature, in agreement with the experimental study [G. Algara-Siller et al. Nature 519(7544), 443 (2015)].

IR spectral assignments for the hydrated excess proton in liquid water.

The local environmental sensitivity of infrared (IR) spectroscopy to a hydrogen-bonding structure makes it a powerful tool for investigating the structure and dynamics of excess protons in water. Although of significant interest, the line broadening that results from the ultrafast evolution of different solvated proton-water structures makes the assignment of liquid-phase IR spectra a challenging task. In this work, we apply a normal mode analysis using density functional theory of thousands of proton-water clusters taken from reactive molecular dynamics trajectories of the latest generation multistate empirical valence bond proton model (MS-EVB 3.2). These calculations are used to obtain a vibrational density of states and IR spectral density, which are decomposed on the basis of solvated proton structure and the frequency dependent mode character. Decompositions are presented on the basis of the proton sharing parameter δ, often used to distinguish Eigen and Zundel species, the stretch and bend character of the modes, the mode delocalization, and the vibrational mode symmetry. We find there is a wide distribution of vibrational frequencies spanning 1200-3000 cm-1 for every local proton configuration, with the region 2000-2600 cm-1 being mostly governed by the distorted Eigen-like configuration. We find a continuous red shift of the special-pair O⋯H+⋯O stretching frequency, and an increase in the flanking water bending intensity with decreasing δ. Also, we find that the flanking water stretch mode of the Zundel-like species is strongly mixed with the flanking water bend, and the special pair proton oscillation band is strongly coupled with the bend modes of the central H5O2+moiety.

Delocalization and stretch-bend mixing of the HOH bend in liquid water.

Liquid water's rich sub-picosecond vibrational dynamics arise from the interplay of different high- and low-frequency modes evolving in a strong yet fluctuating hydrogen bond network. Recent studies of the OH stretching excitations of H2O indicate that they are delocalized over several molecules, raising questions about whether the bending vibrations are similarly delocalized. In this paper, we take advantage of an improved 50 fs time-resolution and broadband infrared (IR) spectroscopy to interrogate the 2D IR lineshape and spectral dynamics of the HOH bending vibration of liquid H2O. Indications of strong bend-stretch coupling are observed in early time 2D IR spectra through a broad excited state absorption that extends from 1500 cm-1 to beyond 1900 cm-1, which corresponds to transitions from the bend to the bend overtone and OH stretching band between 3150 and 3550 cm-1. Pump-probe measurements reveal a fast 180 fs vibrational relaxation time, which results in a hot-ground state spectrum that is the same as observed for water IR excitation at any other frequency. The fastest dynamical time scale is 80 fs for the polarization anisotropy decay, providing evidence for the delocalized or excitonic character of the bend. Normal mode analysis conducted on water clusters extracted from molecular dynamics simulations corroborate significant stretch-bend mixing and indicate delocalization of δHOH on 2-7 water molecules.

Molecular modeling and assignment of IR spectra of the hydrated excess proton in isotopically dilute water.

Infrared (IR) spectroscopy of the water O-H stretch has been widely used to probe both the local hydrogen-bonding structure and dynamics of aqueous systems. Although of significant interest, the IR spectroscopy of excess protons in water remains difficult to assign as a result of extensive and strong intermolecular interactions in hydrated proton complexes. As an alternate approach, we develop a mixed quantum-classical model for the vibrational spectroscopy of the excess proton in isotopically dilute water that draws on frozen proton-water clusters taken from reactive molecular dynamics trajectories of the latest generation multi-state empirical valence bond proton model (MS-EVB 3.2). A semi-empirical single oscillator spectroscopic map for the instantaneous transition frequency and transition dipole moment is constructed using potential energy surfaces for the O-H stretch coordinate of the excess proton using electronic structure calculations. Calculated spectra are compared with experimental spectra of dilute H+ in D2O obtained from double-difference FTIR to demonstrate the validity of the map. The model is also used to decompose IR spectra into contributions from different aqueous proton configurations. We find that the O-H transition frequency continuously decreases as the oxygen-oxygen length for a special pair proton decreases, shifting from Eigen- to Zundel-like configurations. The same shift is accompanied by a shift of the flanking water stretches of the Zundel complex to higher frequency than the hydronium O-H vibrations.

Diffusion on a rugged energy landscape with spatial correlations.

Rugged energy landscapes find wide applications in diverse fields ranging from astrophysics to protein folding. We study the dependence of diffusion coefficient (D) of a Brownian particle on the distribution width (ɛ) of randomness in a Gaussian random landscape by simulations and theoretical analysis. We first show that the elegant expression of Zwanzig [Proc. Natl. Acad. Sci. U.S.A. 85, 2029 (1988)] for D(ɛ) can be reproduced exactly by using the Rosenfeld diffusion-entropy scaling relation. Our simulations show that Zwanzig's expression overestimates D in an uncorrelated Gaussian random lattice - differing by almost an order of magnitude at moderately high ruggedness. The disparity originates from the presence of "three-site traps" (TST) on the landscape - which are formed by the presence of deep minima flanked by high barriers on either side. Using mean first passage time formalism, we derive a general expression for the effective diffusion coefficient in the presence of TST, that quantitatively reproduces the simulation results and which reduces to Zwanzig's form only in the limit of infinite spatial correlation. We construct a continuous Gaussian field with inherent correlation to establish the effect of spatial correlation on random walk. The presence of TSTs at large ruggedness (ɛ ≫ k(B)T) gives rise to an apparent breakdown of ergodicity of the type often encountered in glassy liquids.

Significance of the Disulfide Bridge in the Structure and Stability of Metalloprotein Azurin.

Metalloproteins make up a class of proteins that incorporate metal ions into their structures, enabling them to perform essential functions in biological systems, such as catalysis and electron transport. Azurin is one such metalloprotein with copper cofactor, having a ß-barrel structure with exceptional thermal stability. The copper metal ion is coordinated at one end of the ß-barrel structure, and there is a disulfide bond at the opposite end. In this study, we explore the effect of this disulfide bond in the high thermal stability of azurin by analyzing both the native S-S bonded and S-S nonbonded (S-S open) forms using temperature replica exchange molecular dynamics (REMD). Similar to experimental observations, we find a 35 K decrease in denaturation temperature for S-S open azurin compared to that of the native holo form (420 K). As observed in the case of native holo azurin, the unfolding process of the S-S open form also started with disruptions of the α-helix. The free energy surfaces of the unfolding process revealed that the denaturation event of the S-S open form progresses through different sets of conformational ensembles. Subsequently, we compared the stabilities of individual ß-sheet strands of both the S-S bonded and the S-S nonbonded forms of azurin. Further, we examined the contacts between individual residues for the central structures from the free energy surfaces of the S-S nonbonded form. The microscopic origin of the lowering in the denaturation temperature is further supplemented by thermodynamic analysis.

One-Pot Interference-Based Colorimetric Detection of Melamine in Raw Milk via Green Tea-Modified Silver Nanostructures.

Detection of melamine has proven to be a challenge, requiring the use of complex analytical techniques. This study introduces an innovative, straightforward one-pot technique for qualitative assessment of the milk adulterant melamine. Originally, silver nanoparticles (AgNPs) were synthesized by utilizing green tea extract, which acted as both a reducing and sensing element. The synthesized AgNPs were characterized using UV-vis spectroscopy, X-ray diffraction, zeta potential, transmission electron microscopy, field emission scanning electron microscopy, and Fourier transform infrared spectroscopy. Melamine, rich in -NH2 groups, interacts with the biopolyphenols of green tea extract through hydrogen bonding. This interaction inhibits the formation of nanoparticles, resulting in a noticeable colorimetric response. The data obtained were confirmed by a standard UV-vis spectrophotometer and validated by the high-performance liquid chromatography technique. The limit of detection achieved by this scheme was quite low, falling below the permissible levels recommended by government bodies, e.g., the Food Safety and Standards Authority of India (FSSAI).

Highly sensitive biofunctionalized nanostructures for paper-based colorimetric sensing of hydrogen peroxide in raw milk.

Hydrogen Peroxide (H2O2) is a highly hazardous, toxic, and carcinogenic chemical compound utilised in various industries-based applications. Despite strict restriction, they are deliberately added to food items such as milk as preservatives to increase its shelf life. Herein, we have formulated a green rapid colorimetric nanosensor for detection of H2O2 in milk using cotton leaves as both reducing and functionalizing agent for synthesis of silver nanoparticles (AgNPs). UV-Vis spectra exhibit a strong plasmonic peak at around 434 nm. X-Ray Diffraction (XRD) analysis was performed to determine the crystallinity of the nanoparticles. Field Emission Scanning Electron Microscope (FESEM) and Transmission Electron Microscope (TEM) characterizations revealed spherical morphology with size approximately âˆ¼16 nm. This functionalized nanoparticle could colorimetrically sense presence of H2O2 in milk samples both in liquid media and on paper substrates with Limit of Detection (LOD) of 8.46 ppm even in presence of other interfering substances in milk. This inexpensive route will pave the way for in depth research.

Integrated microfluidic platforms for heavy metal sensing: a comprehensive review.

Heavy metals are found naturally; however, anthropogenic activities such as mining, inappropriate disposal of industrial waste, and the use of pesticides and fertilizers containing heavy metals can cause their unwanted release into the environment. Conventionally, detection of heavy metals is performed using atomic absorption spectrometry, electrochemical methods and inductively coupled plasma-mass spectrometry; however, they involve expensive and sophisticated instruments and multistep sample preparation that require expertise for accurate results. In contrast, microfluidic devices involve rapid, cost-efficient, simple, and reliable approaches for in-laboratory and real-time monitoring of heavy metals. The use of inexpensive and environment friendly materials for fabrication of microfluidic devices has increased the manufacturing efficiency of the devices. Different types of techniques used in heavy metal detection include colorimetry, absorbance-based, and electrochemical detection. This review provides insight into the detection of toxic heavy metals such as mercury (Hg), cadmium (Cd), lead (Pb), and arsenic (As). Importance is given to colorimetry, optical, and electrochemical techniques applied for the detection of heavy metals using microfluidics and their modifications to improve the limit of detection (LOD).

Perturbation of Fermi Resonance on Hydrogen-Bonded > C═O: 2D IR Studies of Small Ester Probes.

We utilized linear and 2D infrared spectroscopy to analyze the carbonyl stretching modes of small esters in different solvents. Particularly noteworthy were the distinct carbonyl spectral line shapes in aqueous solutions, prompting our investigation of the underlying factors responsible for these differences. Through our experimental and theoretical calculations, we identified the presence of the hydrogen-bond-induced Fermi resonance as the primary contributor to the varied line shapes of small esters in aqueous solutions. Furthermore, our findings revealed that the skeletal deformation mode plays a crucial role in the Fermi resonance for all small esters. Specifically, the first overtone band of the skeletal deformation mode intensifies when hydrogen bonds form with the carbonyl group of esters, whereas such coupling is rare in aprotic organic solvents. These spectral insights carry significant implications for the utilization of esters as infrared probes in both biological and chemical systems.

Dietary Acrylamide: A Detailed Review on Formation, Detection, Mitigation, and Its Health Impacts.

In today's fast-paced world, people increasingly rely on a variety of processed foods due to their busy lifestyles. The enhanced flavors, vibrant colors, and ease of accessibility at reasonable prices have made ready-to-eat foods the easiest and simplest choice to satiate hunger, especially those that undergo thermal processing. However, these foods often contain an unsaturated amide called 'Acrylamide', known by its chemical name 2-propenamide, which is a contaminant formed when a carbohydrate- or protein-rich food product is thermally processed at more than 120 °C through methods like frying, baking, or roasting. Consuming foods with elevated levels of acrylamide can induce harmful toxicity such as neurotoxicity, hepatoxicity, cardiovascular toxicity, reproductive toxicity, and prenatal and postnatal toxicity. This review delves into the major pathways and factors influencing acrylamide formation in food, discusses its adverse effects on human health, and explores recent techniques for the detection and mitigation of acrylamide in food. This review could be of interest to a wide audience in the food industry that manufactures processed foods. A multi-faceted strategy is necessary to identify and resolve the factors responsible for the browning of food, ensure safety standards, and preserve essential food quality traits.

Resistive Memory-Switching Behavior in Solution-Processed Trans, trans-1,4-bis-(2-(2-naphthyl)-2-(butoxycarbonyl)-vinyl) Benzene-PVA-Composite-Based Aryl Acrylate on ITO-Coated PET.

Resistive switching memories are among the emerging next-generation technologies that are possible candidates for in-memory and neuromorphic computing. In this report, resistive memory-switching behavior in solution-processed trans, trans-1,4-bis-(2-(2-naphthyl)-2-(butoxycarbonyl)-vinyl) benzene-PVA-composite-based aryl acrylate on an ITO-coated PET device was studied. A sandwich configuration was selected, with silver (Ag) serving as a top contact and trans, trans-1,4-bis-(2-(2-naphthyl)-2-(butoxycarbonyl)-vinyl) benzene-PVA-composite-based aryl acrylate and ITO-PET serving as a bottom contact. The current-voltage (I-V) characteristics showed hysteresis behavior and non-zero crossing owing to voltages sweeping from positive to negative and vice versa. The results showed non-zero crossing in the devices' current-voltage (I-V) characteristics due to the nanobattery effect or resistance, capacitive, and inductive effects. The device also displayed a negative differential resistance (NDR) effect. Non-volatile storage was feasible with non-zero crossing due to the exhibition of resistive switching behavior. The sweeping range was -10 V to +10 V. These devices had two distinct states: 'ON' and 'OFF'. The ON/OFF ratios of the devices were 14 and 100 under stable operating conditions. The open-circuit voltages (Voc) and short-circuit currents (Isc) corresponding to memristor operation were explained. The DC endurance was stable. Ohmic conduction and direct tunneling mechanisms with traps explained the charge transport model governing the resistive switching behavior. This work gives insight into data storage in terms of a new conception of electronic devices based on facile and low-temperature processed material composites for emerging computational devices.

A phase 3 non-inferiority trial of locally manufactured Meningococcal ACWY vaccine 'Ingovax ACWY' among Bangladeshi adults.

Meningococcal disease is caused by Neisseria meningitidis or meningococcus. Every year globally around 1.2 million people are affected and approximately 120,000 deaths occur due to meningitis. The disease can be prevented by a single dose of meningococcal vaccine. We carried out a randomized observer-blinded non-inferiority trial to evaluate and compare the immunogenicity and safety of a local meningococcal polysaccharide vaccine 'Ingovax ACWY' (test) with Quadri MeningoTM (comparator), an approved meningococcal polysaccharide vaccine in India. A total of 88 healthy adults (18-45 years old) were randomized at a 1:1 ratio in two vaccine groups receiving a single dose vaccine subcutaneously. All participants were followed until three months post-vaccination. Blood for clinical parameters (hematology and biochemistry) and serum bactericidal assay (SBA) was collected prior to vaccination and one-month post-vaccination. Solicited adverse events (AEs) were assessed up to 6 days following vaccination and unsolicited AEs were monitored throughout the follow-up period. There was no significant difference in rates of AE between the two groups. The commonest solicited AE was injection site pain. No serious AEs were reported. There was no significant difference (p<0.05) in seroconversion rate as well as pre and post-vaccination SBA geometric mean titers (GMT)between test and comparator vaccine. The post-vaccination GMT ratio (GMR) of the test and comparator vaccine was found to be 0.9, 1, 1.29, and 0.85 for serogroup A, C, W135, and Y respectively. For all the serogroups, lower limit of 95% CI of the GMR was found to be greater than the pre-defined 0.5 non-inferiority margin suggesting that Ingovax ACWY is similar to Quadri MeningoTM vaccine. We observed the immunogenicity and safety of Ingovax ACWY is non-inferior to comparator vaccine. The development of facilities for manufacturing polysaccharide ACWY vaccines locally will further lead to capacity building in the field of vaccines for Bangladesh.

Layerwise decomposition of water dynamics in reverse micelles: a simulation study of two-dimensional infrared spectrum.

We present computer simulation study of two-dimensional infrared spectroscopy (2D-IR) of water confined in reverse micelles (RMs) of various sizes. The present study is motivated by the need to understand the altered dynamics of confined water by performing layerwise decomposition of water, with an aim to quantify the relative contributions of different layers water molecules to the calculated 2D-IR spectrum. The 0-1 transition spectra clearly show substantial elongation, due to inhomogeneous broadening and incomplete spectral diffusion, along the diagonal in the surface water layer of different sized RMs. Fitting of the frequency fluctuation correlation functions reveal that the motion of the surface water molecules is sub-diffusive and indicate the constrained nature of their dynamics. This is further supported by two peak nature of the angular analogue of van Hove correlation function. With increasing system size, the water molecules become more diffusive in nature and spectral diffusion almost completes in the central layer of the larger size RMs. Comparisons between experiments and simulations establish the correspondence between the spectral decomposition available in experiments with the spatial decomposition available in simulations. Simulations also allow a quantitative exploration of the relative role of water, sodium ions, and sulfonate head groups in vibrational dephasing. Interestingly, the negative cross correlation between force on oxygen and hydrogen of O-H bond in bulk water significantly decreases in the surface layer of each RM. This negative cross correlation gradually increases in the central water pool with increasing RMs size and this is found to be partly responsible for the faster relaxation rate of water in the central pool.

Chitosan-the miracle biomaterial as detection and diminishing mediating agent for heavy metal ions: A mini review.

Proliferation of heavy metal ions as aquatic pollutants has been a matter of growing concern now a days. Several anthropogenic activities have fueled higher concentration of heavy metal ions in aquatic bodies above threshold values, as set by World Health Organization. Of late, chitosan for its exquisite properties has been widely used in tackling this burning problem of aquatic pollution caused by heavy metal ions. Accordingly, this mini review appraises the detection as well as diminution activities where chitosan plays the major contributing part. Starting from the intrinsic properties of chitosan, the detection strategy via chitosan composites is comprehensively delineated. Likewise, the removal activities via chitosan mediating agents are also overviewed, followed by future recommendations. It is believed that this mini review will give researchers a brief appraisal of two prominent activities related to controlling of heavy metal ion pollution.

Role of Metal Cofactor in Enhanced Thermal Stability of Azurin.

Metal cofactors are critical centers for different biochemical processes of metalloproteins, and often, this metal coordination renders additional structural stability. In this study, we explore the additional stability conferred by the copper ion on azurin by analyzing both the apo and holo forms using temperature replica exchange molecular dynamics (REMD) data. We find a 14 K decrease in denaturation temperature for apo (406 K) azurin relative to that of holo (420 K), indicating a copper ion-induced additional thermal stability for holo azurin. The unfolding of apo azurin begins with the melting of α-helix and ß-sheet V, similar to that of holo form. ß-Sheets IV, VII, and VIII are comparatively more stable than other ß-strands and melt at higher temperatures. Similar to holo azurin, the strong hydrophobic interactions among the apolar residues in the protein core is the key factor that renders high stability to apo protein as well. We construct free energy surfaces at different temperatures to capture the major conformations along the unfolding basins of the protein. Using contact maps from different basins we show the changes in the interaction between different residues along the unfolding pathway. Furthermore, we compare the Cα root-mean-square fluctuations (Cα-RMSF) and B-factor of all residues of apo and holo forms to understand the flexibility of different regions. The concerted displacement of α-helix and ß-sheets V and VI from the protein core is another distinction we observe for apo compared to the holo form, where ß-sheet VI was relatively stable.