Modulation of biliverdin dynamics and spectral properties by Sandercyanin.

Biliverdin IX-alpha (BV), a tetrapyrrole, is found ubiquitously in most living organisms. It functions as a metabolite, pigment, and signaling compound. While BV is known to bind to diverse protein families such as heme-metabolizing enzymes and phytochromes, not many BV-bound lipocalins (ubiquitous, small lipid-binding proteins) have been studied. The molecular basis of binding and conformational selectivity of BV in lipocalins remains unexplained. Sandercyanin (SFP)-BV complex is a blue lipocalin protein present in the mucus of the Canadian walleye (Stizostedion vitreum). In this study, we present the structures and binding modes of BV to SFP. Using a combination of designed site-directed mutations, X-ray crystallography, UV/VIS, and resonance Raman spectroscopy, we have identified multiple conformations of BV that are stabilized in the binding pocket of SFP. In complex with the protein, these conformers generate varied spectroscopic signatures both in their absorption and fluorescence spectra. We show that despite no covalent anchor, structural heterogeneity of the chromophore is primarily driven by the D-ring pyrrole of BV. Our work shows how conformational promiscuity of BV is correlated to the rearrangement of amino acids in the protein matrix leading to modulation of spectral properties.

Erythroid spectrin binding modulates peroxidase and catalase activity of heme proteins.

Hemoglobin oxidation due to oxidative stress and disease conditions leads to the generation of ROS (reactive oxygen species) and membrane attachment of hemoglobin in-vivo, where its redox activity leads to peroxidative damage of membrane lipids and proteins. Spectrin, the major component of the red blood cell (RBC) membrane skeleton, is known to interact with hemoglobin and, here this interaction is shown to increase hemoglobin peroxidase activity in the presence of reducing substrate ABTS (2', 2'-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid). It is also shown that in the absence of reducing substrate, spectrin forms covalently cross-linked aggregates with hemoglobin which display no peroxidase activity. This may have implications in the clearance of ROS and limiting peroxidative damage. Spectrin is found to modulate the peroxidase activity of different hemoglobin variants like A, E, and S, and of isolated globin chains from each of these variants. This may be of importance in disease states like sickle cell disease and HbE-ß-thalassemia, where increased oxidative damage and free globin subunits are present due to the defects inherent in the hemoglobin variants associated with these diseases. This hypothesis is corroborated by lipid peroxidation experiments. The modulatory role of spectrin is shown to extend to other heme proteins, namely catalase and cytochrome-c. Experiments with free heme and Raman spectroscopy of heme proteins in the presence of spectrin show that structural alterations occur in the heme moiety of the heme proteins on spectrin binding, which may be the structural basis of increased enzyme activity.

Comparison of adductor canal block with femoral nerve block for post-operative pain relief after bilateral total knee arthroplasty: A non-randomised controlled trial.

BACKGROUND AND AIMS: Pain perception and pain threshold vary from one individual to another and also differ in the right and left limbs leading to an inter-cerebral pain variability bias and inter-patient pain variability bias. To date, data comparing adductor canal block (ACB) with femoral nerve block (FNB) in the same patient who underwent bilateral total knee arthroplasty (TKA) remain limited. METHODS: We performed a prospective, non-randomised, controlled study. Patients scheduled for bilateral TKA with central neuraxial anaesthesia received every 12-hourly intermittent boluses of 15 mL of ropivacaine 0.5% in two different peripheral nerve blocks (ACB and FNB) in either of the lower limbs postoperatively. The primary outcome was to assess postoperative VAS score, and the secondary outcomes were muscle strength of the quadriceps muscle and degree of flexion at the knee joint. RESULTS: Among the 80 patients assessed for eligibility, 72 patients were enroled, and 69 were included in the final analysis. VAS scores after the two blocks during rest at 30 minutes, 6 hours, 12 hours and 48 hours post-operatively had no significant difference. VAS scores during rest at 24 hours and on doing exercise at 24 hours and 48 hours showed a significant difference. Quadriceps muscle strength and degree of flexion of knee at 24 hours and 48 hours post-operatively did show a significant difference in favour of the ACB over FNB. CONCLUSION: ACB provides equivalent analgesia in comparison with FNB at rest and during passive exercise up to 48 hours post-operatively. ACB significantly preserved motor power of quadriceps muscles when compared with FNB with no added complications.

Divalent Ion-Induced Switch in DNA Cleavage of KpnI Endonuclease Probed through Surface-Enhanced Raman Spectroscopy.

We demonstrate the remarkable ability of surface-enhanced Raman spectroscopy (SERS) to track the allosteric changes in restriction endonuclease KpnI (R.KpnI) caused by metal ions. R.KpnI binds and promiscuously cleaves DNA upon activation by Mg2+ ions. However, the divalent ion Ca2+ induces high fidelity cleavage, which can be overcome by higher concentrations of Mg2+ ions. In the absence of any 3D crystal structure, for the first time, we have elucidated the structural underpinnings of such a differential effect of divalent ions on the endonuclease activity. A combined SERS and molecular dynamics (MD) approach showed that Ca2+ ion activates an enzymatic switch in the active site, which is responsible for the high fidelity activity of the enzyme. Thus, SERS in combination with MD simulations provides a powerful tool for probing the link between the structure and activity of enzyme molecules that play vital roles in DNA transactions.

Improved surface-enhanced Raman and catalytic activities of reduced graphene oxide-osmium hybrid nano thin films.

Reduced graphene oxide-osmium (rGO-Os) hybrid nano dendtrites have been prepared by simple liquid/liquid interface method for the first time. The method involves the introduction of phase-transfered metal organic precursor in toluene phase and GO dispersion in the aqueous phase along with hydrazine hydrate as the reducing agent. Dendritic networks of Os nanoparticles and their aggregates decorating rGO layers are obtained. The substrate shows improved catalytic and surface-enhanced activities comparable with previous reports. The catalytic activity was tested for the reduction of p-nitroaniline into p-phenyldiamine with an excess amount of NaBH4. The catalytic activity factors of these hybrid films are 2.3 s-1 g-1 (Os film) and 4.4 s-1 g-1 (rGO-Os hybrid film), which are comparable with other noble metal nanoparticles such as Au, Ag, but lower than Pd-based catalysts. Surface-enhanced Raman spectroscopy (SERS) measurements have been done on rhodamine 6G (R6G) and methylene blue dyes. The enhancement factor for the R6G adsorbed on rGO-Os thin film is 1.0 × 105 and for Os thin film is 7 × 103. There is a 14-fold enhancement observed for Os hybrids with rGO. The enhanced catalytic and SERS activities of rGO-Os hybrid thin film prepared by simple liquid/liquid interface method open up new challenges in electrocatalytic application and SERS-based detection of biomolecules.

Thermally stable plasmonic nanocermets grown on microengineered surfaces as versatile surface enhanced Raman spectroscopy sensors for multianalyte detection.

Noble metal nanoparticle-based plasmonic sensors, fabricated by top-down and colloidal routes, are widely used for high sensitivity detection of diverse analyte molecules using surface enhanced Raman spectroscopy (SERS). However, most of these sensors do not show stability under harsh environments, which limits their use as versatile SERS substrates. In this work, we report the first use of plasmonic nanocermets, grown on microengineered Si surfaces, as potential candidates for a highly robust SERS sensor. The robustness of the sensor is attributed to the anchoring of the nanoparticles in the nanocermet, which is an important factor for exploiting its reusability. The fairly uniform distribution of nanoparticles in the sensor led to high enhancement factors (10(6)-10(7)) and enabled the detection of low concentrations of a wide range of analytes, including differently charged biomolecules, which is extremely difficult for other SERS sensors. With more precise control over the particle geometry and distribution, plasmonic nanocermets may play an important role in ultrasensitive SERS measurements in adverse conditions such as high temperature.