Porphyrin Production by Corynebacterium diphtheriae Strains from Clinical Isolates.

Porphyrins are intermediate metabolites involved in the biosynthesis of vital molecules, including heme, cobalamin, and chlorophyll. Bacterial porphyrins are known to be proinflammatory and are associated with biofilm formation. This study investigated porphyrin production by strains of Corynebacterium diphtheriae using emission spectroscopy, high-performance liquid chromatography with fluorescence detection, diode array detector, and mass spectrometry. Emission spectroscopy revealed characteristic porphyrin emission spectra in all strains, with coproporphyrin III predominating. Qualitative analysis by different chromatographic methods identified coproporphyrin III, uroporphyrin I, and protoporphyrin IX in all strains. Quantitative analysis revealed strain-dependent coproporphyrin III production. Further studies are required to investigate the relationship between porphyrin production and the virulence potential of Corynebacterium diphtheriae.

Comparison of α and ß-acid isomerization in hops and beer using HPLC, confocal microscopy, spectrofluorimetry and chemical analysis of metabolites and essential oils in flowers of different hop cultivars produced in Brazil.

We used confocal microscopy and spectrofluorescence to characterize the emission spectra in hop flowers, to follow the isomerization processes in different hop preparations, and beers, to compare with HPLC extracted samples. Flowers of different hop cultivars produced in three regions of Brazil, were quantitated by HPLC and GC-MS. The fluorescence spectra showed two characteristic emission bands evaluated from different preparations. The isomerization process leads to a gradual decrease in fluorescence intensity as the reaction progresses. This demonstrates the valuable use of confocal microscopy and fluorescence spectroscopy for analysis of the correlation between bitter acid indices with fluorescence intensity and lifetime microscopy. Such techniques can be used directly in the flowers allowing rapid monitoring of the brewing process. Twenty-nine substances were characterized in the essential oils and some cultivars presented quantities of bitter acids and essential oil levels close to those expected for plants after more than three years of cultivation.

Aggregation-Induced Emission (AIE) in Polymers: Effect of Polymer size on the Fluorescence of Low Molecular Weight PEG and PPG.

Aggregation-induced emission (AIE) is a fascinating phenomenon where specific molecules exhibit enhanced fluorescence upon aggregation. This unique property has revolutionized the design and development of new fluorescent materials for different applications, from biosensors and organic light-emitting diodes (OLEDs) to biomedical imaging and diagnostics. Researchers are creating sensitive and selective sensing platforms, opening new avenues in material science and engineering by harnessing the potential of AIE. To expand the knowledge in this field, this study explored the aggregation-induced emission (AIE) properties of two polymers, namely polyethylene glycol (PEG) and polypropylene glycol (PPG) of low molecular weight (MW) using fluorescence spectroscopy and absorbance (UV). PEG-300 and PPG-725 were the most fluorescent polymers at UV of the ten investigated. Interestingly, AIE did not correlate linearly with molecular weight (MW), and monobutyl ether substitution in PEG with a similar MW substantially altered its AIE. Furthermore, fluorescence precisely quantified low polymer concentrations in water, and non-aqueous solvents suppressed AIE, suggesting potential for AIE manipulation. These findings enhance our understanding of AIE in polymers, fostering the development of novel materials for applications such as biosensors.

The role of staphyloxanthin in the regulation of membrane biophysical properties in Staphylococcus aureus.

Staphylococcus aureus is an opportunistic pathogen that is considered a global health threat. This microorganism can adapt to hostile conditions by regulating membrane lipid composition in response to external stress factors such as changes in pH and ionic strength. S. aureus synthesizes and incorporates in its membrane staphyloxanthin, a carotenoid providing protection against oxidative damage and antimicrobial agents. Staphyloxanthin is known to modulate the physical properties of the bacterial membranes due to the rigid diaponeurosporenoic group it contains. In this work, preparative thin layer chromatography and liquid chromatography mass spectrometry were used to purify staphyloxanthin from S. aureus and characterize its structure, identifying C15, C17 and C19 as the main fatty acids in this carotenoid. Changes in the biophysical properties of models of S. aureus membranes containing phosphatidylglycerol, cardiolipin, and staphyloxanthin were evaluated. Infrared spectroscopy shows that staphyloxanthin reduces the liquid-crystalline to gel phase transition temperature in the evaluated model systems. Interestingly, these shifts are not accompanied by strong changes in trans/gauche isomerization, indicating that chain conformation in the liquid-crystalline phase is not altered by staphyloxanthin. In contrast, headgroup spacing, measured by Laurdan GP fluorescence spectroscopy, and lipid core dynamics, measured by DPH fluorescence anisotropy, show significant shifts in the presence of staphyloxanthin. The combined results show that staphyloxanthin reduces lipid core dynamics and headgroup spacing without altering acyl chain conformations, therefore decoupling these normally correlated effects. We propose that the rigid diaponeurosporenoic group in staphyloxanthin and its positioning in the membrane is likely responsible for the results observed.

Distribution of photoactive ß-carboline alkaloids across Passiflora caerulea floral organs.

This study reports valuable information regarding the presence and concentration of a series of photoactive ß-carboline (ßCs) alkaloids (norharmane, harmane, harmine, harmol, harmaline, and harmalol) and their distribution across the floral age and organs of Passiflora caerulea. UHPLC-MS/MS data reported herein reveal that the ßCs' content ranged from 1 to 110 µg kg-1 , depending on the floral organ and age. In certain physiologically relevant organs, such as anthers, ßCs' content was one order of magnitude higher than in other organs, suggesting a special role for ßCs in this specific organ. ßCs' content also varied in a structure-dependent manner. Alkaloids bearing a hydroxyl group at position C(7) of the main ßC ring were present at concentrations one order of magnitude higher than other ßC derivatives investigated. UV-visible and fluorescence spectroscopy of the flower extracts provided complementary information regarding other biologically relevant groups of chromophores (phenolic/indolic derivatives, flavonoids/carotenes, and chlorophylls). Since flowers are constantly exposed to solar radiation, the presence of photoactive ßCs in floral organs may have several (photo)biological implications that are further discussed.

Direct spectrofluorimetric methods as alternatives to compendial ones used for the quality control of biopharmaceuticals: development, validation and application

Abstract A simple, rapid, precise, accurate and sustainable spectrofluorimetric method (SFM) was developed, validated and applied for the determination of 4-aminobenzoic acid and aromatic amino acids (phenylalanine, tryptophan and tyrosine). These compounds are used in biopharmaceutical formulations and therefore must be analyzed by quality control laboratories to meet the criteria established in pharmacopoeias. In general, potentiometric titration (PT) is described in the compendia as the official analytical technique. However, this method showed low sensitivity and selectivity, and moreover was performed with a non-aqueous solvent (acetic acid), which led to higher consumption of reagents and consequently to the formation of residues. Therefore, the SFM was developed in aqueous medium at pH 7.2 using phosphate buffer. It was successfully validated according to the ICH guidelines and showed good linearity range (r>0.999), specificity, accuracy and precision (within and between days) and robustness. The test results were compared between the SFM and PT using raw material samples, while according to the F- and t-tests at 95% confidence level, no statistical difference was found between the methods

Staphylococcus aureus Modulates Carotenoid and Phospholipid Content in Response to Oxygen-Restricted Growth Conditions, Triggering Changes in Membrane Biophysical Properties.

Staphylococcus aureus membranes contain carotenoids formed during the biosynthesis of staphyloxanthin. These carotenoids are considered virulence factors due to their activity as scavengers of reactive oxygen species and as inhibitors of antimicrobial peptides. Here, we show that the growth of S. aureus under oxygen-restricting conditions downregulates carotenoid biosynthesis and modifies phospholipid content in biofilms and planktonic cells analyzed using LC-MS. At oxygen-restrictive levels, the staphyloxanthin precursor 4,4-diapophytofluene accumulates, indicating that the dehydrogenation reaction catalyzed by 4,4'-diapophytoene desaturases (CrtN) is inhibited. An increase in lysyl-phosphatidylglycerol is observed under oxygen-restrictive conditions in planktonic cells, and high levels of cardiolipin are detected in biofilms compared to planktonic cells. Under oxygen-restriction conditions, the biophysical parameters of S. aureus membranes show an increase in lipid headgroup spacing, as measured with Laurdan GP, and decreased bilayer core order, as measured with DPH anisotropy. An increase in the liquid-crystalline to gel phase melting temperature, as measured with FTIR, is also observed. S. aureus membranes are therefore less condensed under oxygen-restriction conditions at 37 °C. However, the lack of carotenoids leads to a highly ordered gel phase at low temperatures, around 15 °C. Carotenoids are therefore likely to be low in S. aureus found in tissues with low oxygen levels, such as abscesses, leading to altered membrane biophysical properties.

Foliar Calcium Absorption by Tomato Plants: Comparing the Effects of Calcium Sources and Adjuvant Usage.

The deficiency of calcium (Ca) reduces the quality and shelf life of fruits. In this scenario, although foliar spraying of Ca2+ has been used, altogether with soil fertilization, as an alternative to prevent deficiencies, little is known regarding its absorption dynamics by plant leaves. Herein, in vivo microprobe X-ray fluorescence was employed aiming to monitor the foliar absorption of CaCl2, Ca-citrate complex, and Ca3(PO4)2 nanoparticles with and without using adjuvant. We also investigated whether Sr2+ can be employed as Ca2+ proxy in foliar absorption studies. Moreover, the impact of treatments on the cuticle structure was evaluated by scanning electron microscopy. For this study, 45-day-old tomato (Solanum lycopersicum L., cv. Micro-Tom) plants were used as a model species. After 100 h, the leaves absorbed 90, 18, and 4% of aqueous CaCl2, Ca-citrate, and Ca3(PO4)2 nanoparticles, respectively. The addition of adjuvant increased the absorption of Ca-citrate to 28%, decreased that of CaCl2 to 77%, and did not affect Ca3(PO4)2. CaCl2 displayed an exponential decay absorption profile with half-lives of 15 h and 5 h without and with adjuvant, respectively. Ca-citrate and Ca3(PO4)2 exhibited absorption profiles that were closer to a linear behavior. Sr2+ was a suitable Ca2+ tracer because of its similar absorption profiles. Furthermore, the use of adjuvant affected the epicuticular crystal structure. Our findings reveal that CaCl2 was the most efficient Ca2+ source. The effects caused by adjuvant suggest that CaCl2 and Ca-citrate were absorbed mostly through hydrophilic and lipophilic pathways.

Physiological responses of plants to in vivo X-ray damage from X-ray fluorescence measurements: insights from anatomical, elemental, histochemical, and ultrastructural analyses.

X-ray fluorescence spectroscopy (XRF) is a powerful technique for the in vivo assessment of plant tissues. However, the potential X-ray exposure damages might affect the structure and elemental composition of living plant tissues, leading to artefacts in the recorded data. Herein, we exposed in vivo soybean (Glycine max (L.) Merrill) leaves to several X-ray doses through a polychromatic benchtop microprobe X-ray fluorescence spectrometer, modulating the photon flux density by adjusting either the beam size, current, or exposure time. Changes in the irradiated plant tissues' structure, ultrastructure, and physiology were investigated through light and transmission electron microscopy (TEM). Depending on X-ray exposure dose, decreased K and X-ray scattering intensities and increased Ca, P, and Mn signals on soybean leaves were recorded. Anatomical analysis indicated the necrosis of epidermal and mesophyll cells on the irradiated spots, where TEM images revealed the collapse of cytoplasm and cell wall breaking. Furthermore, the histochemical analysis detected the production of reactive oxygen species and the inhibition of chlorophyll autofluorescence in these areas. Under certain X-ray exposure conditions, e.g. high photon flux density and long exposure time, XRF measurements may affect the soybean leaves structures, elemental composition, and cellular ultrastructure, inducing programmed cell death. Our characterization shed light on the plant's responses to the X-ray-induced radiation damage and might help to establish proper X-ray radiation limits and novel strategies for in vivo benchtop-XRF analysis of vegetal materials.

Evaluation of Steady-State and Time-Resolved Fluorescence Spectroscopy as a Method for Assessing the Impact of Photo-Oxidation on Refined Soybean Oils.

The type of material used in packaging, lighting, and storage time can impact food quality during storage. This study aimed to investigate the progress of photosensitized oxidation in refined soybean oil using steady-state and time-resolved fluorescence spectroscopy. The experiment was conducted through accelerated photo-oxidation with Light-Emitting Diode (LED) in samples stored for ten days at room temperature (26.0 ± 2.0 °C) in clear polyethylene terephthalate (PET) packaging of different colors and different transmission spectra in the UV and visible range. Emission spectra were obtained with excitation at 373, 405, and 500 nm, resulting in two main emission peaks: the first with maximum emission between 430 and 555 nm and the second at around 660 nm. Fluorescence decay curves were obtained with excitation at 340 and 405 nm. The results indicated that transparent PET bottles are not effective in protecting soybean oil from photosensitized oxidation under the studied conditions. Strong correlations were observed between fluorescence parameters and peroxide and conjugated diene values, indicators of lipid oxidation progress. Fluorescence spectroscopy has several advantages over traditional methods as it is a simple, fast, low-cost, and low-waste technique.

Copper-flavonoid family of complexes involved in alkaline phosphatase activation.

The flavonoid naringenin and a family of naringenin derivative Cu(II) complexes having phenanthroline-based second ligands were selected to study alkaline phosphatase activation. This enzyme plays a critical role in tissue formation, increasing the inorganic phosphate formation, favoring mineralization, and being essential to producing bone mineralization. The effects of those compounds on the function and structure of the enzyme were evaluated by kinetic measurements, fluorescence, FTIR, and UV-Vis spectroscopies. The results showed that naringenin did not affect alkaline phosphatase activity, having a value of the Michaelis-Menten-constant close to the enzyme (Km = 3.07 × 10-6). The binary complex, Cu(II)-naringenin, and the ternary complex Cu(II)-naringenin-phenanthroline behaved as an enzyme activator in all the concentrations range used in this study. Those complexes increased in c.a. 1.9% the catalytic efficiency concerning enzyme and naringenin. The ternary complex Cu(II)-naringenin-bathophenanthroline, provokes an activator mixed effect, dependent on the substrate concentrations. The different kinetic behavior can be correlated with different conformational changes observed under the interaction with ALP. Fluorescence experiments showed a raising of the binding constant with temperature. FTIR determinations showed that the complex with bathophenanthroline modifies the ALP structure but maintains the helical structure. The other copper complexes provoked a structural unfolding, decreasing the α-helix content. None of them affect the dephosphorylation enzyme ability. Even though the interactions and structural modifications on ALP are different, it is evident that the presence of copper favors enzymatic activity. The observed electrostatic interactions probably benefit the dissociation of the bound phosphate. The results suggest potential biological applications for the studied compounds.

Cardiolipin Strongly Inhibits the Leakage Activity of the Short Antimicrobial Peptide ATRA-1 in Comparison to LL-37, in Model Membranes Mimicking the Lipid Composition of Staphylococcus aureus.

Cardiolipin is one of the main phospholipid components of Staphylococcus aureus membranes. This lipid is found at varying concentrations in the bilayer, depending on the growth stage of the bacteria, and as a response to environmental stress. Cardiolipin is an anionic phospholipid with four acyl chains, which modulates the bending properties of the membrane due to its inverted conical shape. It has been shown to inhibit the pore forming activity of several antimicrobial peptides, in general doubling the peptide concentration needed to induce leakage. Here we find that the short snake-derived antimicrobial peptide ATRA-1 is inhibited by several orders of magnitude in the presence of cardiolipin in saturated membranes (DMPG) compared to the human cathelicidin LL-37, which is only inhibited two-fold in its leakage-inducing concentration. The ATRA-1 is too short to span the membrane and its leakage activity is likely related to detergent-like alterations of bilayer structure. Fluorescence spectroscopy shows only a minor effect on ATRA-1 binding to DMPG membranes due to the presence of cardiolipin. However, FTIR spectroscopy shows that the acyl chain structure of DMPG membranes, containing cardiolipin, become more organized in the presence of ATRA-1, as reflected by an increase in the gel to liquid-crystalline phase transition temperature. Instead, a depression in the melting temperature is induced by ATRA-1 in DMPG in the absence of cardiolipin. In comparison, LL-37 induces a depression of the main phase transition of DMPG even in the presence of cardiolipin. These data suggest that cardiolipin inhibits the penetration of ATRA-1 into the membrane core, impeding its capacity to disrupt lipid packing.

A major conformational change of N-terminal helices of Bacillus thuringiensis Cry1Ab insecticidal protein is necessary for membrane insertion and toxicity.

Pore forming toxins rely on oligomerization for membrane insertion to kill their targets. Bacillus thuringiensis produces insecticidal Cry-proteins composed of three domains that form pores that kill the insect larvae. Domain I is involved in oligomerization and membrane insertion, whereas Domains II and III participate in receptor binding and specificity. However, the structural changes involved in membrane insertion of these proteins remain unsolved. The most widely accepted model for membrane insertion, the 'umbrella model', proposed that the α-4/α-5 hairpin of Domain I swings away and is inserted into the membrane. To determine the topology of Cry1Ab in the membrane, disulfide bonds linking α-helices of Domain I were introduced to restrict their movement. Disulfide bonds between helices α-2/α-3 or α-3/α-4 lost oligomerization and toxicity, indicating that movement of these helices is needed for insecticidal activity. By contrast, disulfide bonds linking helices α-5/α-6 did not affect toxicity, which contradicts the 'umbrella model'. Additionally, Föster resonance energy transfer closest approach analyses measuring distances of different points in the toxin to the membrane plane and collisional quenching assays analysing the protection of specific fluorescent-labeled residues to the soluble potassium iodide quencher in the membrane inserted state were performed. Overall, the data show that Domain I from Cry1Ab may undergo a major conformational change during its membrane insertion, where the N-terminal region (helices α-1 to α-4) participates in oligomerization and toxicity, probably forming an extended helix. These data break a paradigm, showing a new 'folding white-cane model', which better explains the structural changes of Cry toxins during insertion into the membrane.

Oocyte quality assessment in marine invertebrates: a novel approach by fluorescence spectroscopy.

BACKGROUND: The assessment of oocyte quality is, nowadays, a major challenge in aquaculture, oocyte cryopreservation, and environmental science. Oocyte quality is a determining factor in fertilization and embryo development; however, there is still a lack of rapid and sensitive cellular markers for its assessment. Currently, its estimation is predominantly based on morphological analysis, which is subjective and does not consistently reflect the developmental competence of the oocytes. Despite several recent studies investigating molecular markers related to oocyte quality, methods currently available for their determination pose various technical challenges and limitations. In this study, we developed a novel approach based on fluorescence spectroscopy to assess different intrinsic physiological parameters that can be employed to evaluate egg quality in marine invertebrates that are widely used as animal models such as sea urchins and mussels. RESULTS: Different physiological parameters, such as viability, mitochondrial activity, intracellular ROS levels, plasma membrane lipid peroxidation, and intracellular pH, for egg quality evaluation have been successfully assessed in sea urchins and mussels by using specific fluorescent dyes and detecting the fluorescent signals in eggs through fluorescence spectroscopy. CONCLUSIONS: Based on our findings, we propose these physiological markers as useful predictors of egg quality in marine invertebrates; they can be estimated rapidly, selectively, and sensitively by employing this novel approach, which, due to the speed of analysis, the low cost, and easy use can be considered a powerful analytical tool for the egg quality assessment.

A Computational-Experimental Investigation of the Molecular Mechanism of Interleukin-6-Piperine Interaction.

Herein, we elucidate the biophysical aspects of the interaction of an important protein, Interleukin-6 (IL6), which is involved in cytokine storm syndrome, with a natural product with anti-inflammatory activity, piperine. Despite the role of piperine in the inhibition of the transcriptional protein NF-κB pathway responsible for activation of IL6 gene expression, there are no studies to the best of our knowledge regarding the characterisation of the molecular interaction of the IL6-piperine complex. In this context, the characterisation was performed with spectroscopic experiments aided by molecular modelling. Fluorescence spectroscopy alongside van't Hoff analyses showed that the complexation event is a spontaneous process driven by non-specific interactions. Circular dichroism aided by molecular dynamics revealed that piperine caused local α-helix reduction. Molecular docking and molecular dynamics disclosed the microenvironment of interaction as non-polar amino acid residues. Although piperine has three available hydrogen bond acceptors, only one hydrogen-bond was formed during our simulation experiments, reinforcing the major role of non-specific interactions that we observed experimentally. Root mean square deviation (RMSD) and hydrodynamic radii revealed that the IL6-piperine complex was stable during 800 ns of simulation. Taken together, these results can support ongoing IL6 drug discovery efforts.

Direct solid sample analysis using synchronous fluorescence spectroscopy coupled with chemometric tools for the geographical discrimination of coffee samples.

This work aims the study chemometric methods for the classification of the origin of coffee samples. Samples of finely pulverized coffee grains were analyzed by synchronous molecular fluorescence spectroscopy to carry out the classification. The spectral data of the samples were obtained in triplicate in two offsets: 10 nm (with emission wavelengths from 240 nm to 600 nm) and 40 nm (from 240 nm to 560 nm), all with 1 nm resolution. Different strategies were performed using the spectra obtained with the offsets of 10 nm and 40 nm and fused data at mid-level (10 nm + 40 nm). The performances of linear and nonlinear methods were compared, the best results were obtained from the raw data from the fusion at low-level of the 10 nm and 40 nm offset spectra with the Pareto optimization criterion.

Detection of adulterations in a valuable Brazilian honey by using spectrofluorimetry and multiway classification.

Spectrofluorimetry combined with multiway chemometric tools were applied to discriminate pure Aroeira honey samples from samples adulterated with corn syrup, sugar cane molasses and polyfloral honey. Excitation emission spectra were acquired for 232 honey samples by recording excitation from 250 to 500 nm and emission from 270 to 640 nm. Parallel factor analysis (PARAFAC), partial least squares discriminant analysis (PLS-DA), unfolded PLS-DA (UPLS-DA) and multilinear PLS-DA (NPLS-DA) methods were used to decompose the spectral data and build classification models. PLS-DA models presented poor classification rates, demonstrating the limitation of the traditional two-way methods for this dataset, and leading to the development of three-way classification models. Overall, UPLS-DA provided the best classification results with misclassification rates of 4% and 8% for the training and test sets, respectively. These results showed the potential of the proposed method for routine laboratory analysis as a simple, reliable, and affordable tool.

Biomineralization induced by chitosan and collagen-based materials with fluoride for dentin coverage: Chemical and morphological analysis.

The prevention and treatment of erosive tooth wear are becoming increasingly important due to its increasing prevalence. The use of natural solutions to modify dental surfaces has become an area of research. Organic materials such as chitosan and hydrolyzed collagen may be a promising option to treat dentin. This in vitro study aimed to evaluate the influence of chitosan or hydrolyzed collagen, alone or combined with acidulated phosphate fluoride (APF) gel, on the composition and morphology of dentin after erosion. Bovine dentin samples were prepared (n = 84) and treated with artificial saliva (AS, negative control); APF gel (F, positive control); chitosan solution (Chi); hydrolyzed collagen solution (Col); fluoride/chitosan composition (F_Chi); and fluoride/hydrolyzed collagen composition (F_Col). Erosive cycles (six cycles of immersion in orange juice for 1 min, followed by immersion in AS for 1 hr) were performed. The materials were characterized by their morphology, composition, and particle size distribution. Micro-energy dispersive X-ray fluorescence spectroscopy and scanning electron were used to evaluate the dentin's inorganic chemical composition and morphology. The F_Col and F groups had a reduction in calcium loss by 17 and 26%, respectively (p < .001). Both of these groups still had a covering layer of agglomerates at the dentin surface after the erosive cycles. The fluoridated chitosan or collagen solutions improved the dentin resistance to erosion as a novel hybrid-fluoride-based material approach to provide surface protection from erosion.

Oocyte quality assessment in marine invertebrates: a novel approach by fluorescence spectroscopy

BACKGROUND: The assessment of oocyte quality is, nowadays, a major challenge in aquaculture, oocyte cryopreservation, and environmental science. Oocyte quality is a determining factor in fertilization and embryo development; however, there is still a lack of rapid and sensitive cellular markers for its assessment. Currently, its estimation is pre-dominantly based on morphological analysis, which is subjective and does not consistently reflect the developmental competence of the oocytes. Despite several recent studies investigating molecular markers related to oocyte quality, methods currently available for their determination pose various technical challenges and limitations. In this study, we developed a novel approach based on fluorescence spectroscopy to assess different intrinsic physiological parameters that can be employed to evaluate egg quality in marine invertebrates that are widely used as animal models such as sea urchins and mussels. RESULTS: Different physiological parameters, such as viability, mitochondrial activity, intracellular ROS levels, plasma membrane lipid peroxidation, and intracellular pH, for egg quality evaluation have been successfully assessed in sea urchins and mussels by using specific fluorescent dyes and detecting the fluorescent signals in eggs through fluorescence spectroscopy. CONCLUSIONS: Based on our findings, we propose these physiological markers as useful predictors of egg quality in marine invertebrates; they can be estimated rapidly, selectively, and sensitively by employing this novel approach, which, due to the speed of analysis, the low cost, and easy use can be considered a powerful analytical tool for the egg quality assessment.

Unravelling the Interaction of Piperlongumine with the Nucleotide-Binding Domain of HSP70: A Spectroscopic and In Silico Study.

Piperlongumine (PPL) is an alkaloid extracted from several pepper species that exhibits anti-inflammatory and anti-carcinogenic properties. Nevertheless, the molecular mode of action of PPL that confers such powerful pharmacological properties remains unknown. From this perspective, spectroscopic methods aided by computational modeling were employed to characterize the interaction between PPL and nucleotide-binding domain of heat shock protein 70 (NBD/HSP70), which is involved in the pathogenesis of several diseases. Steady-state fluorescence spectroscopy along with time-resolved fluorescence revealed the complex formation based on a static quenching mechanism. Van't Hoff analyses showed that the binding of PPL toward NBD is driven by equivalent contributions of entropic and enthalpic factors. Furthermore, IDF and Scatchard methods applied to fluorescence intensities determined two cooperative binding sites with Kb of (6.3 ± 0.2) × 104 M-1. Circular dichroism determined the thermal stability of the NBD domain and showed that PPL caused minor changes in the protein secondary structure. Computational simulations elucidated the microenvironment of these interactions, showing that the binding sites are composed mainly of polar amino acids and the predominant interaction of PPL with NBD is Van der Waals in nature.