Prolonged cold-preservation of domestic cat ovarian tissue is improved by extracellular solution but impaired by the fragmentation of ovary.

For domestic cats ovaries, recommended cold-storage limit is 24 h in Phosphate Buffered Saline (PBS) or Dulbecco`s PBS (DPBS). Here, we attempted to verify wheatear cat ovaries may benefit from more complex solutions during prolonged cold-storage (>24 h). First, the preservation capabilities of extracellular (SP+), intracellular (UW) solutions and DPBS supplemented with glutathione (DPBS+GSH) were compared using ovary fragments from the same ovary (n=10). Intact ovary stored in DPBS served as a control. Ovaries were kept at 4 °C for 48 h, and 72 h. In the second experiment, first ovary was stored in DPBS, second in SP+ or UW solution for 48 h (n = 12). Ovaries pairs stored in DPBS for 24 h served as a control (n=8). Tissue samples were evaluated directly after cold-storage and after following 24 h in vitro culture. Ovarian follicle morphology, apoptosis rates (cleaved caspase-3, TUNEL), and follicular growth activation (Ki-67) were assessed. Ovary fragmentation impaired follicular morphology preservation upon cold-storage comparing to intact ovary. However, ovarian fragments stored in UW for 48 h and in SP+ for 72 h presented better morphology than DPBS+GSH group. Comparison of intact ovaries cold-storage for 48 h showed that SP+ provided superior follicular morphology over DPBS, and it was comparable to the outcome of 24-hour storage. No follicular activation after in vitro culture was observed. Nevertheless, tissue culture increased considerably caspase-3 cleavage and TUNEL detection. The ovary fragmentation prior to cold-storage is not recommended in domestic cats. Replacement of DPBS with SP+ solution for whole ovary and UW solution for ovarian tissue fragments improves follicular structure preservation during 48-hour cold-storage.

Structural Characterization of Cu(I)/Zn(II)-metallothionein-3 by Ion Mobility Mass Spectrometry and Top-Down Mass Spectrometry.

Mammalian zinc metallothionein-3 (Zn7MT3) plays an important role in protecting against copper toxicity by scavenging free Cu(II) ions or removing Cu(II) bound to ß-amyloid and α-synuclein. While previous studies reported that Zn7MT3 reacts with Cu(II) ions to form Cu(I)4Zn(II)4MT3ox containing two disulfides (ox), the precise localization of the metal ions and disulfides remained unclear. Here, we undertook comprehensive structural characterization of the metal-protein complexes formed by the reaction between Zn7MT3 and Cu(II) ions using native ion mobility mass spectrometry (IM-MS). The complex formation mechanism was found to involve the disassembly of Zn3S9 and Zn4S11 clusters from Zn7MT3 and reassembly into Cu(I)xZn(II)yMT3ox complexes rather than simply Zn(II)-to-Cu(I) exchange. At neutral pH, the ß-domain was shown to be capable of binding up to six Cu(I) ions to form Cu(I)6Zn(II)4MT3ox, although the most predominant species was the Cu(I)4Zn(II)4MT3ox complex. Under acidic conditions, four Zn(II) ions dissociate, but the Cu(I)4-thiolate cluster remains stable, highlighting the MT3 role as a Cu(II) scavenger even at lower than the cytosolic pH. IM-derived collision cross sections (CCS) reveal that Cu(I)-to-Zn(II) swap in Zn7MT3 with concomitant disulfide formation induces structural compaction and a decrease in conformational heterogeneity. Collision-induced unfolding (CIU) experiments estimated that the native-like folded Cu(I)4Zn(II)4MT3ox conformation is more stable than Zn7MT3. Native top-down MS demonstrated that the Cu(I) ions are exclusively bound to the ß-domain in the Cu(I)4Zn(II)4MT3ox complex as well as the two disulfides, serving as a steric constraint for the Cu(I)4-thiolate cluster. In conclusion, this study enhances our comprehension of the structure, stability, and dynamics of Cu(I)xZn(II)yMT3ox complexes.

Ion mobility mass spectrometry and molecular dynamics simulations unravel the conformational stability of zinc metallothionein-2 species.

Ion mobility-mass spectrometry (IM-MS) unraveled different conformational stability in Zn4-7-metallothionein-2. We introduced a new molecular dynamics simulation approach that permitted the exploration of all of the conformational space confirming the experimental data, and revealed that not only the Zn-S bonds but also the α-ß domain interactions modulate protein unfolding.

Non-Conserved Amino Acid Residues Modulate the Thermodynamics of Zn(II) Binding to Classical ßßα Zinc Finger Domains.

Classical zinc fingers domains (ZFs) bind Zn(II) ion by a pair of cysteine and histidine residues to adopt a characteristic and stable ßßα fold containing a small hydrophobic core. As a component of transcription factors, they recognize specific DNA sequences to transcript particular genes. The loss of Zn(II) disrupts the unique structure and function of the whole protein. It has been shown that the saturation of ZFs under cellular conditions is strictly related to their affinity for Zn(II). High affinity warrants their constant saturation, while medium affinity results in their transient structurization depending on cellular zinc availability. Therefore, there must be factors hidden in the sequence and structure of ZFs that impact Zn(II)-to-protein affinities to control their function. Using molecular dynamics simulations and experimental spectroscopic and calorimetric approaches, we showed that particular non-conserved residues derived from ZF sequences impact hydrogen bond formation. Our in silico and in vitro studies show that non-conserved residues can alter metal-coupled folding mechanisms and overall ZF stability. Furthermore, we show that Zn(II) binding to ZFs can also be entropically driven. This preference does not correlate either with Zn(II) binding site or with the extent of the secondary structure but is strictly related to a reservoir of interactions within the second coordination shell, which may loosen or tighten up the structure. Our findings shed new light on how the functionality of ZFs is modulated by non-coordinating residues diversity under cellular conditions. Moreover, they can be helpful for systematic backbone alteration of native ZF ßßα scaffold to create artificial foldamers and proteins with improved stability.

Current Landscape of Non-Small Cell Lung Cancer: Epidemiology, Histological Classification, Targeted Therapies, and Immunotherapy.

Non-small cell lung cancer (NSCLC) is a subtype of the most frequently diagnosed cancer in the world. Its epidemiology depends not only on tobacco exposition but also air quality. While the global trends in NSCLC incidence have started to decline, we can observe region-dependent differences related to the education and the economic level of the patients. Due to an increasing understanding of NSCLC biology, new diagnostic and therapeutic strategies have been developed, such as the reorganization of histopathological classification or tumor genotyping. Precision medicine is focused on the recognition of a genetic mutation in lung cancer cells called "driver mutation" to provide a variety of specific inhibitors of improperly functioning proteins. A rapidly growing group of approved drugs for targeted therapy in NSCLC currently allows the following mutated proteins to be treated: EGFR family (ERBB-1, ERBB-2), ALK, ROS1, MET, RET, NTRK, and RAF. Nevertheless, one of the most frequent NSCLC molecular sub-types remains without successful treatment: the K-Ras protein. In this review, we discuss the current NSCLC landscape treatment focusing on targeted therapy and immunotherapy, including first- and second-line monotherapies, immune checkpoint inhibitors with chemotherapy treatment, and approved predictive biomarkers.

An Integrated Mass Spectrometry and Molecular Dynamics Simulations Approach Reveals the Spatial Organization Impact of Metal-Binding Sites on the Stability of Metal-Depleted Metallothionein-2 Species.

Mammalian metallothioneins (MTs) are a group of cysteine-rich proteins that bind metal ions in two α- and ß-domains and represent a major cellular Zn(II)/Cu(I) buffering system in the cell. At cellular free Zn(II) concentrations (10-11-10-9 M), MTs do not exist in fully loaded forms with seven Zn(II)-bound ions (Zn7MTs). Instead, MTs exist as partially metal-depleted species (Zn4-6MT) because their Zn(II) binding affinities are on the nano- to picomolar range comparable to the concentrations of cellular Zn(II). The mode of action of MTs remains poorly understood, and thus, the aim of this study is to characterize the mechanism of Zn(II) (un)binding to MTs, the thermodynamic properties of the Zn1-6MT2 species, and their mechanostability properties. To this end, native mass spectrometry (MS) and label-free quantitative bottom-up and top-down MS in combination with steered molecular dynamics simulations, well-tempered metadynamics (WT-MetaD), and parallel-bias WT-MetaD (amounting to 3.5 µs) were integrated to unravel the chemical coordination of Zn(II) in all Zn1-6MT2 species and to explain the differences in binding affinities of Zn(II) ions to MTs. Differences are found to be the result of the degree of water participation in MT (un)folding and the hyper-reactive character of Cys21 and Cys29 residues. The thermodynamics properties of Zn(II) (un)binding to MT2 are found to differ from those of Cd(II), justifying their distinctive roles. The potential of this integrated strategy in the investigation of numerous unexplored metalloproteins is attested by the results highlighted in the present study.

Mass Spectrometry-Based Structural Analysis of Cysteine-Rich Metal-Binding Sites in Proteins with MetaOdysseus R Software.

Identification of metal-binding sites in proteins and understanding metal-coupled protein folding mechanisms are aspects of high importance for the structure-to-function relationship. Mass spectrometry (MS) has brought a powerful adjunct perspective to structural biology, obtaining from metal-to-protein stoichiometry to quaternary structure information. Currently, the different experimental and/or instrumental setups usually require the use of multiple data analysis software, and in some cases, they lack some of the main data analysis steps (MS processing, scoring, identification). Here, we present a comprehensive data analysis pipeline that addresses charge-state deconvolution, statistical scoring, and mass assignment for native MS, bottom-up, and native top-down with emphasis on metal-protein complexes. We have evaluated all of the approaches using assemblies of increasing complexity, including free and chemically labeled proteins, from low- to high-resolution MS. In all cases, the results have been compared with common software and proved how MetaOdysseus outperformed them.

Metal- and Affinity-Specific Dual Labeling of Cysteine-Rich Proteins for Identification of Metal-Binding Sites.

Here, using human metallothionein (MT2) as an example, we describe an improved strategy based on differential alkylation coupled to MS, assisted by zinc probe monitoring, for identification of cysteine-rich binding sites with nanomolar and picomolar metal affinity utilizing iodoacetamide (IAM) and N-ethylmaleimide reagents. We concluded that an SN2 reaction provided by IAM is more suitable to label free Cys residues, avoiding nonspecific metal dissociation. Afterward, metal-bound Cys can be easily labeled in a nucleophilic addition reaction after separation by reverse-phase C18 at acidic pH. Finally, we evaluated the efficiency of the method by mapping metal-binding sites of Zn7-xMT species using a bottom-up MS approach with respect to metal-to-protein affinity and element(al) resolution. The methodology presented might be applied not only for MT2 but to identify metal-binding sites in other Cys-containing proteins.

A chemometric-assisted voltammetric analysis of free and Zn(II)-loaded metallothionein-3 states.

We focused on the application of mass spectrometry and electrochemical methods combined with a chemometric analysis for the characterization of partially metallothionein-3 species. The results showed decreased Cat1 and Cat2 signals for the Zn(II)-loaded MT3 species with respect to the metal-free protein, which might be explained by the arrangement of tetrahedral metal-thiolate coordination environments and the formation of metal clusters. Moreover, there was a decrease in the Cat1 and Cat2 signals, and a plateau was reached with 4-5 Zn(II) ions that corresponded to the formation of the C-terminal α-domain. Regarding the Zn7-xMT3 complexes, we observed three different electrochemical behaviours for the Zn1-2MT3, Zn3-6MT3 and Zn7MT3 species. The difference for Zn1-2MT3 might be explained by the formation of independent ZnS4 cores in this stage that differ with respect to the formation of ZnxCysy clusters with an increased Zn(II) loading. The binding of the third Zn(II) ion to MT3 resulted in high sample heterogeneity due the co-existence of Zn3-6MT3. Finally, the Zn7MT3 protein showed a third type of behaviour. The fact that there were no free Cys residues might explain this phenomenon. Thus, this research identifies the major proteins responsible for zinc buffering in the cell.

The Glutathione/Metallothionein System Challenges the Design of Efficient O2 -Activating Copper Complexes.

Copper complexes are of medicinal and biological interest, including as anticancer drugs designed to cleave intracellular biomolecules by O2 activation. To exhibit such activity, the copper complex must be redox active and resistant to dissociation. Metallothioneins (MTs) and glutathione (GSH) are abundant in the cytosol and nucleus. Because they are thiol-rich reducing molecules with high CuI affinity, they are potential competitors for a copper ion bound in a copper drug. Herein, we report the investigation of a panel of CuI /CuII complexes often used as drugs, with diverse coordination chemistries and redox potentials. We evaluated their catalytic activity in ascorbate oxidation based on redox cycling between CuI and CuII , as well as their resistance to dissociation or inactivation under cytosolically relevant concentrations of GSH and MT. O2 -activating CuI /CuII complexes for cytosolic/nuclear targets are generally not stable against the GSH/MT system, which creates a challenge for their future design.

Reactivity of Cu(ii)-, Zn(ii)- and Fe(ii)-thiosemicarbazone complexes with glutathione and metallothionein: from stability to dissociation to transmetallation.

Thiosemicarbazones (TSCs) are a class of strong metal ion ligands, which are currently being investigated for several applications, such as anticancer treatment. In addition to these ligands only, which exert their activity upon interaction with metal ions in cells, preformed metal-TSC complexes are also widely studied, predominantly with the essential metal ions iron, copper and zinc. Currently, it is unclear what the active species are, which complexes are present and what are their biological targets. Herein, we study the complexes of copper(ii), zinc(ii) and iron(ii) with three TSCs, PT, 3-AP (triapine) and Dp44mT, (latter two are currently in clinical trials), concerning their reactivity with glutathione (GSH) and Zn7-metallothionein (Zn7MT-1, 2 and 3). These two cysteine-containing molecules can have a major impact on metal-TSC complexes because they are abundant in the cytosol and nucleus, they are strong metal ligands and have the potential to reduce Cu(ii) and Fe(iii). Our results indicate that Fe(ii)-TSC is stable in the presence of typical cytosolic concentrations of GSH and Zn7MT. In contrast, all three Cu(ii)-TSCs react rapidly due to the reduction of Cu(ii) to Cu(i), which is then transferred to MT. This suggests that Cu(ii)-TSCs are rapidly dissociated in a cytosolic-type environment and the catalytic generation of reactive oxygen species by Cu(ii)-TSCs is stopped. Moreover, in the case Cu(ii)-Dp44mT, transmetallation with Zn(ii) from MT occurs. The reaction of Zn(ii)-TSCs is ligand dependent, from predominant dissociation for PT and 3-AP, to very little dissociation of Zn(ii)-Dp44mT2. These results indicate that GSH and Zn7MT may be important factors in the fate of Cu(ii)- and Zn(ii)-TSCs. In particular, for Cu, its chemistry is complex, and these reactions may also occur for other families of Cu-complexes used in cancer treatment or for other applications.

Chemometrics-assisted optimization of liquid chromatography-quadrupole-time-of-flight mass spectrometry analysis for targeted metabolomics.

Mass spectrometry-based metabolomics is characterized by a vast number of variables leading to a great degree of complexity. In this work, we aimed to simplify this process with a stepped chemometric optimization of the both funnel technology (funnel exit DC, FDC; funnel RF LP, FLC; funnel RF HP, FRP) and ion source parameters (Octopolo, Oct; and Fragmentor, Frag) of a quadrupole-time of flight (qTOF) for a human urinary metabolites. The workflow comprised a Box-Behnken experimental design with 47 experiments followed by the identification and quantification of a set of metabolites using high-resolution full-scan MS mode and feature extraction with an inclusion list. Metabolite peak areas were grouped according to abundance (high and low) and modeled by Random Forest regression (variance explained >85%). The full three-level factorial design consisting in 243 experiments was predicted and top 10 solutions for desirability function and those comprising the Pareto front were extracted and investigated. To guarantee the quality of results, we compared the Pareto front solutions with those achieved by standard instrumental parameters suggested by the manufacturer. A set of five solutions were identified that increased the mean peak area by 56-59% and 17%, for high- and low-abundance metabolites, respectively. The optimal parameters were determined to be: FLP, 100 V; FDC, 40 and 30 V; Frag, 275 and 400 V; and Oct, 600 and 800 V. The methodology applied throughout this work represents a flexible strategy to optimize instrumental parameters and exploit the performance of a qTOF MS detector.

Multiobjective optimization of liquid chromatography-triple-quadrupole mass spectrometry analysis of underivatized human urinary amino acids through chemometrics.

Optimization of instrumental settings of a triple-quadrupole mass analyzer was performed by Box-Behnken design, support vector machines, and a Pareto-optimality approach. This time-saving, stepped chemometric strategy was used to model the signal response of underivatized human urinary amino acids. Drying gas flow, nebulizer pressure, sheath gas flow, and capillary voltage settings were exhaustively studied beyond the parameters conventionally optimized in triple-quadrupole devices (multiple reaction monitoring transitions, fragmentor and collision energy voltages). The results indicate that the best signal response for high-abundance and low-abundance underivatized amino acids was achieved with drying gas flow of 9 L/min, nebulizer pressure of 60 psi, sheath gas flow of 13 L/min, and capillary voltage of 3000 V. Compared with the widely standardized settings tested, chemometric analysis led to signal intensities 74% and 68% higher for high-abundance and low-abundance amino acids, respectively. The flexibility, speed, and efficiency of this method allows its affordable implementation in all mass spectrometry-based research to obtain superior results compared with those obtained with conventionally optimized mass spectrometry instrumental parameters.

Crosstalk of the structural and zinc buffering properties of mammalian metallothionein-2.

Metallothioneins (MTs), small cysteine-rich proteins, present in four major isoforms, are key proteins involved in zinc and copper homeostasis in mammals. To date, only one X-ray crystal structure of a MT has been solved. It demonstrates seven bivalent metal ions bound in two structurally independent domains with M4S11 (α) and M3S9 (ß) clusters. Recent discoveries indicate that Zn(ii) ions are bound with MT2 with the range from nano- to picomolar affinity, which determines its cellular zinc buffering properties that are demonstrated by the presence of partially Zn(ii)-depleted MT2 species. These forms serve as Zn(ii) donors or acceptors and are formed under varying cellular free Zn(ii) concentrations. Due to the lack of appropriate methods, knowledge regarding the structure of partially-depleted metallothionein is lacking. Here, we describe the Zn(ii) binding mechanism in human MT2 with high resolution with respect to particular Zn(ii) binding sites, and provide structural insights into Zn(ii)-depleted MT species. The results were obtained by the labelling of metal-free cysteine residues with iodoacetamide and subsequent top-down electrospray ionization analysis, MALDI MS, bottom-up nanoLC-MALDI-MS/MS approaches and molecular dynamics (MD) simulations. The results show that the α-domain is formed sequentially in the first stages, followed by the formation of the ß-domain, although both processes overlap, which is in contrast to the widely investigated cadmium MT. Independent ZnS4 cores are characteristic for early stages of domain formation and are clustered in later stages. However, Zn-S network rearrangement in the ß-domain upon applying the seventh Zn(ii) ion explains its lower affinity. Detailed analysis showed that the weakest Zn(ii) ion associates with the ß-domain by coordination to Cys21, which was also found to dissociate first in the presence of the apo-form of sorbitol dehydrogenase. We found that Zn(ii) binding to the isolated ß-domain differs significantly from the whole protein, which explains its previously observed different Zn(ii)-binding properties. MD results obtained for Zn(ii) binding to the whole protein and isolated ß-domain are highly convergent with mass spectrometry data. This study provides a comprehensive overview of the crosstalk of structural and zinc buffering related-to-thermodynamics properties of partially metal-saturated mammalian MT2 and sheds more light on other MT proteins and zinc homeostasis.

RpeakChrom: Novel R package for the automated characterization and optimization of column efficiency in high-performance liquid chromatography analysis.

Characterization of chromatographic columns using the traditional van Deemter method is limited by the necessity of calculating extra-column variance, issue particularly relevant when modeling asymmetrical peaks eluted from monolithic columns. A novel R package that implements Parabolic Variance Modified Gaussian approach for accurate peak modeling, van Deemter equation and two alternatives approaches, based on van Deemter, has been developed to calculate the height equivalent to a theoretical plate (HETP). To assess package capabilities conventional packed reverse-phase and monolithic HPLC columns were characterized. Peaks eluted from the monolithic column showed a high value of factor asymmetry due, in part, to the contribution of extra-column factors. Such deviation can be circumvented by the two alternatives approaches implemented in the R-package. Furthermore, increased values of eddy diffusion and mass transfer kinetics terms in HETP were observed for the packed column, while accuracy was below 9% in all cases. These results showed the usefulness of the R-package for both modeling chromatographic peaks and assessing column efficiency. The RpeakChrom package could become a helpful tool for testing new stationary phases during column development and to evaluate column during its lifetime. This R tool is freely available from CRAN (https://CRAN.R-project.org/package=RpeakChrom).

A lipidomic cell-based assay for studying drug-induced phospholipidosis and steatosis.

Phospholipidosis and steatosis are two toxic effects, which course with overaccumulation of different classes of lipids in the liver. MS-based lipidomics has become a powerful tool for the comprehensive determination of lipids. LC-MS lipid profiling of HepG2 cells is proposed as an in vitro assay to study and anticipate phospholipidosis and steatosis. Cells with and without preincubation with a mixture of free fatty acids (FFA; i.e. oleic and palmitic) were exposed to a set of well-known steatogenic and phospholipidogenic compounds. The use of FFA preloading accelerated the accumulation of phospholipids, thus leading to a better discrimination of phospholipidosis, and magnified the lipidomic alterations induced by steatogenic drugs. Phospholipidosis was characterized by increased levels of phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, and phosphatidylinositols, while steatosis induced alterations in FA oxidation and triacylglyceride (TG) synthesis pathways (with changes in the levels of FFA, acylcarnitines, monoacylglycerides, diacylglycerides, and TG). Interestingly, palmitic and oleic acids incorporation into lipids differed. A characteristic pattern was observed in the fold of change of particular TG species in the case of steatosis (TG(54:3) > TG(52:2) > TG(50:1) > TG(48:0)). Based on the levels of those lipids containing only palmitic and/or oleic acid moieties a partial least squares-discriminant analysis model was built, which showed good discrimination among nontoxic, phospholipidogenic and steatogenic compounds. In conclusion, it has been shown that the use of FFA preincubation together with intracellular LC-MS based lipid profiling could be a useful approach to identify the potential of drug candidates to induce phospholipidosis and/or steatosis.