An indole-based near-infrared fluorescent "Turn-On" probe for H2O2: Selective detection and ultrasensitive imaging of zebrafish gallbladder.

Fluorescent probes play essential roles in medical imaging, where the researchers can select one of many molecules to use to help monitor the status of living systems under investigation. To date, a few scaffolds that allow the in vivo detection of H2O2 are available only. Herein, we provide a highly sensitive and selective near-infrared fluorescent probe that detects H2O2 based on the ICT sensing mechanism. We report the first indole-incorporated fluorescent probe Indo-H2O2 that allows H2O2 detection with a LOD of 25.2 nM featuring a boronate group conjugated to an indole scaffold; the boronate cleaves upon reaction with H2O2. A 5-membered malononitrile derivative was incorporated; Indo-H2O2 has near-infrared (NIR) properties and the reaction time is low (∼25 min) compared to other related probes. Indo-H2O2 was successfully employed in both endogenous and exogenous imaging trials of H2O2 in living cells. Indo-H2O2 also allows the real-time monitoring of H2O2in vivo. It preferentially accesses the gallbladder of zebrafish. Our findings support Indo-H2O2 as a highly sensitive fluorescent NIR probe for detecting H2O2, and an idea to incorporate a central indole unit in future fluorescent probe designs.

Highly sensitive and rapid detection of hypochlorous acid in aqueous media and its bioimaging in live cells and zebrafish using an ESIPT-driven mycophenolic acid-based fluorescent probe.

Excessive production of potent biological oxidants such as HOCl has been implicated in numerous diseases. Thus, it is crucial to develop highly specific and precise methods to detect HOCl in living systems, preferably with molecules that can show a distinct therapeutic effect. Our study introduces the synthesis and application of a highly sensitive fluorescence "turn-on" probe, Myco-OCl, based on the mycophenolic acid scaffold with exceptional water solubility. The ESIPT-driven mechanism enables Myco-OCl to specifically and rapidly detect (<5 s) HOCl with an impressive Stokes shift of 105 nm (λex = 417 nm, λem = 522 nm) and a sub-nanomolar (97.3 nM) detection limit with the detection range of 0 to 50 µM. The potential of Myco-OCl as an excellent biosensor is evident from its successful application for live cell imaging of exogenous and endogenous HOCl. In addition, Myco-OCl enabled us to detect HOCl in a zebrafish inflammatory animal model. These underscore the great potential of Myco-OCl for detecting HOCl in diverse physiological systems. Our findings thus offer a highly promising tool for detecting HOCl in living organisms.

Nontoxic Levels of Se-Containing Compounds Increase Survival by Blocking Oxidative and Inflammatory Stresses via Signal Pathways Whereas High Levels of Se Induce Apoptosis.

Selenium is a main group element and an essential trace element in human health. It was discovered in selenocysteine (SeC) by Stadtman in 1974. SeC is an encoded natural amino acid hailed as the 21st naturally occurring amino acid (U) present in several enzymes and which exquisitely participates in redox biology. As it turns out, selenium bears a U-shaped toxicity curve wherein too little of the nutrient present in biology leads to disorders; concentrations that are too great, on the other hand, pose toxicity to biological systems. In light of many excellent previous reviews and the corpus of literature, we wanted to offer this current review, in which we present aspects of the clinical and biological literature and justify why we should further investigate Se-containing species in biological and medicinal contexts, especially small molecule-containing species in biomedical research and clinical medicine. Of central interest is how selenium participates in biological signaling pathways. Several clinical medical cases are recounted; these reports are mainly pertinent to human cancer and changes in pathology and cases in which the patients are often terminal. Selenium was an option chosen in light of earlier chemotherapeutic treatment courses which lost their effectiveness. We describe apoptosis, and also ferroptosis, and senescence clearly in the context of selenium. Other contemporary issues in research also compelled us to form this review: issues with CoV-2 SARS infection which abound in the literature, and we described findings with human patients in this context. Laboratory scientific studies and clinical studies dealing with two main divisions of selenium, organic (e.g., methyl selenol) or inorganic selenium (e.g., sodium selenite), are discussed. The future seems bright with the research and clinical possibilities of selenium as a trace element, whose recent experimental clinical treatments have so far involved dosing simply and inexpensively over a set of days, amounts, and time intervals.

"Lighting up" fluoride: cellular imaging and zebrafish model interrogations using a simple ESIPT-based mycophenolic acid precursor-based probe.

The discovery and implementation of media that derive from bioinspired designs and bear optical readouts featuring large Stokes shifts are of continued interest to a wide variety of researchers and clinicians. Myco-F, a novel mycophenolic acid precursor-based probe features a cleavable tert-butyldimethylsiloxy group to allow for fluoride detection. Myco-F exhibits high selectivity and specificity towards F- (Stokes shift = 120 nm). All measurements were performed in complete aqueous media (LOD=0.38 µM). Myco-F enables detection of fluoride ions in living HEK293 cells and localizes in the eye region (among other regions) of the zebrafish. DFT calculations support the proposed ESIPT working photomechanism.

Milestones in corrole chemistry: historical ligand syntheses and post-functionalization.

Corroles are synthetic porphyrin analogs that contain one meso carbon atom lesser and bear a trianionic N4 metal-chelating core. They require in-depth preparative chemistry, demonstrate unique coordination chemistry and have impressive and diverse physical properties, and these are commonly compared to their respective porphyrins. The corrole's macrocyclic system is inherently electron rich and chelates metal ions in a more compact, less symmetric tetranitrogen cavity compared to that of porphyrins. Herein, we cover the highlights of the corrole research through the decades by first reviewing, in a chronological sense, multi-step syntheses; some routes have since been discontinued. This is followed by describing post-functionalization of already formed corroles via reactions performed on either the macrocycle's periphery or the inner nitrogen atoms or on the existing substituents. We do also mention milestones in literature reviewing, publication of encyclopedias, and the creation of professional organizations and conferences (ICPP) which make up the corrole/porphyrin research landscape. Also highlighted are still existing challenges and future perspectives.

A foundational theoreticalAl12E12(E = N, P) adsorption and quinolone docking study: cage-quinolone pairs, optics and possible therapeutic and diagnostic applications.

This combined Al12E12 (E = N, P) surface adsorption and docking study describes the new possibility of prospective potential probing(photophysical/optical) and therapy(medicinal/biochemical) with these adsorbent conjugates. DFT investigations were undertaken herein to help generate geometrical models and better understand the possible favorable adsorption energetics. We attempt to explain their adsorption behaviors and docking involving SARS-CoV-2 viruses (PDB)to assess their possible pharmaceutical potential against the pandemic virus (COVID-19). The adsorption behavior of 8-hydroxy-2-methylquinoline (MQ) and its halogenated derivatives, 5,7-diiodo-8-hydroxy-2-methylquinoline (MQI), 5,7-dichloro-8-hydroxy-2-methylquinoline (MQCl), and 5,7-dibromo-8-hydroxy-2-methylquinoline (MQBr), with aluminum-nitrogen (AlN), and aluminum-phosphorous (AlP) fullerene-like nanocages is reported. A decrease in the hardness of the nanoclusters when adsorbed with drug molecules resulted in an incrementally improved chemical softness (see e.g., Hard-Soft Acid Base theory) indicating that reactivity of the drug molecule in the resulting complex increases upon cluster chemical adsorption. The energy gap is found to be maximized for AlN-MQ and minimized for AlP-MQI; the reduced density gradient (RDG) iso-surfaces and AIM studies also corroborated this. Therefore, these two were found, respectively, to be the least and most electrically conductive of the species under study. We selected a simple medicinal building block (chelator)in addition to selecting the cluster based on previous literature reports. Important parameters such as gap energies and global indices were determined. We assessed NLO properties. The SARS-CoV-2 virus PDB docking data for 6VW1, 6VYO, 6WKQ, 7AD1, 7AOL, 7B3C, were enlisted as ligand targets for studies of docking (PatchDock Server) using the requisite PDB geometries (For the structure of 6VW1, kindly see reference, 2020; For the structure of 6VYO kindly see reference, 2020; For the structure of 6WKQ kindly see reference, 2020; For the structure of 7AD1 kindly see reference, 2021; For the structure of 7AOL kindly see reference, 2021; For the structure of 7B3C kindly see reference, 2021). Such findings indicate that the AlN-drug conjugation have inhibitory effect against these selected receptors.Communicated by Ramaswamy H. Sarma.

Theoretical study of glycoluril by highly symmetrical magnesium oxide Mg12O12 nanostructure: adsorption, detection, SERS enhancement, and electrical conductivity study.

Using metal substrates that are nanoscale in size, surface-enhanced Raman scattering (SERS) is a technique for enhancing the Raman signal of biomolecules. Numerous industries including sensing materials, adsorption and medical devices, use nanomaterials like nanocages and nanoclusters. To discover a possible novel sensor platform involving a small metal cluster and a curved rigid substrate, we used density functional theoretical (DFT) simulations to explore the adsorption of glycoluril (GLC), a prospective drug intermediate, on a pure magnesium oxide cage (Mg12O12). This well defined cage was used as (i) an exact probable structure that could be used as well as (ii) a general model for MgO nanostructures. We also investigated the mono Al-doped Mg12O12 nanocage version Mg11AlO12. All computations were performed at the M06-2X level of theory. The GLC binds to the Mg12O12 nanocage by way of strong donor-acceptor interactions. The adsorption is releasing - 45.80 kcal mol-1 of energy. Due to Al doping, the energy gap of GLC-Mg11AlO12 (1.91 eV) is reduced from that of GLC-Mg12O12 (4.28 eV) and hence there is an increase in electrical conductivity of GLC-Mg11AlO12. The electronic change in the nanocage's conductivity can be transformed into an electrical signal which can be used to detect the presence of the drug analyte. In addition, when a GLC molecule is present, the work function of the nanocage is also reduced. The MgO nanocage, we conclude, is a work function type as well as a possible electronic sensor for GLC drug detection. GLC desorption from the Mg11AlO12 surface recovers more quickly in comparison with Mg12O12 recovery time. The AIM and NCIs assessed in this study were performed to help analyze the electronic structures of the complexes. Our findings pave the possibility for Mg11AlO12 nanostructures to be used in drug recognition.

Macro and micro thermal investigation of nanoarchitectonics-based coatings on cotton fabric using new quaternized starch.

Implementation of a new cationizing reagent and its incorporation onto the backbone of starch was performed successfully, confirmed from the remarkable micro- and macro anti-flammable properties. The morphologies and localized compositional analysis of the modified starch-based LBL coatings on the cotton surface were carried out using LV-SEM and EDX: highly uniform coating layers and uptake of solution species for intermediate implant reagent concentrations were confirmed. The subject samples were further analyzed through thermogravimetric analysis (TGA), microcombustion experiments (MCC), flame testing (VFT) and afterburn measurements. The peak range of the degradation was highly improved from the lower range to the higher range (329.92-394.48 °C), together with significant mass residue for TBAB-0.7-17.02%. Moreover, a significant decrease in the absolute heat loss (THR ∼ 30%), heat dissipation competence (HRC ∼ 27.86%), and peak heat output (PHRR ∼ 23%) was achieved for a TBAB loading of ∼0.7 g. The results were further confirmed from the increase in the limiting oxygen index (LOI) to a higher rate of ∼23.2, improved structural integrity and higher quality of char obtained in the VFT and after-burn analysis.

Theoretical model study of adsorbed antimalarial-graphene dimers: doping effects, photophysical parameters, intermolecular interactions, edge adsorption, and SERS.

Future diagnostics and therapy applications are in part riding on the discovery and implementation of new optical techniques and strategies (which often derive from dyads) for example, prediction of features in surface-enhanced Raman spectroscopy requires the study of chromophore-chromophore interactions involve intermolecular forces, drug delivery, and photo mechanisms which are of great interest. New matches between chromophore systems (i.e. FRET), and π-delocalized surfaces are important to study. We explore low-molecular weight drug molecules and their interaction with the reporter material/surface of graphene. Bonding, charge transfer and orbital interactions for 2-amino-5-(1-methyl-5-nitro-2-imidazolyl)-1,3,4-thiadiazole (megazol or AMIT) on graphene were carried out. The graphene model substrate was monotonically/monatomically substituted (doped) with one neutral heteroatom (N/O/S/B) in place of one carbon center; chemical adsorption of AMIT is due to charge transfer from doped graphene to AMIT (DFT). Our AMIT-nanocluster studies show that the nanoclusters will act as a sensor component for the detection of drugs due to SERS. Our findings identified that the greater the energy of the charge transfer, the stronger the calculated chemical adsorption. Additionally, charge transfer is highest for the N-doped systems and least for pristine graphene, resulting in a stronger adsorption energy for N-doped graphene. Mulliken charge analysis of structures confirms enhancement found in QD-AMIT systems.Communicated by Ramaswamy H. Sarma.

Phosphinate-benzoindocyanin fluorescent probe for endogenous mitochondrial peroxynitrite detection in living cells and gallbladder access in inflammatory zebrafish animal models.

Potent oxidants such as peroxynitrite (ONOO-) play important roles in the regulation of different physiopathological processes; their overproduction is thought to potentially cause several diseases in living organisms. Hence, the precise and selective monitoring of ONOO- is imperative for elucidating its interplay and roles in pathological and physiological processes. Herein, we present a novel diphenyl phosphinate-masked benzoindocyanin "turn-on" fluorogenic probe to help detect mitochondrial ONOO- in living cells and zebrafish models. A pale yellow color solution of BICBzDP turns rose-red upon the addition of ONOO-, selectively, contrary to that of other competitive bioactive molecules. BICBzDP displays an ultra-sensitivity detection limit (47.8 nM) with outstanding selectivity and sensitivity towards mitochondrial ONOO- and possesses a notable 68-fold fluorescence enhancement involving a large redshift of 91 nm. Importantly, further biological experimental investigations with BICBzDP indicate specific sensitivity and reliability of the probe to track the ONOO- level, not only in live cells, but also demonstrates dynamic fluctuations in the inflammatory zebrafish animal models. Thus, BICBzDP could be employed as a future potential biological tool for exploiting the role of ONOO- in a variety of different physiological systems.

Adsorption properties of dacarbazine with graphene/fullerene/metal nanocages - Reactivity, spectroscopic and SERS analysis.

Drug delivery devices are an effective way to minimize anticancer drug toxicity and nanostructures are used in the targeted drug delivery. In the present work, adsorption and interaction behavior of 4-(dimethylaminodiazenyl)-1H-imidazole-5-carboxamide (DAIC) with nano complexes (graphene, fullerene and fullerene like metal cages) are reported theoretically. From the reactivity studies, the electrophilicity index of DAIC-nanoclusters are increasing and this gives the bioactivity of the nanocluster systems. Adsorption energy is highest in the case of AlP and lowest in the case of BP clusters. Mulliken charge distribution of all systems is an evidence for chemical enhancement. DAIC adsorption over nanocages causes changes in electronic properties resulting in chemical enhancement and variation in Raman spectra which suggests that nanocages could be a good candidate for DAIC detection.

LBL generated fire retardant nanocomposites on cotton fabric using cationized starch-clay-nanoparticles matrix.

In this work, starch-clay-TiO2-based nanocomposites were deposited on cotton fabric through layer-by-layer (LBL) process and their effect on the flame retardancy, inhibition of pyrolysis and combustion processes were discussed in details. Polyelectrolyte solutions/suspensions of cationized starch and VMT (vermiculite)/TiO2 nanoparticles were used to deposit these nanocomposites in the form of multi-layered coatings (5, 7, 10 and 15 bilayers). Uniform fabric coverage and presence of electrolytes was imaged by scanning electron microcopy (LV-SEM), attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and EDX characterizations. The greatest pyrolysis reduction was found for the StVT-7 sample (7 bilayers); ~30% and 21%, based on microscale combustion calorimetry (MCC) and thermogravimetric analysis (TGA). When using MCC, the improved values of the PHRR ~ 193 W/g, THR ~ 10.7 kJ/g), HRC ~ 390 J/g∙K and LOI ~ 22.2% were found for the StVT-7 sample which was strongly supported by the UL-94 test.

Solvent Effects on the Phosphorescence of Gold(III) Complexes Chelated by ß-Multisubstituted Corroles.

A set of gold corrole complexes containing four different ß-substituent groups (Br/I/CF3), namely, 4Br-Au, 4I-Au, and 4CF3-Au, were investigated; all showed room temperature phosphorescence. The phosphorescence quantum yields of the corroles were determined using tetraphenylporphyrin as a reference: Φph (4I-Au, 0.75%) > Φph (4Br-Au, 0.64%) > Φph (4CF3-Au, 0.38%). 4CF3-Au exhibited near-IR emission (858 nm, aerobic); absorbance intensity for the Q-band was higher than that for the Soret band. Complex 4I-Au showed a longer phosphorescence lifetime (82 µs) compared to those of 4Br-Au (53 µs) and 4CF3-Au (28 µs; N2, tol). Thermally activated delayed fluorescence (TADF) emission of 4I/Br-Au complexes was observed: stronger emission intensity correlated with increasing temperature. Good negative correlations for 4I/Br-Au were observed between the Soret band absorption energy and the solvent polarizability: excited states of 4I/Br-Au are more polar than their ground states. TD-DFT calculations revealed very fast intersystem crossing (ISC) rate constants, 2.20 × 1012 s-1 (4CF3-Au) > 1.96 × 1011 s-1 (4Br-Au) > 1.15 × 1011 s-1 (4I-Au), and importantly, the reverse intersystem crossing (rISC) rate constants are determined as 1.68 × 107 s-1 (4I-Au) > 2.40 × 103 s-1 (4Br-Au) ≫ 8.09 × 10-8 s-1 (4CF3-Au). The exceptionally low rISC rate constant of 4CF3-Au is attributed to its more steric and deformed structure bearing a larger energy gap between the S1 and T1 states.

Bisphenol-based cyanide sensing: Selectivity, reversibility, facile synthesis, bilateral "OFF-ON" fluorescence, C2ν structural and conformational analysis.

A structurally characterized novel dual-pocketed tetra-conjugated bisphenol-based chromophore (fluorescence = 652 nm) was synthesized in gram scale in ~90% yield from its tetraaldehyde. Highly selective, naked-eye detection of CN- (DMSO/H2O) was confirmed by interferent testing. A detection limit of 0.38 µM, within the permissible limit of CN- concentration in drinking water was achieved as mandated by WHO. The "reversibility" study shows potential applicability and reusability of Sen. Moreover, cost-effective and on-site interfaces, application tools such as fabricated cotton swabs, plastic Petri dishes, and filter papers further demonstrated the specific selectivity of Sen for the toxic CN-. In addition, an easily available and handy smartphone-assisted "Color Picker" app was utilized to help estimate the concentration of CN- ion present. A dual phenol deprotonation mechanism is active and supported by 1H NMR spectroscopic data and DFT calculation results.

Minding our P-block and Q-bands: paving inroads into main group corrole research to help instil broader potential.

Main group chemistry is often considered less "dynamic" than transition metal (TM) chemistry because of predictable VSEPR-based central atom geometries, relatively slower redox switching and lack of electronic d-d transitions. However, we delineate what has been made possible with main group chemistry to give it its proper due and up-to-date treatment. The huge untapped potential regarding photophysical properties and functioning hereby spurred us to review a range of corrole reports addressing primarily photophysical trends, synthetic aspects, and important guidelines regarding substitution and inorganic principles. We also look at Ag and Au systems and also consider substitutions such as CF3, halogens, additives and also counterions. Throughout, as well as at the end of this review, we suggest various future directions; further future industrial catalytic and health science research is encouraged.

Inexpensive water soluble methyl methacrylate-functionalized hydroxyphthalimide: variations of the mycophenolic acid core for selective live cell imaging of free cysteine.

Evident from numerous studies, cysteine plays a crucial role in cellular function. Reactions with analyte also enables for molecular recognition to adhere to molecular therapeutic potential; integration between synthetic probes therefore allows for a potentially deep therapy-related interogation of biological systems (theranostics). The development of molecular cysteine probes with extremely accurate detection is still a key challenge for the field. The development of water-soluble organic molecular fluorescent probes able to efficiently distinguish common biothiols such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) by chemical recognition means i.e. by (binding, cleavage) in biological systems is a greatly sought research challenge due to the similarity of the small sulfhydryl-containing species. Herein, we have developed a water-soluble and highly cell viable fluorescent organic molecule (log P = 0.82) for the selective detection of cysteine. The probe (Myco-Cys) shows a "turn-on" response with the cleavage ester linkage of the methacrylate as cysteine is encountered in solution. The probe shows strong fluorescence enhancement (16.5-fold) when treated with Cys (1 equiv., 10 µM) compared to closely related species such as amino acids, including HCy/GSH, and the limit of detection was determined as 45.0 nM. DFT calculations helped confirm the photomechanism of Myco-Cys. Furthermore, the sensing ability of the probe was demonstrated by living cell assays through the use of confocal fluorescence microscopy. Myco-Cys could selectively detect cysteine among biothiols. Myco-Cys was able to monitor the cysteine level, apart from the oxidative stress present in the form of H2O2 in A549 cells.

Discussions of Fluorescence in Selenium Chemistry: Recently Reported Probes, Particles, and a Clearer Biological Knowledge.

In this review from literature appearing over about the past 5 years, we focus on selected selenide reports and related chemistry; we aimed for a digestible, relevant, review intended to be usefully interconnected within the realm of fluorescence and selenium chemistry. Tellurium is mentioned where relevant. Topics include selenium in physics and surfaces, nanoscience, sensing and fluorescence, quantum dots and nanoparticles, Au and oxide nanoparticles quantum dot based, coatings and catalyst poisons, thin film, and aspects of solar energy conversion. Chemosensing is covered, whether small molecule or nanoparticle based, relating to metal ion analytes, H2S, as well as analyte sulfane (biothiols-including glutathione). We cover recent reports of probing and fluorescence when they deal with redox biology aspects. Selenium in therapeutics, medicinal chemistry and skeleton cores is covered. Selenium serves as a constituent for some small molecule sensors and probes. Typically, the selenium is part of the reactive, or active site of the probe; in other cases, it is featured as the analyte, either as a reduced or oxidized form of selenium. Free radicals and ROS are also mentioned; aggregation strategies are treated in some places. Also, the relationship between reduced selenium and oxidized selenium is developed.

Adapting educational experiences for the chemists of tomorrow.

Technical universities are constantly experimenting with innovative student-centred classroom approaches. We describe flipped and linked classroom approaches in the context of our ongoing chemical education theme of catalysis.

Clean Ar-Me conversion to Ar-aldehyde with the aid of carefully designed metallocorrole photocatalysts.

Toluene, p-xylene and mesitylene were cleanly converted to their corresponding monoaldehydes via mild photooxygenation utilizing transition metal and main group ß-CF3-substituted corroles. Aldehyde yield increased as more electron-donating CH3 groups are present on the substrate. 4-P was most efficient (TON ∼ 1072, mesitylene) via the singlet oxygen vis the superoxide mechanism.

Didactic approach recounting advances and limitations in novel glutathione and cysteine detection (reduced GSH probe) with mixed coumarin, aldehyde, and phenyl-selenium chemistry.

We describe the pertinent research steps and analysis, many of which are chemical, to achieve a novel molecular probe for glutathione (GSH) which has been published and patented based on two recent articles: "Exceptional time response, stability and selectivity in doubly-activated phenyl selenium-based glutathione-selective platform" and "Enhanced Doubly Activated Dual Emission Fluorescent Probes for Selective Imaging of Glutathione or Cysteine in Living Systems" (Kim et al., 2015; Mulay et al., 2018). The papers involve coumarin probes. Reaction/detection unfolds with aminothiol attack at an electrophilic ring carbon position. An adjacent -CHO group is heavily involved in resonance aspects of the C-Se position, as well as the binding of the pendant N-group; the coumarin lactone carbonyl also allows for resonance to be achieved (vide infra). The leaving group, -SePh, while precedented in some systems, depends on electronic tuning (Fig. 1). For 1, the response times with GSH was ~100ms; a 100-fold fluorescence increase is observed (Compound 1). The probe also reacts with cysteine (Cys) and homocysteine (Hcy), albeit differently. For glutathione probing, the greater wavelength maxima (1: 550nm, DACP-1: 555nm, DACP-2: 590nm) enabled eventual cell studies (confocal microscopy) and animal studies. The limits of detection (LOD, 1: 270nM DACP-1: 10.1nM DACP-2: 17.0nM), as measured using the 3σ/k method. We provide a didactic presentation from probe conception to probe in vivo testing, etc., with additional considerations presented; a variety of factors/issues (2.1-2.28) help maintain a realistic sequence, a flow from wider to narrower, of the factors that go into developing medical, biological and neurodegenerative disease-related probes, meant to help other researchers follow our intention, gain perspective, and overcome current limitations.