Puerarin dry powder inhaler formulations for pulmonary delivery: Development and characterization.

This study aims at developing and characterizing the puerarin dry powder inhaler (DPI) formulations for pulmonary delivery. The inhalable particles size (<2 µm) was accomplished by micronization and its morphology was examined by scanning electron microscopy (SEM). The puerarin-excipient interaction in powder mixtures was analyzed by using Fourier transform infrared spectroscopy (FTIR), Raman confocal microscopy, X-Ray powder Diffraction (XRD), and differential scanning calorimetry (DSC) methods. Using a Twin stage impinger (TSI), the in-vitro aerosolization of the powder formulations was carried out at a flow rate of 60 L/min and the drug was quantified by employing a validated HPLC method. No significant interactions between the drug and the excipients were observed in the powder formulations. The fine particle fraction (FPF) of the drug alone was 4.2% which has increased five to six-fold for the formulations with aerosolization enhancers. Formulation containing lactose as large carriers produced 32.7% FPF, which further increased with the addition of dispersibility enhancers, leucine and magnesium stearate (40.8% and 41.2%, respectively). The Raman and FTIR techniques are very useful tool for understanding structural integrity and stability of the puerarin in the powder formulations. The puerarin was found to be compatible with the excipients used and the developed DPI formulation may be considered as an efficient formulation for pulmonary delivery for the management of various diseases at a very low dose.

Dispersal and transport of microplastics in river sediments.

Rivers are viewed as major pathways of microplastic transport from terrestrial areas to marine ecosystems. However, there is paucity of knowledge on the dispersal pattern and transport of microplastics in river sediments. In this study, a three dimensional hydrodynamic and particle transport modelling framework was created to investigate the dispersal and transport processes of microplastic particles commonly present in the environment, namely, polyethylene (PE), polypropylene (PP), polyamide (PA), and polyethylene terephthalate (PET) in river sediments. The study outcomes confirmed that sedimental microplastics with lower density would have higher mobility. PE and PP are likely to be transported for a relatively longer distance, while PA and PET would likely accumulate close to source points. High water flow would transport more microplastics from source points, and high flow velocity in bottom water layer are suggested to facilitate the transport of sedimental microplastics. Considering the limited dispersal and transport, the study outcomes indicated that river sediments would act as a sink for microplastic pollutants instead of being a transport pathway. The patchiness associated with the hotspots of different plastic types is expected to provide valuable information for microplastic source tracking.

Development and characterization of meropenem dry powder inhaler formulation for pulmonary drug delivery.

Meropenem (MPN), a broad spectrum ß-lactam antibiotic, has been increasingly used in the treatment of moderate to severe bacterial infections. However, due to its short plasma half-life and chemical instability in solution form, it has been challenging to use in the intravenous formulation. This study aims to develop and characterize MPN dry powder inhaler (DPI) formulation for pulmonary delivery. The inhalable MPN particles (1-5 µm) were prepared by micronization. Lactose, L-leucine and magnesium stearate (MgSt) were used in the powder formulation as carriers and dispersibility enhancers. The formulations were characterized by Scanning electron microscopy (SEM), Transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), Raman confocal microscopy, X-Ray powder diffraction analysis (PXRD), and differential scanning calorimetry (DSC) methods. The concentration of MPN was determined by using a validated HPLC method. The Fine Particle Fraction (FPF) of meropenem from powder mixtures was determined by a Twin Stage Impinger (TSI) at a flow rate of 60 L/min. The FPF of the original MPN was 1.91% which was significantly increased to 37.5% for the formulations with excipients. No physical interactions between the drug and the excipients observed. This study revealed the potential of a stable meropenem DPI formulation for pulmonary delivery.

Characterisation of iron oxide encrusted microbial fossils.

Robust methods for the characterisation of microbial biosignatures in geological matrices is critical for developing mineralogical biosignatures. Studying microbial fossils is fundamental for our understanding of the role microorganisms have played in elemental cycling in modern and ancient environments on Earth and potentially Mars. Here, we aim to understand what promotes the fossilisation of microorganisms after the initial stages of biomineralisation, committing bacteriomorphic structures to the geological record within iron-rich environments. Mineral encrusted cell envelope structures were routinely identified within a goethite-rich vein that cross-cut the saprolite (iron ore) of a weathered banded iron formation (BIF) system in the Quadrilátero Ferrífero, Brazil. The preservation of potential organic and mineralogical biosignatures associated with these fossils was characterised using the following high-resolution analytical techniques: scanning and transmission electron microscopy, focused ion beam scanning electron microscopy, nanoscale secondary ion mass spectrometry, synchrotron-based Fourier transform infrared spectroscopy and Raman spectroscopy. Electron microscopy demonstrated that mineral nucleation associated with a range of cell envelope structures typically followed the extant cell templates. These biologically-influenced iron-rich minerals are microcrystalline with minimal secondary growth. In contrast, intracellular mineralisation formed larger minerals that grew inward from the cell membrane to infill intracellular voids after cell death. A three dimensional reconstruction of encrusted cell envelopes in a fossilised biofilm suggests that microorganisms may be able to replicate, during the initial stages of mineralisation. Carbon and nitrogen signatures are preserved associated with the cell envelope structures; however, there were no conclusive mineralogical biosignatures associated with the mineralised cell envelopes highlighting the classical importance of morphology and elemental biosignatures in determining the biogenicity of bacteriomorphic structures.

Influential factors on microplastics occurrence in river sediments.

Rivers are regarded as sinks and pathways of plastic pollutants from terrestrial environments to various other aquatic systems such as lakes and oceans. Although extensive research has been conducted in recent years on microplastic pollution in river sediments, knowledge on the relationships between potential influential factors such as catchment characteristics and sediment properties, and microplastics occurrence in river sediments, is still considered an under-researched area. This study evaluated the influence of land use, population, and sediment particle size on the presence of microplastics abundance and types. Based on a Bayesian Network modelling approach to characterise the correlations between influencing factors and microplastics occurrence, it was evident that microplastics type had a positive correlation with different land use types and population. Catchment characteristics were found to play a more important role in influencing microplastics type than microplastics concentration. A statistically significant positive relationship was observed between microplastics concentration and clay particles which suggested that the occurrence of microplastics in sediments can be impacted by the presence of clay particles.

Influence of microplastics on nutrients and metal concentrations in river sediments.

Microplastics pose threats to aquatic environments because they serve as hard-substrate for microbial community colonization and biofilm formation due to their long-life span and hydrophobic surface which can impact on aquatic ecosystems. However, the association between microplastics and other pollutants, particularly nutrients and metals in river sediments are largely unknown. In this study, microplastics abundance and hazard scores which are the risks arising from chemical compounds used for plastics manufacture, and the correlations between microplastics and the concentrations of total carbon (TC), total nitrogen (TN), total phosphorus (TP) and metals commonly present in the urban environment such as Al, As, Cr, Co, Cu, Fe, Mg, Mn, Ni, Cd, Se, Sr, Zn, Pb, in Brisbane River sediments were investigated. The study confirmed that the risk associated with microplastics is based on their monomer composition rather than the quantities present. Sediments having relatively higher abundance of microplastics with a relatively lower hazard score result in higher nutrient concentrations. The concentrations of metals in river sediments are more dependent on their original sources rather than the concentration of microplastics. Nevertheless, leachate from plastics should be considered in risk assessment in relation to the association between metals and plastics in aquatic environments.

Microplastic pollution in a stormwater floating treatment wetland: Detection of tyre particles in sediment.

Synthetic rubber particles released from car tyres are expected to be an important type of microplastics in the environment, with road runoff and stormwater likely to transport tyre particles to the aquatic environment. Stormwater treatment wetlands are one of the key methods for treating road runoff and stormwater, but the presence and concentration of synthetic rubber microplastics from tyre particles in wetlands are largely unknown. In addition, constructed floating wetlands can be built using recycled PET plastic bottles, raising concerns about potential release of microplastics to the environment. In this study, we measured the concentrations of microplastics in water and sediment from the inlet and outlet of a stormwater floating treatment wetland on Queensland's Gold Coast. An average of 0.9 ± 0.3 and 4.0 ± 2.4 microplastic particles/L were detected in the water phase in the inlet and outlet samples, respectively. The sediment contained an average of 595 ± 120 and 320 ± 42 microplastic particles/kg dry sediment in inlet and outlet sediments, respectively. Between 15 and 38% of microplastics in the sediment were identified by FTIR as synthetic rubber-carbon filled particles, most likely derived from car tyres. The presence of synthetic rubber microplastics confirms that tyres can contribute to microplastic pollution in stormwater, with road runoff likely to be an important pathway. No microplastics with the same characteristics and polymer composition as the floating wetland construction material were detected in the water and sediment samples, indicating that the microplastics in the water and sediment detected here did not originate from the floating wetland's material. However, further investigation of older treatment wetlands is required to better understand the potential role of floating treatment wetlands as a source of microplastics.

Abundance, distribution patterns, and identification of microplastics in Brisbane River sediments, Australia.

Plastic pollutants in aquatic ecosystems have received wide attention and research endeavours since early 1970s. However, in comparison to marine environments, the occurrence of microplastics in a tidal river system remains largely unknown, especially in river sediments. Sediment samples taken from twenty-two sampling points along Brisbane River over the four different seasons revealed relatively high concentrations of microplastics in river sediments, with abundance ranging from 0.18 to 129.20 mg kg-1, or 10 to 520 items kg-1. Unfortunately, varied methods and units used for reporting do not allow the accurate comparison between related studies. The spatial distributions of microplastics hotspots indicated that microplastics abundance is distance-dominated caused by flow velocities. Lower and higher concentrations of microplastics abundance mostly occurred in dry and wet seasons, respectively. Significant temporal variations of microplastics concentrations was observed in residential and commercial areas. Polyethylene (PE), polyamide (PA) and polypropylene (PP), were the three main polymer types found in the Brisbane River sediments. Other polymer types such as polyethylene terephthalate (PET) were also detected. The majority of the detected microplastic particles were found to be <3 mm. This study reveals the abundance, spatial and temporal distribution patterns, and characteristics of microplastic pollutants in Brisbane River sediments, and provides systematic data for further research on microplastics in estuarine environments worldwide.

Aberrant activation of Wnt signaling pathway altered osteocyte mineralization.

Mineralization of bone is a dynamic process, involving a complex interplay between cells, secreted macromolecules, signaling pathways, and enzymatic reactions; the dysregulation of bone mineralization may lead to serious skeletal disorders, including hypophosphatemic rickets, osteoporosis, and rheumatoid arthritis. Very few studies have reported the role of osteocytes - the most abundant bone cells in the skeletal system and the major orchestrators of bone remodeling in bone mineralization, which is owed to their nature of being deeply embedded in the mineralized bone matrix. The Wnt/ß-catenin signaling pathway is actively involved in various life processes including osteogenesis; however, the role of Wnt/ß-catenin signaling in the terminal mineralization of bone, especially in the regulation of osteocytes, is largely unknown. This research demonstrates that during the terminal mineralization process, the Wnt/ß-catenin pathway is downregulated, and when Wnt/ß-catenin signaling is activated in osteocytes, dendrite development is suppressed and the expression of dentin matrix protein 1 (DMP1) is inhibited. Aberrant activation of Wnt/ß-catenin signaling in osteocytes leads to the spontaneous deposition of extra-large mineralized nodules on the surface of collagen fibrils. The altered mineral crystal structure and decreased bonding force between minerals and the organic matrix indicate the inferior integration of minerals and collagen. In conclusion, Wnt/ß-catenin signaling plays a critical role in the terminal differentiation of osteocytes and as such, targeting Wnt/ß-catenin signaling in osteocytes may serve as a potential therapeutic approach for the management of bone-related diseases.

Excipient Interactions in Glucagon Dry Powder Inhaler Formulation for Pulmonary Delivery.

PURPOSE: This study describes the development and characterization of glucagon dry powder inhaler (DPI) formulation for pulmonary delivery. Lactose monohydrate, as a carrier, and L-leucine and magnesium stearate (MgSt) were used as dispersibility enhancers for this formulation. METHODS: Using Fourier-transform infrared (FTIR) spectroscopy, Differential Scanning Calorimetry (DSC), and Raman confocal microscopy, the interactions between glucagon and all excipients were characterized. The fine particle fractions (FPFs) of glucagon in different formulations were determined by a twin stage impinger (TSI) using a 2.5% glucagon mixture, and the glucagon concentration was measured by a validated LC-MS/MS method. RESULTS: The FPF of the glucagon was 6.4%, which increased six-fold from the formulations with excipients. The highest FPF (36%) was observed for the formulation containing MgSt and large carrier lactose. The FTIR, Raman, and DSC data showed remarkable physical interactions of glucagon with leucine and a minor interaction with lactose; however, there were no interactions with MgSt alone or mixed with lactose. CONCLUSION: Due to the interaction between L-leucine and glucagon, leucine was not a suitable excipient for glucagon formulation. In contrast, the use of lactose and MgSt could be considered to prepare an efficient DPI formulation for the pulmonary delivery of glucagon.

Preparation of Ibuprofen Microparticles by Antisolvent Precipitation Crystallization Technique: Characterization, Formulation, and In Vitro Performance.

This study demonstrates the preparation and characterization of ibuprofen (IBP) microparticles with some excipients by a controlled crystallization technique with improved dissolution performance. Using the optimum concentrations pluronic F127, hydroxypropyl methyl cellulose, D-mannitol, and l-leucine in aqueous ethanol, the IBP microparticles were prepared. The dissolution tests were performed in phosphate buffer saline using a United States Pharmacopoeia dissolution tester at 37°C. The Raman spectroscopy was used to investigate the interactions and distribution of the IBP with the additives in the microcrystals. The prepared IBP microparticles showed higher dissolution compared to that of the smaller sized original IBP particles. The Raman data revealed that the excipients with a large number of hydroxyl groups distributed around the IBP particle in the crystal enhanced the dissolution of the drug by increasing the drug-solvent interaction presumably through hydrogen bonding. The Raman mapping technique gave an insight into the enhanced dissolution behavior of the prepared IBP microparticles, and such information will be useful for developing pharmaceutical formulations of hydrophobic drugs. The controlled crystallization was a useful technique to prepare complex crystals of IBP microparticles along with other additives to achieve the enhanced dissolution profile.

Wastewater treatment plants as a pathway for microplastics: Development of a new approach to sample wastewater-based microplastics.

Wastewater effluent is expected to be a pathway for microplastics to enter the aquatic environment, with microbeads from cosmetic products and polymer fibres from clothes likely to enter wastewater treatment plants (WWTP). To date, few studies have quantified microplastics in wastewater. Moreover, the lack of a standardized and applicable method to identify microplastics in complex samples, such as wastewater, has limited the accurate assessment of microplastics and may lead to an incorrect estimation. This study aimed to develop a validated method to sample and process microplastics from wastewater effluent and to apply the developed method to quantify and characterise wastewater-based microplastics in effluent from three WWTPs that use primary, secondary and tertiary treatment processes. We applied a high-volume sampling device that fractionated microplastics in situ and an efficient sample processing procedure to improve the sampling of microplastics in wastewater and to minimize the false detection of non-plastic particles. The sampling device captured between 92% and 99% of polystyrene microplastics using 25 µm-500 µm mesh screens in laboratory tests. Microplastic type, size and suspected origin in all studied WWTPs, along with the removal efficiency during the secondary and tertiary treatment stages, was investigated. Suspected microplastics were characterised using Fourier Transform Infrared spectroscopy, with between 22 and 90% of the suspected microplastics found to be non-plastic particles. An average of 0.28, 0.48 and 1.54 microplastics per litre of final effluent was found in tertiary, secondary and primary treated effluent, respectively. This study suggests that although low concentrations of microplastics are detected in wastewater effluent, WWTPs still have the potential to act as a pathway to release microplastics given the large volumes of effluent discharged to the aquatic environment. This study focused on a single sampling campaign, with long-term monitoring recommended to further characterise microplastics in wastewater.

Nickel(II) meso-Hydroxyporphyrin Complexes Revisited: Palladium-Catalysed Synthesis, Electronic Structures of Derived Oxy Radicals, and Oxidative Coupling to a Dioxoporphodimethene Dyad.

We report the synthesis and characterisation of new examples of meso-hydroxynickel(II) porphyrins with 5,15-diphenyl and 10-phenyl-5,15-diphenyl/diaryl substitution. The OH group was introduced by using carbonate or hydroxide as nucleophile by using palladium/phosphine catalysis. The NiPor-OHs exist in solution in equilibrium with the corresponding oxy radicals NiPor-O. . The 15-phenyl group stabilises the radicals, so that the 1 H NMR spectra of {NiPor-OH} are extremely broad due to chemical exchange with the paramagnetic species. The radical concentration for the diphenylporphyrin analogue is only 1 %, and its NMR line-broadening was able to be studied by variable-temperature NMR spectroscopy. The EPR signals of NiPor-O. are consistent with somewhat delocalised porphyrinyloxy radicals, and the spin distributions calculated by using density functional theory match the EPR and NMR spectroscopic observations. Nickel(II) meso-hydroxy-10,20-diphenylporphyrin was oxidatively coupled to a dioxo-terminated porphodimethene dyad, the strongly red-shifted electronic spectrum of which was successfully modelled by using time-dependent DFT calculations.

Encapsulation of nanoparticles into single-crystal ZnO nanorods and microrods.

One-dimensional single crystal incorporating functional nanoparticles of other materials could be an interesting platform for various applications. We studied the encapsulation of nanoparticles into single-crystal ZnO nanorods by exploiting the crystal growth of ZnO in aqueous solution. Two types of nanodiamonds with mean diameters of 10 nm and 40 nm, respectively, and polymer nanobeads with size of 200 nm have been used to study the encapsulation process. It was found that by regrowing these ZnO nanorods with nanoparticles attached to their surfaces, a full encapsulation of nanoparticles into nanorods can be achieved. We demonstrate that our low-temperature aqueous solution growth of ZnO nanorods do not affect or cause degradation of the nanoparticles of either inorganic or organic materials. This new growth method opens the way to a plethora of applications combining the properties of single crystal host and encapsulated nanoparticles. We perform micro-photoluminescence measurement on a single ZnO nanorod containing luminescent nanodiamonds and the spectrum has a different shape from that of naked nanodiamonds, revealing the cavity effect of ZnO nanorod.

A systematic theoretical study of the electronic structures of porphyrin dimers: DFT and TD-DFT calculations on diporphyrins linked by ethane, ethene, ethyne, imine, and azo bridges.

Theoretical calculations of the geometries, electronic structures and electronic absorption spectra of a series of covalently-linked porphyrin dimers are reported. The diporphyrins comprise 5,10,15-triphenylporphyrinatozinc(II) (ZnTriPP) units linked through the meso carbons by two-atom bridges, namely 1,2-ethanediyl (1), trans-1,2-ethenediyl (2), ethynediyl (3), 1,2-iminomethenediyl (4), and transdiazenediyl (5). The structures were optimised in toluene solvent by Density Functional Theory (DFT), using the integral equation formalism variant of the polarizable continuum model. The calculations were performed using the B3LYP functional and the 6-31G(d,p) basis set. The complete molecules were modelled, with no substitution of smaller groups on the periphery. In parallel, the compounds 2­5 were prepared by known or novel synthetic routes, to enable comparisons of experimental electronic absorption spectra with those calculated using time dependent-DFT at the same level of theory. As the ethane dimer 1 is not yet synthetically accessible, the model monomer meso-2-phenylethylZnTriPP was used for comparisons with the theoretical predictions. The results form a self-consistent set, enabling for the first time legitimate comparisons of the electronic structures of the series, especially regarding the degree to which the porphyrin p-systems interact by conjugation across the bridges. The theoretical calculations of the electronic transitions match the observed spectra in toluene to a remarkable degree, especially with respect to the peak maximum of the Q band, which represents to a large degree the energy of the HOMO­LUMO transition. The imine 4 is intrinsically polar due to the asymmetric bridge, and the HOMO is located almost exclusively on the ZnTriPP unit attached to the nitrogen of the imine, and the LUMO on the C-attached ring. Thus the Q-band transition is mapped as a comprehensive charge-transfer from the former ring to the latter. This may have consequences for the non-linear optical properties of the system. The azoporphyrin 5 exhibits the largest splittings between the interacting MOs via the conjugated bridge, vindicating a prediction by Anderson and co-workers in 2002, and confirmed experimentally by our synthesis of 5. The collected results also indicate that this level of theory is more thanadequate as a model with which to handle these large delocalised molecules.

Functionalised polycaprolactone films and 3D scaffolds via gamma irradiation-induced grafting.

Polycaprolactone (PCL) is frequently used as the base polymer in scaffolds targeted for tissue engineering applications. However, in the absence of further surface modification, the lack of functional moieties on the PCL chain results in non-ideal surface properties of such scaffolds, especially in terms of the inability to tailor the presentation of functional ligands for directed cell adhesion and growth. The current study investigates gamma irradiation-induced grafting as a means of improving the biofunctionality of the PCL surface. The surface presentation of carboxylic acid groups on 2D PCL films could be tailored by changing the acrylic acid (AAc) concentration and/or the solvent during grafting, as evaluated from X-ray photoelectron spectroscopy (XPS). From data obtained using Raman spectroscopy, it was concluded that the penetration depth of the grafted pAAc was affected by the solvent system with a mixed water-methanol system yielding high penetration. Grafted samples displayed a decreased elastic modulus of the surface correlating with pAAc penetration depth, as shown by nano-indentation using atomic force microscopy (AFM). The most promising grafting conditions found for the 2D PCL films were then applied to 3D thermally induced phase separation (TIPS) scaffolds and it was demonstrated using XPS that equivalent levels of grafting of pAAc could be achieved throughout the whole depth of the scaffold. The scaffolds maintained their overall integrity after grafting, even though we observed a decrease in the compressive modulus by 20% after surface modification. These combined studies confirm the utility of this surface modification methodology for scaffolds targeted at tissue engineering and cell culture applications.

Raman, infrared and XPS study of bamboo phytoliths after chemical digestion.

Raman, infrared and X-ray photoelectron spectroscopies have been used to examine the effect of various chemical digestion methods on the composition of bamboo phytoliths. Intact bilobate phytoliths, suitable for interrogation by Raman microprobe analysis, were isolated by a microwave wet ashing technique using hydrogen peroxide with nitric and hydrochloric acids. The occluded phytolith carbon presented evidence of cellulose, lignin and carboxylic acids. Nitrate from the nitric acid used in the digestion was observed in homogenized samples of the isolated phytoliths; in addition to nitrogen of plant origin occluded within the phytolith, which was observed as amine nitrogen and ammonia. Intact bilobate phytoliths were not observed following an exothermic hydrogen peroxide/sulfuric acid digest, suggesting that these structures ruptured during this digestion procedure. The silicate network was significantly altered during isolation using the exothermic hydrogen peroxide/sulfuric digest, with surface hydroxyls undergoing condensation to form a SiO(3) ring structure.

Halogen bonding between an isoindoline nitroxide and 1,4-diiodotetrafluorobenzene: new tools and tectons for self-assembling organic spin systems.

Radical assembly: Halogen bonding has been observed for the first time between an isoindoline nitroxide and an iodoperfluorocarbon (see figure), which cocrystallize to form a discrete 2:1 supramolecular compound in which N--O(.)I halogen bonding is the dominant intermolecular interaction. This illustrates the potential use of halogen bonding and isoindoline nitroxide tectons for the assembly of organic spin systems.The isoindoline nitroxide 1,1,3,3-tetramethylisoindolin-2-yloxyl (TMIO) and 1,4-diiodotetrafluorobenzene readily form a discrete 2:1 complex that shows evidence of relatively strong N--O(.)I halogen bonding. This interaction was characterized in the solid state by single-crystal X-ray analysis, thermal analysis, and vibrational spectroscopy (IR and Raman), backed by density functional theory calculations. EPR spectroscopy performed on a solution of TMIO in pentafluoroiodobenzene, a halogen-bonding donor, indicates that halogen bonding induces an increase in electron density at the nitroxide nitrogen nucleus and an increase in the nitroxide rotational correlation time. Our findings demonstrate the potential of utilizing halogen-bonding interactions to promote the self-assembly of new isoindoline nitroxide tectons for the preparation of organic spin systems.

Micro-attenuated total reflection spectral imaging in archaeology: application to Maya paint and plaster wall decorations.

Fourier transform infrared (FT-IR) attenuated total reflection (ATR) imaging has been successfully used to identify individual mineral components of ancient Maya paint. The high spatial resolution of a micro FT-IR-ATR system in combination with a focal plane array detector has allowed individual particles in the paint to be resolved and identified from their spectra. This system has been used in combination with micro-Raman spectroscopy to characterize the paint, which was found to be a mixture of hematite and silicate particles with minor amounts of calcite, carbon, and magnetite particles in a sub-micrometer hematite and calcite matrix. The underlying stucco was also investigated and found to be a combination of calcite with fine carbon particles, making a dark sub-ground for the paint.