Development of New Dermato-Cosmetic Therapeutic Formulas with Extracts of Vinca minor L. Plants from the Dobrogea Region.

A new trend in the use of indole alkaloids from natural products is the preparation of topical pharmaceutical formulations with applications in the field of regenerative medicine. These formulations can be characterized through the ease of administration, the proven healing action of indole alkaloids, the protection of skin lesions, and the assurance of oxygen permeability. Based on the numerous benefits that indole compounds extracted from the Vinca minor plant show externally, the purpose of this study was to develop new semi-solid biocomposites for topical application obtained from hydroalcoholic macerates of 40%, 70%, and 96% concentrations from the stems and leaves of the Vinca minor L. plant from the Dobrogea area. A total of 12 pharmaceutical formulations (named P1-P12) were prepared for which the physicochemical properties, pH, thermal stability, spreading capacity, and rheological behavior were determined. The optimal formulas with antioxidant and antimicrobial capacity were evaluated and determined (P3, P4, P9, and P10). Antioxidant activity was elicited using the photochemiluminescence method. The microorganisms used for the evaluation of antimicrobial activity were Gram-positive Staphylococcus aureus (ATCC 25923), Gram-negative Escherichia coli (ATCC 25922), and a fungal species, Candida albicans (ATCC 900288). The study of the rheological profile for the obtained composites revealed Newtonian, pseudoplastic, and thixotropic fluid behaviors. Following determinations using the photochemiluminescence method, the best antioxidant activity was obtained in the P3 and P9 preparations. The results of the antimicrobial analysis confirmed that both the leaves and the stems of the Vinca minor plant represent a valuable source of antibacterial substances, and the biocomposites analyzed may represent an alternative in the realization of new pharmaceutical preparations with topical applications based on hydroalcoholic macerates obtained from the Vinca minor plant.

Biogenic synthesis of gold nanoparticles using dual extract of tulsi-Vinca for breast cancer tumor regression in mice.

Aim: This work aims to synthesize the gold nanoparticles (GNPs) using a dual extract of tulsi and Vinca (T+V-Gold) for breast cancer tumor regression. Methods: The GNPs were synthesized and characterized for their microscopic, spectroscopic and crystalline properties. Further, the GNPs were investigated for in vitro and in vivo studies for the treatment of the 4T1-induced triple-negative breast cancer murine model. Results: The GNPs for 4T1 tumor-challenged mice resulted in delayed tumor development and lower tumor burden, with T+V-Gold demonstrating the highest prevention of tumor spread. The antitumor effect of T+V-Gold is highly significant in the glutathione family antioxidants glutathione S-transferase and glutathione in tumor tissue samples. Conclusion: The bioefficacy and anticancer outcomes of T+V-Gold nanoformulation can be used as therapeutic agents and drug-delivery vehicles.

Unveiling novel RNA viruses in trematodes parasitizing common periwinkle: Implications for host-parasite interactions.

This study characterized novel RNA viruses, parasites of parasites, or hyperparasites identified during transcriptomic analyses of two trematode species, Cryptocotyle lingua and Himasthla elongata, infecting a sea snail, Littorina littorea. According to the viral genome structures and phylogenetic analysis, Cryptolin alternavirus (ClRNAV1), Cryptolin calicivirus (ClRNAV2) and Himastelon rhabdovirus 1 (HeRNAV1) were respectively classified within the families Alternaviridae, Caliciviridae and Rhabdoviridae. They replicate species-specifically in two studied phases of trematode live cycle: intramolluscan parthenogenetic rediae and free-swimming cercariae. ClRNAV1 showed significantly higher expression in C. lingua cercariae relative to rediae. HeRNAV1's similarity to rabies viruses raises questions about its potential effects on the nervous system of H. elongata. This 'trematode rabies' could enable the use of genetically modified viruses for developing new methods to control the spread and intensity of diseases caused by trematodes.

Phytochemical Analysis and In Vitro Antileukemic Activity of Alkaloid-Enriched Extracts from Vinca sardoa (Stearn) Pignatti.

Vinca sardoa (Stearn) Pignatti, known as Sardinian periwinkle, is widely diffused in Sardinia (Italy). This species contains indole alkaloids, which are known to have a great variety of biological activities. This study investigated the antileukemic activity against a B lymphoblast cell line (SUP-B15) of V. sardoa alkaloid-rich extracts obtained from plants grown in Italy, in Iglesias (Sardinia) and Rome (Latium). All the extracts showed a good capacity to induce reductions in cell proliferation of up to 50% at the tested concentrations (1-15 µg/mL). Moreover, none of the extracts showed cytotoxicity on normal cells at all the studied concentrations.

Determination of Active Ingredients, Mineral Composition and Antioxidant Properties of Hydroalcoholic Macerates of Vinca minor L. Plant from the Dobrogea Area.

In recent decades, new alternative therapies using drugs containing active ingredients of natural origin have been a hot topic for medical research. Based on the confirmed therapeutic potential of the Vinca minor plant, considered in the specialized literature to be of pharmaceutical interest, the purpose of this study is to determine the chemical and mineral composition of the Vinca minor plant grown in the Dobrogea area, with a view to its use in the formulation of dermal preparations. For this purpose, plant materials were collected from the mentioned area and hydroalcoholic macerates of different concentrations were obtained: 40%, 70% and 96% from leaves (F40, F70, F96) and stems (T40, T70, T96) of Vinca minor plant to determine the optimal extraction solvent. The hydroalcoholic macerates were analyzed via the HPLC method for the identification and quantification of the main bioactive compounds, and two methods were used to evaluate their antioxidant properties: the DPPH radical scavenging test and the photochemiluminescence method. HPLC analysis showed the presence of four indole alkaloids: vincamine, 1,2-dehydroaspidospermidine, vincaminoreine and eburnamonine. Vincamine was the alkaloid found in the highest concentration in Vinca leaves (2.459 ± 0.035 mg/100 g d.w.). The antioxidant activity of Vinca minor hydroalcoholic macerates showed values between 737.626-1123.500 mg GAE/100 g d.w (DPPH test) and 77.439-187.817 mg TE/100 g d.w (photochemiluminescence method). The concentrations of toxic metals Cd, Cu, Ni, Pb in dried leaves and stems of Vinca minor, determined by AAS, were below detection limits.

Development of PSA@PS-TiO2 nanocomposite photocatalyst: structure, mechanism, and application using response surface designs and molecular modeling.

Using periwinkle shell ash (PSA) and polystyrene (PS), a new-fangled PSA@PS-TiO2 photocatalyst was fabricated. The morphological images of all the samples studied using a high-resolution transmission electron microscope (HR-TEM) showed a size distribution of 50-200 nm for all samples. The SEM-EDX showed that the membrane substrate of PS was well dispersed, confirming the presence of anatase/rutile phases of TiO2, and Ti and O2 were the major composites. Given the very rough surface morphology (atomic force microscopy (AFM)) due to PSA, the main crystal phases (XRD) of TiO2 (rutile and anatase), low bandgap (UVDRS), and beneficial functional groups (FTIR-ATR), the 2.5 wt.% of PSA@PS-TiO2 exhibited better photocatalytic efficiency for methyl orange degradation. The photocatalyst, pH, and initial concentration were investigated and the PSA@PS-TiO2 was reused for five cycles with the same efficiency. Regression modeling predicted 98% efficiency and computational modeling showed a nucleophilic initial attack initiated by a nitro group. Therefore, PSA@PS-TiO2 nanocomposite is an industrially promising photocatalyst for treating azo dyes, particularly, methyl orange from an aqueous solution.

Dietary Exposure to Particles of Polytetrafluoroethylene (PTFE) and Polymethylmethacrylate (PMMA) Induces Different Responses in Periwinkles Littorina brevicula.

The marine and ocean water pollution with different-sized plastic waste poses a real threat to the lives of the next generations. Plastic, including microplastics, is found in all types of water bodies and in the organisms that live in them. However, given the chemical diversity of plastic particles, data on their toxicity are currently incomplete. Moreover, it is clear that different organisms, depending on their habitat and feeding habits, are at different risks from plastic particles. Therefore, we performed a series of experiments on feeding the gastropod scraping mollusk Littorina brevicula with two types of polymeric particles-polymethylmethacrylate (PMMA) and polytetrafluoroethylene (PTFE)-using a special feeding design. In the PMMA-exposed group, changes in gastrointestinal biochemical parameters such as increases in malondialdehyde (MDA) and protein carbonyls (PC) were detected, indicating the initiation of oxidative stress. Similarly, a comet assay showed an almost twofold increase in DNA damage in digestive gland cells compared to the control group. In mollusks fed with PTFE-containing food, no similar changes were recorded.

Bio-evaluation of Untapped Alkaloids from Vinca minor Enriched by Methyl-jasmonate-induced Stress: an Integrated Approach.

The low amount of metabolites isolated from natural products is one of the challenges preventing their biological evaluation. The modulation of biosynthetic pathways by stimulating stress-induced responses in plants was proven to be a valuable tool for diversification of already known natural products. Recently, we reported the dramatic effect of methyl jasmonate (MeJA) on Vinca minor alkaloids distribution. In this study, three compounds identified as 9-methoxyvincamine, minovincinine, and minovincine are successfully isolated in good yield and subjected to several bioassays based on a network pharmacology study. The extracts and isolated compounds show weak to moderate antimicrobial and cytotoxic activities. Also, they are found to significantly promote wound healing in scratch assay, and transforming growth factor-ß (TGF-ß) modulation is suggested to be the potential pathway based on bioinformatic analysis. Hence, Western blotting is used to assess the expression of several markers related to this pathway and wound healing. The extracts and isolated compounds are able to increase the expression of Smad3 and Phosphatidylinositol-3-kinase (PI3K), while downregulating the levels of cyclin D1 and the mammalian target of rapamycin (mTOR) except for minovincine, which increases the mTOR expression, inferring that it might act through a different mechanism. Molecular docking is used to give insights on the ability of isolated compounds to bind with different active sites in mTOR. Collectively, the integrated phytochemical, in silico, and molecular biology approach reveal that V. minor and its metabolite could be repurposed for the management of dermatological disorders where these markers are dysregulated, which opens the gate to develop new therapeutics in the future.

Mercury Exposure and Habitat Fragmentation Affect the Movement, Foraging Behavior, and Search Efficiency of the Marsh Periwinkle (Littorina irrorata).

The interactions between habitat fragmentation and other stressors are considered a key knowledge gap. The present study tested the hypotheses that mercury enhances the effects of fragmentation by (1) reducing the cumulative daily movement of organisms, (2) shifting their foraging behavior, and (3) altering the vertical movement of the marsh periwinkle (Littoraria irrorata) in a field experiment. Random walk simulations were used to access how changes in movement affect the search efficiency of organisms in the long term. Eighteen 1.5 m2 plots were constructed in a salt marsh where landscapes characteristics were manipulated to reach three different levels of habitat cover. Daily movement of 12 marked control and mercury-exposed snails were measured in each plot. Bayesian models were used to analyze the data and evidence ratios were used to test the hypotheses. The results showed that the effects of fragmentation were consistent in both control and exposed treatments, with an increase in the cumulative displacement of organisms. However, mercury significantly reduced the movement of organisms in all levels of fragmentation, shifting their foraging behavior (evidence ratio > 1000). Exposed snails were more likely to be found inactive in comparison with the control treatment (evidence ratio > 1000). Fragmentation also reduced the vertical movement of organisms in both treatments. In contrast, mercury increased the vertical movement of organisms (evidence ratio > 1000). The search efficiency of organisms also increased in a highly fragmentated landscape, suggesting that changes in foraging behavior are likely due to reduced resources and consequently increase in foraging effort. The present study shows that mercury exposure can enhance the effects of habitat fragmentation by changing organisms' movement, foraging behavior, and search efficiency. Environ Toxicol Chem 2023;42:1971-1981. © 2022 SETAC.

Degradation of micro-nano-sized polytetrafluoroethylene and acrylic fluorinated copolymer particles in the periwinkle digestive tract.

The aim of the work was to investigate the possible biodegradation of such stable plastics as fluorated acrylic copolymer Protacryl-M and polytetrafluoroethylene-teflon (PTFE)-that are widely applied in medicine for prosthetics in dentistry and orthopedics. Our tasks were as follows: 1. To select a suitable marine biological object (multicellular invertebrate) 2. To develop a method for delivering microplastic particles (MP) of selected plastics to the gastrointestinal tract (GIT) of an experimental animal 3. To develop a technique for separating MP particles from animal feces 4. To develop a technology for multiple passages of MP particles through the gastrointestinal tract of an animal 5. To select methods and techniques for determining the degradation of the surface of MP particles after passing through the gastrointestinal tract of the animal The effect of a biological agent on the types of plastic above is considered using marine gastropods of the genus Littorina (in particular, the periwinkle L. brevicula) as a model organism widely distributed in the marine intertidal zone.

Structural and vibrational investigations and molecular docking studies of a vinca alkoloid, vinorelbine.

Vinorelbine, a vinca alkaloid, is an antimitotic drug that inhibits polymerisation process of tubulins to microtubules, and is widely used in cancer chemotherapy. Due to the importance of the structure-activity relationship, in this work the conformational preferences of the vinorelbine molecule were surched by PM3 method. The obtained lowest energy conformer was then optimized at DFT/B3LYP/6-31G(d,p) level of theory and the structural characteristics were determined. Frontier orbital (HOMO, LUMO) and molecular electrostatic potential (MEP) analyses were performed for the optimized structure. The experimental FT-IR, Raman and UV-VIS spectral data of vinorelbine along with the theoretical DFT/B3LYP/6-31G(d,p) calculations were investigated in detail. The vibrational wavenumbers were assigned based on the calculated potential energy distribution (PED) of the vibrational modes. To shed light into the anticancer property of vinorelbine as microtubule destabilizer, the most favourable binding mode and the interaction details between vinorelbine and tubulin were revealed by molecular docking studies of vinorelbine into the α,ß-tubulin (PDB IDs: 4O2B; 1SA0; 7CNN) and binding free energies were calculated by the combination of Molecular Mechanics/Generalized Born Surface Area (MMGBSA) and Molecular Mechanics/Poisson-Boltzmann Surface Area (MM-PBSA) methods {MM/PB(GB)SA}. The calculated vinorelbine-7CNN binding free energy, using by MM/PB(GB)SA approach, was found to be the best (-50.39 kcal/mol), and followed by vinorelbine-4O2B (-28.5 kcal/mol) and vinorelbine-1SA0 (-17.59 kcal/mol) systems. Moreover, the interaction of vinorelbine with the cytochrome P450 enzymes (CYP), which are known to help in the metabolism of many drugs in the body, was investigated by docking studies against CYP2D6 and CYP3A4 targets.Communicated by Ramaswamy H. Sarma.

The Vinca minor genome highlights conserved evolutionary traits in monoterpene indole alkaloid synthesis.

Vinca minor, also known as the lesser periwinkle, is a well-known species from the Apocynaceae, native to central and southern Europe. This plant synthesizes monoterpene indole alkaloids, which are a class of specialized metabolites displaying a wide range of bioactive- and pharmacologically important properties. Within the almost 50 monoterpene indole alkaloids it produces, V. minor mainly accumulates vincamine, which is commercially used as a nootropic. Using a combination of Oxford Nanopore Technologies long read- and Illumina short-read sequencing, a 679,098 Mb V. minor genome was assembled into 296 scaffolds with an N50 scaffold length of 6 Mb, and encoding 29,624 genes. These genes were functionally annotated and used in a comparative genomic analysis to establish gene families and to investigate gene family expansion and contraction across the phylogenetic tree. Furthermore, homology-based monoterpene indole alkaloid gene predictions together with a metabolic analysis across 4 different V. minor tissue types guided the identification of candidate monoterpene indole alkaloid genes. These candidates were finally used to identify monoterpene indole alkaloid gene clusters, which combined with synteny analysis allowed for the discovery of a functionally validated vincadifformine-16-hydroxylase, reinforcing the potential of this dataset for monoterpene indole alkaloids gene discovery. It is expected that access to these resources will facilitate the elucidation of unknown monoterpene indole alkaloid biosynthetic routes with the potential of transferring these pathways to heterologous expression systems for large-scale monoterpene indole alkaloid production.

Thermal Traits Vary with Mass and across Populations of the Marsh Periwinkle, Littoraria irrorata.

AbstractPhysiological processes influence how individuals perform in various environmental contexts. The basis of such processes, metabolism, scales allometrically with body mass and nonlinearly with temperature, as described by a thermal performance curve. Past studies of thermal performance curves tend to focus on effects of temperature on a single body size or population, rather than variation in the thermal performance curve across sizes and populations. Here, we estimate intraspecific variation in parameters of the thermal performance curve in the salt marsh gastropod Littoraria irrorata. First, we quantify the thermal performance curve for respiration rate as a function of both temperature and body size in Littoraria and evaluate whether the thermal parameters and body size scaling are interdependent. Next, we quantify how parameters in the thermal performance curve for feeding rate vary between three Littoraria populations that occur along a latitudinal gradient. Our work suggests that the thermal traits describing Littoraria respiration are dependent on body mass and that both the thermal traits and the mass scaling of feeding vary across sites. We found limited evidence to suggest that mass scaling of Littoraria feeding or respiration rates depends on temperature. Variation in the thermal performance curves interacts with the size structure of the Littoraria population to generate divergent population-level responses to temperature. These results highlight the importance of considering variation in population size structure and physiological allometry when attempting to predict how temperature change will affect physiological responses and consumer-resource interactions.

Agrobacterium-Mediated in Planta Transformation in Periwinkle.

Madagascar periwinkle (Catharanthus roseus, family Apocynaceae) is a reservoir of more than 130 monoterpene indole alkaloids (MIAs) including the famous anti-neoplastic dimeric MIAs vinblastine and vincristine, and anti-hypertensive monomeric MIAs ajmalicine and serpentine. Understanding the biosynthetic steps and regulatory factors leading to the formation of MIAs is crucial for rational engineering to achieve targeted enhancement of different MIAs. Due to its highly recalcitrant nature, C. roseus is considered genetically non-tractable for transformation at the whole-plant level. Though few reports have demonstrated tissue culture-mediated regeneration and transformation of C. roseus at whole-plant level recently, the efficiency and reproducibility of these protocols have been a major challenge. To overcome this, we have developed a tissue-culture-independent Agrobacterium-mediated in planta transformation method in C. roseus. Using this method, we were able to efficiently generate stable transgenic plants without relying on the cumbersome methods of tissue-culture regeneration and transformation. Moreover, the transformed plants obtained through this in planta method exhibited stability in subsequent generations. Our method is useful not only for the elucidation of biosynthetic and regulatory steps involved in MIA formation through transgenic plant approach but also for metabolic engineering at the whole-plant level in C. roseus.

Structural convergence for tubulin binding of CPAP and vinca domain microtubule inhibitors.

Microtubule dynamics is regulated by various cellular proteins and perturbed by small-molecule compounds. To what extent the mechanism of the former resembles that of the latter is an open question. We report here structures of tubulin bound to the PN2-3 domain of CPAP, a protein controlling the length of the centrioles. We show that an α-helix of the PN2-3 N-terminal region binds and caps the longitudinal surface of the tubulin ß subunit. Moreover, a PN2-3 N-terminal stretch lies in a ß-tubulin site also targeted by fungal and bacterial peptide-like inhibitors of the vinca domain, sharing a very similar binding mode with these compounds. Therefore, our results identify several characteristic features of cellular partners that bind to this site and highlight a structural convergence of CPAP with small-molecule inhibitors of microtubule assembly.

Joint Effects of Fragmentation and Mercury Contamination on Marsh Periwinkle (Littoraria irrorata) Movement.

There are different ways contaminants can interact and enhance the effects of habitat fragmentation, such as modifying the movement of organisms. The present study tested the hypothesis that mercury exacerbates the effects of fragmentation by affecting the movement of the marsh periwinkle Littoraria irrorata and reducing the probability of snails crossing fragmented microlandscape experimental systems. How these changes could affect the search efficiency of organisms in the long term was assessed using hidden Markov models and random walks simulations. Bayesian nonlinear models were used to analyze the effects of fragmentation and contamination on the mean speed and mean directional change of organisms. Snail movement for control and two mercury-exposure treatments were recorded in microlandscapes with six different levels of habitat cover and three landscape replicates. The results indicated that exposed organisms had lower probabilities of crossing the landscape, reduced speed, and shifts in step length distributions. Both mercury exposure and habitat fragmentation affected the movement of the marsh periwinkle. Mercury exacerbated the effects of habitat fragmentation by affecting the cognition (e.g., route planning, orientation, and spatial learning) and movement of L. irrorata. Hence, the interaction of these stressors could further reduce the functional connectivity of landscapes and reduce the search efficiency of organisms. Environ Toxicol Chem 2022;41:1742-1753. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Detecção de 'CA. Phytoplasma phoenicium' em plantas de pervinca (Cathranthus roseus) em Uttar Pradesh, Índia

Cathranthus roseus also known as periwinkle, an ornamental plant contains several medicinal values, was found with the symptoms of little leaf and witches' broom at Shahjahanpur location with the incidence of up to 8%. The phytoplasma etiology was confirmed through scanning electron microscopy examination in all the four-leaf samples. Molecular analysis through PCR with universal primer pairs P1/P6 followed by nested PCR with R16F2n/R16r2 primers yielded ~1.2kbp amplicons in all the four symptomatic leaf samples. One amplicon was eluted, purified, sequenced, and used in BLASTn searches, which showed maximum identity of periwinkle isolate with several isolates of 16SrIX group of phytoplasma. Further, phylogenic analysis and in silico RFLP confirmed the association of 16SrIX-C subgroup phytoplasma in little leaf and witches broom plants which is the first report from India.

Fluorometric quantification of alkaloids in the homeopathic mother tinctures of Vinca minor L. and Fumaria officinalis L.

To assess the toxic potential of the alkaloids, a quantification method is necessary. An ion pair extraction method was used for quantitative fluorometric determination of vincamine, protopine and all contained alkaloids in the mother tinctures of Vinca minor and Fumaria officinalis. The non-fluorescent alkaloids were transformed into an ion pair with sodium-9,10-dimethoxy-anthracene-sulfonate and then fluorometrically determined and quantified in this study. The applicable ion pair was extracted in a suitable organic solvent, where dichloromethane has proven to be beneficial. Conditions for the ion pairing and fluorometric quantification are given. The recovery rate was used to investigate the quality of determinability and the influence of the mother tincture matrix. The method was applied to determine the concentration of protopine in the range 0.1 - 15 µg/ml and of vincamine in the range of 0.5 - 20 µg/ml. The limit of detection was < 0.3 µg/ml, and the limit of quantification < 0.9 µg/ml for both alkaloids.

Monoterpene indole alkaloids from Vinca minor L. (Apocynaceae): Identification of new structural scaffold for treatment of Alzheimer's disease.

One undescribed indole alkaloid together with twenty-two known compounds have been isolated from aerial parts of Vinca minor L. (Apocynaceae). The chemical structures of the isolated alkaloids were determined by a combination of MS, HRMS, 1D, and 2D NMR techniques, and by comparison with literature data. The NMR data of several alkaloids have been revised, corrected, and missing data have been supplemented. Alkaloids isolated in sufficient quantity were screened for their in vitro acetylcholinesterase (AChE; E.C. 3.1.1.7) and butyrylcholinesterase (BuChE; E.C. 3.1.1.8) inhibitory activity. Selected compounds were also evaluated for prolyl oligopeptidase (POP; E.C. 3.4.21.26), and glycogen synthase 3ß-kinase (GSK-3ß; E.C. 2.7.11.26) inhibition potential. Significant hBuChE inhibition activity has been shown by (-)-2-ethyl-3[2-(3-ethylpiperidinyl)-ethyl]-1H-indole with an IC50 value of 0.65 ± 0.16 µM. This compound was further studied by enzyme kinetics, along with in silico techniques, to reveal the mode of inhibition. This compound is also predicted to cross the blood-brain barrier (BBB) through passive diffusion.

Of herds and societies-Seasonal aspects of Vinca culture herding and land use practices revealed using sequential stable isotope analysis of animal teeth.

Late Neolithic Vinca communities, spread over much of central and northern Balkans during the late sixth to mid-fifth millennium BC and characterised by unusually large and densely population centres, would have required highly organised food production systems. Zooarchaeological analysis indicates that domesticate livestock were herded, but little is known about the seasonal husbandry practices that helped ensure a steady supply of animal products to Vinca farming communities. Here, we present new stable carbon (δ13C) and oxygen (δ18O) isotopic measurements of incremental bioapatite samples from the teeth of domesticated livestock and wild herbivore teeth from two late Neolithic Vinca culture sites: Vinca-Belo brdo and Stubline (Serbia). Our results show a low variation overall within sheep and goats in terms of pasture type that may have been composed of seasonal halophyte plant communities, which have higher δ13C values due to the saline rich growing environments. Cattle feeding strategies were more variable and provided with supplementary forage, such as cut branches or leafy hay, during winter. The sharp distinction in the management of cattle and sheep/goat may be associated with the development of herding strategies that sought to balance livestock feeding behaviours with available forage or, more provocatively, the emergence of household-based control over cattle-an animal that held a central economic and symbolic role in Vinca societies.