Search Results (135)

Almond decline and dieback have become significant challenges in newly established orchards, with symptoms including internal necrosis, canker, and external gummosis. This work aims to explore the potential fungal and bacterial causative agents through metabarcoding and traditional culture plate isolation across six almond cultivars. Our results emphasize the multifactorial nature of almond decline and dieback, with possible co-infections by opportunistic fungi and bacteria playing a central role. Classical isolation identified 47 fungal species or genera, including Diaporthe amygdali, Diplodia corticola, Phytophthora sp., and several Fusarium species. Almond metabarcoding revealed a more diverse microbial community, highlighting the prevalence of soilborne pathogens such as Neocosmospora rubicola, Dactylonectria estremocensis, and Plectosphaerella niemeijerarum. Soil metabarcoding suggested that these pathogens likely originate from nursery substrates or soils shared with other crops, such as olives and vineyards, that serve as a source of inoculum. ‘Soleta’ generally presented lower richness when compared to the other tested cultivars, suggesting a higher degree of biotic stress and decreased plant resilience. This study highlights the value of integrating NGS approaches to comprehensively study complex diseases and the need for further research on pathogen interactions and cultivar susceptibility for the future development of new sustainable, targeted management strategies in almond orchards. Full article

Gastrointestinal (GI) parasitic infections in non-human primates are of growing concern due to their implications for both veterinary and public health. Long-tailed macaques (Macaca fascicularis), commonly found in peri-urban and temple environments in Southeast Asia, may act as reservoirs for zoonotic parasites, posing risks to humans and domestic animals. This study investigated the prevalence and species diversity of GI parasites in free-ranging macaques from four provinces in Northeast Thailand (Loei, Khon Kaen, Bueng Kan, and Sisaket). A cross-sectional study was conducted between April and May 2025. A total of 445 fecal samples were examined using two parasitological techniques: agar plate culture (APC) and the formalin–ethyl acetate concentration technique (FECT). The overall prevalence of parasitic infection was 86.5%, with Strongyloides sp. (65.2%) as the most prevalent helminth and Balantioides coli-like (29.5%) and Entamoeba histolytica-like (28.8%) as the predominant protozoa. Other parasites identified included helminths (Trichuris sp., Ascaris sp.) and protozoa (Blastocystis sp., Iodamoeba bütschlii, Entamoeba coli, and Chilomastix mesnili). Mixed infections were frequently observed, with both helminths and protozoa co-occurring in 37.3% of cases. The high infection rates and parasite diversity reflect substantial environmental contamination and sustained transmission cycles. These findings underscore the importance of integrated surveillance in wildlife populations and the need for One Health-based approaches to minimize zoonotic transmission risks at the human–animal–environment interface. Full article

The global market size of animal parasiticides was valued at USD 12.9 billion in 2024. Animal deworming only results in temporary cures with little to no preventive effects; therefore, a strategy that combines animal deworming with prevention is essential in improving the control of helminths. The effectiveness of co-administrating curative and preventive agents and their compatibility were considered based on the parasitophagous fungus Mucor circinelloides, which was developed in edible agar–agar (red seaweed)-carrying dewormers. Accordingly, Petri dishes were prepared with either a biopolymer alone (control, G-C) or with the anthelmintic piperazine (550, 1102, 2210, and 5500 mg/plate) or levamisole (37.5, 75, 150, and 300 mg/plate) and were used to culture the fungus Mucor circinelloides. Strong fungal growth and high numbers of spores were observed in the presence of the anthelmintics. No differences were measured between the control plates and those containing parasiticide drugs. Similar mycelial growth patterns and sporogenesis rates were recorded for different amounts of each anthelmintic. In conclusion, this novel formulation based on biopolymers containing anthelmintics and enriched with the parasitophagous fungus represents a highly promising tool to consider for jointly deworming animals and minimizing the risks of helminth infection. Further studies are in progress to confirm these in vitro results. Full article

Poloxamer (P) 188 attenuates myocardial ischemia/reperfusion injury through cell membrane stabilization. Cell–cell interactions between endothelial cells (ECs) and cardiomyocytes (CMs) further protect CMs: co-cultures showed that, at an optimal density, ECs protected CMs against hypoxia/reoxygenation (HR) injury. The mechanism of interaction with P188 still requires exploration. We examined if N(ω)-nitro-L-arginine methyl ester (LNAME), a non-specific nitric oxide (NO) synthase inhibitor, abolishes protection in the presence or absence of P188 and/or ECs. We co-cultured mouse coronary artery ECs in an insert atop mouse CMs plated at confluency on the bottom of a well. Normoxic controls remained in complete media while HR groups were exposed to 24 h hypoxia at 0.01% O₂ in serum- and glucose-free media, followed by 2 h reoxygenation in complete media. P188 (300 μM), LNAME (40 mM), or vehicle were administered upon reoxygenation. ECs at the used lower density did not decrease HR-triggered lactate dehydrogenase release or calcium overload in CMs by themselves. P188 reduced both indicators after HR by 16/18% without and by 22/25% with ECs, respectively. LNAME abrogated CM protection by P188. Neither intervention had an effect under normoxia. Our co-culture data indicates that P188 requires NO, not necessarily of endothelial origin, to elicit CM protection. Full article

Three-dimensional (3D) cell culture models are widely used to provide a more physiologically relevant microenvironment in which to host and study desired cell types. These models vary in complexity and cost, ranging from simple and inexpensive to highly sophisticated and costly systems. In this study, we introduce a novel translucent multi-compartmentalized stacked multilayered nanocellulose scaffold and describe its fabrication, characterization, and potential application for co-culturing multiple cell types. The scaffold consists of bacterial nanocellulose (BNC) layers separated by interlayers of a lower density of nanocellulose fibers. Using this system, we co-cultured the MDA-MB-231 cell line with two tumor-associated cell types, namely BC-CAFs and M2 macrophages, to simulate the tumor microenvironment (TME). Cells remained viable and metabolically active for up to 15 days. Confocal microscopy showed no signs of cell invasion. However, BC-CAFs and MDA-MB-231 cells were frequently observed within the same layer. The expression of breast cancer-related genes was analyzed to assess the downstream functionality of the cells. We found that the E-cadherin expression was 20% lower in cancer cells co-cultured in the multi-compartmentalized scaffold than in those cultured in 2D plates. Since E-cadherin plays a critical role in preventing the initial dissociation of epithelial cells from the primary tumor mass and is often downregulated in the tumor microenvironment in vivo, this finding suggests that our scaffold more effectively recapitulates the complexity of a tumor microenvironment. Full article

The International Agency for Research on Cancer has studied and classified 1045 potential substances. It is therefore important to develop rapid screening methods to identify the mutagenicity of compounds and, further on, the intensity and number of individual mutagenic substances in complex sample mixtures. The current in vitro Ames assay in the microtiter plate format (MPF) uses a pH-sensitive detection as endpoint, however, acidic substances in complex mixtures may interfere the mutagenicity result. Hence, it was transferred to the planar assay format to be more selective for complex mixture testing. The co-culture of Salmonella Typhimurium strains TA98 and TA100 with an optical density of 0.4 at 600 nm was applied on a high-performance thin-layer chromatography silica gel 60 chromatogram and on-surface incubated for 5 h, which period was limited due to zone diffusion. Various positive controls were tested, and 4-nitrochinolin-N-oxide with a limit of detection of 100 ng was established as a positive control. However, due to the shorter incubation time, no mutagenic compounds were detectable or differentiable in the tested perfumes, herbal teas, margarines, and hand creams. This does not mean that the samples are mutagen-free, but it suggests that further improvements to the bioassay are urgently needed to increase the sensitivity and selectivity of the response. Compared to conventional sum value assays, a planar Ames assay performed on the separated and adsorbed sample components advances toxicology research because mutagenic compounds are separated from interfering molecules due to the integrated separation. It thus would provide a more selective detection of mutagens in complex mixtures and allow testing of large sample volumes or concentrated samples without matrix interference. Full article

Biofilms are emerging platforms for the production of valuable compounds. The present study is the first to assess the capacity of Escherichia coli biofilms to produce curcumin through the expression of a biosynthetic pathway involving three genes: 4-coumarate-CoA ligase (4CL), diketide-CoA synthase (DCS), and curcumin synthase (CURS). The effects of chemical induction with isopropyl β-d-1-thiogalactopyranoside (IPTG) and ferulic acid (FA), and the incubation temperature on biofilm formation and curcumin production were evaluated. Biofilms were formed in 12-well microtiter plates over three days and then induced with 1 mM IPTG and FA at 2 or 8 mM. After induction, the samples were incubated for two days at 26 or 30 °C. Total and culturable planktonic and biofilm cells, as well as biofilm thickness and volumetric and specific curcumin production, were assessed on days 3, 4, and 5. The results demonstrated that biofilms produced up to 10-fold higher curcumin levels (0.9–2.2 fg·cell⁻¹) than their planktonic counterparts (0.1–0.3 fg·cell⁻¹). The highest specific curcumin production (2.2 fg·cell⁻¹) was achieved using 8 mM FA. However, no significant differences in curcumin production were observed between the induced samples incubated at the tested temperatures. These results validated the potential of biofilm systems for expressing a complete exogenous biosynthetic pathway using metabolic engineering, particularly for curcumin production. Full article

Healthcare organisations in the United Kingdom must comply with national standards for food and drink, including sustainable sourcing and minimisation, mitigation, and management of food waste. Despite this, an estimated one in six plates of food served in hospitals are wasted daily, producing 12% of the UK’s food waste, equating to 6% of carbon dioxide emissions (CO₂e) nationally, and a waste-management cost of GBP 230 m per annum. Within healthcare, there is a move towards the implementation of “plant-based diets by default” to reduce the environmental impact, improve nutritional outcomes, and reduce costs. However, plant-based diets are often perceived as being difficult to prepare by caterers, less enjoyable, and potentially resulting in more food waste. We conducted a scoping review to examine the influence of the social, medical, and physical environment on food intake during inpatient admission to a mental health hospital. Fourteen studies were included. We identified five critical knowledge areas: (i) food and socio-cultural environment, (ii) evidence-based measures and strategies to reduce food waste, (iii) economic food environment, (iv) inevitability of weight gain, and (v) applications of theoretical models for behaviour change. Future research should explore the development of a behaviour-change framework inclusive of training, education, and goal-setting components for staff, patients, and visitors. Full article

Background/Objectives: Numerous intrinsic and extrinsic stressors can disrupt the balance of the skin microbiome, leading to the development of various skin diseases. It has been proven that coagulase-negative staphylococci (CoNS) are important commensals for maintaining skin microbiome homeostasis and fighting cutaneous pathogens such as Staphylococcus aureus (S. aureus). Here, we examined the influence of polyphenol-rich enzymatic blackcurrant extract (EBCE) on pathogenic coagulase-positive S. aureus strains and beneficial CoNS, like Staphylococcus epidermidis (S. epidermidis), to explore its potential for rebalancing the skin microbiota. Methods: The polyphenol profile of EBCE was determined by ultra-high-pressure liquid chromatography–tandem mass spectrometry. Microwell plate assays were employed to study the effect of EBCE on five S. aureus strains isolated from the skin of atopic dermatitis patients. An in vitro human stratum corneum model was used to test its effect on mixed bacterial cultures. Results: EBCE inhibited the growth of all tested S. aureus strains by 80–100% at the highest tested concentration after 7 h. No microbial growth was observed at the highest tested EBCE concentration using the stratum corneum model inoculated with one selected pathogen (S. aureus SA-DUS-017) and one commensal laboratory strain (S. epidermidis DSM 20044). The lowest tested concentration did not interfere with S. aureus growth but strongly stimulated the growth of S. epidermidis (~300-fold colony forming unit increase). In addition, low EBCE concentrations strongly stimulated CoNS growth in microbiome samples taken from the armpits of healthy volunteers that were spiked with S. aureus SA-DUS-017. Conclusions: These preclinical data support further testing of EBCE-enriched topical preparations as potential cutaneous prebiotics in human studies. Full article

Background: Poultry litter is the main waste of poultry farming and is widely used as an agricultural fertilizer. However, owing to the use of antimicrobials in animal production, it can accumulate antimicrobial residues, antimicrobial-resistant bacteria (ARB), and antimicrobial resistance genes (ARGs). This study aimed to evaluate the impact of poultry litter use on the microbiome and resistome of agricultural soils. Methods: Soil samples from fertilized and unfertilized plots were collected from two horticultural farms that intensively use poultry litter. Microbiome composition was assessed using 16S rRNA sequencing. A culture-dependent method was used to isolate resistant strains on CHROMagar plates supplemented with sulfamethoxazole or ciprofloxacin. ARGs and integrase-encoding genes were identified by PCR. Results: Microbiome analysis revealed significant differences in structure and composition between poultry litter-fertilized and unfertilized soils. Fertilized soils exhibited greater alpha diversity and richness. Bacillota, commonly found in the avian gastrointestinal tract, were more abundant in fertilized soils. A total of 62 resistant strains were isolated, and 23 clinically relevant strains harbored ARGs, including fluoroquinolone (qnrA and qnrB) and β-lactam (bla_GES, bla_TEM, and bla_SHV) resistance genes. Class 1 and 2 integron-associated genes (intI1 and intI2) were also detected. Notably, the rare bla_GES gene was detected in Bacillus sp. from unfertilized soil. Similarly, qnrA co-occurred with bla_SHV in a Bosea sp. strain from unfertilized soil. Conclusions: These findings highlight the potential for ARB dissemination in agricultural environments, where ARB and ARGs, once introduced into soils, may spread by weathering and other environmental factors, complicating negative control selection in in situ studies. Full article

Coagulase-negative bacteria of the Staphylococcus genus are currently frequent food contaminants. The increase in antibiotic resistance means that these microorganisms are becoming the cause of many serious infections and toxications. Their resistance to routinely used chemical compounds has led to the search for alternative methods to combat food-borne pathogens. For this purpose, plant extracts rich in phenolic compounds are increasingly used. The aim of this study was to assess the effect of extracts obtained from the pseudo-fruits and flesh of Rosa canina, Rosa rugosa and Rosa pomifera ‘Karpatia’ on the growth dynamics of bacterial strains of the Staphylococcus genus (72-h co-culture; plate inoculation method). The conducted studies allowed us to conclude that extracts from Rosa spp. show high antistaphylococcal activity. However, it is not proportional to the dose used. Rosa spp. extracts already at concentrations of ¼ MIC limit the growth of the biomass of bacteria of the Staphylococcus genus. The above-described dependencies are very individual—strain-specific, not species-specific. However, based on SEM analysis, it can be observed that the antistaphylococcal mechanism of action of Rosa spp. extracts is associated with the coating of cell walls by the extracts and the disintegration of cell membranes, as a result of which the cells are destroyed. Full article

Cell culture in two dimensions has been the main instrument in cellular and molecular biology. But there are limitations to two-dimensional culture when it comes to tissue engineering and in vivo reproduction. Tissue engineering technology enabled the creation of biomedical scaffolds, which are mostly utilized to biofabricate different artificial human organs. Tissue architecture that encourage cell proliferation can be produced using direct bioprinting technology. The development of bioinks for 3D bioprinting is consistently seen as a problem in the domains of biofabrication and tissue engineering. This study aimed to determine if Fibroblasts and Keratinocytes could grow on hydrogel scaffolds as efficiently as they can in the culture plates. Melanocytes were co-cultured, and the production of melanin was assessed in a two- and three-dimensional culture system. Scaffolds were fabricated using 8% alginate and 6% gelatin and 3D-printed using a cell link printer. FTIR was used to determine the precise composition of the gels. SEM analysis was performed for the cells present in gel and the topology of the cells. In addition, 8% alginate and 6% alginate gel scaffolds were analyzed for swelling and degradation over time in the cell growth medium and PBS. Furthermore, a gene expression study of cell cultures on scaffolds was performed through qPCR. A live/dead assay was performed to determine cell viability for cells grown on scaffolds for 7, 14, and 21 days. Most of the cells were shown to be viable, similar to the control cells grown on a plate. The findings from the SEM showed that cells were grown on the gel surface, remained viable even after 21 days, and displayed circular cells stacked three-dimensionally on the gel surface in the 3D scaffold. The MTT assay was performed to check the viability of cells cultured on a 3D-printed scaffold for 1, 5, and 15 days. We observed about 40% viable cells after 15 days, as shown by the MTT assay. Furthermore, a co-culture study with Melanocyte showed an increased production of melanin in a 3D culture as compared to a 2D culture. Our findings suggest that an alginate and gelatin polymer can be used as a cellular matrix for epithelial cell culture. Further, in vivo and ex vivo experiments are needed to validate the results for future applications in tissue engineering for wound healing and other tissue engineering domains. Full article

Background/Objectives: The viral pandemic caused by the SARS-CoV-2 virus has affected millions of people. However, it was noticed that high mortality was often a result of bacterial co-infections. One of the main pathogens responsible for secondary infections in patients with viral respiratory tract infections, including COVID-19, is Staphylococcus aureus. In recent years, the number of infections caused by drug-resistant strains of S. aureus has been growing rapidly, often exceeding the number of infections caused by antibiotic-sensitive strains. In addition, biofilm-related infections are more difficult to treat due to the lower sensitivity of biofilm structure to antibiotics. Bacteriophages are seen as alternative treatment of bacterial infections. Therefore, in our work, we have analyzed the efficacy of three Kayviruses against S. aureus strains isolated from COVID-19 patients. Methods: We analyzed the ability of tested phages to remove S. aureus biofilm both from polystyrene plates as well as from the surface of pulmonary epithelial cells. Results: We have observed that tested Kayviruses had a broad host range. Furthermore, phages were able to effectively reduce biofilm biomass and number of viable cells in pure culture. During our research, none of the tested phages was shown to have a negative effect on cell viability and were able to inhibit the negative effect S. aureus had on cell condition. Conclusions: Our results show tested phages were effective in reducing the biofilm of S. aureus strains isolated from COVID-19 patients, had no adverse effect on lung epithelial cell viability. Therefore, it should be recognized that the properties of three studied Kayviruses give them an advantage in the selection of phages for treatment of staphylococcal infections. Full article

The engraftment of transplanted islets depends on the rapid establishment of a novel vascular network. The present study evaluated the effects of cord blood-derived blood outgrowth endothelial cells (BOECs) on the viability of neonatal porcine islets (NPIs) and the post-transplant outcome of grafted NPIs. Dispersed NPIs and human BOECs were reaggregated on microwell cell culture plates and tested for their anti-apoptotic and pro-angiogenic capacity by qRT-PCR and immunohistochemistry. The in vivo functionality was analyzed after transplantation into diabetic NOD-SCID IL2rγ^−/− (NSG) mice. The spheroids, which contained reaggregated neonatal porcine islet cells (REPIs) and BOECs, exhibited enhanced viability and a significantly elevated gene expression of VEGFA, angiopoetin-1, heme oxygenase-1, and TNFAIP3 (A20) in vitro. The development of normoglycemia was significantly faster in animals transplanted with spheroids in comparison to the only REPI group (median 51.5 days versus 60 days) (p < 0.05). Furthermore, intragraft vascular density was substantially increased (p < 0.01). The co-transplantation of prevascularized REPI-BOEC spheroids resulted in superior angiogenesis and accelerated in vivo function. These findings may provide a novel tool to enhance the efficacy of porcine islet xenotransplantation. Full article

Lipotoxicity, resulting from the buildup of excess lipids in non-adipose tissues, is increasingly recognized as a major contributor to the progression of kidney disease, highlighting the need for alternative models to assess its effects on renal cells. The main aim of this study was to investigate the usefulness of Caki-1, a human proximal tubule (PT) and renal cell carcinoma (RCC) representative cell line, as a 3D model system for studying free fatty acid-induced PT lipotoxicity. Caki-1 spheroids were generated and maintained on ultra-low attachment plates and characterized regarding time-dependent morphology changes. In optimal 3D culture conditions, Caki-1 cells formed well-defined large compact spheroids with uniform morphology, good circularity, and increased diameter from days 4–12. Chronic exposure to saturated palmitate resulted in dose- and time-dependent spheroid disintegration and cell death, including dispersed and flattened spheroid morphology, with increased dead cells in the peripheral layers and decreased spheroid core. Moreover, palmitate-treated spheroids showed a significant increase in cleaved poly(ADP-ribose) polymerase (PARP) and active caspase-3. Palmitate-induced PARP cleavage, as well as endoplasmic reticulum (ER) stress and autophagy dysfunction, were blunted by triacsin C, an inhibitor of long-chain acyl-CoA synthetases. In addition, co-incubation with unsaturated oleate prevented palmitate-induced spheroid disintegration and apoptotic cell death in Caki-1 3D culture. While fatty acid overload upregulated lipid droplet protein perilipin 2 in Caki-1 cells, knockdown of perilipin 2 by siRNAs resulted in an exacerbation of palmitate-induced cell death. Together, these results indicate that the 3D Caki-1 spheroid model is a simple and reproducible in vitro system for studying renal lipotoxicity and lipid metabolism that gives useful readouts at the molecular, cellular, and multicellular levels. Full article