Search Results (29)

Noncommunicable diseases (NCDs) like diabetes and cancer drive demand for therapeutic functional foods. This study developed freeze-dried fermented camel milk (FCM) with Ajwa date pulp (ADP), evaluating its physical and functional properties, probiotic survival, and potential benefits for diabetes and cancer. To achieve this target, six FCM formulations were prepared using ABT-5 starter culture (containing Lactobacillus acidophilus, Bifidobacterium bifidum, and Streptococcus thermophilus) with or without Lacticaseibacillus rhamnosus B-1937 and ADP (12% or 15%). The samples were freeze-dried, and their functional properties, such as water activity, dispersibility, water absorption capacity, water absorption index, water solubility index, insolubility index, and sedimentation, were assessed. Reconstitution properties such as density, flowability, air content, porosity, loose bulk density, packed bulk density, particle density, carrier index, Hausner ratio, porosity, and density were examined. In addition, color and probiotic survivability under simulated gastrointestinal conditions were analyzed. Also, antidiabetic potential was assessed via α-amylase and α-glucosidase inhibition assays, while cytotoxicity was evaluated using the MTT assay on Caco-2 cells. The results show that ADP supplementation significantly improved dispersibility (up to 72.73% in FCM15D+L). These improvements are attributed to changes in particle size distribution and increased carbohydrate and mineral content, which facilitate powder rehydration and reduce clumping. All FCM variants demonstrated low water activity (0.196–0.226), indicating good potential for shelf stability. The reconstitution properties revealed that FCM powders with ADP had higher bulk and packed densities but lower particle density and porosity than controls. Including ADP reduced interstitial air and increased occluded air within the powders, which may minimize oxidation risks and improve packaging efficiency. ADP incorporation resulted in a significant decrease in lightness (L*) and increases in redness (a*) and yellowness (b*), with greater pigment and phenolic content at higher ADP levels. These changes reflect the natural colorants and browning reactions associated with ADP, leading to a more intense and visually distinct product. Probiotic survivability was higher in ADP-fortified samples, with L. acidophilus and B. bifidum showing resilience in intestinal conditions. The FCM15D+L formulation exhibited potent antidiabetic effects, with IC₅₀ values of 111.43 μg mL⁻¹ for α-amylase and 77.21 μg mL⁻¹ for α-glucosidase activities, though lower than control FCM (8.37 and 10.74 μg mL⁻¹, respectively). Cytotoxicity against Caco-2 cells was most potent in non-ADP samples (IC₅₀: 82.22 μg mL⁻¹ for FCM), suggesting ADP and L. rhamnosus may reduce antiproliferative effects due to proteolytic activity. In conclusion, the study demonstrates that ADP-enriched FCM is a promising functional food with enhanced probiotic viability, antidiabetic potential, and desirable physical properties. This work highlights the potential of camel milk and date synergies in combating some NCDs in vitro, suggesting potential for functional food application. Full article

Induced pluripotent stem cell (iPSC)-derived neurons (iNs) have been widely used as models of neurodevelopment and neurodegenerative diseases. Coating cell culture vessels with extracellular matrixes (ECMs) gives structural support and facilitates cell communication and differentiation, ultimately enhances neuronal functions. However, the relevance of different ECMs to the natural environment and their impact on neuronal differentiation have not been fully characterized. In this study, we report the use of four commonly used extracellular matrixes, poly-D-lysine (PDL), poly-L-ornithine (PLO), Laminin and Matrigel, which we applied to compare the single-coating and double-coating conditions on iNs differentiation and maturation. Using the IncuCyte live-cell imaging system, we found that iNs cultured on single Matrigel- and Laminin-coated vessels have significantly higher density of neurite outgrowth and branch points than PLO or PDL but produce abnormal highly straight neurite outgrowth and larger cell body clumps. All the four double-coating conditions significantly reduced the clumping of neurons, in which the combination of PDL+Matrigel also enhanced neuronal purity. Double coating with PDL+Matrigel also tended to improve dendritic and axonal development and the distribution of pre and postsynaptic markers. These results demonstrate that the extracellular matrix contributes to the differentiation of cultured neurons and that double coating with PDL+Matrigel gives the best outcomes. Our study indicates that neuronal differentiation and maturation can be manipulated, to a certain extent, by adjusting the ECM recipe, and provides important technical guidance for the use of the ECM in neurological studies. Full article

Induced pluripotent stem cells (iPSCs) are a promising cell source because of their pluripotency and self-renewal abilities. However, there is a risk of pluripotency loss during cell expansion. Particularly, cell passaging is associated with a higher risk of decreasing cell quality. There are two iPSC passaging methods: single-cell and clump passaging. Single-cell passaging is a rapid and simple method for cell manipulation, whereas clump passaging is superior for maintaining iPSC pluripotency. Therefore, clump passaging is a robust method for expanding iPSCs while maintaining their pluripotency. However, clump size control during clump passaging is difficult because colony fragmentation is performed manually by pipetting the colonies detached from the cell culture substrates. In this study, the effect of pipetting on iPSC colony fragmentation was evaluated and the relationship between iPSC clump size and pluripotency was clarified. An automated pipetting device was developed to standardize the clump passage process. The effect of clump size on the pluripotency and proliferative capacity of mouse iPSCs was investigated. Clump size was controlled by varying the number of pipetting cycles, and pluripotency and proliferation were assessed via alkaline phosphatase staining and flow cytometry. Our results revealed that a decrease in clump size corresponded to an increase in cell proliferation, while pluripotency maintenance was optimized under specific clump sizes. These results underscore the significance of clump size for stem cell quality, emphasizing the need for a balanced approach to maintain pluripotency while fostering proliferation in the cell expansion culture for iPSCs. Full article

Within the last decade, a wide variety of protocols have emerged for the generation of retinal organoids. A subset of studies have compared protocols based on stem cell source, the physical features of the microenvironment, and both internal and external signals, all features that influence embryoid body and retinal organoid formation. Most of these comparisons have focused on the effect of signaling pathways on retinal organoid development. In this study, our aim is to understand whether starting cell conditions, specifically those involved in embryoid body formation, affect the development of retinal organoids in terms of differentiation capacity and reproducibility. To investigate this, we used the popular 3D floating culture method to generate retinal organoids from stem cells. This method starts with either small clumps of stem cells generated from larger clones (clumps protocol, CP) or with an aggregation of single cells (single cells protocol, SCP). Using histological analysis and gene-expression comparison, we found a retention of the pluripotency capacity on embryoid bodies generated through the SCP compared to the CP. Nonetheless, these early developmental differences seem not to impact the final retinal organoid formation, suggesting a potential compensatory mechanism during the neurosphere stage. This study not only facilitates an in-depth exploration of embryoid body development but also provides valuable insights for the selection of the most suitable protocol in order to study retinal development and to model inherited retinal disorders in vitro. Full article

It is critical that we move our understanding of the ecosystem services (ESs) produced by landscapes from the present abundance of analysis to a fundamental basis of design. This involves enhancing the ability to understand and model the interconnected, coevolving system of humans and the rest of nature, thus contributing to the design of sustainable landscapes. In this paper, we hypothesise that the spatial configuration of landscape components (the size and arrangement of tree clumps, paddocks, crops, water features, etc.) impacts the production of regulating ESs, which in turn have a leveraging effect on provisioning and cultural ESs. Drawing on the precepts of Ecological Field Theory, we present the development and implications of a conceptual Geographic Information System (GIS)-based model, ESMAX, that utilises the idiosyncratic distance-decay characteristics of regulating ESs. These ‘ES fields’ are visualised as radiating into the landscape from their source components, addressing a gap in biophysical reality that has been identified as a shortcoming of existing ES modelling based on landcover proxies. Hypothetical landscape arrangements of simplified landscape components are tested with ESMAX across three regulating ESs: cooling effect, nitrogen retention, and habitat provision. The model calculates the overall ES performance of each landscape arrangement by tabulating the ES fields produced and, critically, the nonlinear response where fields overlap. The results indicate a primary sensitivity to the size of components and a secondary sensitivity to the arrangement of components. Consequently, ESMAX can be used to design landscape configurations that (1) maximise the production of specific regulating ESs and (2) improve the utilisation of natural ES-producing resources. Full article

Background: Cells typically function and behave within a three-dimensional (3D) environment. Mesenchymal stem cells (MSCs), known for their self-renewal, multi-lineage differentiation capabilities, and paracrine effects, have garnered significant medical interest. MSC spheroid culture is widely adopted to study the biological properties of MSCs in a 3D context. In contrast, we previously developed 3D clumps of MSC/ECM complexes termed C-MSCs. C-MSCs consisted of cells and self-produced ECM proteins, allowing grafting into tissue defects without any artificial scaffolds. This present study aimed to elucidate the fundamental biological distinctions between 3D MSC spheroids and C-MSCs. Methods: MSC spheroids and C-MSCs are generated from human bone-marrow-derived MSCs. The physical properties, histological structures, and gene expression patterns were compared in vitro. Results: Macroscopic and histological examinations revealed that, whereas MSC spheroids are dense cell clusters primarily formed through Cadherin-mediated cell–cell interactions, C-MSCs are cell aggregates anchored by the ECM component COL1, enabling them to form larger structures. Furthermore, transcriptome analysis showed that C-MSCs possess enhanced capacities to produce immunomodulatory and cytoprotective factors, a prominent biological characteristic of MSCs. Conclusion: Recognizing the distinct attributes of each cell aggregate offers insights into the potential evolution of 3D cell culture techniques and possible therapeutic implications. Full article

Graphene oxide (GOX) has become attractive due to its unique physicochemical properties. This nanomaterial can associate with other dendrimers, making them more soluble and allowing better interaction with biomacromolecules. The present study aimed to investigate, by real-time microscopy, the behavior of human breast cancer cells exposed to particles of materials based on graphene oxide. The MCF-7 cell line was exposed to GOX, GOX associated with Polypropylenimine hexadecaamine Dendrimer, Generation 3.0—DAB-AM-16 (GOXD) and GOX associated with polypropyleneimine—PAMAM (GOXP) in the presence or absence of fetal bovine serum (FBS). GOX, GOXD and GOXP were taken up by the cells in clusters and then the clusters were fragmented into smaller ones inside the cells. Real-time microscopy showed that the presence of FBS in the culture medium could allow a more efficient internalization of graphene materials. After internalizing the materials, cells can redistribute the clumps to their daughter cells. In conclusion, the present study showed that the particles can adhere to the cell surface, favoring their internalization. The presence of FBS contributed to the formation of smaller aggregates of particles, avoiding the formation of large ones, and thus transmitted a more efficient internalization of the materials through the interaction of the particles with the cell membrane. Full article

The widespread applications of ZnO NPs in the different areas of science, technology, medicine, agriculture, and commercial products have led to increased chances of their release into the environment. This created a growing public concern about the toxicological and environmental effects of the nanoparticles. The impact of these NPs on the genetic materials of living organisms is documented in some cultured cells and plants, but there are only a few studies regarding this aspect in animals. In view of this, the present work regarding the assessment of the genotoxicity of zinc oxide nanoparticles using the mosquito Culex quinquefaciatus has been taken up. Statistically significant chromosomal aberrations over the control are recorded after the exposure of the fourth instar larvae to a dose of less than LD₂₀ for 24 h. In order to select this dose, LD₂₀ of ZnO NPs for the mosquito is determined by Probit analysis. Lacto-aceto-orcein stained chromosomal preparations are made from gonads of adult treated and control mosquitoes. Both structural aberrations, such as chromosomal breaks, fragments, translocations, and terminal fusions, resulting in the formation of rings and clumped chromosomes, and numerical ones, including hypo- and hyper-aneuploidy at metaphases, bridges, and laggards at the anaphase stage are observed. The percentage frequency of abnormalities in the shape of sperm heads is also found to be statistically significant over the controls. Besides this, zinc oxide nanoparticles are also found to affect the reproductive potential and embryo development as egg rafts obtained from the genetic crosses of ZnO nanoparticle-treated virgin females and normal males are small in size with a far smaller number of eggs per raft. The percentage frequencies of dominant lethal mutations indicated by the frequency of unhatched eggs are also statistically significant (p < 0.05) over the control. The induction of abnormalities in all of the three short-term assays studied during the present piece of work indicates the genotoxic potential of ZnO NPs, which cannot be labeled absolutely safe, and this study pinpoints the need to develop strategies for the protection of the environment and living organisms thriving in it. Full article

Leishmania are an understudied genus of parasitic protozoans causing significant health problems for people, particularly in tropical climates. To better understand the growth of Leishmania and potential drug sensitivity implications, the effects of motion on cells grown in vitro were probed. Using a stock Leishmania tarentolae cell culture, cells were placed in 10 mL of a Brain–Heart Infusion medium in either a non-moving (static) environment or on a flat platform of one of two lab rockers (set at a minimal speed) in a dark environment for 13 days. Also, the addition of 0.5 M of L-Proline was evaluated. Microscopy, cell clumping, cell viability, and secreted acid phosphatase (SAP) activity data were collected. Results show that a constant slow rocking motion changed cell growth, clumping behavior, and detectable SAP activity relative to the no-motion cultures, but this change was dependent on which rocker was used, indicating a complexity in the growth of these cells in culture. Thus, continuous motion affects the stresses placed on the cells during a growth curve under some conditions. The implications of this study lead to questions about the effects of motion on the efficacy of pharmaceutical testing in vitro. Further study of the effects of motion on Leishmania is important. Full article

Centella asiatica or gotu kola has long been used as a traditional medicinal plant. Here, immersion times and culture systems on growth and biomass production of C. asiatica were investigated using a twin-bottle, temporary immersion system. Results indicated that all immersion times gave 100% survival, with a 5 min immersion 12 times/day, providing the highest number of new shoots (3.6 shoots/explant), leaves (10.2 leaves/explant), roots (8.3 roots/explant), and fresh and dry weights of clumps (5.06 g fresh weight and 0.48 g dry weight/clump). The temporary immersion system resulted in more than a three-fold increase in biomass accumulation, with the highest average number of new shoots, leaves, and roots compared to a semi-solid system. Full article

Conventional stirred-tank bioreactor (STR) designs are optimised for cultures of bacteria but not fungal cultures; therefore, a new Air-L-Shaped Bioreactor (ALSB) was fabricated. The ALSB was designed to eliminate the wall growth and clumping of fungal mycelium in STRs. Ganoderma lucidum was used as a fungal model and its biomass and exopolysaccharide (EPS) production were maximised by optimising the agitation rate, glucose concentration, initial pH, and aeration via response surface methodology (RSM). The ALSB system generated 7.8 g/L of biomass (biomass optimised conditions: 110 rpm, 24 g/L glucose, pH 5.6, and 3 v/v of aeration) and 4.4 g/L of EPS (EPS optimised conditions: 90 rpm, 30 g/L glucose, pH 4, and 2.5 v/v of aeration). In combination, for both optimised conditions, biomass (7.9 g/L) and EPS (4.6 g/L) were produced at 110 rpm, 30 g/L glucose, pH 4, and 3 v/v of aeration with minimal wall growth. The data prove that the ALSB is a blueprint for efficient economical fungal cultivation. Full article

The work aims to investigate biofilm formation and biofilm/adhesion-encoding genes in coagulase-negative staphylococci (CoNS) species recovered from blood culture isolates. Eighty-nine clinical CoNS were confirmed using the VITEK 2 system, and antibiotic susceptibility testing of isolates was conducted using the Kirby-Bauer disk diffusion method against a panel of 20 antibiotics. Isolates were qualitatively screened using the Congo red agar medium. Quantitative assays were performed on microtiter plates, where the absorbances of the solubilised biofilms were recorded as optical densities and quantified. In all, 12.4% of the isolates were strong biofilm formers, 68.5% had moderate biofilm capacity, and 17.9% showed weak capacity. A subset of 18 isolates, mainly methicillin-resistant S. epidermidis, were investigated for adherence-related genes using whole-genome sequencing and bioinformatics analysis. The highest antibiotic resistance rates for strongly adherent isolates were observed against penicillin (100%) and cefoxitin (81.8%), but the isolates showed no resistance to linezolid (0.0%) and tigecycline (0.0%). The icaABC genes involved in biofilm formation were detected in 50% of the screened isolates. Other adherence-related genes, including autolysin gene atl (88.8%), elastin binding protein gene ebp (94.4%), cell wall-associated fibronectin-binding protein gene ebh (66.7%), clumping factor A gene clfA (5.5%), and pili gene ebpC (22.2%) were also found. The insertion sequence IS256, involved in biofilm formation, was found in 10/18 (55.5%) screened isolates. We demonstrate a high prevalence of biofilm-forming coagulase-negative staphylococci associated with various resistance phenotypes and a substantial agreement between the possession of biofilm-associated genes and the biofilm phenotype. Full article

Ranging from simple food ingredients to complex pharmaceuticals, value-added products via microbial fermentation have many advantages over their chemically synthesized alternatives. Some of such advantages are environment-friendly production pathways, more specificity in the case of enzymes as compared to the chemical catalysts and reduction of harmful chemicals, such as heavy metals or strong acids and bases. Fungal fermentation systems include yeast and filamentous fungal cells based on cell morphology and culture conditions. However, filamentous fungal fermentation has gained attention in the past few decades because of the diversity of microbial products and robust production of some of the most value-added commodities. This type of fungal fermentation is usually carried out by solid-state fermentation. However, solid-state fermentation poses problems during the scale-up for industrial production. Therefore, submerged fermentation for value-added products is usually preferred for scaling-up purposes. The main problem with submerged fungal fermentation is the formation of complex mycelial clumps or pellets. The formation of such pellets increases the viscosity of the media and hinders the efficient transfer of oxygen and nutrient resources in the liquid phase. The cells at the center of the clump or pellet start to die because of a shortage of resources and, thus, productivity decreases substantially. To overcome this problem, various morphological engineering techniques are being researched. One approach is the use of microparticles. Microparticles are inert particles with various size ranges that are used in fermentation. These microparticles are shown to have positive effects, such as high enzyme productivity or smaller pellets with fungal fermentation. Therefore, this review provides a background about the types of microparticles and summarizes some of the recent studies with special emphasis on the fungal morphology changes and microparticle types along with the applications of microparticles in filamentous fungal fermentations. Full article

Peatlands in Indonesia are one of the world’s largest carbon sinks, helping to regulate greenhouse gas emissions and global climate change. Lophostemon suaveolens is a relatively unexplored plant found in Papua’s endemic peat ecosystem that grows well in wet areas with low fertility. It is geographically dispersed and has the potential for peatland rehabilitation. Seed is one of materials for the reproduction of L. suaveolens. However, the difficulty in seed collection and the limitation in seed production has become a current problem for its cultivation. Seed multiplication by using an in vitro method would be one of the mechanisms to overcome the problem. We present an efficient and reproducible protocol for in vitro multiplication of plantlets using nodal segments and shoot apices collected from plantlets. After 3 months of the culture initiation stage, the elongated axillary shoots were separated from the clumps and further multiplied using Murashige and Skoog (MS) media supplemented with (1) BAP (0.5 mL/L) as single PGR, (2) NAA (0.1 mL/L) as a single PGR, and (3) a combination of two types of PGR BAP (0.5 mL/L) and NAA (0.1 mL/L). Up to an incubation period of 6 months, the efficiency of leaf axillary shoot propagation was determined by counting the number of nodule multiplication coefficient (NMC), shoot length, root length, and number of leaves (six consecutive subcultures). The higher the NMC, the higher the plantlets obtained, increasing shoot regeneration from nodules physiologically increasing evapotranspiration in vitro. The highest of NMC (8.4) was observed in MS medium with a combination of 0.5 mL/L BAP and 0.1 mL/L NAA (double PGRs), with the longest shoots (5.91 cm), the longest root length (8.83 cm), and the most leaves (32). When a combination of BAP and NAA were used simultaneously, the plantlets during acclimatization were the highest survived. It was concluded that MS in combination with 0.5 mL/L BAP and 0.1 mL/L NAA is the most appropriate protocol for the success of in vitro multiplication of L. suaveolens. This is the first report of L. suaveolens in vitro multiplication, and the protocol could be used to propagate this peatland species on a large scale. The authors acknowledge the limitations of the experimental work and recommend further work to increase the sample size and complete the field-testing phase to help verify the initial findings presented in this paper. Full article

The in vitro propagation and ex situ conservation strategy provides new means for the conservation and mass propagation of economically and medicinally important plants. The present work aimed to observe the main characteristics of the in vitro propagation of Garcinia livingstonei from matured seed segments. Successful multiple shoots were induced on a woody plant (WP) medium supplemented with cytokinins. An average of 13.0 shoots per explant were grown from matured seed segments on a WP medium containing 15.0 µM BAP after 12 weeks of culture. The shoot elongation and multiplication were achieved using a repeated and periodic subculturing of shoot clumps in the same medium. The optimum in vitro rooting of shoots was obtained on the half-strength WP medium supplemented with IBA (5.0 µM). The regenerated plantlets were successfully transplanted to pots containing soil, sand, and farmyard manure (1:1:1) and were maintained in a greenhouse with a survival frequency was 80%. Full article