This chapter introduces the increasing significance of mesenchymal stromal/stem cell (MSC) production in regenerative medicine and cellular therapeutics, outlines the growing interest in MSCs for various medical applications, and highlights their potential in advanced therapy medicinal products (ATMPs) and the advancements in cell culture technologies that have facilitated large-scale MSC production under Good Manufacturing Practices (GMP), ensuring safety and efficacy. This chapter describes an optimized upstream protocol for laboratory-scale MSC production from different tissue sources. This protocol, conducted in flasks, controls critical parameters and lays the foundation for downstream processing to generate ATMPs. This comprehensive approach underscores the potential of MSCs in clinical applications and the importance of tailored production processes.

Biomaterials like chitosan, hydroxyapatite have been used in biomedical and pharmaceutical field, due to its valuable biochemical and physiological properties. In current work firstly our group has isolated a polysaccharide chitosan along with hydroxyapatite biomaterial from the same source by varying the process condition via greener approach. We have adapted greener approach for the isolation of chitosan within a short period of time and this is the very first report for the isolation of both chitosan and hydroxyapatite simultaneously from the same waste edible garden snail shells. Both these materials were thoroughly characterized by using UV, FT-IR, SEM techniques. Among synthetic colourants, congo red dye is recognized as carcinogens, which are usually used in the textile manufacturing. Interestingly, one of our biomaterial hydroxyapatite has shown good selectivity towards Congo red dye. The sensitivity range was obtained from 10 to 100 μM within the LOD of 101.52 nM. The developed sensor has been tested for various industrial effluents and shown good agreement with our results. Meanwhile these chitosan and hydroxyapatite have also been used as capping agent for the preparation of stable gold nanoparticles.

BACKGROUND: This study investigates seafarers\' loneliness as a mediating variable between psychological well-being and resilience, and tests resilience as a mediating variable between psychological well-being and loneliness. It also examines the challenges faced by seafarers on different types of ships and evaluates the mediating roles of resilience and loneliness.
METHODS: The research uses descriptive data analysis, reliability analysis, correlation analysis, and mediation analysis with bootstrap-based regression models. An online survey was conducted with 471 active Turkish seafarers using a sociodemographic questionnaire and three standardized scales measuring psychological well-being (PW), loneliness at work (LAW), and psychological resilience (PR). Data were collected between 01/07/2023 and 01/09/2023.
RESULTS: The findings indicate that ship type is a significant factor in the mediating roles of loneliness at work and psychological resilience. There is a partial mediating role of loneliness at work and resilience in psychological well-being across different ship types. Specifically, loneliness at work partially mediates the relationship between psychological resilience and psychological well-being among seafarers on tanker or bulk carrier vessels, but not on container vessels.
CONCLUSIONS: The study concludes with suggestions to address the mental health challenges faced by seafarers, emphasizing the importance of ship type in the mediating roles of loneliness and resilience. Regarding the mediating role of loneliness at work, it has been determined that there is partial mediation between psychological resilience and psychological well-being among seafarers working on tanker or bulk carrier vessels. However, on container vessels, loneliness at work does not mediate the relationship between psychological well-being and psychological resilience. Based on these findings, the authors conclude by offering a range of helpful solutions to address this problem.

The COVID-19 pandemic was characterized by loneliness, especially among teenagers. This study explored the coping mechanisms, activities, challenges, places visited, and sources of support that predicted teenagers feeling connected to others during COVID-19. Data come from surveys administered in the United States and Canada in summer 2022. Multivariate logistic regression showed that producing personal protective equipment, supporting siblings, getting involved in the local community, becoming more politically active, and taking language classes were positively associated with connectedness. In addition, teens in Canada were more likely to feel connected to others than teens in the United States. These findings can inform policies to enhance resilience in teenagers during protracted crises.

Extracellular vesicles (EVs) are nanosized vesicles with a lipid bilayer that are secreted by cells and play a critical role in cell-to-cell communication. Despite the promising reports regarding their diagnostic and therapeutic potential, the utilization of EVs in the clinical setting is limited due to insufficient information about their cargo and a lack of standardization in isolation and analysis methods. Considering protein cargos in EVs as key contributors to their therapeutic potency, we conducted a tandem mass tag (TMT) quantitative proteomics analysis of three subpopulations of mesenchymal stem cell (MSC)-derived EVs obtained through three different isolation techniques: ultracentrifugation (UC), high-speed centrifugation (HS), and ultracentrifugation on sucrose cushion (SU). Subsequently, we checked EV marker expression, size distribution, and morphological characterization, followed by bioinformatic analysis. The bioinformatic analysis of the proteome results revealed that these subpopulations exhibit distinct molecular and functional characteristics. The choice of isolation method impacts the proteome of isolated EVs by isolating different subpopulations of EVs. Specifically, EVs isolated through the high-speed centrifugation (HS) method exhibited a higher abundance of ribosomal and mitochondrial proteins. Functional apoptosis assays comparing isolated mitochondria with EVs isolated through different methods revealed that HS-EVs, but not other EVs, induced early apoptosis in cancer cells. On the other hand, EVs isolated using the sucrose cushion (SU) and ultracentrifugation (UC) methods demonstrated a higher abundance of proteins primarily involved in the immune response, cell-cell interactions and extracellular matrix interactions. Our analyses unveil notable disparities in proteins and associated biological functions among EV subpopulations, underscoring the importance of meticulously selecting isolation methods and resultant EV subpopulations based on the intended application.

Jack bean (JB), Canavalia ensiformis (L.) DC, is a commonly cultivated legume in Indonesia. It is rich in protein, which can be hydrolyzed, making it potentially a good source of bioactive peptides. Intestinal inflammation is associated with several diseases, and the production of interleukin-8 (IL-8) in intestinal epithelial cells induced by tumor necrosis factor (TNF)-α has an important role in inflammatory reaction. The present study investigated the anti-inflammatory effects of peptides generated from enzymatic hydrolysis of JB protein on human intestinal Caco-2BBe cells. Additionally, in silico approaches were used to identify potential bioactive peptides. JB protein hydrolysate (JBPH) prepared using pepsin and pancreatin reduced the IL-8 expression at protein and mRNA levels in Caco-2BBe cells stimulated with TNF-α. Immunoblot analysis showed that the JBPH reduced the TNF-α-induced phosphorylation of c-Jun-NH(2)-terminal kinase, nuclear factor kappa B (NF-κB), and p38 proteins. Anti-inflammatory activity was observed in the 30% acetonitrile fraction of JBPH separated on a Sep-Pak C18 column. An ultrafiltration method revealed that relatively small peptides (< 3 kDa) had a potent inhibitory effect on the IL-8 production. Purification of the peptides by reversed-phase and anion-exchange high performance chromatography produced three peptide fractions with anti-inflammatory activities. A combination of mass spectrometry analysis and in silico approaches identified the potential anti-inflammatory peptides. Peptides derived from JB protein reduces the TNF-α-induced inflammatory response in Caco-2BBe cells via NF-κB and mitogen-activated protein kinase signaling pathways. Our results may lead to a novel therapeutic approach to promote intestinal health.

Extracellular vesicles (EVs) have the potential to provide new insights into skeletal muscle (SM) physiology and pathophysiology. However, current isolation protocols often do not eliminate co-isolated components such as lipoproteins and RNA binding proteins that could confound outcomes and hinder downstream clinical translation. In this study, we validated an EV isolation protocol that combined size-exclusion chromatography (SEC) with ultrafiltration (UF) to increase sample throughput, scalability and purity, while providing the very first analysis of the effects of UF column choice and fraction window on EV recovery. C2C12 myotube conditioned medium was pre-concentrated using either Amicon® Ultra 15 or Vivaspin®20 100 KDa UF columns and processed by SEC (IZON, qEV 70 nm). The resulting thirty fractions obtained were individually analysed to identify an optimal fraction window for EV recovery. The EV marker TSG101 could be detected from fractions 5 to 14, while CD9 and Annexin A2 only up to fraction 6. ApoA1+ lipoprotein co-isolates were detected from fraction 6 onwards for both protocols. Strikingly, Amicon and Vivaspin UF concentration protocols led to qualitative and quantitative variations in EV marker profiles and purity. Eliminating lipoprotein co-isolation by reducing the SEC fraction window resulted in a net loss of particles, but increased measures of sample purity and had only a negligible impact on the presence of EV marker proteins. In conclusion, our study developed an effective UF+SEC protocol for the isolation of EVs based on sample purity (fractions 1-5) and total EV abundance (fractions 2-10). We provide evidence to demonstrate that the choice of UF column can affect the composition of the resulting EV preparation and needs to be considered when being applied in EV isolation studies in SM. The resulting protocols will be valuable in isolating highly pure EV preparations for applications in a range of therapeutic and diagnostic studies.

Extracellular vesicle-derived microRNAs (EV-miRNAs) are promising biomarkers for early cancer diagnosis. However, existing EV-miRNA extraction technologies have a complex two-step process that results in low extraction efficiency and inconsistent results. This study aimed to develop and evaluate a new single-step extraction method, called miRQuick, for efficient and high-recovery extraction of EV-miRNAs from samples. The miRQuick method involves adding positively charged substances to the sample, causing negatively charged EVs to quickly aggregate and precipitate. A membrane lysate is then added to extract only miRNA. The entire process can be completed within an hour using standard laboratory equipment. We validated the miRQuick method using various analytical techniques and compared its performance to other methods for plasma, urine and saliva samples. The miRQuick method demonstrated significantly higher performance than other methods, not only for blood plasma but also for urine and saliva samples. Furthermore, we successfully extracted and detected nine biomarker candidate miRNAs in the plasma of breast cancer patients using miRQuick. Our results demonstrate that miRQuick is a rapid and efficient method for EV-miRNA extraction with excellent repeatability, making it suitable for various applications including cancer diagnosis.

Understanding which factors shape and maintain biodiversity is essential to understand how ecosystems respond to crises. Biodiversity observed in ecological communities is a result of the interaction of various factors which can be classified as either neutral- or niche-based. The importance of these processes has been debated, but many scientists believe that both processes are important. Here, we use unique ecosystems in groundwater-filled lava caves near Lake Mývatn, to examine the importance of neutral- versus niche-based factors for shaping invertebrate communities. We studied diversity in benthic and epibenthic invertebrate communities and related them to ecological variables. We hypothesized that if neutral processes are the main drivers of community structure we would not see any clear relationship between the structure of community within caves and ecological factors. If niche-based processes are important we should see clear relationships between community structure and variation in ecological variables across caves. Both communities were species poor, with low densities of invertebrates, showing the resource limited and oligotrophic nature of these systems. Unusually for Icelandic freshwater ecosystems, the benthic communities were not dominated by Chironomidae (Diptera) larvae, but rather by crustaceans, mainly Cladocera. The epibenthic communities were not shaped by environmental variables, suggesting that they may have been structured primarily by neutral processes. The benthic communities were shaped by the availability of energy, and to some extent pH, suggesting that niche-based processes were important drivers of community structure, although neutral processes may still be relevant. The results suggest that both processes are important for invertebrate communities in freshwater, and research should focus on understanding both of these processes. The ponds we studied are representative of a number of freshwater ecosystems that are extremely vulnerable for human disturbance, making it even more important to understand how their biodiversity is shaped and maintained.

This article develops a multi-perspective view on motivations and methods for tobamovirus purification through the ages and presents a novel, efficient, easy-to-use approach that can be well-adapted to different species of native and functionalized virions. We survey the various driving forces prompting researchers to enrich tobamoviruses, from the search for the causative agents of mosaic diseases in plants to their increasing recognition as versatile nanocarriers in biomedical and engineering applications. The best practices and rarely applied options for the serial processing steps required for successful isolation of tobamoviruses are then reviewed. Adaptations for distinct particle species, pitfalls, and \'forgotten\' or underrepresented technologies are considered as well. The article is topped off with our own development of a method for virion preparation, rooted in historical protocols. It combines selective re-solubilization of polyethylene glycol (PEG) virion raw precipitates with density step gradient centrifugation in biocompatible iodixanol formulations, yielding ready-to-use particle suspensions. This newly established protocol and some considerations for perhaps worthwhile further developments could serve as putative stepping stones towards preparation procedures appropriate for routine practical uses of these multivalent soft-matter nanorods.