Analytical and computational studies predict negligible risk of cell death from eddy generation off flat surfaces in cell culture flow systems.

Cell-based therapies represent the current frontier of biomedical innovations, with the technologies required underpinning treatments as broad as CAR-T cell therapies, stem cell treatments, genetic therapies and mRNA manufacture. A key bottleneck in the manufacturing process for each of these lies in the expansion of cells within a bioreactor vessel, requiring by far the greatest share of time for what are often time-critical therapies. While various designs, culture feeding and mixing methods are employed in these bioreactors, a common concern among manufacturers and researchers lies in whether shear stresses generated by culture media flow will damage cells and inhibit expansion. This study develops an analytical tool to link macro-scale measures of flow to risk of cell death using relationships with eddy size and dissipation rates, from eddies generated off flat surfaces. This analytical tool was then employed using computational fluid dynamics (CFD) to replicate a range of generic bioreactor geometries and flow conditions. We found that no combination of flow condition or design parameter was predicted by the tool to cause cell death within eddies, indicating negligible risk of cell death due to eddy formation within cell culture systems. While this requires experimental validation, and does not apply when cells are expanded using microcarriers, this tool nonetheless provides reassurance and accessible prediction of bioreactor design parameters that could result in cell death. Finally, our findings show that bioreactor design can be tailored such that the shear stress stimulation of cells can be selectively altered through small changes in flow rate.

Landscape design of urban culture transmission based on the regional information security of Internet of Things.

In the context of the rapid development of Internet of Things technology, urban cultural communication and information security have become a new focus in the field of landscape design. This paper innovatively discusses the landscape design of urban cultural communication based on Internet of Things regional information security, aiming at building a safe and culturally rich urban landscape environment. Taking the unique regional culture of Zhangjiajie as an example, this study evaluated the cultural communication effect of landscape design under information security guarantee through in-depth case analysis and field investigation, combined with Internet of Things information security technology. The results show that the cluster head node strategy has significant advantages in resisting physical capture attacks, especially when the number of sensor nodes captured is less than 2000, the information loss rate is less than 0.1. This discovery not only improves the level of information security in the Internet of Things environment, but also provides technical support for the effective dissemination of urban culture. In addition, through the detailed analysis and evaluation of landscape, this study further reveals the important role of landscape design in regional cultural inheritance. To sum up, this study not only provides a new perspective for urban landscape design, but also provides practical guidance for the protection and dissemination of urban culture in the era of Internet of Things.

Innovative Management of Blood Culture-Negative Endocarditis With Pulmonary Septic Emboli in a Patient With a Biventricular Pacemaker and Implantable Cardioverter Defibrillator (BiV-ICD) and Psychosocial Stressors: A Case Report.

Blood culture-negative endocarditis (BCNE) poses significant diagnostic and therapeutic challenges and is associated with notable morbidity and mortality. When presented concurrently with other comorbidities, these challenges and the chances of morbidity and mortality significantly increase. This case presents right-sided BCNE accompanied by pulmonary cavitary lesions in a patient with a history of supraventricular tachycardias (SVT), a biventricular pacemaker and implantable cardioverter-defibrillator (BiV-ICD), alcohol use, and anticoagulant noncompliance. The patient missed follow-up appointments for six months after the death of his wife, leading to increased alcohol use and noncompliance with medications. During this period, his home monitoring device was offline. Once reconnected, it detected several episodes of SVT and ventricular tachycardia (VT), prompting a wellness check. He presented to the cardiology clinic with shortness of breath and a cough producing brown-tinged sputum. Evaluation revealed cavitary lesions in the lingula and left lower lobe, a vegetation on his tricuspid valve, and vegetations on his endocardial leads, despite negative blood cultures. Tuberculosis testing was negative, while sputum cultures were positive for Haemophilus influenzae. After ruling out other possible infectious causes of the cavitary lesions, septic emboli were suspected as the cause. Broad-spectrum antibiotics were begun and surgical intervention was done to replace the tricuspid valve and remove the endocardial leads. This procedure was complicated by fibrosis of the leads at the coronary sinus, necessitating their cutting at the superior vena cava and leaving them inside the patient until laser therapy could be performed for their removal. The patient's history of bradycardia and SVTs required the ongoing use of a pacemaker. Inventory discrepancy during the placement of the new pacemaker epicardial leads lead to complications warranting an alternative approach to lead implantation. A traditionally used epicardial lead was placed on the right ventricle for pacing, and an innovative technique was employed to place an endocardial lead on the right atrium epicardium for sensing. This case underscores the importance of thorough evaluation and collaborative management strategies to optimize outcomes for patients with concomitant cardiac and pulmonary pathologies, particularly in the context of underlying psychosocial stressors. Additionally, it demonstrates solutions to challenges that can arise during surgery and presents an alternative lead placement technique for physicians who have only one epicardial lead available after removing infected endocardial leads. This is illustrated by the innovative use of an endocardial lead as an epicardial sensing lead.

Leadership as a determinant of need fulfillment: implications for meta-theory, methods, and practice.

Of all the most prominent business concepts (e.g., DE&I, employee well-being, employee engagement, organizational culture, etc.) none rivals leadership in terms of public interest and annual monetary investment. Despite the obvious importance of leadership as a determinant of many important outcomes, the concept of leadership has been surprisingly hard to pin down, lacking consensus as to its precise meaning. As numerous authors introduce ever more constructs (e.g., servant leadership, toxic leadership, sustainable leadership, transformational leadership, etc.), the leadership concept has become emblematic of the problem of construct proliferation. Like the related fields of employee engagement, subjective well-being, and organizational culture, the leadership field is in desperate need of a clearly articulated meta-theory to house its many constructs, allowing theory and measurement to build up instead of continuing to pile up. This paper argues for grounding the concept of leadership within the psychological literature on human needs. In reviewing the leading definitions of leadership in the literature we find that they are reducible to a core set of follower needs that can be facilitated or inhibited by leaders. We propose that there is substantial value in adopting a comprehensive needs-based taxonomy over current approaches. We consider the impact of setting the concepts of leadership within existing need constructs for each of the following: (a) theory, especially the development of leadership frameworks and particularly how the concept of leadership relates to the concepts of organizational culture, employee well-being, and employee engagement; (b) methods, including the value of applying a comprehensive, structured model; and (c) practice, where we emphasize the practical advantages of clear operational definitions.

Religion and mental health: an Eastern Mediterranean region perspective.

In the last decade, spirituality gained increasing recognition, with an individualized approach to the significance of symptoms and the purpose of treatment. Many psychiatrists started adopting the term "biopsychosocial-spiritual" management. Modern psychological therapies have drawn from the principles of ancient Eastern traditions. However, the spiritual beliefs within Islam and their impact on mental well-being have yet to be studied sufficiently. The Eastern Mediterranean region is largely Muslim and shares similar cultural beliefs. This paper explores some of these beliefs and their impact on perceptions of mental health and help-seeking behavior. The paper will focus primarily on the religious ideologies, the concepts of punishment and reward from Allah (God) in the context of the high stigma associated with mental illness in the region, and the knowledge, attitudes, and perceptions toward people with mental illness. We will explore cultural beliefs in possession, evil eye, and black magic and how they influence treatment adherence. Traditional and faith healers remain very popular in this part of the world and are often the first line of treatment for people presenting with mental illness. Suicide and self-harming are particularly sensitive areas due to their religious implications in life and death, as well as the afterlife. Despite the global interest in the Eastern Mediterranean region, there still is a shortage of research on the intercultural aspects of this region, especially regarding mental health assessments. In this regard, we will explore some published literature from Qatar and draw comparisons with regional findings.

Protocol for quantifying drug sensitivity in 3D patient-derived ovarian cancer models.

Three-dimensional (3D) ex vivo cultures allow the study of cancer progression and drug resistance mechanisms. Here, we present a protocol for measuring on-target drug sensitivity in a scaffold-free 3D culture system through quantification of apoptotic tumor cells. We provide detailed steps for sample processing, immunofluorescence staining, semi-high-throughput confocal imaging, and imaged-based quantification of 3D cultures. This protocol is versatile and can be applied in principle to any patient-derived material.

Comparing Physical Activity and Exercise Experiences, Values, and Beliefs of Latino, Latina, and/or Latine People and Non-Latino, Non-Latina, and/or Non-Latine People with Parkinson Disease: A Qualitative Study.

OBJECTIVE: The objective of this study was to examine and compare the beliefs, values, experiences, and barriers to and facilitators of exercise in Latino/a/e (Latino, Latina, and/or Latine) people and non-Latino/a/e (non-Latino, non-Latina, and/or non-Latine) people with Parkinson disease (PD). METHODS: This study involved a qualitative research design, with data collection via focus groups of Latino/a/e and non-Latino/a/e people with PD. A semistructured interview protocol was used, and a theoretical (social ecological model) thematic analysis was used for data analysis. RESULTS: Twenty-five individuals (15 non-Latino/a/e and 10 Latino/a/e) participated across 5 focus groups. While there were several common themes among both groups, 7 themes related to exercise in the management of PD were unique to Latino/a/e participants: generalized versus specific knowledge of exercise components; family can have both facilitating and impeding roles; finances influence access to exercise and physical therapy; prior emotional experiences related to exercise and PD affect the future use of exercise; group exercise can have both facilitating and impeding roles; availability and access to a variety of modes and stratification of exercise classes for people with PD facilitate exercise; and cultural beliefs and expectations can impede the use of exercise at the population level. CONCLUSION: Latino/a/e people with PD have overlapping and unique themes related to the use of exercise. Distinctive factors must be addressed in physical activity interventions and during physical therapists' care of people with PD to maximize the use of exercise and ultimately improve health equity in this overburdened and underserved population. IMPACT: Physical therapists should provide information and education to their Latino/a/e patients with PD and family members on the frequency, intensity, type, and time of exercise and how to safely participate in physical activity in the home and community to best manage PD. Physical therapists and exercise professionals should work at the environmental level to increase access to affordable, culturally tailored, PD-specific exercise services and enhance community knowledge of PD.

GTF2H5 Identified as a crucial synthetic lethal target to counteract chemoresistance in colorectal cancer.

BACKGROUND: Synthetic lethality (SL) emerges as a novel concept being explored to combat cancer progression and resistance to conventional therapy. Despite the efficacy of chemotherapy in select cases of colorectal cancer (CRC), a substantial proportion of patients encounter challenges, leading to an adverse prognosis of CRC patients. CRC-related SL genes offer a potential avenue for identifying therapeutic targets. METHODS: CRC-related SL genes were obtained from the SynLethDB database. The bulk RNA sequencing data, mutation data, and clinical information for treated and untreated CRC patients were enrolled from the UCSC and GEO databases. The Tumor Immunology Single Cell Center database served as the repository for collecting and analyzing single-cell RNA sequencing data. The synergistic killing effect of SL genes and chemotherapeutic drugs on resistant cells was experimentally verified. RESULTS: In the present study, pivotal SL genes associated with chemoresistance identified by using WGCNA and CRC patients categorized into two groups based on these genes. Variations between the groups were most pronounced in pathways associated with extracellular matrix remodeling. Further by integrating mutation data, five potential SL genes were discerned, which were highly expressed in the presence of TP53 or KRAS mutations, leading to a severely poor prognosis. Subsequent time series analysis revealed that the expression of GTF2H5 was gradually elevated at different stages of the transition from sensitive to resistant in CRC cells. Finally, it was preliminarily verified by experiments that GTF2H5 may play a key role in driving the drug-resistant transition within CRC cells. CONCLUSIONS: The identification of SL genes that collaboratively interact with chemotherapeutic agents could provide new insights into solving the issue of chemotherapy resistance in CRC patients. And GTF2H5 wields a fundamental influence in inducing chemoresistance in CRC, which provided a potential therapeutic target for CRC.

Osteoblast and osteoclast activity on collagen-based 3D printed scaffolds enriched with strontium-doped bioactive glasses and hydroxyapatite nanorods for bone tissue engineering.

Bone tissue engineering (BTE) aims to promote bone regeneration by means of the synergistic effect of biomaterials, cells, and other factors, as potential alternative to conventional treatments for bone fractures. To this aim, a composite material was developed, based on collagen type I, strontium-enriched mesoporous bioactive glasses, and hydroxyapatite nanoparticles as bioactive and biomimetic components. Nanostructured scaffolds were 3D printed and subsequently chemically crosslinked with genipin to improve mechanical properties and stability. The developed nanostructured system was maintained in culture until 3 weeks with a co-culture of human bone cells to provide an ex vivo model of bone microenvironment and examine the cellular crosstalk and signaling pathways through paracrine cell activities. Human osteoblasts (OBs), derived from trabecular bone, and human osteoclast precursors (OCs), isolated from buffy coat samples were involved, with OBs seeded on the scaffold and OCs precursors seeded in a transwell device. When compared to the material without inorganic components, the bioactive and biomimetic scaffold positively influenced cell proliferation and cell metabolic activity, boosting alkaline phosphatase activity of osteoblasts, and reducing osteoclast differentiation. Thus, the bioactive and biomimetic system promoted an enhanced cellular response, highlighting its potential application in bone tissue engineering. .

Investigating immune-modulatory function of α-glucopyranose-rich compound polysaccharides by MC38-N4/OT-I co-culture system.

The potential antitumor function of polysaccharides is well accepted, it is unclear whether polysaccharides have immunoregulatory effect on CD8+ T lymphocyte cells to attack tumor cells. To evaluate the CD8+ T function enhancing role of polysaccharide compounds, the MC38-N4/OT-I co-culture system was established. The synergistic and complementary immune effect of α-glucopyranose-rich compound polysaccharides can be achieved by manipulating the antigen-specific T-cell expansion capacity and efficacy. This study was designed to investigate the antitumor-enhancement activity of a α-glucopyranose-rich compound polysaccharides by determining the activation of CD8+ T cells in a co-culture system. Compared to the control group (42.5 % ±â€¯0.72 %), the specific α-glucopyranose-rich compound polysaccharides, comprising Agaricus blazei Murill, Grifola frondosa and Pericarpium Citri Reticulatae, demonstrated a significant decrease (20.4 % ±â€¯1.23 %, p < 0.05) in the survival rate of MC38-N4 cells in the co-culture system. Additionally, the α-glucopyranose-rich compound polysaccharides resulted in a substantial increase (p < 0.01) in the proportion of CD8+ T cells and CD62L+ central memory T cells, which is a less differentiated T cell subset with high immune activity. Collectively, we reported that specific polysaccharide combination, which remodel the function of cytotoxic T cells and provided a basis for improving immune functions by using the specific types of polysaccharides.

MUL1 identified as mitochondria-linked biomarker promoting cisplatin resistance in OC cells.

Ovarian cancer (OC) ranks among the prevalent tumors affecting the female reproductive system. The aim of this study was to evaluate mitochondria-associated platinum resistance genes using organoid models. Univariate Cox regression, LASSO and multivariate Cox regression analyses were performed on The Cancer Genome Atlas (TCGA) database to construct 2-gene prognostic signature (MUL1 and SSBP1), and GSE26712 dataset was used for external validation. In addition, the relationship between MUL1 and platinum resistance was examined by organoid culture, lentiviral transduction, CCK8 assay, and Western blot. The results showed that patients in the high-risk group exhibited significantly worse OS (P = 0.002, P = 0.017). Drug sensitivity analysis revealed that platinum resistance increased with the upregulation of MUL1 expression (Cor = 0.5154, P = 0.02). Our experimental findings demonstrated that knockout of the MUL1 gene significantly increased apoptosis and enhanced the sensitivity of the OC cell line A2780 to cisplatin. Through this study, we have provided strong evidence for further research on prognostic risk factors and individualized treatment in OC patients, and provided new insights into addressing platinum resistance in OC.

Tapping the potential of Calotropis procera hairy roots for cardiac glycosides production and their identification using UHPLC/QTOF-MS.

The present work deals with the establishment of hairy root cultures from different explants of C. procera using Agrobacterium rhizogenes strain A4. A high transformation frequency (95%) was obtained from leaves followed by cotyledons (81.6%) and hypocotyls (38.3%). Genetic transformation of hairy roots was confirmed through PCR by amplifying a 400 bp fragment of the rolB gene. Hairy roots were highly branched, possessed plagiotropic and rapid growth on hormone-free ½ B5 medium. Ten cardiac glycosides, including calotropagenin, calotoxin, frugoside, coroglaucigenin, calotropin, calactin, uzarigenin, asclepin, uscharidin, and uscharin, based on their specific masses and fragmentation properties were identified in ethanolic extracts of hairy roots by ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry UHPLC/QTOF-MS. This protocol could be used as a powerful tool for large-scale in vitro production of highly valued cardiac glycosides and for further transcriptomics or metabolomics studies.

Analytical methods, risk assessment, and mitigation strategies for furan in processed foods in various countries.

This article provides an overview of analytical methods for measuring furan levels in food. Given the potential carcinogenicity of furans in humans, several studies have focused on assessing furan levels in various food products. In this review, we specifically examine furan levels in foods that are central to regional culinary traditions and summarize the results of country-specific risk assessments. Consequently, we have identified foods that contribute significantly to dietary furan exposure in each region. Coffee and baby foods, regardless of region, emerged as the primary sources of furan intake among adults and infants, respectively. Several previous studies have been conducted to develop various mitigation strategies aimed at reducing exposure to furan through food intake. Therefore, in this paper, we categorize effective mitigation strategies into two main groups: alterations to processing conditions and the addition or removal of food additives and ingredients.

Spheroid trilineage differentiation model of primary mesenchymal stem/stromal cells under hypoxia and serum-free culture conditions.

Due to their unique properties, human mesenchymal stem/stromal cells (MSCs) possess tremendous potential in regenerative medicine, particularly in cell-based therapies where the multipotency and immunomodulatory characteristics of MSCs can be leveraged to address a variety of disease states. Although MSC-based cell therapeutics have emerged as one of the most promising medical treatments, the clinical translation is hampered by the variability of MSC-based cellular products caused by tissue source-specific differences and the lack of physiological cell culture approaches that closely mimic the human cellular microenvironment. In this study, a model for trilineage differentiation of primary adipose-, bone marrow-, and umbilical cord-derived MSCs into adipocytes, chondrocytes and osteoblasts was established and characterized. Differentiation was performed in spheroid culture, using hypoxic conditions and serum-free and antibiotics-free medium. This platform was characterized for spheroid diameter and trilineage differentiation capacity reflecting functionality of differentiated cells, as indicated by lineage-specific extracellular matrix (ECM) accumulation and expression of distinct secreted markers. The presented model shows spheroid growth during the course of differentiation and successfully supports trilineage differentiation for MSCs from almost all tissue sources except for osteogenesis of umbilical cord-derived MSCs. These findings indicate that this platform provides a suitable and favorable environment for trilineage differentiation of MSCs from various tissue sources. Therefore, it poses a promising model to generate highly relevant biological data urgently required for clinical translation and therefore might be used in the future to generate in vitro microtissues, building blocks for tissue engineering or as disease models.

Assessing inoculation's effectiveness in motivating resistance to conspiracy propaganda in Finnish and United States samples.

Introduction: This study tested the motivational power of inoculation to foster resistance to conspiracy propaganda (9/11 Truth Movement), comparing inoculation effects across United States and Finnish study participants. Method: We used a 2 inoculation (treatment vs. control) × 2 national culture (American vs. Finnish) independent groups design (N = 319), while examining the effects of motivational threat and thinking modes-analytic vs. intuitive-on the inoculation process. To test the effectiveness of the inoculation strategy, we used an excerpt from a conspiracy film Loose Change as a counterattitudinal attack message. Results: Our results indicated that inoculation was effective at motivating resistance regardless of national culture. Inoculation effects emerged mostly as a direct effect on resistance and two indirect effects wherein motivational threat mediated the relationship between inoculation and resistance as well as inoculation and analytic mode of message processing. Although we found that an increase in analytic mode of processing facilitated resistance and intuitive processing increased conspiracy-theory endorsement, the indirect effects between inoculation and resistance via message processing modes were not significant. Finally, the data revealed national culture differences in analytic mode and cultural-context differences mostly pertaining to the relationships between thinking styles, media literacy, and modes of thinking. Discussion: These results offer important theoretical implications for inoculation scholarship and suggest viable practical solutions for efforts to mitigate misinformation and conspiratorial beliefs.

The contribution of quality management practices to student performance: Mediated by school culture.

School management is responsible and accountable for implementing educational policies into practice effectively and efficiently to provide quality education. Simultaneously, school management can grasp the core features of the whole school process and identify the relationship among three variables: quality management practices, school culture, and student performance. The current study aims to explore the school principals' perception about quality management practices and its relationship with school culture and student performance in the public secondary schools of Punjab province, Pakistan. In order to achieve the objectives of the study, the study adopted an exploratory sequential mix-methods research design. The researcher conducted a systematic literature review of sixty-three previous studies and interviews with eleven school principals for the qualitative data. Based on results obtained from the qualitative phase, a questionnaire was prepared and dispatched to 150 school principals to get quantitative data. Successively 120 valid responses were received. SEM analysis was performed to get quantitative results. The study's preliminary conclusion demonstrated a positive connection between quality management and student performance in public secondary schools, and quality management was also a significant predictor of school culture. Further, school culture served as a complete mediator between quality management and student performance.

Generation of mouse testicular organoids with highly compartmentalized tubular lumen structure and their cryopreservation.

Testicular organoids have great potential for maintaining male fertility and even restoring male infertility. However, existing studies on generating organoids with testis-specific structure and function are scarce and come with many limitations. Research on cryopreservation of testicular organoids is even more limited, and inappropriate cryopreservation methods may result in the loss of properties in resuscitated or regenerated organoids, rendering them unsuitable for clinical or research needs. In this paper, we investigated the effects of mouse age and cell number on the self-aggregation of testicular cells into spheres in low-adsorption plates. Various media compositions, culture systems, and cell numbers were used to culture cell spheres for 14 days to form testicular organoids, and the self-organization of the organoids was assessed by histological and immunofluorescence staining. We determined the appropriate cryopreservation conditions for testicular cells, cell spheres, and tissues. Subsequently, organoids derived from cryopreserved testicular tissues, testicular cells, and testicular cell spheres were compared and evaluated by histological and immunofluorescence staining. The results indicate that testicular cell spheres consisting of 30 × 104 testicular cells from 2-week-old mice were able to form organoids highly similar to the luminal structure and cell distribution of natural mouse testicular tissues. This transformation occurred over 14 days of incubation in α-MEM medium containing 10 % knockout serum replacer (KSR) using an agarose hydrogel culture system. Additionally, the Sertoli cells were tightly connected to form a blood-testis barrier. The relative rates of tubular area, germ cells, Sertoli cells, and peritubular myoid cells were 36.985 % ±â€¯0.695, 13.347 % ±â€¯3.102, 47.570 % ±â€¯0.379, and 27.406 % ±â€¯1.832, respectively. The optimal cryopreservation protocol for primary testicular cells involved slow freezing with a cryoprotectant consisting of α-MEM with 10 % dimethyl sulfoxide (DMSO). Slow freezing with cryoprotectants containing 5 % DMSO and 5 % ethylene glycol (EG) was optimal for all different volumes of testicular cell spheres. Compared to testicular organoids generated from frozen testicular tissue and cell spheres, freezing testicular cells proved most effective in maintaining organoid differentiation characteristics and cell-cell interactions. The findings of this study contribute to a "universal" testicular organoid in vitro culture protocol with promising applications for fertility preservation and restoration in prepubertal cancer patients and adult infertile patients.

Multiparameter imaging flow cytometry-based cytokinesis-block micronucleus assay: Reduction of culture time and blood volume for improved efficiency.

In the event of a large-scale incident involving radiological or nuclear exposures, there is a potential for large numbers of individuals to have received doses of radiation sufficient to cause adverse health effects. It is imperative to quickly identify these individuals in order to provide information to the medical community to assist in making decisions about their treatment. The cytokinesis-block micronucleus assay is a well-established method for performing biodosimetry. This assay has previously been adapted to imaging flow cytometry and has been validated as a high-throughput option for providing dose estimates in the range of 0-10 Gy. The goal of this study was to test the ability to further optimize the assay by reducing the time of culture to 48 h from 68 h as well as reducing the volume of blood required for the analysis to 200 µL from 2 mL. These modifications would provide efficiencies in time and ease of processing impacting the ability to manage large numbers of samples and provide dose estimates in a timely manner. Results demonstrated that either the blood volume or the culture time could be reduced while maintaining dose estimates with sufficient accuracy for triage analysis. Reducing both the blood volume and culture time, however, resulted in poor dose estimates. In conclusion, depending on the needs of the scenario, either culture time or the blood volume could be reduced to improve the efficiency of analysis for mass casualty scenarios.

The impact of raw material form and starter culture inoculation on bacterial communities and metabolites in fermented anchovy sauce.

This study explored the dynamics of anchovy sauce fermentation and investigated how the raw material form and the use of starter cultures affect bacterial and metabolite profiles. Using a comprehensive approach, we examined the fermentation process using anchovies in two forms (whole and ground) and three different starter cultures. The use of ground anchovies resulted in an accelerated fermentation process for anchovy sauce; however, the increased diversity of bacterial phylotypes and altered accumulation of biogenic amines were observed. Inoculation of starter cultures resulted in a shift from spontaneous to controlled fermentation, highlighting their ability to regulate bacterial communities. Despite a slightly reduced fermentation rate, inoculation with Tetragenococcus halophilus was shown to be a potent method for reducing biogenic amines and affecting metabolite profiles. As the industry strives to balance fermentation speed and quality, our research could provide insights for improving the efficiency, safety, and quality of anchovy sauce production.

Novel three-dimensional live skin-like in vitro composite for bioluminescence reporter gene assay.

We genetically manipulated HaCaT cells, a spontaneously immortalised normal keratinocyte cell line, to stably express two different coloured luciferase reporter genes, driven by interleukin 8 (IL-8) and ubiquitin-C (UBC) promoters, respectively. Subsequently, we generated a three-dimensional (3D) skin-like in vitro composite (SLIC) utilising these cells, with the objective of monitoring bioluminescence emitted from the SLIC. This SLIC was generated on non-woven silica fibre membranes in differentiation medium. Immunohistochemical analyses of skin differentiation markers in the SLIC revealed the expression of keratins 2 and 10, filaggrin, and involucrin, indicating mature skin characteristics. This engineered SLIC was employed for real-time bioluminescence monitoring, allowing the assessment of time- and dose-dependent responses to UV stress, as well as to hydrophilic and hydrophobic chemical loads. Notably, evaluation of responses to hydrophobic substances has been challenging with conventional 2D cell culture methods, suggesting the need for a new approach, which this technology could address. Our observations suggest that engineered SLIC with constitutively expressing reporters driven by selected promoters which are tailored to specific objectives, significantly facilitates assays exploring the physiological functions of skin cells based on genetic response mechanisms. It also highlights new avenues for evaluating the physiological impacts of various compounds designed for topical application to human skin.