Nonetheless, thermogenic activity has frequently been evaluated using indirect methods, such as monitoring oxygen consumption. For the purpose of elucidating the mechanisms of heat production in BACs, recent developments in fluorescent nanothermometers allow direct measurement of intracellular temperatures. In this chapter, we introduce a protocol that directly measures temperature within primary cultured BACs, utilizing a cationic fluorescent polymeric thermometer. We foresee this protocol contributing substantially to the understanding of the thermogenesis mechanism in BAC cultures.
In the pursuit of novel anti-obesity treatments, the induction of thermogenesis in brown and beige adipocytes presents a key target, consequently demanding the development of precise techniques for measuring heat production within these cells. Modern isothermal microcalorimetric methods enable the high-throughput, quantitative assessment of cellular heat production, even with a restricted quantity of sample material. Genetic affinity For the purpose of determining thermogenesis in adipocytes, both in floating and adherent forms, isolated from diverse murine depots and human cell lines, this technique is elucidated.
High-resolution respirometry is a prevalent technique for measuring mitochondrial respiratory rates. To derive the rate of oxygen consumption (JO2), a polarographic electrode within the respirometry chamber detects changes in oxygen concentration. A modified protocol for studying the bioenergetic function of mitochondria from mouse brown adipose tissue (BAT) is described in the following. Analyzing energy transduction via oxidative phosphorylation (OXPHOS) in brown adipose tissue (BAT) mitochondria, with their uncoupling protein 1 (UCP1), requires unique considerations and opportunities when applying high-resolution respirometry.
Determining the respiratory capacity of brown adipocyte mitochondria outside the body provides essential insights into the cellular control mechanisms of mitochondrial uncoupling within brown adipose tissue. We present a detailed description of two protocols used to isolate brown preadipocytes from mice, directing their ex vivo development into mature brown adipocytes and then quantifying their mitochondrial uncoupling capacity using respirometry techniques.
Metabolic abnormalities accompany the onset of obesity, stemming from dysfunction within adipocyte expansion processes. Evaluating the metabolic status of adipose tissue requires an assessment of adipocyte size and abundance. This document illustrates three different ways to measure adipocyte size in tissue specimens obtained from both human and rodent models. While the presented primary method demonstrates greater resilience, it incorporates osmium, a toxic heavy metal, which necessitates specific handling protocols, disposal procedures, and specialized equipment. Researchers can employ two more techniques, elaborated below, to be beneficial.
Brown adipose tissue (BAT) is essential for the maintenance of appropriate energy levels in the body. In vitro studies on brown adipose tissue are facilitated by the use of primary brown adipocyte cultures, a powerful and biologically relevant approach. This document outlines a thorough procedure for the separation and maturation of adipocyte precursors originating from newborn murine interscapular brown adipose tissue (iBAT).
In their developmental journey, fibroblastic preadipocyte precursors eventually become terminally differentiated adipocytes. Using a defined method, we isolate and proliferate preadipocytes from murine subcutaneous white adipose tissue, which are then cultured to differentiate into mature adipocytes; these are designated primary in vitro differentiated preadipocytes (PPDIVs). In contrast to adipogenic cell lines, the metabolic processes of PPDIV and the secretion of adipokines demonstrate a closer alignment with the biological mechanisms observed in vivo adipocytes. Mature, primary adipocytes, while crucial for in vivo studies, are challenging to work with due to their fragility and tendency to float, making them unsuitable for many cell culture-based procedures. Genetically modified adipocytes can be produced by PPDIVs, taking advantage of transgenic and knockout mouse models. Thusly, PPDIVs are a valuable resource for research into the behavior and function of adipocytes within cultured cells.
The therapeutic target of increasing brown adipose tissue (BAT) mass and activating it offers a potential strategy for preventing and treating obesity and its associated health complications. Due to obesity and diabetes, patients typically possess lower quantities of brown adipose tissue (BAT), rendering it imperative to identify and implement effective means of expanding their BAT reserves. The development, differentiation, and optimal activation of human BAT remain largely unknown. The process of accessing human brown adipose tissue (BAT) is complicated by its infrequent occurrence and scattered locations within the body. VT103 mw The constraints in place make any in-depth investigation into the developmental and functional mechanisms of BAT in human subjects virtually impossible. We've created a groundbreaking, chemically defined protocol for turning human pluripotent stem cells (hPSCs) into authentic brown adipocytes (BAs), which surpasses the limitations of existing methods. Human brown adipose tissue's physiological developmental pathway is methodically and sequentially outlined in this protocol.
Cancer treatment's precision medicine approach, although promising, is mostly applied to tumors with clinically relevant genetic mutations. Precision medicine benefits from expanded scope by using gene expression signatures to forecast responses to standard cytotoxic chemotherapy without the need to assess mutational status. A novel signature extraction technique, drawing inspiration from the principle of convergent phenotypes, is presented. This principle posits that tumors, despite differing genetic origins, can independently develop similar phenotypic characteristics. This method, drawing inspiration from evolutionary processes, enables the creation of consensus signatures, allowing for the prediction of responses to over 200 chemotherapeutic drugs cataloged in the Genomics of Drug Sensitivity in Cancer (GDSC) Database. We illustrate its application by isolating the Cisplatin Response Signature (CisSig) in this example. Utilizing the GDSC database, we demonstrate this signature's predictive capacity for cisplatin response within carcinoma-based cell lines, a capacity further confirmed by its alignment with clinical trends seen in independent tumor sample datasets from The Cancer Genome Atlas (TCGA) and Total Cancer Care (TCC). Lastly, we demonstrate initial validation of CisSig's applicability to muscle-invasive bladder cancer, projecting overall survival in a small cohort of patients undergoing cisplatin-containing chemotherapy. This methodology yields robust signatures capable of predicting traditional chemotherapeutic responses, a prospect that, upon further clinical validation, could dramatically expand the reach of personalized medicine in oncology.
The Covid-19 pandemic's global impact became apparent at the close of 2019, and the utilization of a variety of vaccine platforms became a critical approach to its eventual resolution. Indonesia's research efforts resulted in the creation of an adenovirus-based Covid-19 vaccine candidate, addressing the global disparity in vaccine technology access. The pAdEasy vector was engineered to incorporate the SARS-CoV-2 Spike (S) gene. Recombinant adenovirus was generated by transfecting the serotype 5 adenovirus (AdV S) recombinant genome into AD293 cells. The PCR-based characterization method identified the spike gene. The expression of the S protein was confirmed by transgene expression analysis in AD293 and A549 cells that were infected with AdV S. Viral production optimization revealed the highest titer at an MOI of 0.1 and 1 after 4 days of incubation. By injecting Balb/c mice with 35107 ifu of purified adenovirus, the in vivo study was undertaken. Following a single dose of AdV S, S1-specific IgG levels were notably elevated up to 56 days post-administration. Remarkably, AdV S treatment in Balb/c mice led to a substantial rise in S1 glycoprotein-specific IFN- ELISpot readings. The AdV S vaccine candidate, produced at a laboratory scale, demonstrated immunogenicity, and caused no severe inflammation in Balb/c mice. As a pioneering endeavor, this Indonesian study paves the way for adenovirus-based vaccine production.
The development of tumors is influenced by chemokines, a group of small cytokines, which demonstrate chemotactic capability. The contributions of chemokines to anti-tumor immunity are subjects of intense research interest. Within the category of chemokines, CXCL9, CXCL10, and CXCL11 are particularly significant molecules. It is well documented that these three chemokines can engage with their common receptor CXCR3, thereby modulating immune cell differentiation, migration, and infiltration of tumors, ultimately affecting the rate of tumor growth and metastasis. In this paper, we describe the impact of the CXCL9/10/11-CXCR3 axis on the tumor microenvironment and discuss the latest research assessing its prognostic significance for different types of cancer. Moreover, immunotherapy contributes to improved survival rates among oncology patients, though drug resistance remains a challenge for some. Studies have demonstrated that the control of CXCL9/10/11-CXCR3 interaction in the tumor microenvironment impacts the process of immunotherapy resistance. Molecular Biology Services We also detail novel methods of revitalizing immune checkpoint inhibitor responsiveness via the CXCL9/10/11-CXCR3 pathway in this report.
The heterogeneous nature of childhood asthma is evident in the diverse clinical presentations stemming from persistent airway inflammation. Asthma, categorized as nonallergic, is differentiated by the absence of allergic sensitization. The exploration of both the clinical features and the immunopathological pathways of non-allergic childhood asthma has been notably understudied. We sought to compare clinical characteristics between non-allergic and allergic childhood asthma, employing microRNA analysis to probe the mechanistic underpinnings of non-allergic childhood asthma.