Specific Gene Expression Research Articles

Practical laboratory class to assess gene silencing using CRISPR interference (CRISPRi) technology in the archaeon Haloferax volcanii.

Perturbation of gene expression using RNA interference (RNAi) or CRISPR interference (CRISPRi) is a useful strategy to explore the function of essential genes. In the archaeon Haloferax volcanii, the CRISPR-Cas system has been adapted as a CRISPRi tool to silence the expression of specific genes. We developed a laboratory class (LC) to conceptualize gene silencing through inactivation of the H. volcanii LonB protease gene, a negative regulator of carotenoid pigments biosynthesis, using CRISPRi. This LC has been successfully applied in the Biology and Biochemistry of Microorganisms course for undergraduate students of Biology in 2022 and 2023. The following objectives were proposed: (a) generate H. volcanii mutant strains with reduced expression of the lonB gene using CRISPRi; (b) examine the effect of lonB gene silencing on cell pigmentation and growth rate; (c) assess lonB gene repression by Western blotting (WB). This LC allows students to obtain and screen CRISPRi silenced-mutants by means of simple procedures using a non-pathogenic organism as well as handle basic microbiology, biochemistry and molecular biology protocols. Additionally, the LC fosters social actions through collaborative work (experimental work), the interpretation and discussion of data and the ability to communicate outcomes orally and in a written format (scientific report).

Exploring the genetic expression of Wickerhamomyces anomalus during biosurfactant production from waste cooking oil.

Biosurfactants are valuable eco-friendly compounds with broad industrial applications, particularly when produced sustainably using yeast and renewable carbon sources. Despite the potential of yeast in biosurfactant synthesis, little is known about the specific gene expression changes underlying this process. This study investigates the genetic response of Wickerhamomyces anomalus CCMA 0358 to biosurfactant production using waste cooking oil (WCO) as a low-cost carbon source. During a 0-12 hour fermentation period, RNA sequencing revealed 829 differentially expressed genes in W. anomalus grown with WCO, suggesting targeted metabolic adaptations. Pathway analysis showed WCO's significant impact on glycolysis, gluconeogenesis, and lipid biosynthesis. Gene ontology annotations further indicated adaptive responses in ribosome biogenesis and lipid metabolism, which are crucial for the efficient utilization of WCO. Notably, WCO induced the upregulation of very-long-chain fatty acid (VLCFA) precursors and adjustments in glycolytic enzyme expression, both essential for biosurfactant production. This study reveals, for the first time, the specific genetic pathways and metabolic adjustments that W. anomalus employs to produce biosurfactants from WCO. The increased expression of lipid metabolism enzymes and cell membrane components highlights a tailored adaptive mechanism for lipid-rich waste substrates, positioning W. anomalus as a promising candidate for sustainable biosurfactant production.

Hidden features: CD36/SR-B2, a master regulator of macrophage phenotype/function through metabolism

Once thought to be in a terminally differentiated state, macrophages are now understood to be highly pliable, attuned and receptive to environmental cues that control and align responses. In development of purpose, the centrality of metabolic pathways has emerged. Thus, macrophage inflammatory or reparative phenotypes are tightly linked to catabolic and anabolic metabolism, with further fine tuning of specific gene expression patterns in specific settings. Single-cell transcriptome analyses have revealed a breadth of macrophage signatures, with some new influencers driving phenotype. CD36/Scavenger Receptor B2 has established roles in immunity and lipid metabolism. Macrophage CD36 is a key functional player in metabolic expression profiles that determine phenotype. Emerging data show that alterations in the microenvironment can recast metabolic pathways and modulate macrophage function, with the potential to be leveraged for therapeutic means. This review covers recent data on phenotypic characterization of homeostatic, atherosclerotic, lipid-, tumor- and metastatic-associated macrophages, with the integral role of CD36 highlighted.

Bone metastasis in endocrine-related cancer: unravelling invasion and destruction.

Bone is a common and debilitating site for metastatic cancer cell expansion. Skeletal metastasis is a multistage process, with primary stages of circulating tumour cells, progressing to a dormant state in vasculature and bone marrow niches, followed by tumorigenic reactivation, proliferation, and finally bone destruction. The frequency of bone metastasis is reconciled in Paget's "seed and soil" hypothesis, where a conducive microenvironment (bone niche) is essential for cancer cell colonisation. Cancer cells can mimic bone cells (osteomimicry) and interact with the bone marrow's vascular architecture, utilising pathways akin to hematopoietic stem cell expansion. Current research suggests that each phase of bone metastasis is associated with specific gene expression and protein abundance patterns. For example, E-selectin, CXCR-4, and CXCL-12 are crucial for cancer cell homing, dormancy, and colonisation of bone tissue. In contrast, different primary cancers appear to have unique staging profiles. In prostate cancer, dormancy is modulated by the CXCR-4/CXCL-12, ANXA2/CXCL12, and GAS6 pathways, while in breast cancer, dormancy involves ERK1/2, p38, MSK1, LIF, BMP-7, TGF-β1/2, and bone resorption factors. Conversely, osteoblastic metastasis in both breast and prostate cancers is characterised by ET-1, Dkk1 suppression, and the release of IL-6, MCP-1, VEGF, FGF, and IGF, while osteolytic metastasis primarily depends on PTHrP, RANKL, OPG, TGF-β, IGF, TNF-α, IL-1, and IL-7. Understanding the complex molecular mechanisms facilitating cancer cell colonisation and expansion in bone tissues is essential for developing effective treatments to prevent bone metastases . In this review, we discuss current theories linking bone remodelling with bone.

The Mechanisms of Cadmium Toxicity in Living Organisms

Cadmium (Cd) is a toxic metal primarily found as a by-product of zinc production. Cd was a proven carcinogen, and exposure to this metal has been linked to various adverse health effects, which were first reported in the mid-19th century and thoroughly investigated by the 20th century. The toxicokinetics and dynamics of Cd reveal its propensity for long biological retention and predominant storage in soft tissues. Until the 1950s, Cd pollution was caused by industrial activities, whereas nowadays, the main source is phosphate fertilizers, which strongly contaminate soil and water and affect human health and ecosystems. Cd enters the human body mainly through ingestion and inhalation, with food and tobacco smoke being the primary sources. It accumulates in various organs, particularly the kidney and liver, and is known to cause severe health problems, including renal dysfunction, bone diseases, cardiovascular problems, and many others. On a cellular level, Cd disrupts numerous biological processes, inducing oxidative stress generation and DNA damage. This comprehensive review explores Cd pollution, accumulation, distribution, and biological impacts on bacteria, fungi, edible mushrooms, plants, animals, and humans on a molecular level. Molecular aspects of carcinogenesis, apoptosis, autophagy, specific gene expression, stress protein synthesis, and ROS formation caused by Cd were discussed as well. This paper also summarizes how Cd is removed from contaminated environments and the human body.

Alteration in the balance between ABA and GA signaling mediates genetic variation in induction and retention of dormancy during seed maturation in wheat

Induction and retention of dormancy are among the physiological processes that take place during seed maturation; however, the molecular mechanisms underlying these events are poorly understood in wheat. This study revealed that seed maturation in wheat is associated with decreases in abscisic acid (ABA) and gibberellin (GA) levels irrespective of dormancy level exhibited by the seeds mainly via expression of specific ABA (TaCYP707A1) and GA (TaGA3ox2, TaGA2ox3 and TaGA2ox6) metabolism genes. Consistently, ABA to GA level ratio decreased during maturation in both highly dormant and low-dormant seeds with no apparent difference in the ratio of their levels between the two seed samples. Our data, however, showed a close association between the induction and retention of dormancy during seed maturation and modulation of the balance between ABA and GA signaling via expression of specific genes that acts as positive regulators seed response to ABA (TaPYL5 and TaABI5) and GA (TaGAMyb). Consistently, the highly dormant and low-dormant seeds exhibited substantial variation in their sensitivity to ABA and GA during their maturation. The findings of this study highlight that genetic variation in induction and retention of dormancy during wheat seed maturation can be mediated by a shift in balance between seed sensitivity to ABA and GA independent of a shift in balance between their levels.

Inhibition of Mitochondrial Bioenergetics and Hypoxia to Radiosensitize Diffuse Intrinsic Pontine Glioma.

Diffuse Intrinsic Pontine Gliomas (DIPG) and other H3K27M-mutated diffuse midline gliomas (DMGs) are brain tumors that primarily affect children. Radiotherapy is the standard of care but only provides temporary symptomatic relief due to radioresistance. While hypoxia is a major driver of radioresistance in other tumors, there is no definitive evidence that DIPGs are hypoxic. DIPGs often contain histone mutations, which alter tumor metabolism and are also associated with radioresistance. Our objective was to identify the metabolic profiles of DIPG cells, detect hypoxia signatures, and uncover metabolism-linked mechanisms of radioresistance to improve tumor radiosensitivity. Using DIPG models combined with clinical datasets, we examined mitochondrial metabolism and signatures of hypoxia. We explored DIPG reliance on mitochondrial metabolism using extracellular flux assays and targeted metabolomics. In vitro and in vivo models were used to explore the mechanisms of targeting mitochondrial bioenergetics and hypoxia for radiosensitization. Treatment-induced transcriptomics and metabolomics were also investigated. Comprehensive analyses of DIPG cells show signatures of enhanced oxidative phosphorylation (OXPHOS). We also identified increased expression of specific OXPHOS related genes and signatures of hypoxia gene expression in datasets obtained from DIPG patients. We found the presence of hypoxia in orthotopic mouse models bearing DIPG tumors. These findings enabled us to develop a proof-of-concept treatment strategy to enhance radiosensitivity of DIPGs in vitro and in animal models. DIPG cells rely on mitochondrial metabolism for growth, and targeting mitochondria disrupts bioenergetics, alleviates hypoxia, and enhances radiosensitivity. These findings warrant further exploration of OXPHOS inhibition as a radiosensitizing strategy for DIPG treatment.

CosGeneGate selects multi-functional and credible biomarkers for single-cell analysis.

Selecting representative genes or marker genes to distinguish cell types is an important task in single-cell sequencing analysis. Although many methods have been proposed to select marker genes, the genes selected may have redundancy and/or do not show cell-type-specific expression patterns to distinguish cell types. Here, we present a novel model, named CosGeneGate, to select marker genes for more effective marker selections. CosGeneGate is inspired by combining the advantages of selecting marker genes based on both cell-type classification accuracy and marker gene specific expression patterns. We demonstrate the better performance of the marker genes selected by CosGeneGate for various downstream analyses than the existing methods with both public datasets and newly sequenced datasets. The non-redundant marker genes identified by CosGeneGate for major cell types and tissues in human can be found at the website as follows: https://github.com/VivLon/CosGeneGate/blob/main/marker gene list.xlsx.

Wnt signaling aberrant activation drives ameloblastoma invasion and recurrence: bioinformatics and in vitro insights

ObjectiveThis study aims to explore the regulatory mechanisms of Wnt signaling in the invasion and recurrence of ameloblastoma (AM) to provide a new theoretical basis for its treatment.MethodsBulk RNA sequencing was employed to analyze samples from AM patients, and identify differentially expressed genes. Subsequently, bioinformatics methods such as Weighted Gene Co-Expression Network Analysis (WGCNA), DESeq2, and KEGG enrichment analysis were utilized to construct gene co-expression networks and identify pathways associated with invasion and recurrence. Furthermore, in vitro experiments, including Cell Counting Kit-8 (CCK-8), Wound healing assays, Western blotting, and qPCR were conducted to validate the effects of Wnt signaling on AM biological functions and the expression of related genes and proteins.ResultsBioinformatics analysis revealed significant activation of the Wnt signaling pathway during AM invasion and recurrence, and differential gene analysis identified specific gene expression patterns associated with the Wnt signaling pathway. In vitro experiments further demonstrated that the standard Wnt/β-catenin pathway activator, Laduviglusib significantly activated Wnt signaling, leading to a marked increase in the mRNA and protein expression levels of TCF7, β-catenin, WNT2B, and LEF1, thereby enhancing the proliferation and migration capabilities of AM cells.ConclusionThis study reveals the critical role of aberrant Wnt signaling activation in AM proliferation and migration, identifying it as a key driver of AM invasion and recurrence. The findings provide new insights into the mechanisms underlying AM invasion and recurrence, laying the foundation for developing novel therapeutic strategies.

Gut metatranscriptomics based de novo assembly reveals microbial signatures predicting immunotherapy outcomes in non-small cell lung cancer.

Advanced-stage non-small cell lung cancer (NSCLC) poses treatment challenges, with immune checkpoint inhibitors (ICIs) as the main therapy. Emerging evidence suggests the gut microbiome significantly influences ICI efficacy. This study explores the link between the gut microbiome and ICI outcomes in NSCLC patients, using metatranscriptomic (MTR) signatures. We utilized a de novo assembly-based MTR analysis on fecal samples from 29 NSCLC patients undergoing ICI therapy, segmented according to progression-free survival (PFS) into long (> 6months) and short (≤ 6months) PFS groups. Through RNA sequencing, we employed the Trinity pipeline for assembly, MMSeqs2 for taxonomic classification, DESeq2 for differential expression (DE) analysis. We constructed Random Forest (RF), Support Vector Machine (SVM), and Extreme Gradient Boosting (XGBoost) machine learning (ML) algorithms and comprehensive microbial profiles. We detected no significant differences concerning alpha-diversity, but we revealed a biologically relevant separation between the two patient groups in beta-diversity. Actinomycetota was significantly overrepresented in patients with short PFS (vs long PFS, 36.7% vs. 5.4%, p < 0.001), as was Euryarchaeota (1.3% vs. 0.002%, p = 0.009), while Bacillota showed higher prevalence in the long PFS group (66.2% vs. 42.3%, p = 0.007), when comparing the abundance of corresponding RNA reads. Among the 120 significant DEGs identified, cluster analysis clearly separated a large set of genes more active in patients with short PFS and a smaller set of genes more active in long PFS patients. Protein Domain Families (PFAMs) were analyzed to identify pathways enriched in patient groups. Pathways related to DNA synthesis and Translesion were more enriched in short PFS patients, while metabolism-related pathways were more enriched in long PFS patients. E. coli-derived PFAMs dominated in patients with long PFS. RF, SVM and XGBoost ML models all confirmed the predictive power of our selected RNA-based microbial signature, with ROC AUCs all greater than 0.84. Multivariate Cox regression tested with clinical confounders PD-L1 expression and chemotherapy history underscored the influence of n = 6 key RNA biomarkers on PFS. According toML models specific gut microbiome MTR signatures' associate with ICI treated NSCLC outcomes. Specific gene clusters and taxa MTR gene expression might differentiate long vs shortPFS.

The E3 ubiquitin ligase COP1 and transcription factors HY5 and RHD6 integrate light signaling and root hair development.

Light signaling plays a substantial role in regulating plant development, including the differentiation and elongation of single-celled tissue. However, the identity of the regulatory machine that affects light signaling on root hair cell (RHC) development remains unclear. Here, we investigated how darkness inhibits differentiation and elongation of RHC in Arabidopsis (Arabidopsis thaliana). We found that light promotes the growth and development of RHC. RNA-seq analysis showed that light signaling regulates the differentiation of RHC by promoting the expression of specific genes in the root epidermis associated with cell wall remodeling, JA, auxin, and ethylene signaling pathways. Together, these genes integrate light and phytohormone signals with root hair development. Our investigation also revealed that the core light signal factor ELONGATED HYPOCOTYL 5 (HY5) directly interacts with the key root hair development factor ROOT HAIR DEFECTIVE6 (RHD6), which promotes the transcription of RSL4. However, CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) repressed RHD6 function through the COP1-HY5 complex. Our genetic studies confirm associations between RHD6, HY5, and COP1, indicating that RHD6 largely depends on HY5 for root hair development. Ultimately, our work suggests a central COP1-HY5-RHD6 regulatory module that integrates light signaling and root hair development with several downstream pathways, offering perspectives to decipher single-celled root hair development.

Transcriptional regulators ensuring specific gene expression and decision-making at high TGFβ doses.

TGFβ-signaling regulates cancer progression by controlling cell division, migration, and death. These outcomes are mediated by gene expression changes, but the mechanisms of decision-making toward specific fates remain unclear. Here, we combine SMAD transcription factor imaging, genome-wide RNA sequencing, and morphological assays to quantitatively link signaling, gene expression, and fate decisions in mammary epithelial cells. Fitting genome-wide kinetic models to our time-resolved data, we find that most of the TGFβ target genes can be explained as direct targets of SMAD transcription factors, whereas the remainder show signs of complex regulation, involving delayed regulation and strong amplification at high TGFβ doses. Knockdown experiments followed by global RNA sequencing revealed transcription factors interacting with SMADs in feedforward loops to control delayed and dose-discriminating target genes, thereby reinforcing the specific epithelial-to-mesenchymal transition at high TGFβ doses. We identified early repressors, preventing premature activation, and a late activator, boosting gene expression responses for a sufficiently strong TGFβ stimulus. Taken together, we present a global view of TGFβ-dependent gene regulation and describe specificity mechanisms reinforcing cellular decision-making.

What Do We Know About CapsicumVolatilome?

The Capsicum genus includes several cultivated species that release complex blends of volatile organic compounds (VOCs) associated with their unique aroma. These VOCs are essential info-chemicals in ecological interactions. In this review, we describe how the volatilomic profiling naturally varies based on specific plant organs and genotypes as well as how non-beneficial organisms affect VOCs biosynthesis and accumulation in pepper plants. Also, we show evidence about VOCs variation under the pressure of different abiotic factors such as water stress, soil type and nutrient availability. The contribution of specific metabolic pathways and gene expression related to the biosynthesis of particular VOCs is addressed. We highlighted the utility of VOCs as chemical markers for quality control in the food industry, breeding programs to generate resistant plants and to improve aroma innovation. Herein we present a database containing 2734 VOCs, revealing 113 as the basic core of the volatilome from five Capsicum species.

Transcriptome and genome-wide analysis of the mango glycosyltransferase family involved in mangiferin biosynthesis

Mangiferin, a C-glucosyl xanthone, is a biologically active glycoside naturally synthesized in mango. Glycosyltransferase can catalyze the biosynthesis of mangiferin. In this study, we identified 221 members of the UGT glycosyltransferase family in mango. The 221 MiUGT genes were grouped into 13 subfamilies through phylogenetic tree analysis with Arabidopsis, Chinese bayberry, and mango. All UGT family members in mango were unevenly distributed on 17 chromosomes and found that tandem duplication dominated the expansion of UGT family members in mango. Purification selection primarily influenced the evolution of the mango UGT family members. In addition, cis-element analysis of the mango UGT gene family revealed the presence of MYB binding sites, which are involved in flavonoid biosynthesis; which further supports the role of UGT family members in the synthesis of flavonoids. To verify these results, we analyzed the expression of UGT family members in mango leaves, stems, and different developmental stages of fruit peel. The RNA-seq and qRT-PCR results showed significant differences in the expression patterns of MiUGT genes in various tissues and developmental stages of mango. We identified MiUGT gene-specific expression at different stages of fruit development. These results lay a theoretical foundation for research on the relationship between members of the mango UGT family and the synthesis of flavonoids, mangiferin.

Deciphering temporal gene expression dynamics during epilepsy development using a rat model of focal neocortical epilepsy.

Epilepsy involves significant changes in neural cells during epileptogenesis. Although the molecular mechanism of epileptogenesis remains obscure, changes in gene regulation play a crucial role in the evolution of epilepsy. This study aimed to compare changes in a subset of specific genes during epilepsy development, focusing on the period after the first spontaneous seizure, to identify critical time windows for targeting different regulators. Using a rat model of acquired focal neocortical epilepsy induced by tetanus toxin, we characterized gene expression at acute, subacute, and chronic stages (48-72 h, 2 weeks, and 30 days after first spontaneous seizure, respectively), focusing on genes' potential contribution to epilepsy progression. We observed dynamic changes in the expression of these genes throughout the period after the first spontaneous seizure. Astrocytic reactions primarily occur early, before epilepsy is well established. Changes in Mtor (mammalian target of rapamycin) and Rest (repressor element 1 silencing transcription factor) signaling pathways are highly dynamic and correlated with the progression of epilepsy development. Ccl2 (chemokine C-C-motif ligand) is upregulated at the chronic stage, indicating activation of the neuroinflammatory pathway. Finally, Gabra5 (γ-aminobutyric acidergic signaling) is downregulated at the late stage after epilepsy is established. Surprisingly, changes in the expression of specific genes are linked to the time since the first seizure, rather than seizure frequency or duration. These results suggest that the regulation of specific genes is essentially stage-dependent during the development of epilepsy, highlighting the importance of targeting specific genes at appropriate stages of epilepsy development.

CSIG-21. THE ROLE OF GUT MICROBIOTA IN GLIOBLASTOMA: INSIGHTS FROM MICROBIOME-GENE EXPRESSION INTERACTIONS

Abstract Glioblastoma, the most aggressive form of primary brain tumor, remains a formidable clinical challenge due to its invasiveness and resistance to therapy. Emerging evidence suggests that the gut microbiome plays a crucial role in modulating cancer progression through complex interactions with host physiology. In this study, we investigated the influence of gut microbiota and tumor gene expression by analyzing stool and tissue samples from glioblastoma patients. Utilizing 16S rRNA sequencing of stool samples and NanoString gene expression analysis of tumor tissue samples, we explored the relationship between gut microbiome taxa and gene expression levels in tissue. Pathway analysis using KEGG and Gene Ontology databases was employed to elucidate the functional implications of these interactions. Our findings reveal contrasting effects of specific gut microbiota taxa and gene expression, with Fusobacterium demonstrating a potential oncogenic role, while Bifidobacteriaceae and Akkermansiaceae display potential tumor-suppressive effects. Fusobacterium is associated with the upregulation of key oncogenes and pathways promoting cancer cell survival, growth, and metabolism, whereas Bifidobacteriaceae and Akkermansiaceae are linked to the downregulation of genes involved in pathways promoting cancer cell survival and proliferation, such as MAPK signaling and stress response pathways, potentially inhibiting cancer cell growth. These results highlight the intricate interplay between gut microbiota composition and cancer biology and underscore the potential of gut microbiota modulation as a therapeutic strategy in glioblastoma management.

Train and Reprogram Your Brain: Effects of Physical Exercise at Different Stages of Life on Brain Functions Saved in Epigenetic Modifications.

Multiple studies have demonstrated the significant effects of physical exercise on brain plasticity, the enhancement of memory and cognition, and mood improvement. Although the beneficial impact of exercise on brain functions and mental health is well established, the exact mechanisms underlying this phenomenon are currently under thorough investigation. Several hypotheses have emerged suggesting various possible mechanisms, including the effects of hormones, neurotrophins, neurotransmitters, and more recently also other compounds such as lactate or irisin, which are released under the exercise circumstances and act both locally or/and on distant tissues, triggering systemic body reactions. Nevertheless, none of these actually explain the long-lasting effect of exercise, which can persist for years or even be passed on to subsequent generations. It is believed that these long-lasting effects are mediated through epigenetic modifications, influencing the expression of particular genes and the translation and modification of specific proteins. This review explores the impact of regular physical exercise on brain function and brain plasticity and the associated occurrence of epigenetic modifications. It examines how these changes contribute to the prevention and treatment of neuropsychiatric and neurological disorders, as well as their influence on the natural aging process and mental health.

Spatial Transcriptomics and Single-Nucleus Multi-Omics Analysis Revealing the Impact of High Maternal Folic Acid Supplementation on Offspring Brain Development.

Background: Folate, an essential vitamin B9, is crucial for diverse biological processes, including neurogenesis. Folic acid (FA) supplementation during pregnancy is a standard practice for preventing neural tube defects (NTDs). However, concerns are growing over the potential risks of excessive maternal FA intake. Objectives/Methods: Here, we employed a mouse model and spatial transcriptomic and single-nucleus multi-omics approaches to investigate the impact of high maternal FA supplementation during the periconceptional period on offspring brain development. Results: Maternal high FA supplementation affected gene pathways linked to neurogenesis and neuronal axon myelination across multiple brain regions, as well as gene expression alterations related to learning and memory in thalamic and ventricular regions. Single-nucleus multi-omics analysis revealed that maturing excitatory neurons in the dentate gyrus (DG) are particularly vulnerable to high maternal FA intake, leading to aberrant gene expressions and chromatin accessibility in pathways governing ribosomal biogenesis critical for synaptic formation. Conclusions: Our findings provide new insights into specific brain regions, cell types, gene expressions and pathways that can be affected by maternal high FA supplementation.

Specific human gene expression in response to infection is an effective marker for diagnosis of latent and active tuberculosis

RNA sequencing and microarray analysis revealed transcriptional markers expressed in whole blood can differentiate active pulmonary TB (ATB) from other respiratory diseases (ORDs), and latent TB infection (LTBI) from healthy controls (HC). Here we describe a streamlined reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assay that could be applied at near point-of-care for diagnosing and distinguishing ATB from ORDs and LTBI from HC. A literature review was undertaken to identify the most plausible host-gene markers (HGM) of TB infection. Primers, and dual labelled hydrolysis probes were designed and analytically evaluated for accuracy in an in-vitro model of infection using a lung fibroblast cell-line. Best performing genes were multiplexed into panels of 2–4 targets and taken forward for clinical evaluation. Mycobacteria Growth Indicator Tube and QuantiFERON-TB Gold Plus were used as reference tests for ATB and LTBI respectively. A total of 16 HGM were selected and incorporated into five multiplex RT-qPCR panels. PCR assay efficiency of all evaluated targets was ≥ 90% with a median analytical sensitivity of 292 copies/µl [IQR: 215.0-358.3 copies/µl], and a median limit of quantification of 61.7 copies/µl [IQR: 29.4-176.3 copies/µl]. Clinically, ATB was characterised by higher gene expression than ORDs, while LTBI was associated with lower gene expression than HC, Kruskal-Wallis p < 0.0001. Crucially, BATF2, CD64, GBP5, C1QB, GBP6, DUSP3, and GAS6 exhibited high differentiative ability for ATB from ORDs, LTBI or HC while KLF2, PTPRC, NEMF, ASUN, and ZNF296 independently discriminated LTBI from HC. Our results show that different HGM maybe required for ATB and LTBI differentiation from ORDs or HC respectively and demonstrate the feasibility of host gene-based RT-qPCR to diagnose ATB and LTBI at near point-of-care.

Bioengineered skin-substitutes incorporating rete-ridges using a bioprinting process

Abstract Bioprinting is a widely used technique for creating three-dimensional, complex, and heterogeneous artificial tissue constructs that are biologically and biophysically similar to natural tissues. The skin is composed of several layers including the epidermis, basement membrane (BM), and dermis. However, the unique undulating structure of basement membranes (i.e. rete ridges) and the function of BM have not been extensively studied in the fabrication of engineered skin substitutes. In this study, a novel engineered skin substitute incorporating an artificially designed rete ridge (i.e. mogul-shape) was developed using bioprinting and bioinks prepared using collagen and fibrinogen. To mimic the structure of the rete ridges of skin tissue, we developed a modified bioprinting technique, controlling rheological property of bioink to create a mogul-shaped layer. In vitro cellular activities, including the expression of specific genes (those encoding vimentin, laminin-5, collagen IV, and cytokeratins), demonstrated that the engineered skin substitute exhibited more potent cellular responses than the normally bioprinted control owing to the favorable biophysical BM structure and the bioink microenvironment. Additionally, the feasibility of utilizing the bioprinted skin-structure was evaluated in a mouse model, and in vivo results demonstrated that the bioprinted skin substitutes effectively promoted wound healing capabilities. Based on these results, we suggest that bioprinted skin tissues and the bioprinting technique for mimicking rete ridges can be used not only as potential lab-chip models for testing cosmetic materials and drugs, but also as complex physiological models for understanding human skin.