Changes In Transcript Levels Research Articles

CSIG-29. CLINICAL AND TRANSCRIPTOMIC DATASET IDENTIFIES LARGE CHANGES IN IMMUNE AND HORMONAL SIGNALING AMONGST RECURRENT PATIENTS WITH CRANIOPHARYNGIOMA

Abstract INTRODUCTION Craniopharyngioma is a low-grade suprasellar brain tumor that often produces deficiencies of pituitary hormones, including growth hormone. Although epidemiological data are reassuring that growth hormone does not lead to craniopharyngioma recurrence, the theoretical risk is cause for concern. To address this and other questions related to the care of individuals with recurrent craniopharyngioma, we analyzed gene expression in craniopharyngiomas in the context of clinical data. We envision insights that could guide decisions about hormone replacement and tumor-focused treatment alternatives. METHODS Clinical information was abstracted for a cohort of individuals diagnosed with craniopharyngioma at the Children’s Hospital of Philadelphia between 2016 - 2020. In parallel, tumor transcriptomic data for patients with craniopharyngioma were collected from the Children’s Brain Tumor Network (CBTN) and matched to clinical data where applicable. Bulk RNA-sequencing analysis, pathway analysis, and immune deconvolution (xCell) of samples was performed in R to identify transcript-level changes that distinguish recurrent from initial craniopharyngioma. RESULTS CBTN data was available for 54 initial tumor samples and 17 recurrent tumor samples, with matched clinical data available for 33 samples (26 initial, 7 recurrent). We did not find differences in clinical characteristics between individuals with versus without recurrence. Pathway analysis notably suggested altered levels of antigen presentation (NES: 2.97), Programmed cell death (NES: 2.34), WNT signaling (NES: 1.97), and Growth Hormone Signaling (NES: -1.277) amongst recurrent samples when compared against initial samples. Deconvolution of samples showed altered levels of M2-like macrophages (39.2% increased) and CD4+ T-helper-2 cells (-54.7% decreased) in recurrent samples compared against initial samples. Preliminary work is being completed to frame these results in the context of available clinical neuroendocrine data. CONCLUSION This work highlights marked changes in immune and hormonal signaling in samples from recurrent craniopharyngioma tumors. These insights have potential therapeutic implications that deserve further investigation.

Effect of anterior quadratus lumborum block with ropivacaine on the immune response after laparoscopic surgery in colon cancer: a substudy of a randomized clinical trial

BackgroundSurgery induces a temporal change in the immune system, which might be modified by regional anesthesia. Applying a bilateral preoperative anterior quadratus lumborum block has proven to be a safe...

Assessing the Role of Post-Translational Modifications of Mitochondrial RNA Polymerase.

The mitochondrial proteome is subject to abundant post-translational modifications, including lysine acetylation and phosphorylation of serine, threonine, and tyrosine. The biological function of the majority of these protein modifications is unknown. Proteins required for the transcription and translation of mitochondrial DNA (mtDNA) are subject to modification. This suggests that reversible post-translational modifications may serve as a regulatory mechanism for mitochondrial gene transcription, akin to mechanisms controlling nuclear gene expression. We set out to determine whether acetylation or phosphorylation controls the function of mitochondrial RNA polymerase (POLRMT). Mass spectrometry was used to identify post-translational modifications on POLRMT. We analyzed three POLRMT modification sites (lysine 402, threonine 315, threonine 993) found in distinct structural regions. Amino acid point mutants that mimic the modified and unmodified forms of POLRMT were employed to measure the effect of acetylation or phosphorylation on the promoter binding ability of POLRMT in vitro. We found a slight decrease in binding affinity for the phosphomimic at threonine 315. We did not identify large changes in viability, mtDNA content, or mitochondrial transcript level upon overexpression of POLRMT modification mimics in HeLa cells. Our results suggest minimal biological impact of the POLRMT post-translational modifications studied in our system.

The misregulation of mitochondria-associated genes caused by GAGA-factor lack promotes autophagic germ cell death in Drosophila testes.

The Drosophila GAGA-factor encoded by the Trithorax-like (Trl) gene is DNA-binding protein with unusually wide range of applications in diverse cell contexts. In Drosophila spermatogenesis, reduced GAGA expression caused by Trl mutations induces mass autophagy leading to germ cell death. In this work, we investigated the contribution of mitochondrial abnormalities to autophagic germ cell death in Trl gene mutants. Using a cytological approach, in combination with an analysis of high-throughput RNA sequencing (RNA-seq) data, we demonstrated that the GAGA deficiency led to considerable defects in mitochondrial ultrastructure, by causing misregulation of GAGA target genes encoding essential components of mitochondrial molecular machinery. Mitochondrial anomalies induced excessive production of reactive oxygen species and their release into the cytoplasm, thereby provoking oxidative stress. Changes in transcription levels of some GAGA-independent genes in the Trl mutants indicated that testis cells experience ATP deficiency and metabolic aberrations, that may trigger extensive autophagy progressing to cell death.

Prohibitin StPHB3 affects the browning of fresh-cut potatoes via influencing antioxidant capacity and polyphenol oxidase activation

Enzymatic browning causes a decline in consumer’s acceptance of fresh-cut products. Previous study showed reactive oxygen species (ROS) is associated with enzymatic browning, and prohibitin (PHB) is involved in ROS formation. However, whether PHB influences the enzymatic browning is still unknown. In this paper, changes of StPHB3 transcript levels after cutting were investigated, then stphb3 mutants were constructed through CRISPR/Cas9 and the browning difference between stphb3 mutants and wild type was compared, finally the mechanism of PHB influencing the browning was explored. The results showed that the transcript levels of StPHB3 were enhanced in potato (cultivar Desiree) tubers after fresh cutting. Mutation of StPHB3 caused lower browning, less production of soluble quinones, decreased malondialdehyde (MDA) content, increased 1,1-Diphenyl-2-picrylhydrazyl (DPPH) inhibition, and increased total phenol contents. Additionally, polyphenol oxidase (PPO) activity and StPOT32 gene transcript level were also reduced after disturbing StPHB3 gene expression. StPHB3 was found to be located within the chloroplast same as the predominant location and activation site of PPO protein. Yeast two-hybrid (Y2H) and Co-Immunoprecipitation (Co-IP) assays showed StPHB3 interacted with PPO. And mature PPO contents were decreased in stphb3. These results suggest that suppression of StPHB3 can alleviate the browning of fresh-cut potatoes by enhancing the antioxidant activity and inhibiting PPO activation.

SAT263 Osteocalcin Promotes Cortisol Production In Human Adrenal Cells

Abstract Disclosure: R. Scheske: None. D. van Rooyen: None. W.E. Rainey: None. Background: Osteocalcin (OCN) is a peptide exclusively produced by osteoblasts in bone. In an uncarboxylated form, OCN has been shown to function as a hormone affecting physiological processes such as reproduction and energy metabolism in mice and humans. Murine studies have shown that uncarboxylated OCN upregulates fetal mouse glucocorticoid production through a unique regulatory axis between osteoblasts and adrenal cortex cells. The increase in adrenal steroid production has been attributed to OCN heightening adrenocorticotropic hormone (ACTH). The influence of adrenal glucocorticoids on glucose metabolism and bone composition is well studied but the effect of OCN on human adrenal steroidogenesis remains unknown. Herein, we hypothesized OCN would stimulate cortisol production in human adrenal cells. Methods: OCN’s effect on adrenal glucocorticoid production was investigated using primary cultures of human adrenal cells (PAC). Cells were treated for 24h with 1000 nM OCN with/without cAMP/protein kinase A (PKA) signaling pathway agonists (ACTH, forskolin, 8-bromo cAMP (8Br-cAMP), and dibutyryl cyclic AMP (dbcAMP)). Cortisol levels were quantified using an enzyme-linked immunosorbent assay. RNA was isolated and assessed by quantitative real-time PCR (RT-qPCR) to identify changes in transcript levels of enzymes catalyzing glucocorticoid production: cholesterol side-chain cleavage enzyme (CYP11A1), 17α-hydroxylase/17,20-lyase (CYP17A1), and 11β-hydroxylase (CYP11B1). Results: Cortisol production in PAC were significantly induced by all tested agonists; ACTH (22-fold), forskolin (27-fold), 8Br-cAMP (26-fold), and dbcAMP (30-fold) relative to basal. Although OCN, alone, did not significantly increase cortisol in this cell model, co-treatment with ACTH increased cortisol levels 1.4-fold (40.1 ng/mL; p<0.0001) compared to ACTH (28.2 ng/mL). Similarly, co-treatment with forskolin or 8Br-cAMP increased cortisol, relative to the respective agonists, by 1.3-fold (p<0.001) and dbcAMP by 1.2-fold (p<0.01). RT-qPCR analysis showed no changes in CYP11A1 or CYP17A1 transcript levels in co-treatments with OCN and ACTH relative to ACTH. However, CYP11B1 mRNA levels, relative to the respective controls, were increased in samples co-treated with OCN and ACTH/-agonist. OCN co-treatments with ACTH, forskolin, 8Br-cAMP and dbcAMP increased CYP11B1 levels by 1.8-, 1.4-, 1.3-, and 1.2-fold, respectively. Conclusion: Data presented in this study show stimulatory effect of OCN on human adrenal cells. OCN, in the presence of ACTH and other PKA agonists, increased cortisol production via upregulating CYP11B1 transcript levels. Defining the mechanism and receptor interaction through which OCN functions in human adrenal cells remain to be determined. Presentation: Saturday, June 17, 2023

OR12-01 Blocking The Maladaptive Integrated Stress Response Increases Immunosuppressive PD-L1 In Pancreatic β-Cells

Abstract Disclosure: C. Muralidharan: None. F.H. huang: None. S.C. May: None. J. Enriquez: None. J.B. Nelson: None. E. Wang: None. A. Chakraborthy: None. K.T. Figatner: None. S.N. Navitskaya: None. S.A. Tersey: None. R.G. Mirmira: None. In type 1 diabetes (T1D), the immune system destroys the insulin-secreting β-cells. This autoimmune attack may be triggered by the β-cells themselves when they encounter viral infections or changes in gut microbiome or diet, years before clinical diagnosis of T1D. In response to these stressors, four kinases (PERK, PKR, GCN2, and HRI) activate the integrated stress response (ISR), which temporarily halts protein translation to promote cellular survival. However, if prolonged, the ISR can render β-cells dysfunctional. Therefore, we hypothesized that inhibiting the ISR under chronic inflammatory stress would suppress β-cell autoimmunity and reduce T1D risk. To test this, we pre-treated human islets with ISR inhibitors (ISRIB or a novel inhibitor of PERK, HC-PERKi) and then exposed them to proinflammatory cytokines (50 IU/mL IL-1β + 1000 IU/mL IFNγ) for 24h. As expected, cytokine treatment blocked protein translation, as revealed by polyribosomal profiling. Consistent with this, we saw reduced total protein synthesis (50% decrease) by puromycin incorporation assay. These effects were partially reversed by pretreating with ISR inhibitors. To determine if ISR inhibition delays or prevents diabetes development, we treated female pre-diabetic non-obese diabetic (NOD) mice with vehicle or HC-PERKi for four weeks (6-10 weeks of age). Compared to controls, NOD mice that received HC-PERKi showed a significant delay in diabetes development. Single cell RNA-sequencing analysis of the pancreatic islets revealed alterations in pathways involved in antigen processing and presentation in the β-cell clusters of HC-PERKi islets. Consistent with this observation, spatial proteomics analysis of the pancreatic β-cell area of HC-PERKi islets revealed a striking upregulation of programmed death-ligand 1 (PD-L1), an immune checkpoint regulator that suppresses adaptive T-cell immunity. Notably, there was no change in transcript levels of PD-L1, suggesting that PD-L1 levels are post-translationally regulated. Collectively, our studies show that inhibition of the ISR activates general protein synthesis, reduces immunogenicity via increased β-cell PD-L1 levels, and decreases T1D risk. Whereas the ISR is an adaptive response, our work emphasizes how the ISR could become maladaptive under chronic inflammation and promote β-cell autoimmunity. Presentation: Friday, June 16, 2023

How Does Mitochondrial Protein-Coding Gene Expression in Fejervarya kawamurai (Anura: Dicroglossidae) Respond to Extreme Temperatures?

Unusual climates can lead to extreme temperatures. Fejervarya kawamurai, one of the most prevalent anurans in the paddy fields of tropical and subtropical regions in Asia, is sensitive to climate change. The present study focuses primarily on a single question: how do the 13 mitochondrial protein-coding genes (PCGs) respond to extreme temperature change compared with 25 °C controls? Thirty-eight genes including an extra tRNA-Met gene were identified and sequenced from the mitochondrial genome of F. kawamurai. Evolutionary relationships were assessed within the Dicroglossidae and showed that Dicroglossinae is monophyletic and F. kawamurai is a sister group to the clade of (F. multistriata + F. limnocharis). Transcript levels of mitochondrial genes in liver were also evaluated to assess responses to 24 h exposure to low (2 °C and 4 °C) or high (40 °C) temperatures. Under 2 °C, seven genes showed significant changes in liver transcript levels, among which transcript levels of ATP8, ND1, ND2, ND3, ND4, and Cytb increased, respectively, and ND5 decreased. However, exposure to 4 °C for 24 h was very different in that the expressions of ten mitochondrial protein-coding genes, except ND1, ND3, and Cytb, were significantly downregulated. Among them, the transcript level of ND5 was most significantly downregulated, decreasing by 0.28-fold. Exposure to a hot environment at 40 °C for 24 h resulted in a marked difference in transcript responses with strong upregulation of eight genes, ranging from a 1.52-fold increase in ND4L to a 2.18-fold rise in Cytb transcript levels, although COI and ND5 were reduced to 0.56 and 0.67, respectively, compared with the controls. Overall, these results suggest that at 4 °C, F. kawamurai appears to have entered a hypometabolic state of hibernation, whereas its mitochondrial oxidative phosphorylation was affected at both 2 °C and 40 °C. The majority of mitochondrial PCGs exhibited substantial changes at all three temperatures, indicating that frogs such as F. kawamurai that inhabit tropical or subtropical regions are susceptible to ambient temperature changes and can quickly employ compensating adjustments to proteins involved in the mitochondrial electron transport chain.

High-throughput, fluorescent-aptamer-based measurements of steady-state transcription rates for the Mycobacterium tuberculosis RNA polymerase.

The first step in gene expression is the transcription of DNA sequences into RNA. Regulation at the level of transcription leads to changes in steady-state concentrations of RNA transcripts, affecting the flux of downstream functions and ultimately cellular phenotypes. Changes in transcript levels are routinely followed in cellular contexts via genome-wide sequencing techniques. However, in vitro mechanistic studies of transcription have lagged with respect to throughput. Here, we describe the use of a real-time, fluorescent-aptamer-based method to quantitate steady-state transcription rates of the Mycobacterium tuberculosis RNA polymerase. We present clear controls to show that the assay specifically reports on promoter-dependent, full-length RNA transcription rates that are in good agreement with the kinetics determined by gel-resolved, α-32P NTP incorporation experiments. We illustrate how the time-dependent changes in fluorescence can be used to measure regulatory effects of nucleotide concentrations and identity, RNAP and DNA concentrations, transcription factors, and antibiotics. Our data showcase the ability to easily perform hundreds of parallel steady-state measurements across varying conditions with high precision and reproducibility to facilitate the study of the molecular mechanisms of bacterial transcription.

Environmental factors drive the succession of microbial community structure during wheat Qu fermentation

Environmental factors significantly affected the succession of microbial communities during the fermentation of Huangjiu Raw wheat Qu (RWQ). Influence of environmental factors on microbial community during fermentation of Chinese Huangjiu RWQ. The interaction of environmental factors and microorganisms affects the changes of microbial communities and key enzyme transcript levels in different stages during the production of Huangjiu RWQ. This study combined environmental factor analysis and high-throughput sequencing technology to comprehensively analyze the specific changes of microbial communities and environmental factors at each stage of Huangjiu production. In addition, the influence of the interaction of environmental factors and microorganisms on microbial succession was investigated by correlation analysis, and the enzymes related to saccharification and ethanol fermentation during the production of RWQ were explored. The results showed that Bacillus, Weisseria, Thermophilic actinomycetes and Lactobacillus were the dominant bacteria in RWQ, while Aspergillus, Thermomyces, Fusarium and Alternaria were the dominant fungi. Fungi were active in the early stage of RWQ fermentation, while bacteria were active in the middle and late stage of fermentation. Microbial community succession was closely related to environmental humidity and CO2. These environmental factors were positively correlated with most fungi, but negatively correlated with Thermophilic actinomycetes, Glycopolyspora and Acinetobacter. In fungi, the enzymes related to saccharification and fermentation mainly came from Thermophilic ascomycetes and thermophilic fungi, while in Bacteria, these two enzymes mainly came from weisseria. This study revealed the relationship between RWQ microbe and environmental factors, which was helpful to clarify the succession law of RWQ microbe.

Comparison of carotenoid, chlorophyll concentrations and their biosynthetic transcript levels in different coloured cauliflower

Carotenoids and chlorophylls are among the most widely distributed pigments in nature that play essential roles in the photosynthetic apparatus and confer diverse colours in plants. Among all vegetables, cauliflower (Brassica oleracea L. ssp. var. botrytis) is rich in phytochemicals and is an important crop grown all over the world. This study investigates carotenoid and chlorophyll concentrations in differently pigmented cultivars and elucidates the role of transcriptional regulation of carotenoid accumulation including lutein and β-carotene. Here, we characterised changes in pigments by UHPLC-DAD-ToF-MS and changes in transcript levels of carotenoid metabolic genes by qRT-PCR in florets and leaves of orange (‘Jaffa’ and ‘Sunset’), purple (‘Di Sicilia Violetto’ and ‘Graffiti’), green (‘Trevi’) and white (‘Clapton’) cultivars. Transcript levels of all carotenoid metabolic genes showed different transcript level patterns in the leaves and florets. Compared to the other cultivars, the orange cultivars had the highest levels of β-carotene in the florets and lutein in the leaves resulting in changes lutein/β-carotene ratios. In the green cultivar, higher transcript levels were also found, especially for phytoene synthase and phytoene desaturase genes of the core biosynthesis pathway. However, no increased carotenoid concentrations were observed, possibly due to a higher carotenoid turnover induced by the carotenoid cleavage dioxygenase 4 in the green cultivar. In the white (‘Clapton’) and purple (‘Di Sicilia Violetto’ and ‘Graffiti’) cultivars the phytoene desaturase transcript levels as well as carotenoid concentrations were low. Chlorophyll concentrations changed in trend comparable to the carotenoid concentrations and were only significantly lower in the leaves of the orange cultivar ‘Jaffa’. Also, the chlorophyll a/b ratio changed in ‘Jaffa’. In florets the highest chlorophylls concentrations were observed for the green cultivar (‘Trevi’) and the purple cultivar (‘Di Sicilia Violetto’). Taken together, the study demonstrates the complex source-sink relationship of carotenoid accumulation in different coloured cauliflower.

Transcriptomic analysis of biofilm formation by Bacillus cereus under different carbon source conditions

Abstract Background Previous studies found differences in the utilization of different carbon sources during biofilm formation by Bacillus cereus. Illumina HiSeq high-throughput sequencing technology was used to investigate the changes in gene transcript levels in Bacillus cereus biofilm bacteria under different carbon source conditions. Results Compared with the control group, the number of differentially expressed genes in the glucose, maltose, lactose, and skim milk-supplemented groups was 351, 1136, 133, and 487, respectively. The results showed that the pathways involved in the differentially expressed genes were mainly distributed in glycolysis and pentose phosphate pathway, tricarboxylic acid cycle, amino acid metabolism, and fatty acid metabolism. The gene expression of enzymes related to acetoin synthesis from pyruvate was mostly upregulated in the glucose-supplemented group. The gene expression of enzymes related to pyruvate synthesis of branched-chain amino acids in the maltose-supplemented group was mostly upregulated. In the lactose-supplemented group, the gene expression of acetoin biosynthesis from pyruvate was upregulated. Pyruvate production through glycolysis pathway increased in the skim milk-supplemented group, but the metabolic capacity of the tricarboxylic acid cycle did not change significantly. Conclusion The content of pyruvate stored by Bacillus cereus biofilm bacteria through glycolysis or pentose phosphate pathway increased, but the carbon flux into the tricarboxylic acid cycle did not increase, which suggested that carbon fluxes in the extracellular polysaccharide synthesis pathway of the biofilm may be increased, resulting in increased biofilm biomass formation.

SIMULATED MICROGRAVITY CHANGES THE NUMBER OF MECHANICALLY GATED AND MECHANOSENSITIVE ION CHANNELS GENES TRANSCRIPTS IN RAT VENTRICULAR CARDIOMYOCYTES

The mechanoelectrical feedback in the heart is based on the work of mechanically gated (MGCs) and mechanosensitive (MSCs) channels. Since microgravity alters the heart’s morphological and physiological properties, we hypothesized that the expression of both MGCs and MSCs would be affected. We employed RNA transcriptome sequencing to investigate changes in the gene transcript levels of MGCs and MSCs in isolated rat ventricular cardiomyocytes under control conditions and in a simulated microgravity environment. For the first time, our findings demonstrated that simulated microgravity induces alterations in the gene transcript levels of specific MGCs, such as TRPM7, TRPV2, TRPP1, TRPP2, Piezo1, TMEM63A, TMEM36B, and known MSCs, including K2P2.1, K2P3.1, Kir6.1, Kir6.2, NaV1.5, CaV1.2, KV7.1. However, other voltage-gated channels and channels lacking a voltage sensor remained unaffected. These findings suggest that the altered expression of MGCs and MSCs could lead to changes in the net currents across the membrane, ultimately impacting the heart’s function.

Impact of Classic Adrenal Secretagogues on mRNA Levels of Urotensin II and Its Receptor in Adrenal Gland of Rats.

Urotensin 2 (Uts2) is a biologically active peptide involved in the regulation of a variety of physiological and pathophysiological processes. In both the human and rat adrenal gland, the expressions of the Uts2 gene and its receptor (Uts2r) have been described. This paper focuses on the description of the hormonal control of the mRNA levels of urotensin II and its receptor in the adrenal gland of the rat, both in vitro and in vivo. The initial in vitro experiments were carried out on freshly isolated rat adrenocortical cells and their primary culture. The obtained results indicated a stimulating PKA-independent effect of ACTH on the Uts2 mRNA level in the tested cells, with no changes in the Uts2r transcript. Subsequent in vivo experiments showed that ACTH-induced adrenal growth was accompanied by an elevated level of the Uts2 mRNA, with unchanged expression of Uts2r. In the other types of in vivo gland growth studied, enucleation-induced adrenal regeneration and compensatory growth of the gland, the mRNA levels of the studied genes showed no significant differences. The only exception was hemiadrenalectomy, which led to a significant increase in Uts2 mRNA expression level 24 h after surgery. In 12-week-old rats of both sexes, gonadectomy led to a significant increase in the level of Uts2 mRNA in the adrenal gland, an effect that was prevented by sex hormones' replacement. No changes in Uts2r transcript levels were observed under these conditions. Thus, this study suggests that the regulation of Uts2 and Uts2r mRNA levels differs significantly in the rat adrenal gland. While Uts2 transcript levels appear to be mainly dependent on ACTH action, Uts2r mRNA levels are not under the control of this hormone.

Initial Weight Loss, Anthropometric Parameters, and Proinflammatory Transcript Levels in Patients with Class I Obesity.

Research into early predictors of effective weight loss could help determine more effective therapeutic interventions. In this study, 106 subjects with class I obesity, genotyped with the fat mass and obesity-associated (FTO) rs9930506 gene variant, were enrolled into a 12-week weight loss program (WLP). Anthropometric and body composition measurements were controlled with bioelectrical impedance analysis (BIA) at baseline and after 4 and 12 weeks. Biopsies of abdominal subcutaneous adipose tissue (AT) and venous blood samples were collected to monitor changes in interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), and nuclear factor kappa B (NF-κB) mRNA levels in white blood cells (WBCs) and to assess if changes in WBC gene expression reflected changes in adipose tissue. The FTO rs9930506 variant had no effect on weight loss and no reduction in proinflammatory transcripts in WBCs or AT. Changes in anthropometric parameters were associated with changes in carbohydrate metabolism. A linear regression model showed that initial weight loss (after 4 weeks of the WLP) was the most predictive factor of weight loss success after 12 weeks of the WLP. Changes in plasma lipids or proinflammatory transcript levels in WBCs or AT were not associated with weight loss effectiveness. However, the gene expression in WBCs did reflect changes occurring in subcutaneous AT.

Addition of Saccharomyces eubayanus to SCOBY fermentations modulates the chemical and volatile compound profiles in kombucha

Kombucha is a fermented beverage derived from a sweetened tea fermentation inoculated with a bacteria-yeast consortium referred to as Symbiotic Culture of Bacteria and Yeast (SCOBY). Different SCOBY cultures can impact the beverage's quality and make the whole process highly variable. Adding Saccharomyces yeast cultures to the fermentation process can avoid stalled fermentations, providing a reproducible beverage. Here, we explored using different Saccharomyces eubayanus strains together with SCOBY in the context of kombucha fermentation. Our results show that yeast x SCOBY co-cultures exhibited a robust fermentation profile, providing ethanol and acetic acid levels ranging from 0,18–1,81 %v/v and 0,35–1,15 g/L, respectively. The kombucha volatile compound profile of co-cultures was unique, where compounds such as Isopentyl acetate where only found in yeast x SCOBY fermentations. Metabarcoding revealed that the SCOBY composition was also dependent on the S. eubayanus genotype, where besides Saccharomyces, amplicon sequence variants belonging to Brettanomyces and Starmerella were detected. These differences concomitated global changes in transcript levels in S. eubayanus related to the metabolism of organic molecules used in kombucha fermentation. This study highlights the potential for exploring different S. eubayanus strains for kombucha fermentation, and the significant yeast genotype effect in the profile differentiation in this process.

Comparative Analysis of miRNA Expression Profiles under Salt Stress in Wheat.

Salt stress is one of the important environmental factors that inhibit the normal growth and development of plants. Plants have evolved various mechanisms, including signal transduction regulation, physiological regulation, and gene transcription regulation, to adapt to environmental stress. MicroRNAs (miRNAs) play a role in regulating mRNA expression. Nevertheless, miRNAs related to salt stress are rarely reported in bread wheat (Triticum aestivum L.). In this study, using high-throughput sequencing, we analyzed the miRNA expression profile of wheat under salt stress. We identified 360 conserved and 859 novel miRNAs, of which 49 showed considerable changes in transcription levels after salt treatment. Among them, 25 were dramatically upregulated and 24 were downregulated. Using real-time quantitative PCR, we detected significant changes in the relative expression of miRNAs, and the results showed the same trend as the sequencing data. In the salt-treated group, miR109 had a higher expression level, while miR60 and miR202 had lower expression levels. Furthermore, 21 miRNAs with significant changes were selected from the differentially expressed miRNAs, and 1023 candidate target genes were obtained through the prediction of the website psRNATarget. Gene ontology (GO) analysis of the candidate target genes showed that the expressed miRNA may be involved in the response to biological processes, molecular functions, and cellular components. In addition, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis confirmed their important functions in RNA degradation, metabolic pathways, synthesis pathways, peroxisome, environmental adaptation, global and overview maps, and stress adaptation and the MAPK signal pathway. These findings provide a basis for further exploring the function of miRNA in wheat salt tolerance.

Transcriptional and proteomic analysis of the innate immune response to microbial stimuli in a model invertebrate chordate.

Inflammatory response triggered by innate immunity can act to protect against microorganisms that behave as pathogens, with the aim to restore the homeostatic state between host and beneficial microbes. As a filter-feeder organism, the ascidian Ciona robusta is continuously exposed to external microbes that may be harmful under some conditions. In this work, we used transcriptional and proteomic approaches to investigate the inflammatory response induced by stimuli of bacterial (lipopolysaccharide -LPS- and diacylated lipopeptide - Pam2CSK4) and fungal (zymosan) origin, in Ciona juveniles at stage 4 of metamorphosis. We focused on receptors, co-interactors, transcription factors and cytokines belonging to the TLR and Dectin-1 pathways and on immune factors identified by homology approach (i.e. immunoglobulin (Ig) or C-type lectin domain containing molecules). While LPS did not induce a significant response in juvenile ascidians, Pam2CSK4 and zymosan exposure triggered the activation of specific inflammatory mechanisms. In particular, Pam2CSK4-induced inflammation was characterized by modulation of TLR and Dectin-1 pathway molecules, including receptors, transcription factors, and cytokines, while immune response to zymosan primarily involved C-type lectin receptors, co-interactors, Ig-containing molecules, and cytokines. A targeted proteomic analysis enabled to confirm transcriptional data, also highlighting a temporal delay between transcriptional induction and protein level changes. Finally, a protein-protein interaction network of Ciona immune molecules was rendered to provide a wide visualization and analysis platform of innate immunity. The in vivo inflammatory model described here reveals interconnections of innate immune pathways in specific responses to selected microbial stimuli. It also represents the starting point for studying ontogeny and regulation of inflammatory disorders in different physiological conditions.

Leukemic Transformation of Longstanding Essential Thrombocythemia

Acute Myeloid Leukemia (AML) represents only 1% of all cancers, despite being the most frequent type of leukemia [1]. Multiple genetic pathways are associated with AML, causing various changes in genetic transcription, apoptosis, chromatin remodeling, and protein levels. One such pathway, the JAK-STAT pathway, governs cellular proliferation, differentiation, and apoptosis, and is linked to both AML and myeloproliferative neoplasms due to mutations [2]. The JAK V617F mutation is the most common mutation in this pathway, occurring in about 50% of Essential Thrombocythemia (ET) patients, as described in this report [2]. While rare, transformation of Essential Thrombocythemia (ET) to AML occurs in 1-4% of cases [3]. This report presents a case of AML in a 76-year-old male with a medical history of ET. Following an abnormal peripheral blood smear, bone marrow biopsy confirmed the diagnosis. The current report aims to emphasize the significance of regular screening and monitoring for patients with long-standing Essential Thrombocythemia (ET), especially those under suppressive therapy. The transformation of ET to acute leukemia is associated with an unfavorable prognosis, underscoring the importance of early diagnosis. Therefore, this case serves to draw attention to the criticality of timely intervention through routine follow-up to achieve optimal outcomes for patients with ET.

Ethanol Enhances Endothelial Rigidity by Targeting VE-Cadherin-Implications for Acute Aortic Dissection.

(1) Background: Acute aortic dissection (AAD) is caused by an endothelial entry tear followed by intimomedial delamination of the outer layers of the vessel wall. The established risk factors include hypertension and smoking. Another rising candidate risk factor is excessive alcohol consumption. This experimental study explores the effects of nicotine (Nic), angiotensin II (Ang II), and ethanol (EtOH) on human aortic endothelial cells (hAoEC). (2) Methods: HAoECs were exposed to Nic, Ang II, and EtOH at different dose levels. Cell migration was studied using the scratch assay and live-cell imaging. The metabolic viability and permeability capacity was investigated using the water-soluble tetrazolium (WST)-1 assay and an in vitro vascular permeability assay. Cell adherence was studied by utilizing the hanging drop assay. The transcriptional and protein level changes were analyzed by RT-qPCR, Western blotting and immunohistochemistry for major junctional complexing proteins. (3) Results: We observed reduced metabolic viability following Ang II and EtOH exposure vs. control. Further, cell adherence was enhanced by EtOH exposure prior to trituration and by all risk factors after trituration, which correlated with the increased gene and protein expression of VE-cadherin upon EtOH exposure. The cell migration capacity was reduced upon EtOH exposure vs. controls. (4) Conclusion: Marked functional changes were observed upon exposure to established and potential risk factors for AAD development in hAoECs. Our findings advocate for an enhanced mechanical rigidity in hAoECs in response to the three substances studied, which in turn might increase endothelial rigidity, suggesting a novel mechanism for developing an endothelial entry tear due to reduced deformability in response to increased shear and pulsatile stress.