Luciferase Reporter Assay for Determining the Signaling Activity of Interferons.

The classic dual luciferase reporter assay has been widely used to rapidly and accurately determine the transcriptional activity of a given promoter induced by certain signal pathways in the cells. In particular, the sensitive characteristics of luciferase highlight its significance in many experiments, such as weak promoter analysis, transfection studies using small amounts of DNA, and detection in cell lines with low transfection efficiency. This chapter presents detailed information and experimental procedures for measuring interferon (IFN)-induced Interferon-Stimulated Response Element (ISRE) promoter activity using the dual luciferase reporter assay.

Engineering artificial cross-species promoters with different transcriptional strengths.

As a fundamental tool in synthetic biology, promoters are pivotal in regulating gene expression, enabling precise genetic control and spurring innovation across diverse biotechnological applications. However, most advances in engineered genetic systems rely on host-specific regulation of the genetic portion. With the burgeoning diversity of synthetic biology chassis cells, there emerges a pressing necessity to broaden the universal promoter toolkit spectrum, ensuring adaptability across various microbial chassis cells for enhanced applicability and customization in the evolving landscape of synthetic biology. In this study, we analyzed and validated the primary structures of natural endogenous promoters from Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, and Pichia pastoris, and through strategic integration and rational modification of promoter motifs, we developed a series of cross-species promoters (Psh) with transcriptional activity in five strains (prokaryotic and eukaryotic). This series of cross species promoters can significantly expand the synthetic biology promoter toolkit while providing a foundation and inspiration for standardized development of universal components The combinatorial use of key elements from prokaryotic and eukaryotic promoters presented in this study represents a novel strategy that may offer new insights and methods for future advancements in promoter engineering.

Machine and Deep Learning Methods for Predicting 3D Genome Organization.

Three-dimensional (3D) chromatin interactions, such as enhancer-promoter interactions (EPIs), loops, topologically associating domains (TADs), and A/B compartments, play critical roles in a wide range of cellular processes by regulating gene expression. Recent development of chromatin conformation capture technologies has enabled genome-wide profiling of various 3D structures, even with single cells. However, current catalogs of 3D structures remain incomplete and unreliable due to differences in technology, tools, and low data resolution. Machine learning methods have emerged as an alternative to obtain missing 3D interactions and/or improve resolution. Such methods frequently use genome annotation data (ChIP-seq, DNAse-seq, etc.), DNA sequencing information (k-mers and transcription factor binding site (TFBS) motifs), and other genomic properties to learn the associations between genomic features and chromatin interactions. In this review, we discuss computational tools for predicting three types of 3D interactions (EPIs, chromatin interactions, and TAD boundaries) and analyze their pros and cons. We also point out obstacles to the computational prediction of 3D interactions and suggest future research directions.

Use of Rgg quorum-sensing machinery to create an innovative recombinant protein expression system in Streptococcus thermophilus.

Streptococcus thermophilus holds promise as a chassis for producing and secreting heterologous proteins. Used for thousands of years to ferment milk, this species has generally recognized as safe (GRAS) status in the USA and qualified presumption of safety (QPS) status in Europe. In addition, it can be easily genetically modified thanks to its natural competence, and it secretes very few endogenous proteins, which means less downstream processing is needed to purify target proteins, reducing costs. Extracellular degradation of heterologous proteins can be eliminated by introducing mutations that inactivate the genes encoding the bacterium's three major surface proteases. Here, we constructed an inducible expression system that utilizes a peptide pheromone (SHP1358) and a transcriptional regulator (Rgg1358) involved in quorum-sensing regulation. We explored the functionality of a complete version of the system, in which the inducer is produced by the bacterium itself, by synthesizing a luciferase reporter protein. This complete version was assessed with bacteria grown in a chemically defined medium but also in vivo, in the faeces of germ-free mice. We also tested an incomplete version, in which the inducer had to be added to the culture medium, by synthesizing luciferase and a secreted form of elafin, a human protein with therapeutic properties. Our results show that, in our system, protein production can be modulated by employing different concentrations of the SHP1358 inducer or other SHPs with closed amino acid sequences. We also constructed a genetic background in which all system leakiness was eliminated. In conclusion, with this new inducible expression system, we have added to the set of tools currently used to produce secreted proteins in S. thermophilus, whose myriad applications include the delivery of therapeutic peptides or proteins.

The homeodomain (PHD) protein, PdbPHD3, confers salt tolerance by regulating squamosa promoter binding protein PdbSBP3 in Populus davidiana × P. bolleana.

Plant homeodomain (PHD) proteins are a family of zinc finger transcription factors that play roles in abiotic stress tolerance. However, their mechanisms in conferring salt tolerance are largely unknown. In this study, we characterized a PHD gene, PdbPHD3, from Populus davidiana × P. bolleana (Shanxin poplar) in response to salt stress. PdbPHD3 is a nuclear protein that is strongly induced by salt and abscisic acid (ABA) treatments. Overexpression of PdbPHD3 conferred salt tolerance, while silencing of PdbPHD3 increased sensitivity to salt. PdbPHD3 could enhance the activities of superoxide dismutase and peroxidase to reduce the abundance of reactive oxygen species, and enhance the osmotic potential by increasing the proline content. Chromatin immunoprecipitation sequencing (ChIP-seq) revealed that PdbPHD3 could bind to various DNA motifs, including the G-box ("CACGTG"), PALBOXAPC ("GGACGG"), and POLLEN1LELAT52 ("TTTCTT"). ChIP-seq combined with RNA sequencing identified a transcription factor gene, squamosa promoter binding protein 3 (PdbSBP3), which is directly regulated by PdbPHD3. Overexpression and silencing of PdbSBP3 improved and decreased salt tolerance, respectively. PdbSBP3 could also regulate all the physiological changes associated with salt tolerance, similar to PdbPHD3. These results suggest that PdbPHD3 confers salt tolerance by regulating PdbSBP3 to reduce ROS accumulation and increase proline content. Therefore, the regulatory axis of PdbPHD3 and PdbSBP3 confers salt tolerance in Shanxin poplar.

CRISPR/Cas9-based editing of NF-YC4 promoters yields high-protein rice and soybean.

Genome editing is a revolution in biotechnology for crop improvement with the final product lacking transgenes. However, most derived traits have been generated through edits that create gene knockouts. Our study pioneers a novel approach, utilizing gene editing to enhance gene expression by eliminating transcriptional repressor binding motifs. Building upon our prior research demonstrating the protein-boosting effects of the transcription factor NF-YC4, we identified conserved motifs targeted by RAV and WRKY repressors in the NF-YC4 promoters from rice (Oryza sativa) and soybean (Glycine max). Leveraging CRISPR/Cas9 technology, we deleted these motifs, resulting in reduced repressor binding and increased NF-YC4 expression. This strategy led to increased protein content and reduced carbohydrate levels in the edited rice and soybean plants, with rice exhibiting up to a 68% increase in leaf protein and a 17% increase in seed protein, and soybean showing up to a 25% increase in leaf protein and an 11% increase in seed protein. Our findings provide a blueprint for enhancing gene expression through precise genomic deletions in noncoding sequences, promising improved agricultural productivity and nutritional quality.

Small Regulatory RNAs of the Rsm Clan in Pseudomonas.

Bacteria of the genus Pseudomonas are ubiquitous on Earth due to their great metabolic versatility and adaptation to fluctuating environments and different hosts. Some groups are important animal/human and plant pathogens, whereas others are studied for their biotechnological applications, including bioremediation, biological control of phytopathogens and plant growth promotion. Notably, their adaptability is mediated by various signal transduction systems, with the post-transcriptional Gac-Rsm cascade playing a key role. This pervasive Pseudomonas pathway controls major transitions at the population level, such as motile/sessile lifestyle, primary/secondary metabolism or replicative/infective behaviour. A hallmark of the Gac-Rsm cascade is the participation of small, regulatory, non-coding RNAs of the Rsm clan. These RNAs are synthetised in response to cell-density-dependent autoinducer signals channelled through the GacS/GacA two-component system, and they counteract, by molecular mimicry, the translational control that RNA-binding proteins of the RsmA family exert over hundreds of mRNAs. Rsm RNAs have been investigated in a few Pseudomonas model species, evidencing the presence of a variable number and families of genes depending on the taxonomic clade. However, the global picture of the distribution of these riboregulators at the genus level was unknown until now. We have undertaken a comprehensive survey and annotation of the vast array of gene sequences encoding members of the Rsm RNA clan in 245 complete genomes that cover 28 phylogenomic clades across the entire genus. The properties of the different families of rsm genes, their phylogenetic radiation, as well as the features of their promoters and adjacent regions, are discussed. The novel insights presented in our manuscript will significantly boost research on the biology of these prevalent RNAs in understudied species of the genus Pseudomonas and closely related genera.

Natural variations in the Cis-elements of GhRPRS1 contributing to petal colour diversity in cotton.

The cotton genus comprises both diploid and allotetraploid species, and the diversity in petal colour within this genus offers valuable targets for studying orthologous gene function differentiation and evolution. However, the genetic basis for this diversity in petal colour remains largely unknown. The red petal colour primarily comes from C, G, K, and D genome species, and it is likely that the common ancestor of cotton had red petals. Here, by employing a clone mapping strategy, we mapped the red petal trait to a specific region on chromosome A07 in upland cotton. Genomic comparisons and phylogenetic analyses revealed that the red petal phenotype introgressed from G. bickii. Transcriptome analysis indicated that GhRPRS1, which encodes a glutathione S-transferase, was the causative gene for the red petal colour. Knocking out GhRPRS1 resulted in white petals and the absence of red spots, while overexpression of both genotypes of GhRPRS1 led to red petals. Further analysis suggested that GhRPRS1 played a role in transporting pelargonidin-3-O-glucoside and cyanidin-3-O-glucoside. Promoter activity analysis indicated that variations in the promoter, but not in the gene body of GhRPRS1, have led to different petal colours within the genus. Our findings provide new insights into orthologous gene evolution as well as new strategies for modifying promoters in cotton breeding.

Promoters Constrain Evolution of Expression Levels of Essential Genes in Escherichia coli.

Variability in expression levels in response to random genomic mutations varies among genes, influencing both the facilitation and constraint of phenotypic evolution in organisms. Despite its importance, both the underlying mechanisms and evolutionary origins of this variability remain largely unknown due to the mixed contributions of cis- and trans-acting elements. To address this issue, we focused on the mutational variability of cis-acting elements, that is, promoter regions, in Escherichia coli. Random mutations were introduced into the natural and synthetic promoters to generate mutant promoter libraries. By comparing the variance in promoter activity of these mutant libraries, we found no significant difference in mutational variability in promoter activity between promoter groups, suggesting the absence of a signature of natural selection for mutational robustness. In contrast, the promoters controlling essential genes exhibited a remarkable bias in mutational variability, with mutants displaying higher activities than the wild types being relatively rare compared to those with lower activities. Our evolutionary simulation on a rugged fitness landscape provided a rationale for this vulnerability. These findings suggest that past selection created nonuniform mutational variability in promoters biased toward lower activities of random mutants, which now constrains the future evolution of downstream essential genes toward higher expression levels.

The regulation of NFKB1 on CD200R1 expression and their potential roles in Parkinson's disease.

BACKGROUND: Overactivated microglia are a key contributor to Parkinson's disease (PD) by inducing neuroinflammation. CD200R1, a membrane glycoprotein mainly found on microglia, is crucial for maintaining quiescence with its dysregulation linked to microglia's abnormal activation. We and other groups have reported a decline in CD200R1 levels in several neurological disorders including PD. However, the mechanism regulating CD200R1 expression and the specific reasons for its reduction in PD remain largely unexplored. Given the pivotal role of transcription factors in gene expression, this study aimed to elucidate the transcriptional regulation of CD200R1 and its implications in PD. METHODS: The CD200R1 promoter core region was identified via luciferase assays. Potential transcription factors were predicted using the UCSC ChIP-seq database and JASPAR. NFKB1 binding to the CD200R1 core promoter was substantiated through electrophoretic mobility shift and chromatin immunoprecipitation assays. Knocking-down or overexpressing NFKB1 validated its regulatory effect on CD200R1. Correlation between decreased CD200R1 and deficient NFKB1 was studied using Genotype-Tissue Expression database. The clinical samples of the peripheral blood mononuclear cells were acquired from 44 PD patients (mean age 64.13 ± 9.78, 43.2% male, median Hoehn-Yahr stage 1.77) and 45 controls (mean age 64.70 ± 9.41, 52.1% male). NFKB1 knockout mice were utilized to study the impact of NFKB1 on CD200R1 expression and to assess their roles in PD pathophysiology. RESULTS: The study identified the CD200R1 core promoter region, located 482 to 146 bp upstream of its translation initiation site, was directly regulated by NFKB1. Significant correlation between NFKB1 and CD200R1 expression was observed in human PMBCs. Both NFKB1 and CD200R1 were significantly decreased in PD patient samples. Furthermore, NFKB1-/- mice exhibited exacerbated microglia activation and dopaminergic neuron loss after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment. CONCLUSION: Our study identified that NFKB1 served as a direct regulator of CD200R1. Reduced NFKB1 played a critical role in CD200R1 dysregulation and subsequent microglia overactivation in PD. These findings provide evidence that targeting the NFKB1-CD200R1 axis would be a novel therapeutic strategy for PD.

Screening high-efficiency promoter to construct trans-vp28 gene Anabaena sp. PCC7120 against white spot syndrome virus of Litopenaeus vannamei.

This study aimed to select high-quality promoters to construct trans-vp28 gene Anabaena sp. PCC7120 and feed Litopenaeus vannamei to assess the effect of L.vannamei against white spot syndrome virus (WSSV). Transgenic algae were created using five plasmids containing PrbcL, Pcpc560, Ptrc, Ptac, and PpsbA. According to the gene expression efficiency and the growth index of transgenic algae, Pcpc560 was determined to be the most efficient promoter. Shrimps were continuously fed trans-vp28 gene Anabaena sp. PCC7120 for one week and then challenged with WSSV. After the challenge, the transgenic algae group (vp28-7120 group) was continuously immunized [continuous immunization for 0 days (vp28-7120-0d); continuous immunization for 2 days (vp28-7120-2d); continuous immunization for 4 days (vp28-7120-4d)]. After seven days, the daily survival rate of each experimental group was continuously tracked. Following the viral challenge, the hepatopancreas samples were assayed for their levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), thioredoxin peroxidase (TPX), acid phosphatase (ACP), and alkaline phosphatase (AKP) at varying time intervals. In comparison to the positive control group (challenge and no vaccination) and the wild-type group (challenge, fed wild-type Anabaena sp. PCC7120), the vp28-7120 group (challenge, fed trans-vp28 gene Anabaena sp. PCC7120) exhibited a remarkable increase in survival rates, reaching 50 % (vp28-7120-0d), 76.67 % (vp28-7120-2d), and 80 % (vp28-7120-4d). Furthermore, the vp28-7120 group consistently displayed significantly higher activities of SOD, CAT, GSH-Px, ACP, and AKP, while exhibiting notably lower TPX activity, when compared to the control group. These results indicate that the Pcpc560 promoter effectively elevated the expression level of the exogenous vp28 gene and spurred the growth of the trans-vp28 gene Anabaena sp. PCC7120. Consequently, trans-vp28 gene Anabaena sp. PCC7120 significantly bolstered the immunity of L.vannamei. Therefore, utilizing the Pcpc560 promoter to develop trans-vp28 gene Anabaena sp. PCC7120 based oral vaccine is highly beneficial for industrial-scale cultivation, advancing its commercialization prospects.

Two decades of progress in glioma methylation research: the rise of temozolomide resistance and immunotherapy insights.

Aims: This study aims to systematically analyze the global trends in glioma methylation research using bibliometric methodologies. We focus on identifying the scholarly trajectory and key research interests, and we utilize these insights to predict future research directions within the epigenetic context of glioma. Methods: We performed a comprehensive literature search of the Web of Science Core Collection (WoSCC) to identify articles related to glioma methylation published from January 1, 2004, to December 31, 2023. The analysis included full-text publications in the English language and excluded non-research publications. Analysis and visualization were performed using GraphPad Prism, CiteSpace, and VOSviewer software. Results: The search identified 3,744 publications within the WoSCC database, including 3,124 original research articles and 620 review articles. The research output gradually increased from 2004 to 2007, followed by a significant increase after 2008, which peaked in 2022. A minor decline in publication output was noted during 2020-2021, potentially linked to the coronavirus disease 2019 pandemic. The United States and China were the leading contributors, collectively accounting for 57.85% of the total research output. The Helmholtz Association of Germany, the German Cancer Research Center (DKFZ), and the Ruprecht Karls University of Heidelberg were the most productive institutions. The Journal of Neuro-Oncology led in terms of publication volume, while Neuro-Oncology had the highest Impact Factor. The analysis of publishing authors revealed Michael Weller as the most prolific contributor. The co-citation network analysis identified David N. Louis's article as the most frequently cited. The keyword analysis revealed "temozolomide," "expression," "survival," and "DNA methylation" as the most prominent keywords, while "heterogeneity," "overall survival," and "tumor microenvironment" showed the strongest citation bursts. Conclusions: The findings of this study illustrate the increasing scholarly interest in glioma methylation, with a notable increase in research output over the past two decades. This study provides a comprehensive overview of the research landscape, highlighting the importance of temozolomide, DNA methylation, and the tumor microenvironment in glioma research. Despite its limitations, this study offers valuable insights into the current research trends and potential future directions, particularly in the realm of immunotherapy and epigenetic editing techniques.

A novel small molecule ZYZ384 targeting SMYD3 for hepatocellular carcinoma via reducing H3K4 trimethylation of the Rac1 promoter.

SMYD3 (SET and MYND domain-containing 3) is a histone lysine methyltransferase highly expressed in different types of cancer(s) and is a promising epigenetic target for developing novel antitumor therapeutics. No selective inhibitors for this protein have been developed for cancer treatment. Therefore, the current study describes developing and characterizing a novel small molecule ZYZ384 screened and synthesized based on SMYD3 structure. Virtual screening was initially used to identify a lead compound and followed up by modification to get the novel molecules. Several technologies were used to facilitate compound screening about these novel molecules' binding affinities and inhibition activities with SMYD3 protein; the antitumor activity has been assessed in vitro using various cancer cell lines. In addition, a tumor-bearing nude mice model was established, and the activity of the selected molecule was determined in vivo. Both RNA-seq and chip-seq were performed to explore the antitumor mechanism. This work identified a novel small molecule ZYZ384 targeting SMYD3 with antitumor activity and impaired hepatocellular carcinoma tumor growth by reducing H3K4 trimethylation of the Rac1 promoter triggering the tumor cell cycle arrest through the AKT pathway.

Characterization and function of promoters of silicon transporter genes PeLsi1-1 and PeLsi1-2 from moso bamboo (Phyllostachys edulis).

KEY MESSAGE: Promoters of moso bamboo silicon transporter genes PeLsi1-1 and PeLsi1-2 contain elements in response to hormone, silicon, and abiotic stresses, and can drive the expression of PeLsi1-1 and PeLsi1-2 in transgene Arabidopsis. Low silicon 1 (Lsi1) transporters from different species have been shown to play an important role in influxing silicon from soil. In previous study, we cloned PeLsi1-1 and PeLsi1-2 from Phyllostachys edulis and verified that PeLsi1-1 and PeLsi1-2 have silicon uptake ability. Furthermore, in this study, the promoters of PeLsi1-1(1910 bp) and PeLsi1-2(1922 bp) were cloned. Deletion analysis identified the key regions of the PeLsi1-1 and PeLsi1-2 promoters in response to hormone, silicon, and abiotic stresses. RT-qPCR analysis indicated that PeLsi1-1 and PeLsi1-2 were regulated by hormones, salt stress and osmotic stress. In addition, we found that the driving activity of the PeLsi1-1 and PeLsi1-2 promoters was regulated by 2 mM K2SiO3 and PeLsi1-1-P3 ~ P4 and PeLsi1-2-P4 ~ 5 were the regions regulated by silicon. Overexpression of PeLsi1-1 or PeLsi1-2 driven by 35S promoter in Arabidopsis resulted in a threefold increase of Si accumulation, whereas transgenic plants showed deleterious symptoms and dwarf seedlings and shorter roots under 2 mM Si treatment. When the 35S promoter was replaced by PeLsi1-1 or PeLsi1-2 promoter, a similar Si absorption was achieved and the transgene plants grew normally. This study, therefore, demonstrates that the promoters of PeLsi1-1 and PeLsi1-2 are indeed effective in driving the expression of moso bamboo Lsi1 genes and leading to silicon uptake.

Exploring the impact of diabetes on aging: insights from TERT and COL1A1 methylation.

Background/aim: Aging, a multifaceted biological process, leads to diminished physical performance, especially in older adults with diabetes, where a mismatch between biological and chronological age is noticeable. Numerous studies have demonstrated that diabetes accelerates aging at the cellular and organ levels. Notable aging markers are telomerase reverse transcriptase (TERT), related to telomere length, and type 1 chain collagen (COL1A1), a key component of skin collagen. Additionally, age-related methylation increases, as revealed through methylation analysis, augmenting aspects of aging. However, the detailed interplay between aging and diabetes, particularly regarding methylation, remains underexplored and warrants further study to elucidate the biological links between the two. Materials and methods: In this study, we elucidate the modulatory influence of diabetes on the aging process, focusing specifically on the modifications in TERT in the kidney and COL1A1 in the skin using mice of Swiss Webster strain as the diabetes model. Specimens were categorized into three distinct chronological cohorts: chronologically young (16 weeks; n = 5), chronologically old (40 weeks; n = 5), and a periodically assessed group (16 weeks; n = 30), from which five mice were systematically sacrificed on a weekly basis. Results: Our findings reveal a marked impact of diabetes on the methylation statuses of TERT and COL1A1, characterized by an elevation in methylation levels within the periodic group (1st-6th week) and a simultaneous, progressive attenuation in the expression of TERT and COL1A1 genes. Conclusion: The observed alterations in the methylation levels of TERT and COL1A1 propound the hypothesis that diabetes potentially expedites the aging process, concomitantly impinging on the production of TERT and COL1A, ostensibly through the mechanism of promoter gene hypermethylation.

Engineered IRES-mediated promoter-free insulin-producing cells reverse hyperglycemia.

Background: Endogenous insulin supplementation is essential for individuals with type 1 diabetes (T1D). However, current treatments, including pancreas transplantation, insulin injections, and oral medications, have significant limitations. The development of engineered cells that can secrete endogenous insulin offers a promising new therapeutic strategy for type 1 diabetes (T1D). This approach could potentially circumvent autoimmune responses associated with the transplantation of differentiated ß-cells or systemic delivery of viral vectors. Methods: We utilized CRISPR/Cas9 gene editing coupled with homology-directed repair (HDR) to precisely integrate a promoter-free EMCVIRES-insulin cassette into the 3' untranslated region (UTR) of the GAPDH gene in human HEK-293T cells. Subsequently quantified insulin expression levels in these engineered cells, the viability and functionality of the engineered cells when seeded on different cell vectors (GelMA and Cytopore I) were also assessed. Finally, we investigated the therapeutic potential of EMCVIRES-based insulin secretion circuits in reversing Hyperglycaemia in T1D mice. Result: Our results demonstrate that HDR-mediated gene editing successfully integrated the IRES-insulin loop into the genome of HEK-293T cells, a non-endocrine cell line, enabling the expression of human-derived insulin. Furthermore, Cytopore I microcarriers facilitated cell attachment and proliferation during in vitro culture and enhanced cell survival post-transplantation. Transplantation of these cell-laden microcarriers into mice led to the development of a stable, fat-encapsulated structure. This structure exhibited the expression of the platelet-endothelial cell adhesion molecule CD31, and no significant immune rejection was observed throughout the experiment. Diabetic mice that received the cell carriers reversed hyperglycemia, and blood glucose fluctuations under simulated feeding stimuli were very similar to those of healthy mice. Conclusion: In summary, our study demonstrates that Cytopore I microcarriers are biocompatible and promote long-term cell survival in vivo. The promoter-free EMCVIRES-insulin loop enables non-endocrine cells to secrete mature insulin, leading to a rapid reduction in glucose levels. We have presented a novel promoter-free genetic engineering strategy for insulin secretion and proposed an efficient cell transplantation method. Our findings suggest the potential to expand the range of cell sources available for the treatment of diabetes, offering new avenues for therapeutic interventions.

The influence of 4G/5G polymorphism in the plasminogen-activator-inhibitor-1 promoter on COVID-19 severity and endothelial dysfunction.

Introduction: Plasminogen activator inhibitor-1 (PAI-1) is linked to thrombosis and endothelial dysfunction in severe COVID-19. The +43 G>A PAI-1 and 4G/5G promoter polymorphism can influence PAI-1 expression. The 4G5G PAI-1 promoter gene polymorphism constitutes the 4G4G, 4G5G, and 5G5G genotypes. However, the impact of PAI-1 polymorphisms on disease severity or endothelial dysfunction remains unclear. Methods: Clinical data, sera, and peripheral blood mononuclear cells (PBMCs) of COVID-19 patients were studied. Results: Comorbidities and clinical biomarkers did not correlate with genotypes in either polymorphism. However, differences between fibrinolytic factors and interleukin-1ß (IL-1ß) were identified in genotypes of the 4G/5G but not the 43 G>A PAI polymorphism. Patients with the 4G4G genotype of the 4G/5G polymorphism showed high circulating PAI-1, mainly complexed with plasminogen activators, and low IL-1ß and plasmin levels, indicating suppressed fibrinolysis. NFκB was upregulated in PBMCs of COVID-19 patients with the 4G4G genotype. Discussion: Mechanistically, IL-1ß enhanced PAI-1 expression in 4G4G endothelial cells, preventing the generation of plasmin and cleavage products like angiostatin, soluble uPAR, and VCAM1. We identified inflammation-induced endothelial dysfunction coupled with fibrinolytic system overactivation as a risk factor for patients with the 5G5G genotype.

C-FOS inhibition promotes pancreatic cancer cell ferroptosis by transcriptionally regulating the expression of SLC7A11.

Cellular proto-oncogene C-Fos forms the AP-1 transcription factor by dimerizing with proto-oncogene c-Jun; this factor upregulates the transcription of genes associated with different malignancies. However, its functions in pancreatic adenocarcinoma (PAAD) remain poorly understood. In this study, the c-Fos was increased in PAAD cells and tissues through bioinformatic analysis, RT-PCR, and WB. In two PAAD cell lines, PANC-1 and BxPC-3, we performed c-Fos knockdown studies using short hairpin RNA (shRNA). Functional analysis indicated that c-Fos depletion in PAAD cells inhibits cell proliferation and promotes ferroptosis. Chromatin Immunoprecipitation (ChIP) and Dual-luciferase experiments showed that c-Fos coupled to the promoter region of SLC7A11 stimulated its transcription, providing mechanistic insight into the process. Moreover, SLC7A11 blocked the decline of proliferation and ferroptosis by c-Fos knockdown in PAAD cells. Furthermore, a xenograft nude mouse model was established to study the impact of c-Fos on tumorigenesis in vivo. Depletion of c-Fos could suppress PC tumor growth and the expressions of SLC7A11, ki-67, and 4HNE, but overexpression of SLC7A11 reversed this process. In summary, our investigation has shown that c-Fos acts as a transcriptional regulator of SLC7A11, which may enhance tumour growth in pancreatic cancer by inhibiting ferroptosis. These results indicate that c-Fos might be a promising target for treating ferroptosis in PAAD.

Dynamically Regulating Homologous Recombination Enables Precise Genome Editing in Ogataea polymorpha.

Methylotrophic yeast Ogataea polymorpha has become a promising cell factory due to its efficient utilization of methanol to produce high value-added chemicals. However, the low homologous recombination (HR) efficiency in O. polymorpha greatly hinders extensive metabolic engineering for industrial applications. Overexpression of HR-related genes successfully improved HR efficiency, which however brought cellular stress and reduced chemical production due to constitutive expression of the HR-related gene. Here, we engineered an HR repair pathway using the dynamically regulated gene ScRAD51 under the control of the l-rhamnose-induced promoter PLRA3 based on the previously constructed CRISPR-Cas9 system in O. polymorpha. Under the optimal inducible conditions, the appropriate expression level of ScRAD51 achieved up to 60% of HR rates without any detectable influence on cell growth in methanol, which was 10-fold higher than that of the wild-type strain. While adopting as the chassis strain for bioproductions, the dynamically regulated recombination system had 50% higher titers of fatty alcohols than that static regulation system. Therefore, this study provided a feasible platform in O. polymorpha for convenient genetic manipulation without perturbing cellular fitness.

Key role of Phyllanthus emblica L. fruit extract promotes ZVI/H2O2 process: rich titratable acid, suitable chelating ability, and antioxidant capacity.

Phyllanthus emblica L. fruit extract (PFE) was introduced to improve ZVI/H2O2 technology, and the efficiency and mechanism of PFE promoting ZVI/H2O2 technology were explored. With the introduction of PFE, the Norfloxacin (NOR) removal rate and kobs of the process were improved by 41.17% and 5.08 times, respectively. In the ZVI/H2O2/PFE process, the degradation of NOR by the attack of ROS is the main pathway for decontamination and is dominated by the heterogeneous reaction on the catalyst surface. PFE contains 13.92 g/L titratable acid and has good complexing ability and antioxidant ability. The mechanism of PFE promoting ZVI/H2O2 technology was based on lowering the pH, complemented by chelation and antioxidant capacity. With the introduction of PFE, the utilization rate of the reagent was significantly increased (7.56 times for ZVI and 3.21 times for H2O2), the applicable pH range was widened (6-9) and the iron sludge was reduced (32.80%). Meanwhile, the concept of UPR is proposed for the first time. The result is the key role to the selection of green promoters in the ZVI/H2O2 process depends on the abundance of titratable acid, followed by a certain chelating ability and antioxidant capacity.