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At least 217 records · Page 12

Circular RNA HIPK3 regulates human lens epithelial cells proliferation and apoptosis by targeting the miR-193a/CRYAA axis

Highlights: • CircRNA sequencing reveals 26 up-regulated and 75 down-regulated circRNAs in ARC. • CircHIPK3 is down-regulated in all three subtypes of ARC. • CircHIPK3 knockdown affects the cellular function in primary cultured HLECs. • CircHIPK3 exerts its role via modulation of circHIPK3/miR-193a/CRYAA axis in HLECs. Circular RNAs (circRNAs) are a novel class of non-coding RNAs generated from back splicing. Accumulating evidence has demonstrated their vital regulation in several biological processes and ocular diseases. However, the role of circRNAs in age-related cataract (ARC), the leading cause of visual impairment worldwide, is still unknown. CircRNA sequencing reveals that 101 circRNAs are differentially expressed between the capsules of transparent and ARC lenses, including 75 down-regulated circRNAs and 26 up-regulated circRNAs transcripts. Eight of 10 differentially expressed circRNAs are further verified by quantitative RT-PCRs. One highly conserved circRNA, circHIPK3, is significantly down-regulated in all cortical, nuclear and posterior subcapsular subtypes of ARC. The silencing of circHIPK3, but not HIPK3 mRNA, significantly accelerates apoptosis development upon oxidative stress and decreases cell viability and proliferation in primary cultured human lens epithelial cells (HLECs). The expression of α-SMA and vimentin was downregulated, while the expression of E-cadherin and ZO-1was upregulated, suggesting the repression of epithelial-mesenchymal transition after circHIPK3 knockdown. CircHIPK3 silencing increases miR-193a expression. miR-193a regulates CRYAA expression by targeting the binding site within the 3′UTR. Moreover, miR-193a decreases the viability and proliferation, and increases the apoptosis of HLECs upon oxidative stress. This study suggests that circRNAs are the potential regulators in cataractogenesis. CircHIPK3 regulates HLECs function through miR-193a-mediated CRYAA expression. This finding would provide a novel insight into the pathogenesis of ARC.

60 APPLIED LIFE SCIENCES↗

Global regulatory function of the low oxygen-induced transcriptional regulator LoiA in Salmonella Typhimurium revealed by RNA sequencing

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major intestinal pathogen that can infect both humans and a variety of animals. LoiA, a novel virulence-regulating protein encoded in Salmonella pathogenicity island (SPI)-14, has been shown to be induced under low oxygen conditions and contribute to S. Typhimurium invasion into intestinal epithetical cells by activating the SPI-1 invasion genes. However, the global regulatory network of LoiA remains unknown. Here, we used high-throughput RNA sequencing (RNA-seq) technology to investigate the regulatory function of LoiA in S. Typhimurium under low oxygen conditions. A total of 1250 genes were differentially expressed between the loiA mutant and the wild-type strain; 413 genes were up-regulated and 837 were down-regulated. SPI-1 gene expression was down-regulated in the loiA mutant, consistent with previous results. SPI-2 gene expression was not affected by deletion of loiA; the expression of most genes involved in flagellar basal body and hook biosynthesis was up-regulated in the loiA mutant, while the expression of genes associated with flagellin, motility, and chemotaxis was down-regulated; the expression of lon, encoding an ATP-dependent protease, was up-regulated in the mutant. This study indicates that LoiA regulates a variety of virulence-associated genes in S. Typhimurium. The negative regulation of Lon protease by LoiA indicates that LoiA can regulates several virulence-associated genes in S. Typhimurium via the Lon protease.

60 APPLIED LIFE SCIENCES↗

Up-regulated miR-548k promotes esophageal squamous cell carcinoma progression via targeting long noncoding RNA-LET

Highlights: • miR-548k is significantly upregulated in ESCC and indicates poor prognosis. • miR-548k promotes the proliferation and migration of ESCC cells. • miR-548k directly targets and represses long noncoding RNA-LET (lncRNA-LET). • lncRNA-LET inhibits the proliferation and migration of ESCC cells. • The roles of miR-548k on ESCC are dependent on the negative regulation of lncRNA-LET. Dysregulated noncoding RNAs have been observed in diverse cancers. MIR458K is frequently amplified in esophageal squamous cell carcinoma (ESCC). However, the expression, clinical significances, and action mechanisms of miR-548k in ESCC are still unclear. In this study, we found that miR-548k is significantly up-regulated in ESCC tissues and cell lines. Up-regulated miR-548k expression is significantly correlated with advanced invasion depth, lymph node metastasis, advanced TNM stage, and poor overall survival. Gain-of- and loss-of-function assays demonstrated that miR-548k promotes the proliferation and migration of ESCC cells in vitro and tumor growth in vivo. Mechanistically, we found that miR-548k directly targets and represses the expression of long noncoding RNA-LET (lncRNA-LET), and further down-regulates p53 and up-regulates NF90. In addition, we found that lncRNA-LET is down-regulated and inversely correlated with miR-548k in ESCC. Down-regulated lncRNA-LET also indicated poor overall survival of ESCC patients. Functional assays demonstrated that lncRNA-LET inhibits the proliferation and migration of ESCC cells, and the effects of miR-548k on ESCC are dependent on the negative regulation of lncRNA-LET. In summary, our data revealed the critical roles of miR-548k-lncRNA-LET regulation axis in ESCC and suggested that the miR-548k-lncRNA-LET regulation axis may be promising prognostic biomarkers and therapeutic targets for ESCC.

60 APPLIED LIFE SCIENCES↗

Tapless Voltage Regulating Transformer. Final Report

Voltage regulation is a common practice in power grid operation. The conventional voltage regulation methods have been facing ever increasing challenges as the penetration of renewable generations deepens in the grid. The intermittent nature of the renewable sources may result in sudden rise or drop of power generation, possibly breaking the generation-load balance. A lack of adequate voltage control not only affects the power quality but is detrimental to the system operation with the potential for large economic losses. Most conventional voltage regulation devices rely on mechanical tap changers to impose the regulation in a step-wise manner. Although sufficient for normal operations, the mechanical tap changers can be easily worn out in the challenging situations created by renewables. In this project, a novel technology called tapless voltage regulating transformer (TAREX) was proposed and investigated. It has the potential to excel at addressing the new voltage regulation challenges and replace the conventional regulating transformers. TAREX is based on the concept of the saturable-core reactor (SCR). The voltage of a TAREX can be smoothly and continuously regulated in a reasonable range by using a low rating power electronics based controller. It uses control of the transformer core magnetization to regulate the voltage on the windings, and thus eliminates the mechanical tap changer. This project is to prove the principle of TAREX, understand the voltage regulation mechanism, and preliminarily understand its impacts on the power systems through modeling, prototyping, and testing.

24 POWER TRANSMISSION AND DISTRIBUTION↗

Onshore U.S. Carbon Pipeline Deployment: Siting, Safety, and Regulation

Carbon capture, utilization, and storage (CCUS) technology has significant potential to reduce greenhouse gas (GHG) emissions and mitigate the impact of climate change, particularly in hard to decarbonize industrial and commercial sectors. CCUS involves capturing carbon dioxide (CO 2 ) from industrial processes or power generation and utilizing it for other purposes, such as enhanced oil recovery (EOR), or storing the captured CO 2 underground. CCUS technology can reduce the environmental impact of continued fossil fuel use while smoothing the transition to a low-carbon economy. CCUS can create new economic opportunities, such as the development of new industries and job creation, and can enhance energy security by diversifying energy sources. For these reasons, enabling CCUS has become a key objective of the Biden-Harris administration’s clean energy policy and has received bipartisan support. Despite its environmental and economic potential, CCUS faces multiple barriers to widespread deployment. One of the main challenges is the high cost and technical difficulty of implementing and operating large-scale CCUS infrastructure. CCUS remains a relatively expensive way to reduce carbon emissions (e.g., compared to solar photovoltaic technology’s displacement of coal generation). Additionally, financial incentives and supportive policies like those enacted to support solar photovoltaic development, especially at the state level, are inconsistent or nonexistent, which can discourage investment in CCUS projects. There are also technical challenges associated with safe and secure underground CO 2 storage and the development of new carbon utilization technologies. Public opposition to various aspects of CCUS technologies, ranging from concerns that CCUS will extend reliance on fossil fuels to CCUS infrastructure being sited in disadvantaged communities, is a growing challenge. This paper focuses on another significant barrier to broad CCUS deployment: the need for considerable expansion of the dedicated land-based CO 2 pipeline network in the United States to meet CCUS goals and the unique regulatory challenges to its development. To reach carbon emissions targets in the United States by 2050, CCUS technology will need to be supported by tens of thousands of miles of CO 2 pipelines. Estimates range from a minimum of roughly 29,000 pipeline miles (according to a 2020 Great Plains Institute study) to 66,000 pipeline miles (as per a 2021 Princeton University–led study). As of October 2022, however, the U.S. Department of Transportation (U.S. DOT) reports fewer than 5,400 miles of U.S. pipelines carrying CO 2 . This deficit—and what it means for the prospect of moving substantially larger quantities of CO 2 from source to use or storage—threatens to stifle the development of CCUS projects and technologies identified as an important tool to meet emissions targets. The current regulatory landscape facing CO 2 pipeline development can best be described as uncertain. At the federal level, the U.S. DOT Pipeline and Hazardous Materials Safety Administration (PHMSA) oversees safety regulation of pipelines transporting hazardous materials, including CO 2 upon commencement of operation. However, PHMSA’s definition of CO 2 as “a fluid consisting of more than 90 percent CO 2 molecules compressed to a supercritical state” has not been updated since its 1991 addition to the Federal Register. Because CO 2 can be transported in a gaseous, liquid, or supercritical state (indeed, the physical state of CO 2 can fluctuate within a single pipeline due to environmental changes), doubts persist about the extent of PHMSA’s purview—and raise questions about what, if anything, states should do to address this apparent gap. PHMSA has begun a major revision of its existing rules, but the agency does not expect a first draft before 2024. Economic oversight of CO 2 pipelines is even less clear. The Federal Energy Regulatory Commission (FERC) and Surface Transportation Board (STB)—which regulate the rates of interstate oil/natural gas and non-energy pipelines, respectively—have both declined jurisdiction over interstate CO 2 pipelines. This presumably leaves economic regulation to state and/or local governments, but few if any states have the laws or resources in place to oversee just and reasonable rates. Further, the interstate nature of CO 2 pipeline development creates questions around how different states should align their rate-making decisions. Onshore U.S. Carbon Pipeline Deployment: Siting, Safety, and Regulation Currently, regulatory responsibilities regarding CO 2 pipeline siting and permitting fall to state and local governments. The variety of laws and regulations across the country, however, creates a maze of requirements for pipeline developers to navigate. To secure necessary permits, most states require pipeline companies to be “common carriers” that provide transport service to the public at uniform rates. However, the specific definition of that term varies. Some states require clear evidence that a pipeline services the public, while others automatically deem any pipeline company transporting energy products or hazardous materials to be a “common carrier”—with little consideration for accessibility to third parties. Other states have eschewed common-carrier terminology entirely, placing private and publicly accessible pipelines on equal footing. Much like the variation in common-carrier requirements, laws governing eminent domain authority to secure rights-of-way (ROW) to commence construction on a planned pipeline route differ by state. Several states have no laws or rules governing CO 2 pipelines. In addition to creating questions about whether long-standing rules for other pipelines (e.g., natural gas or petroleum products) apply to CO 2 , this policy vacuum leaves local governments as the sole authority over sections of pipe within their boundaries. With dozens of counties along a given route, the probability of inconsistent regulation of the same pipeline is significant. Even in states with CO 2 pipeline laws in place, local regulatory attempts to address rising concerns over pipeline routing and safety have triggered lawsuits by pipeline companies seeking to delimit areas of federal, state, and local government responsibility. Meanwhile, legislators across the country have introduced bills to restrict the application of eminent domain to CO 2 pipeline projects, which could threaten a key means of securing ROW that companies cannot secure through negotiation with landowners. Taken separately, any of these regulatory issues—the narrow federal definition of CO 2 , FERC’s and STB’s decisions that CO 2 pipelines are not within their jurisdiction, and the considerable variation in state and local governments’ laws regulating CO 2 pipeline technologies—are extremely difficult to resolve. Adding the required scale of CO 2 pipeline expansion and the currently identified narrow window of time in which to reach climate target goals, the task becomes even more difficult—and raises a host of urgent questions for regulators. How should CO 2 be defined in federal regulations to ensure consistent safety standards across the country? What is the potential impact radius of a CO 2 pipeline rupture, and how should that inform local emergency response? In the absence of centralized federal oversight, what should state legislatures do to increase alignment for interstate CO 2 pipeline projects? This paper intends to serve as a primer for regulators and stakeholders who seek to better understand the regulatory challenges and opportunities facing this critical infrastructure.

42 ENGINEERING↗

Regulatory Design for Transport Security Regulations of Nuclear and Other Radioactive Material

A cornerstone within a nuclear security regime of a country is a legislative and regulatory framework that both regulates the individuals using radioactive and nuclear materials, but also regulates the regulators for their commitments under International Law. When drafting nuclear law and regulations, both technical and legal expertise is needed to ensure the regulation meets the technical requirements for security, but also is legally enforceable within a country’s jurisdiction. While the Primary Nuclear Law, or Enabling Act provides establishes the Competent Authority and responsibilities for nuclear security, regulatory development is where the proverbial, rubber meets the road for requirements. The regulations provide instruction for inspections, authorizations, and managed activities, such as use storage, and transport. Transport Security regulatory development for radioactive and nuclear material is complex because the regulation maybe multi-jurisdictional, multi-modal, and involve multiple stakeholders. Development of transport security regulations requires coordination with other ministerial stakeholders, transport operators, and alignment to international treaties and conventions outside the nuclear domain. This paper provides a brief discussion of the challenges associated with regulatory development for transport security regulations for nuclear and other radioactive materials in transport. The paper will briefly identify key stakeholders, international law involved, and current efforts by the International Atomic Energy Agency (IAEA) in supporting Member States in regulatory development. While active in that regulations are necessary for a robust nuclear security regime, regulatory development is more a slow-burn than an active activity in nuclear security.

Fialkoff, Marc↗

Notch signaling regulates the expression of glycolysis-related genes in a context-dependent manner during embryonic development

Highlights: • Notch does not regulate glycolysis genes during early embryonic development. • Notch regulates glycolysis genes differently in the brain at the larval stage. • Notch-mediated glycolysis gene regulation is context-dependent during development. Glycolysis, the classic pathway for producing energy, has been known to be involved in neural development. Notch signaling also contributes to neural development and regulation of glycolysis in various tissues. However, the role of Notch signaling in glycolysis-related gene regulation during neural development is poorly understood. Here, we analyzed mRNA expression patterns and levels of glucose transporters (GLUT) as well as rate-limiting enzymes in glycolysis using zebrafish mib1{sup ta52b} mutants, in which Notch signaling was deficient at the early embryonic and larval stages. Our results indicated that in neural tissues, Notch signaling positively regulates glut1a and glut3a expression and negatively regulates hk2 expression at the larval stage but may not regulate them during early embryonic stages. Therefore, these results suggest that Notch signaling regulates glycolysis-related gene expression in a context-dependent manner in neural tissues at different developmental stages.

60 APPLIED LIFE SCIENCES↗

Cyber-Physical Dynamic System (CPDS) Modeling for Frequency Regulation and AGC Services of Distributed Energy Resources

The substantial integration of renewable energy brings significant challenges to balance the system in real time because of the variability and intermittency of renewable power. For the reliable system operation, the frequency regulation service is used to stabilize the system frequency through automatically balancing the system generation and load. On one hand, with the substantially increasing deployment of renewable energy in electricity system, the requirement of frequency regulation (FR) services increases significantly. On the other hand, the current main resource of FR services, the controllable conventional generation, is continuously decreasing in the system generation mix. This means that in the future high renewable penetration power system, additional and alternative reliable FR services providers such as distributed energy storage (DES) resources should be explored. Although the capability of utility-scale energy storage to provide FR services has been demonstrated, the integrated control and dynamic modeling of distributed energy resource (DER) providing frequency regulation grid services has been rarely explored. There are several challenges to adopt DERs to provide reliable grid services as illustrated in FERC 755. First, the distributed installation of DES requires a comprehensive cyber-physical dynamic system (CPDS) modeling to fully consider the impacts of the communication latency variability on its real time FR provision capability. Unlike the conventional generators, there are two-layer communication between DERs and system operators which increase the communication delay. Second, unlike the conventional generators whose dynamic models have been comprehensively studied, the difference among individual DER components' power-dynamic characteristic brings challenges in the accurate dynamic AGC modeling of its power-frequency relationship. Third, the temporal dependent state of charge uncertainty of DERs challenges DERs' power and frequency regulation capacities scheduling in the look-ahead generation scheduling. Therefore, the aggregator should optimize the frequency regulation provision from individual DER in real time. To overcome these challenges, this project proposes a cyber-physical dynamic system (CPDS) model to handle the uncertainty of DERs two-layer communication latency and power dynamics. The variability of DERs' communication delay and dynamic constraints will be comprehensively modeled. The DERs' AGC model with communication delay is designed to validate DESs' frequency regulation services. Like current performance-based frequency regulation evaluation, the delivery of DERs' frequency regulation will be assessed through post-analysis of the actual AGC response with respect to the AGC control signal from system operators. Consequently, the reliability improvement with DERs providing reliable frequency regulation services can be evaluated from a comprehensive perspective considering all the dynamics of communication and power.

24 POWER TRANSMISSION AND DISTRIBUTION↗

MicroRNA-378 regulates neural stem cell proliferation and differentiation in vitro by modulating Tailless expression

Previous studies have suggested that microRNAs (miRNAs) play an important role in regulating neural stem cell (NSC) proliferation and differentiation. However, the precise role of miRNAs in NSC remains largely unexplored. In this study, we showed that miR-378 can target Tailless (TLX), a critical regulator of NSC, to regulate NSC proliferation and differentiation. By bioinformatic algorithms, miR-378 was found to have a predicted target site in the 3′-untranslated region of TLX, which was verified by a dual-luciferase reporter assay. The expression of miR-378 was increased during NSC differentiation and inversely correlated with TLX expression. qPCR and Western blot analysis also showed that miR-378 negatively regulated TLX mRNA and protein expression in neural stem cells (NSCs). Intriguingly, overexpression of miR-378 increased NSC differentiation and reduced NSC proliferation, whereas suppression of miR-378 led to decreased NSC differentiation and increased NSC proliferation. Moreover, the downstream targets of TLX, including p21, PTEN and Wnt/β-catenin were also found to be regulated by miR-378. Additionally, overexpression of TLX rescued the NSC proliferation deficiency induced by miR-378 overexpression and abolished miR-378-promoted NSC differentiation. Taken together, our data suggest that miR-378 is a novel miRNA that regulates NSC proliferation and differentiation via targeting TLX. Therefore, manipulating miR-378 in NSCs could be a novel strategy to develop novel interventions for the treatment of relevant neurological disorders. - Highlights: • miR-378 targeted and regulated TLX. • miR-378 was increased during NSC differentiation. • miR-378 regulated NSC proliferation and differentiation. • miR-378 regulated NSC self-renew through TLX.

60 APPLIED LIFE SCIENCES↗

miRNA-152 targets GATA1 to regulate erythropoiesis in Chionodraco hamatus

Highlights: • MiR-152 plays an important role in hematopoiesis during the development of C. hamatus. • The overexpression of miR-152 reduced hematopoiesis was regulated by the miR-152-mediated down-regulation of GATA1. • The miR-152-mediated decrease of hematopoiesis is also suitable for the zebrafish autologous model. Chionodraco hamatus is a teleost within the suborder Notothenioidei, the members of which are known to lack functional erythrocytes with modified hematopoiesis. Hematopoiesis is an essential process during the development of animals, where it is tightly regulated by many different transcription factors, signaling proteins, chromatin modifications, and microRNAs (miRNAs). The miRNAs are known to regulate the expression of their target genes at the post-transcriptional level. However, little is known about the miRNA-mediated regulation of hematopoiesis. In this study, we confirmed that miR-152 plays a crucial role in hematopoiesis during the development of C. hamatus. The overexpression of miR-152 reduced hematopoiesis according to the decreased expression of GATA1 and reduced o-dianisidine staining of hemoglobin. Mechanistically, reduced hematopoiesis was regulated by the miR-152-mediated down-regulated expression of GATA1. Bioinformatics analysis was used to predict the target gene of miR-152. Western blotting as well as dual luciferase and EGFP reporter assays were employed to investigate the expression of GATA1 mediated by miR-152. Finally, verification experiments in the zebrafish autologous model strongly supported the effect of miR-152 on hematopoiesis. In conclusion, we suggest that miR-152 is a novel molecular factor that regulates hematopoiesis during the development of C. hamatus by down-regulating the expression of GATA1.

60 APPLIED LIFE SCIENCES↗

Molecularly targeted anti-cancer drugs inhibit the invasion and metastasis of hepatocellular carcinoma by regulating the expression of MMP and TIMP gene families

Highlights: • The multi-target drugs of liver cancer bringing new hope for advance HCC patients. • Sorafenib, regorafenib, and lenvatinib have inhibitory effect on the metastasis and invasion. • They can down-regulate the expression of MMPs and up-regulate the TIMPs level. To investigate the effect of multi-kinase kinase inhibitors (sorafenib; regorafenib; lenvatinib) on the invasion and metastasis of human hepatocellular carcinoma (HCC) cells, and the outcome of this effect on the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs), yet unclarified. Cells were subjected to four different treatments: blank control group, sorafenib (10 μmol/L) treatment group, regorafenib (20 mmol/L) treatment group, and lenvatinib (4 μmol/L) treatment group. Anti-invasion and anti-metastasis effects were tested using the wound-healing assay and transwell invasion assay. Real-time PCR and Western blot analyses were used to determine the impact of sorafenib, regorafenib, and lenvatinib on the gene expression of MMPs and TIMPs in the two HCC lines (Hep3B and SMMC-7721). Results from the wound-healing and transwell invasion assays showed the three tested anti-cancer drugs to have a significant inhibitory effect on the metastasis and invasion of HCC cells. Real-time PCR and western blot analyses revealed that sorafenib down-regulated the expressions of MMP-7,10,16 and up-regulated those of TIMP-1,3,4, regorafenib down-regulated the expression of MMP-1 and up-regulated TIMP-3 gene expression, and lenvatinib down-regulated the expressions of MMP-1,2,7,9,10,16 and up-regulated those of TIMP-1,3,4. However, these three targeted anti-cancer drugs seem to have no significant regulatory effect on the expressions of other MMPs and TIMPs family genes. In conclusion, sorafenib, regorafenib, and lenvatinib inhibit the invasion and metastasis of HCC cells by regulating MMPs/TIMPs expression levels.

60 APPLIED LIFE SCIENCES↗

Long noncoding RNA LOXL1-AS1 regulates prostate cancer cell proliferation and cell cycle progression through miR-541-3p and CCND1

Highlights: • LOXL1-AS1 regulates proliferation and cell cycle in prostate cancer cells. • miR-541-3p regulates CCND1 expression in prostate cancer cells. • LOXL1-AS1 sponges and regulates the expression of miR-541-3p. • LOXL1-AS1 regulates proliferation and cell cycle through miR-541-3p and CCND1. Prostate cancer is one of the most frequent malignancies affecting men. Long non-coding RNAs (lncRNAs) are involved in the pathogenesis of prostate cancer. LncRNA LOXL1-AS1 participates in the pathogenesis of the exfoliation syndrome. However, the role of LOXL1-AS1 in cancer remains largely unknown. Here, we found that LOXL1-AS1 down-regulation inhibited prostate cancer cell proliferation and cell cycle progression. RNA sequencing analysis revealed that it regulates the expression of cell cycle-related genes. LOXL1-AS1 is predominantly distributed in the cytoplasm, where it interacts with miR-541-3p. In addition, miR-541-3p targets the cell cycle regulator CCND1 in prostate cancer cells. LOXL1-AS1 down-regulation inhibits the expression of CCND1 and cell cycle progression, whereas these effects are abolished upon miR-541-3p suppression. In summary, our study revealed that LOXL1-AS1 regulates prostate cancer cell proliferation and cell cycle progression through miR-541-3p and CCND1. Modulation of their levels may be used to treat prostate cancer.

60 APPLIED LIFE SCIENCES↗

Hedgehog signaling contributes to basic fibroblast growth factor-regulated fibroblast migration

Fibroblast migration is a central process in skin wound healing, which requires the coordination of several types of growth factors. bFGF, a well-known fibroblast growth factor (FGF), is able to accelerate fibroblast migration; however, the underlying mechanism of bFGF regulation fibroblast migration remains unclear. Through the RNA-seq analysis, we had identified that the hedgehog (Hh) canonical pathway genes including Smoothened (Smo) and Gli1, were regulated by bFGF. Further analysis revealed that activation of the Hh pathway via up-regulation of Smo promoted fibroblast migration, invasion, and skin wound healing, but which significantly reduced by GANT61, a selective antagonist of Gli1/Gli2. Western blot analyses and siRNA transfection assays demonstrated that Smo acted upstream of phosphoinositide 3-kinase (PI3K)-c-Jun N-terminal kinase (JNK)-β-catenin to promote cell migration. Moreover, RNA-seq and qRT-PCR analyses revealed that Hh pathway genes including Smo and Gli1 were under control of β-catenin, suggesting that β-catenin turn feedback activates Hh signaling. Taken together, our analyses identified a new bFGF-regulating mechanism by which Hh signaling regulates human fibroblast migration, and the data presented here opens a new avenue for the wound healing therapy. - Highlights: • bFGF regulates Hedgehog (Hh) signaling in fibroblasts. • The Smo and Gli two master regulators of Hh signaling positively regulate fibroblast migration. • Smo facilitates β-catenin nuclear translocation via activation PI3K/JNK/GSK3β. • β-catenin positively regulates fibroblast cell migration and the expression of Hh signaling genes including Smo and Gli.

60 APPLIED LIFE SCIENCES↗

Regulation of alternative splicing in Drosophila by 56 RNA binding proteins

Alternative splicing is regulated by RNA binding proteins (RBPs) that recognize pre-mRNA sequence elements and activate or repress adjacent exons. We used RNA interference and RNA-seq to identify splicing events regulated by 56 Drosophila proteins, some previously unknown to regulate splicing. Nearly all proteins affected alternative first exons, suggesting that RBPs play important roles in first exon choice. Half of the splicing events were regulated by multiple proteins, demonstrating extensive combinatorial regulation. We observed that SR and hnRNP proteins tend to act coordinately with each other, not antagonistically. We also identified a cross-regulatory network where splicing regulators affected the splicing of pre-mRNAs encoding other splicing regulators. In conclusion, this large-scale study substantially enhances our understanding of recent models of splicing regulation and provides a resource of thousands of exons that are regulated by 56 diverse RBPs.

59 BASIC BIOLOGICAL SCIENCES↗

Xylanolytic metabolism is regulated by coordination of transcription factors XynR and XylR in extremely thermophilic Caldicellulosiruptorales

ABSTRACT Global transcription factors (TFs) control metabolic processes in bacteria to efficiently utilize available carbon. The orderCaldicellulosiruptoraleshas drawn interest due to the ability of its members to degrade components of lignocellulosic biomass. Regulatory reconstruction ofAnaerocellum (f. Caldicellulosiruptor) besciiidentified two major global transcription factors for xylan utilization, XynR and XylR, and the corresponding putative transcription factor binding sites. Recombinant versions of XynR (LacI family) and XylR (ROK family) were subjected to fluorescence polarization (FP) and biolayer interferometry (BLI) analysis to confirm the predicted binding sites. Four XynR sites and two XylR sites were validated, accounting for 20 of 26 genes regulated by XynR and six of seven genes regulated by XylR. Bioinformatic analysis of the individual genes controlled by the two regulators showed an inter-dependent scheme for xylan conversion; the transport of xylooligosaccharides (XOS) is dependent on XylR, while enzymes responsible for hydrolysis are controlled by both regulators. For xylose catabolism by the xylose isomerase-xylulose kinase pathway, regulation is also split, with XylR controlling xylose isomerase and XynR controlling xylokinase. The XynR/XylR regulator pair withinA. besciiis conserved in all sequenced species ofCaldicellulosiruptorales, suggesting similarities in regulating linear xylan conversion. In other xylanolytic thermophiles, XylR homologs control xylan degradation, compared to just 6 out of 26 genes forA. bescii. These results show that two separate regulatory schemes (dual repression) are coordinated byA. besciito effectively regulate the hemicellulose inventory and xylan catabolism. IMPORTANCE To take full advantage of extreme thermophiles as platform metabolic engineering microorganisms, the tools for genetic manipulation must be further developed, and strategies that exploit a better understanding of metabolic regulation need to be discerned.Anaerocellum bescii, the most studied of the extremely thermophilic fermentative anaerobic bacteria that can utilize microcrystalline cellulose, can degrade microcrystalline cellulose and hemicellulose and has been metabolically engineered to convert the resulting sugars to products such as ethanol and acetone. For xylan, in particular, two major global transcription factors (TFs), XynR and XylR, play a role in sugar metabolism, although their predicted regulatory interdependence from bioinformatics analysis has not been elucidated experimentally. Here, fluorescence polarization (FP) and biolayer interferometry (BLI) were used to explore this issue to support metabolic engineering efforts aimed at improving carbohydrate processing to industrial chemicals.

Biotechnology & Applied Microbiology↗

Arm-in-Arm Response Regulator Dimers Promote Intermolecular Signal Transduction

ABSTRACT Bacteriophytochrome photoreceptors (BphPs) and their cognate response regulators make up two-component signal transduction systems which direct bacteria to mount phenotypic responses to changes in environmental light quality. Most of these systems utilize single-domain response regulators to transduce signals through unknown pathways and mechanisms. Here we describe the photocycle and autophosphorylation kinetics of RtBphP1, a red light-regulated histidine kinase from the desert bacterium Ramlibacter tataouinensis . RtBphP1 undergoes red to far-red photoconversion with rapid thermal reversion to the dark state. RtBphP1 is autophosphorylated in the dark; this activity is inhibited under red light. The RtBphP1 cognate response regulator, the R. tataouinensis bacteriophytochrome response regulator (RtBRR), and a homolog, AtBRR from Agrobacterium tumefaciens , crystallize unexpectedly as arm-in-arm dimers, reliant on a conserved hydrophobic motif, hFWAhL (where h is a hydrophobic M, V, L, or I residue). RtBRR and AtBRR dimerize distinctly from four structurally characterized phytochrome response regulators found in photosynthetic organisms and from all other receiver domain homodimers in the Protein Data Bank. A unique cacodylate-zinc-histidine tag metal organic framework yielded single-wavelength anomalous diffraction phases and may be of general interest. Examination of the effect of the BRR stoichiometry on signal transduction showed that phosphorylated RtBRR is accumulated more efficiently than the engineered monomeric RtBRR (RtBRR mon ) in phosphotransfer reactions. Thus, we conclude that arm-in-arm dimers are a relevant signaling intermediate in this class of two-component regulatory systems. IMPORTANCE BphP histidine kinases and their cognate response regulators comprise widespread red light-sensing two-component systems. Much work on BphPs has focused on structural understanding of light sensing and on enhancing the natural infrared fluorescence of these proteins, rather than on signal transduction or the resultant phenotypes. To begin to address this knowledge gap, we solved the crystal structures of two single-domain response regulators encoded by a region immediately downstream of that encoding BphPs. We observed a previously unknown arm-in-arm dimer linkage. Monomerization via deletion of the C-terminal dimerization motif had an inhibitory effect on net response regulator phosphorylation, underlining the importance of these unusual dimers for signal transduction.

Baker, Anna W.↗

Structural Basis for DNA Recognition by the Two-Component Response Regulator RcsB

ABSTRACT RcsB is a highly conserved transcription regulator of the Rcs phosphorelay system, a complex two-component signal transduction system (N. Majdalani and S. Gottesman, Annu Rev Microbiol 59:379–405, 2005; A. J. Wolfe, Curr Opin Microbiol 13:204–209, 2010, https://doi.org/10.1016/j.mib.2010.01.002 ; D. J. Clarke, Future Microbiol 5:1173–1184, 2010, https://doi.org/10.2217/fmb.10.83 ). RcsB plays an important role in virulence and pathogenicity in human hosts by regulating biofilm formation. RcsB can regulate transcription alone or together with its auxiliary transcription regulators by forming heterodimers. This complexity allows RcsB to regulate transcription of more than 600 bacterial genes in response to different stresses (D. Wang et al., Mol Plant Microbe Interact 25:6–17, 2012, https://doi.org/10.1094/MPMI-08-11-0207 ). Despite increasing knowledge of RcsB importance, molecular mechanisms that drive the ability of RcsB to control transcription of a large number of genes remain unclear. Here, we present crystal structures of unphosphorylated RcsB in complex with the consensus DNA-binding sequence of 22-mer (DNA22) and 18-mer (DNA18) of the flhDC operon from Escherichia coli determined at 3.15- and 3.37-Å resolution, respectively. The results of our structural analysis combined with the results of in vitro binding assays provide valuable insights to the protein regulatory mechanism, demonstrate how RcsB recognizes target DNA sequences, and reveal a unique oligomeric state that allows RcsB to form homo- and heterodimers. This information will help us understand the complex mechanisms of transcriptional regulation by RcsB in bacteria. IMPORTANCE RcsB is a well-studied two-component response regulator of the Rcs phosphorelay system, conserved within the family Enterobacteriaceae , which includes many pathogens. It is a global regulator, controlling more than 5% of bacterial genes associated with capsule biosynthesis, flagellar biogenesis, cell wall biosynthesis, antibiotic resistance, biofilm formation, and virulence in pathogens. Knowledge of RcsB structure represents a unique opportunity to explore mechanisms that regulate the Rcs phosphorelay system and its role in the family Enterobacteriaceae .

59 BASIC BIOLOGICAL SCIENCES↗

Trainable Gene Regulation Networks with Applications to Drosophila Pattern Formation

This chapter will very briefly introduce and review some computational experiments in using trainable gene regulation network models to simulate and understand selected episodes in the development of the fruit fly, Drosophila melanogaster. For details the reader is referred to the papers introduced below. It will then introduce a new gene regulation network model which can describe promoter-level substructure in gene regulation. As described in chapter 2, gene regulation may be thought of as a combination of cis-acting regulation by the extended promoter of a gene (including all regulatory sequences) by way of the transcription complex, and of trans-acting regulation by the transcription factor products of other genes. If we simplify the cis-action by using a phenomenological model which can be tuned to data, such as a unit or other small portion of an artificial neural network, then the full transacting interaction between multiple genes during development can be modelled as a larger network which can again be tuned or trained to data. The larger network will in general need to have recurrent (feedback) connections since at least some real gene regulation networks do. This is the basic modeling approach taken, which describes how a set of recurrent neural networks can be used as a modeling language for multiple developmental processes including gene regulation within a single cell, cell-cell communication, and cell division. Such network models have been called "gene circuits", "gene regulation networks", or "genetic regulatory networks", sometimes without distinguishing the models from the actual modeled systems.

Mjolsness, Eric↗