Indirect Targets Research Articles

A Systems Biology Approach To Disentangle the Direct and Indirect Effects of Global Transcription Factors on Gene Expression in Escherichia coli.

Delineating the pleiotropic effects of global transcriptional factors (TFs) is critical for understanding the system-wide regulatory response in a particular environment. Currently, with the availability of genome-wide TF binding and gene expression data for Escherichia coli, several gene targets can be assigned to the global TFs, albeit inconsistently. Here, using a systematic integrated approach with emphasis on metabolism, we characterized and quantified the direct effects as well as the growth rate-mediated indirect effects of global TFs using deletion mutants of FNR, ArcA, and IHF regulators (focal TFs) under glucose fermentative conditions. This categorization enabled us to disentangle the dense connections seen within the transcriptional regulatory network (TRN) and determine the exact nature of focal TF-driven epistatic interactions with other global and pathway-specific local regulators (iTFs). We extended our analysis to combinatorial deletions of these focal TFs to determine their cross talk effects as well as conserved patterns of regulatory interactions. Moreover, we predicted with high confidence several novel metabolite-iTF interactions using inferred iTF activity changes arising from the allosteric effects of the intracellular metabolites perturbed as a result of the absence of focal TFs. Further, using compendium level computational analyses, we revealed not only the coexpressed genes regulated by these focal TFs but also the coordination of the direct and indirect target expression in the context of the economy of intracellular metabolites. Overall, this study leverages the fundamentals of TF-driven regulation, which could serve as a better template for deciphering mechanisms underlying complex phenotypes. IMPORTANCE Understanding the pleiotropic effects of global TFs on gene expression and their relevance underlying a specific response in a particular environment has been challenging. Here, we distinguish the TF-driven direct effects and growth rate-mediated indirect effects on gene expression using single- and double-deletion mutants of FNR, ArcA, and IHF regulators under anaerobic glucose fermentation. Such dissection assists us in unraveling the precise nature of interactions existing between the focal TF(s) and several other TFs, including those altered by allosteric effects of intracellular metabolites. We were able to recapitulate the previously known metabolite-TF interactions and predict novel interactions with high confidence. Furthermore, we determined that the direct and indirect gene expression have a strong connection with each other when analyzed using the coexpressed- or coregulated-gene approach. Deciphering such regulatory patterns explicitly from the metabolism point of view would be valuable in understanding other unpredicted complex regulation existing in nature.

Highlights from Recent Cancer Literature.

Breaking Insights| February 03 2023 Highlights from Recent Cancer Literature Author & Article Information Online ISSN: 1538-7445 Print ISSN: 0008-5472 ©2023 American Association for Cancer Research2023American Association for Cancer Research Cancer Res (2023) 83 (3): 349–350. https://doi.org/10.1158/0008-5472.CAN-83-3-BI Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Tools Icon Tools Get Permissions Cite Icon Cite Search Site Article Versions Icon Versions Version of Record February 3 2023 Citation Highlights from Recent Cancer Literature. Cancer Res 1 February 2023; 83 (3): 349–350. https://doi.org/10.1158/0008-5472.CAN-83-3-BI Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest Search Advanced Search Many chemotherapies induce cell death in a subset of tumor cells, but there remains a question of whether this death enables survival of the remaining malignant cells. Using patient-derived colorectal cancer organoids treated with the chemotherapy 5-fluorouracil, Schmitt and colleagues found efficient induction of the mechanistic target of rapamycin (mTOR) pathway in surviving cancer cells, specifically, mTOR complex 1 and its indirect target p-S6. Coadministration of the mTOR inhibitor rapamycin and 5-fluorouracil led to synergistic inhibition of cancer cell survival in organoids and xenografts. Through systematic testing in a mouse model of colorectal cancer, the authors showed that mTOR signaling was driven by paracrine release of ATP from dying cells and required the P2X4 receptor. Inhibition of P2X4, like inhibition of mTOR, greatly decreased tumor graft size when combined with 5-floururacl. Expert Commentary: This work identifies a potential combination therapy not easily predicted by genomic alterations and suggests that profiling... You do not currently have access to this content.

MEK Is a Potential Indirect Target in Subtypes of Head and Neck Cancers.

The poor prognosis of head-and-neck squamous cell carcinoma (HNSCC) is partly due to the lack of reliable prognostic and predictive markers. The Ras/Raf/MEK/ERK signaling pathway is often activated by overexpressed epidermal growth factor receptor (EGFR) and stimulates the progression of HNSCCs. Our research was performed on three human papillomavirus (HPV)-negative HNSCC-cell lines: Detroit 562, FaDu and SCC25. Changes in cell viability upon EGFR and/or MEK inhibitors were measured by the MTT method. The protein-expression and phosphorylation profiles of the EGFR-initiated signaling pathways were assessed using Western-blot analysis. The EGFR expression and pY1068-EGFR levels were also studied in the patient-derived HNSCC samples. We found significant differences between the sensitivity of the tumor-cell lines used. The SCC25 line was found to be the most sensitive to the MEK inhibitors, possibly due to the lack of feedback Akt activation through EGFR. By contrast, this feedback activation had an important role in the FaDu cells. The observed insensitivity of the Detroit 562 cells to the MEK inhibitors might have been caused by their PIK3CA mutation. Among HNSCC cell lines, EGFR-initiated signaling pathways are particularly versatile. An ERK/EGFR feedback loop can lead to Akt-pathway activation upon MEK inhibition, and it is related not only to increased amounts of EGFR but also to the elevation of pY1068-EGFR levels. The presence of this mechanism may justify the combined application of EGFR and MEK inhibitors.

Reinforcement amid genetic diversity in the Candida albicans biofilm regulatory network.

Biofilms of the fungal pathogen Candida albicans include abundant long filaments called hyphae. These cells express hypha-associated genes, which specify diverse virulence functions including surface adhesins that ensure biofilm integrity. Biofilm formation, virulence, and hypha-associated gene expression all depend upon the transcription factor Efg1. This transcription factor has been characterized extensively in the C. albicans type strain SC5314 and derivatives, but only recently has its function been explored in other clinical isolates. Here we define a principal set of Efg1-responsive genes whose expression is significantly altered by an efg1Δ/Δ mutation across 17 clinical isolates. This principal gene set includes 68 direct Efg1 targets, whose 5' regions are bound by Efg1 in five clinical isolates, and 42 indirect Efg1 targets, whose 5' regions are not detectably bound by Efg1. Three direct Efg1 target genes encode transcription factors-BRG1, UME6, and WOR3 -whose increased expression in an efg1Δ/Δ mutant restores expression of multiple indirect and direct principal targets, as well as biofilm formation ability. Although BRG1 and UME6 are well known positive regulators of hypha-associated genes and biofilm formation, WOR3 is best known as an antagonist of Efg1 in the sexual mating pathway. We confirm the positive role of WOR3 in biofilm formation with the finding that a wor3Δ/Δ mutation impairs biofilm formation in vitro and in an in vivo biofilm model. Positive control of Efg1 direct target genes by other Efg1 direct target genes-BRG1, UME6, and WOR3 -may buffer principal Efg1-responsive gene expression against the impact of genetic variation in the C. albicans species.

Building High-Confidence Gene Regulatory Networks by Integrating Validated TF-Target Gene Interactions Using ConnecTF.

Many methods are now available to identify or predict the target genes of transcription factors (TFs) in plants. These include experimental approaches such as in vivo or in vitro TF-target gene-binding assays and various methods for identifying regulated targets in mutants, transgenics, or isolated plant cells. In addition, computational approaches are used to infer TF-target gene interactions from the regulatory elements or gene expression changes across treatments. While each of these approaches has now been applied to a large number of TFs from many species, each method has its own limitations which necessitates that multiple data types are integrated to build the most accurate representation of the gene regulatory networks operating in plants. To make the analyses of TF-target interaction datasets available to the broader research community, we have developed the ConnecTF web platform ( https://connectf.org/ ). In this chapter, we describe how ConnecTF can be used to integrate validated and predicted TF-target gene interactions in order to dissect the regulatory role of TFs in developmental and stress response pathways. Using as our examples KN1 and RA1, two well-characterized maize TFs involved in developing floral tissue, we demonstrate how ConnecTF can be used to (1) compare the target genes between TFs, (2) identify direct vs. indirect targets by combining TF-binding and TF-regulation datasets, (3) chart and visualize network paths between TFs and their downstream targets, and (4) prune inferred user networks for high-confidence predicted interactions using validated TF-target gene data. Finally, we provide instructions for setting up a private version of ConnecTF that enables research groups to store and analyze their own TF-target gene interaction datasets.

A mini-TGA protein modulates gene expression through heterogeneous association with transcription factors.

TGA (TGACG-binding) transcription factors, which bind their target DNA through a conserved basic region leucine zipper (bZIP) domain, are vital regulators of gene expression in salicylic acid (SA)-mediated plant immunity. Here, we investigated the role of StTGA2.1, a potato (Solanum tuberosum) TGA lacking the full bZIP, which we named a mini-TGA. Such truncated proteins have been widely assigned as loss-of-function mutants. We, however, confirmed that StTGA2.1 overexpression compensates for SA-deficiency, indicating a distinct mechanism of action compared with model plant species. To understand the underlying mechanisms, we showed that StTGA2.1 can physically interact with StTGA2.2 and StTGA2.3, while its interaction with DNA was not detected. We investigated the changes in transcriptional regulation due to StTGA2.1 overexpression, identifying direct and indirect target genes. Using in planta transactivation assays, we confirmed that StTGA2.1 interacts with StTGA2.3 to activate StPRX07, a member of class III peroxidases (StPRX), which are known to play role in immune response. Finally, via structural modeling and molecular dynamics simulations, we hypothesized that the compact molecular architecture of StTGA2.1 distorts DNA conformation upon heterodimer binding to enable transcriptional activation. This study demonstrates how protein truncation can lead to distinct functions and that such events should be studied carefully in other protein families.

LINC02323 facilitates development of lung squamous cell carcinoma by miRNA sponge and RBP dysregulation and links to poor prognosis.

The poor outcome of patients with lung squamous cell carcinoma (LUSC) highlights the importance of the identification of novel effective prognostic markers and therapeutic targets. Long noncoding RNAs (lncRNAs) have generally been considered to serve important roles in tumorigenesis and the development of various types of cancer, including LUSC. Here, we aimed to investigate the role of LINC02323 in LUSC and its potential mechanisms by performing comprehensive bioinformatic analyses. LINC02323 was elevated and positively associated with unfavorable prognosis of LUSC patients. LINC02323 exerted oncogenic function by competitively binding to miR-1343-3p and miR-6783-3p, thereby upregulating L1CAM expression. Indeed, we also determined that LINC02323 could interact with the RNA-binding protein DDX3X, which regulates various stages of RNA expression and processing. Taken together, we identified that LINC02323 and its indirect target L1CAM can act as novel biomarkers for determining the prognosis of patients with LUSC and thus deserves further study.

UBE2L3 Reduces TRIM21 Expression and IL-1β Secretion in Epidermal Keratinocytes and Improves Psoriasis-Like Skin

Proinflammatory cytokines, such as IL-1β, are important mediators of psoriasis. UBE2L3, an E2 enzyme, is thought to be an indirect target of IL-1β secretion by binding to ubiquitin ligases such as TRIM21. However, its role in psoriasis remains unknown. In this study, we found that UBE2L3 expression was decreased in psoriatic epidermis, whereas caspase 1 and IL-1β signaling were strongly activated. When normal human epidermal keratinocytes were stimulated with nigericin, adenosine triphosphate, and poly(dA:dT), downregulation of UBE2L3 and increased secretion of IL-1β were observed. Treatment with a caspase 1 inhibitor reversed the decrease in the level of UBE2L3. In addition, UBE2L3 overexpression reduced TRIM21, decreased signal transducer and activator of transcription 3 pathway activity, and reduced the level of the IL-1β precursor (pro‒IL-1β). Consistently, silencing UBE2L3 enhanced TRIM21 expression, signal transducer and activator of transcription 3 activation, and pro‒IL-1β production. Finally, in an imiquimod-induced mouse model, UBE2L3 reduction and caspase 1 activation were localized in the epidermis, whereas overexpression of UBE2L3 ameliorated psoriasis-like lesions and reduced pro‒IL-1β and mature IL-1β levels in the epidermis. Thus, UBE2L3 may be a protective biomarker that regulates IL-1β and inhibits TRIM21 in the epidermis of psoriasis.

Association of oral health knowledge, self-efficacy and behaviours with oral health-related quality of life in Chinese primary school children: a cross-sectional study

ObjectiveAchieving good oral health-related quality of life (OHRQOL) is of particular concern in children. The inter-relations among oral health knowledge, self-efficacy, behaviours and OHRQOL in children groups remain unclear. This...

Revisiting AGAMOUS-LIKE15, a Key Somatic Embryogenesis Regulator, Using Next Generation Sequencing Analysis in Arabidopsis

AGAMOUS-like 15 (AGL15) is a member of the MADS-domain transcription factor (TF) family. MADS proteins are named for a conserved domain that was originally from an acronym derived from genes expressed in a variety of eukaryotes (MCM1-AGAMOUS-DEFICIENS-SERUM RESPONSE FACTOR). In plants, this family has expanded greatly, with more than one-hundred members generally found in dicots, and the proteins encoded by these genes have often been associated with developmental identity. AGL15 transcript and protein accumulate primarily in embryos and has been found to promote an important process called plant regeneration via somatic embryogenesis (SE). To understand how this TF performs this function, we have previously used microarray technologies to assess direct and indirect responsive targets of this TF. We have now revisited this question using next generation sequencing (NGS) to both characterize in vivo binding sites for AGL15 as well as response to the accumulation of AGL15. We compared these data to the prior microarray results to evaluate the different platforms. The new NGS data brought to light an interaction with brassinosteroid (BR) hormone signaling that was "missed" in prior Gene Ontology analysis from the microarray studies.

Bortezomib inhibits NLRP3 inflammasome activation and NF-κB pathway to reduce psoriatic inflammation

The abnormal activation of nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome plays an important role in the pathogenesis of psoriasis. Accordingly, the inhibition of NLRP3 inflammasome may be an effective strategy for psoriasis treatment. However, the NLRP3 inflammasome inhibitors are not available in the clinic. Repurposing FDA-approved drugs is a highly attractive way for identifying new drugs. Here, proteasome inhibitor bortezomib, a marketed drug for treating multiple myeloma, was found to specifically inhibit NLRP3 inflammasome activation at nanomolar concentrations. Mechanistically, bortezomib did not inhibit reactive oxygen species generation, ion efflux, NLRP3 oligomerization, and NLRP3-ASC interactions. Bortezomib reduced ASC oligomerization and ASC speck formation. In addition, bortezomib inhibited the activity of the core subunit β5i in the immunoproteasome and reduced β5i binding to NLRP3. Bortezomib reduced the production of interleukin-1β and attenuated the severity of skin lesions in the imiquimod-induced psoriatic mouse model. Thus, bortezomib is a potential therapeutic drug for psoriasis. Our study also revealed that β5i may be an indirect target for regulating NLRP3 inflammasome activation and treating psoriasis and other NLRP3 inflammasome-related diseases.

Elongator promotes neuritogenesis via regulation of tau stability through acly activity.

The six subunits (Elp1 to Elp6) Elongator complex promotes specific uridine modifications in tRNA's wobble site. Moreover, this complex has been indirectly involved in the regulation of α-tubulin acetylation in microtubules (MTs) via the stabilization of ATP-Citrate Lyase (Acly), the main cytosolic source of acetyl-CoA production in cells, a key substrate used for global protein acetylation. Here, we report additional evidence that Elongator activity is important for proper cytoskeleton remodeling as cells lacking expression of Elp1 show morphology impairment; including distinct neurite process formation and disorganization and instability of MTs. Here, we show that loss of Elongator results in a reduction of expression of the microtubule associated protein Tau (MAPT). Tau, is a well-known key MT regulator in neurons whose lysines can be competitively acetylated or ubiquitylated. Therefore, we tested whether Tau is an indirect acetylation target of Elongator. We found that a reduction of Elongator activity leads to a decrease of lysine acetylation on Tau that favors its proteasomal degradation. This phenotype was prevented by using selective deacetylase or proteasomal inhibitors. Moreover, our data demonstrate that Acly's activity regulates the mechanism underlying Tau mediated neurite morphology defects found in Elp1 KD since both Tau levels and neurites morphology are restored due to Acly overexpression. This suggests a possible involvement of both Tau and Acly dysfunction in Familial Dysautonomia (FD), which is an autosomal recessive peripheral neuropathy caused by mutation in the ELP1 gene that severely affects Elp1 expression levels in the nervous system in FD patients in a similar way as found previously in Elp1 KD neuroblastoma cells.

The comprehensive detection of miRNA and circRNA in the regulation of intramuscular and subcutaneous adipose tissue of Laiwu pig

circRNAs, as miRNA sponges, participate in many important biological processes. However, it remains unclear whether circRNAs can regulate lipid metabolism. This study aimed to explore the competing endogenouse RNA (ceRNA) regulatory network that affects the difference between intramuscular fat (IMF) and subcutaneous fat (SCF) deposition, and to screen key circRNAs and their regulatory genes. In this experiment, we identified 265 differentially expressed circRNAs, of which 187 up-regulated circRNA and 78 down-regulated circRNA in IMF. Subsequently, we annotated the function of DEcircRNA's host genes, and found that DEcircRNA's host genes were mainly involved in GO terms (including cellular response to fatty acids, lysophosphatidic acid acyltransferase activity, R-SMAD binding, etc.) and signaling pathways (fatty acid biosynthesis, Citrate cycle, TGF- β Signal pathway) related to adipogenesis, differentiation and lipid metabolism. By constructing a circRNA-miRNA network, we screened out DEcircRNA that can competitively bind to more miRNAs as key circRNAs (circRNA_06424 and circRNA_08840). Through the functional annotation of indirect target genes and protein network analysis, we found that circRNA_06424 affects the expression of PPARD, MMP9, UBA7 and other indirect target genes by competitively binding to miRNAs such as ssc-miR-339-5p, ssc-miR-744 and ssc-miR-328, and participates in PPAR signaling pathway, Wnt signaling pathway, unsaturated fatty acid and other signaling pathways, resulting in the difference of fat deposition between IMF and SCF. This study provide a theoretical basis for further research investigating the differences of lipid metabolism in different adipose tissues, providing potential therapeutic targets for ectopic fat deposition and lipid metabolism diseases.

Endogenous PTEN-Induced Kinase 1 Regulates Dendritic Architecture and Spinogenesis.

Mutations in PTEN-induced kinase 1 (PINK1) contribute to autosomal recessive Parkinson's disease with cognitive and neuropsychiatric comorbidities. Disturbances in dendritic and spine architecture are hallmarks of neurodegenerative and neuropsychiatric conditions, but little is known of the impact of PINK1 on these structures. We used Pink1 -/- mice to study the role of endogenous PINK1 in regulating dendritic architecture, spine density, and spine maturation. Pink1 -/- cortical neurons of unknown sex showed decreased dendritic arborization, affecting both apical and basal arbors. Dendritic simplification in Pink1 -/- neurons was primarily driven by diminished branching with smaller effects on branch lengths. Pink1 -/- neurons showed reduced spine density with a shift in morphology to favor filopodia at the expense of mushroom spines. Electrophysiology revealed significant reductions in miniature EPSC (mEPSC) frequency in Pink1 -/- neurons, consistent with the observation of decreased spine numbers. Transfecting with human PINK1 rescued changes in dendritic architecture, in thin, stubby, and mushroom spine densities, and in mEPSC frequency. Diminished spine density was also observed in Golgi-Cox stained adult male Pink1 -/- brains. Western blot study of Pink1 -/- brains of either sex revealed reduced phosphorylation of NSFL1 cofactor p47, an indirect target of PINK1. Transfection of Pink1 -/- neurons with a phosphomimetic p47 plasmid rescued dendritic branching and thin/stubby spine density with a partial rescue of mushroom spines, implicating a role for PINK1-regulated p47 phosphorylation in dendrite and spine development. These findings suggest that PINK1-dependent synaptodendritic alterations may contribute to the risk of cognitive and/or neuropsychiatric pathologies observed in PINK1-mutated families.SIGNIFICANCE STATEMENT Loss of PINK1 function has been implicated in both familial and sporadic neurodegenerative diseases. Yet surprisingly little is known of the impact of PINK1 loss on the fine structure of neurons. Neurons receive excitatory synaptic signals along a complex network of projections that form the dendritic tree, largely at tiny protrusions called dendritic spines. We studied cortical neurons and brain tissues from mice lacking PINK1. We discovered that PINK1 deficiency causes striking simplification of dendritic architecture associated with reduced synaptic input and decreased spine density and maturation. These changes are reversed by reintroducing human PINK1 or one of its downstream mediators into PINK1-deficient mouse neurons, indicating a conserved function, whose loss may contribute to neurodegenerative processes.

Alterations of Mitochondrial Structure in Methamphetamine Toxicity.

Recent evidence shows that methamphetamine (METH) produces mitochondrial alterations that contribute to neurotoxicity. Nonetheless, most of these studies focus on mitochondrial activity, whereas mitochondrial morphology remains poorly investigated. In fact, morphological evidence about the fine structure of mitochondria during METH toxicity is not available. Thus, in the present study we analyzed dose-dependent mitochondrial structural alterations during METH exposure. Light and transmission electron microscopy were used, along with ultrastructural stoichiometry of catecholamine cells following various doses of METH. In the first part of the study cell death and cell degeneration were assessed and they were correlated with mitochondrial alterations observed using light microscopy. In the second part of the study, ultrastructural evidence of specific mitochondrial alterations of crests, inner and outer membranes and matrix were quantified, along with in situ alterations of mitochondrial proteins. Neurodegeneration induced by METH correlates significantly with specific mitochondrial damage, which allows definition of a scoring system for mitochondrial integrity. In turn, mitochondrial alterations are concomitant with a decrease in fission/mitophagy protein Fis1 and DRP1 and an increase in Pink1 and Parkin in situ, at the mitochondrial level. These findings provide structural evidence that mitochondria represent both direct and indirect targets of METH-induced toxicity.

Insulinoma-Associated Protein 1 (INSM1): Diagnostic, Prognostic, and Therapeutic Use in Small Cell Lung Cancer

Small cell lung carcinoma (SCLC) is an aggressive and difficult to treat cancer. Although immunohistochemistry is not mandatory for a SCLC diagnosis, it might be required, especially in small samples. Insulinoma-associated protein 1 (INSM1) is expressed in endocrine and nervous tissues during embryogenesis, generally absent in adults and re-expressed in SCLC and other neuroendocrine neoplasms. Its high specificity propelled its use as diagnostic biomarker and an attractive therapeutic target. Herein, we aim to provide a systematic and critical review on the use of INSM1 for diagnosis, prognostication and the treatment of SCLC. An extensive bibliographic search was conducted in PubMed® focusing on articles published since 2015. According to the literature, INSM1 is a highly sensitive (75–100%) and specific (82–100%) neuroendocrine immunohistochemical marker for SCLC diagnosis. It can be used in histological and cytological samples. Although advantageous, its standalone use is currently not recommended. Studies correlating INSM1 expression and prognosis have disclosed contrasting results, although the expression seemed to entail a worse survival. Targeting INSM1 effectively suppressed SCLC growth either as a suicide gene therapy regulator or as an indirect target of molecular-targeted therapy. INSM1 represents a valuable biomarker for a SCLC diagnosis that additionally offers vast opportunities for the development of new prognostic and therapeutic strategies.

Exploring direct and indirect targets of current antileishmanial drugs using a novel thermal proteomics profiling approach.

Visceral leishmaniasis (VL), caused by Leishmania infantum, is an oft-fatal neglected tropical disease. In the absence of an effective vaccine, the control of leishmaniasis relies exclusively on chemotherapy. Due to the lack of established molecular/genetic markers denoting parasite resistance, clinical treatment failure is often used as an indicator. Antimony-based drugs have been the standard antileishmanial treatment for more than seven decades, leading to major drug resistance in certain regions. Likewise, drug resistance to miltefosine and amphotericin B continues to spread at alarming rates. In consequence, innovative approaches are needed to accelerate the identification of antimicrobial drug targets and resistance mechanisms. To this end, we have implemented a novel approach based on thermal proteome profiling (TPP) to further characterize the mode of action of antileishmanials antimony, miltefosine and amphotericin B, as well as to better understand the mechanisms of drug resistance deployed by Leishmania. Proteins become more resistant to heat-induced denaturation when complexed with a ligand. In this way, we used multiplexed quantitative mass spectrometry-based proteomics to monitor the melting profile of thousands of expressed soluble proteins in WT, antimony-resistant, miltefosine-resistant, and amphotericin B-resistant L. infantum parasites, in the presence (or absence) of the above-mentioned drugs. Bioinformatics analyses were performed, including data normalization, melting profile fitting, and identification of proteins that underwent changes (fold change > 4) caused by complexation with a drug. With this unique approach, we were able to narrow down the regions of the L. infantum proteome that interact with antimony, miltefosine, and amphotericin B; validating previously-identified and unveiling novel drug targets. Moreover, analyses revealed candidate proteins potentially involved in drug resistance. Interestingly, we detected thermal proximity coaggregation for several proteins belonging to the same metabolic pathway (i.e., tryparedoxin peroxidase and aspartate aminotransferase in proteins exposed to antimony), highlighting the importance of these pathways. Collectively, our results could serve as a jumping-off point for the future development of innovative diagnostic tools for the detection and evaluation of antimicrobial-resistant Leishmania populations, as well as open the door for new on-target therapies.

Can local environmental constraints improve enterprise's green innovation quality? Evidence from Chinese-listed firms.

The target responsibility system of environmental protection is one of the vital channels to achieve a win-win situation for both economic development and environmental protection. Comprehensively investigating how local environmental target constraints drive enterprises to improve their green innovation quality is of enormous theoretical and practical significance for optimizing the implementation effect of environmental target constraints policies and boosting enterprises' green and high-quality development. We empirically examine the mechanism of the impact of the intensity of different types of local environmental target constraints on the quality of corporate green innovation and the nonlinear relationship between them through innovatively constructing indicators of local environmental target constraint intensity and utilizing the knowledge width of green patents of listed companies as a proxy variable for enterprise green innovation quality. First, the strength of indirect environmental target constraints has a significant positive effect on the quality of green innovation, but further nonlinear characteristics reveal a significant inverse U-shaped relationship between them. Second, indirect environmental target constraint intensity has an inverted U-shaped trend in increasing the intensity of environmental regulation and influencing the digitalization of enterprises, which in turn forms an inverted U-shaped relationship with the quality of green innovation. Third, indirect environmental target constraint intensity works better in areas with policies prioritizing city over province, with mayors less than 57years old, and for enterprises in technology-intensive industries.

Proteomic Identification of Potential Target Proteins of Cathepsin W for Its Development as a Drug Target for Influenza.

ABSTRACTInfluenza A virus (IAV) coopts numerous host factors for efficient replication. The cysteine protease cathepsin W (CTSW) has been identified as one host factor required for IAV entry, specifically for the escape of IAVs from late endosomes. However, the substrate specificity of CTSW and the proviral mechanism are thus far unknown. Here, we show that intracellular but not secreted CTSW promotes viral entry. We reveal 79 potential direct and 31 potential indirect cellular target proteins of CTSW using the high-throughput proteomic approach terminal amine isotopic labeling of substrates (TAILS) and determine the cleavage motif shared by the substrates of CTSW. Subsequent integration with data from RNA interference (RNAi) screens for IAV host factors uncovers first insights into the proviral function of CTSW. Notably, CTSW-deficient mice display a 25% increase in survival and a delay in mortality compared to wild-type mice upon IAV infection. Altogether, these findings support the development of drugs targeting CTSW as novel host-directed antiviral therapies.IMPORTANCE Influenza viruses are respiratory pathogens and pose a constant threat to human health. Although antiviral drugs are available for influenza, the emergence and spread of drug-resistant viruses is cause for concern. Therefore, the development of new antivirals with lower chances of their target viruses acquiring resistance is urgently needed to reduce the high morbidity and mortality caused by influenza. Promising alternatives to drugs targeting viral proteins are those directed against host factors required for viral replication. The cysteine protease cathepsin W (CTSW) is an important host factor for IAV replication, and its proteolytic activity is required for fusion of viral and endosomal membranes. In this work, we identify a number of hitherto unknown CTSW substrates, providing new insights into virus-host interactions, and reveal that CTSW might also play a proviral role in an in vivo model. These results support the development of CTSW as a drug target for next-generation antivirals against influenza.

Political Extravaganzas and a Jonsonian Comedy

Political Extravaganzas and a Jonsonian Comedy