Negative Effector Research Articles

Combined short-term exposure to meteorological, pollution factors and pertussis in different groups from Jining, China.

Previous studies have typically explored daily lagged relationships among pertussis and meteorology, with little assessment of effect and interaction among pollutants mixtures. Our researchers collected pertussis cases data from 2017-2022 as well as meteorological and contaminative factors for the Jining region. First, we reported the application of the Moving Epidemic Method (MEM) to estimate epidemic threshold and intensity level. Then we developed a Weighted Quantile Sum (WQS) regression and Bayesian Kernel Machine Regression (BKMR) model to assess single, multiple effects and interaction of meteorological and pollution factors on pertussis cases for different sex, delayed and epidemic threshold groups. There has been a yearly upward trend in the incidence of pertussis in Jining regions. High prevalence threshold years were in 2018-2019, the epidemic peak was mainly concentrated in 32 weeks. Totally, pertussis infections disease was separately 2.1% (95% confidence Interval (CI) = 1.3, 2.8) and 1.1% (95% CI = 0.3, 1.9) higher per decile increase in temperature and sulphur dioxide (SO2). And pertussis infections disease was 1.1% lower per decile increase in humidity. In the different stratified analyses, air pressure was a strong negative effect in males and in the lagged 11-20 days group, with 7.3 and 14.7%, respectively. Sulphur dioxide had a relatively weak positive effect in males, females and the group after 20 days lag, ranging from 0.5 to 0.6%. The main positive effectors affecting the onset of disease at low and high threshold levels were ozone (O3) and SO2, respectively, while the negative effectors were SO2 and carbon monoxide (CO), respectively. This is the first mathematically based study of seasonal threshold of pertussis in China, which allows accurate estimation of epidemic level. Our findings support that short-term exposure to pollutants is the risk factor for pertussis. We should concentrate on pollutants monitoring and effect modeling.

Modulation of the allosteric and vasoregulatory arms of erythrocytic oxygen transport.

Efficient distribution of oxygen (O2) to the tissues in mammals depends on the evolved ability of red blood cell (RBC) hemoglobin (Hb) to sense not only O2 levels, but metabolic cues such as pH, PCO2, and organic phosphates, and then dispense or take up oxygen accordingly. O2 delivery is the product of not only oxygen release from RBCs, but also blood flow, which itself is also governed by vasoactive molecular mediators exported by RBCs. These vascular signals, including ATP and S-nitrosothiols (SNOs) are produced and exported as a function of the oxygen and metabolic milieu, and then fine-tune peripheral metabolism through context-sensitive vasoregulation. Emerging and repurposed RBC-oriented therapeutics can modulate either or both of these allosteric and vasoregulatory activities, with a single molecule or other intervention influencing both arms of O2 transport in some cases. For example, organic phosphate repletion of stored RBCs boosts the negative allosteric effector 2,3 biphosphoglycerate (BPG) as well as the anti-adhesive molecule ATP. In sickle cell disease, aromatic aldehydes such as voxelotor can disfavor sickling by increasing O2 affinity, and in newer generations, these molecules have been coupled to vasoactive nitric oxide (NO)-releasing adducts. Activation of RBC pyruvate kinase also promotes a left shift in oxygen binding by consuming and lowering BPG, while increasing the ATP available for cell health and export on demand. Further translational and clinical investigation of these novel allosteric and/or vasoregulatory approaches to modulating O2 transport are expected to yield new insights and improve the ability to correct or compensate for anemia and other O2 delivery deficits.

Luminal Polyamines Disrupt Intestinal Barrier and Undergo Complex Regulation by Host and Commensal Microbiota

Background: Polyamines produced by host cells and intestinal microbiota and are critical to overall health. Studies have shown putrescine, a polyamine, can strengthen, or in some instances disrupt, gastrointestinal (GI) barrier integrity, yet the regulation of polyamine concentrations in the GI tract is unknown. Host colonocytes and microbiota both metabolize polyamines, and in GI diseases like irritable bowel syndrome (IBS) where intestinal microbiota is significantly altered, polyamine metabolism may be affected. Aim: Determine regulation of luminal polyamine concentration and its effects on intestinal barrier integrity. Hypothesis: Luminal polyamine concentration is suppressed by commensal intestinal microbiota and increasing concentration of putrescine results in intestinal barrier disruption. Methods: Microbiota effects on luminal polyamine concentrations were assessed by humanizing germ-free (GF) mice with a single oral gavage of commensal human microbiota. Polyamine effect on barrier integrity was assessed in vitro using Caco-2 cells and ex vivo on Ussing using mouse colonic tissue with 300Da fluorescein and 4kDa FITC-Dextran tracers. Changes in transepithelial resistance (TER) and rate of tracer flux was recorded. Untargeted metabolomics with shotgun metagenomics was completed on human fecal samples and sigmoid colonic biopsies were collected for bulk RNAseq. Bray-Curtis distance was determined at the species level using the PERMANOVA method and used to test relationships between beta-diversity and fecal polyamine concentration. Results: Humanization with human commensal microbiota (1.2% C. aerofaciens) lead to a 20-fold increase in cecal putrescine and a 20-fold decrease in fecal putrescine compared to GF controls. In vitro addition of putrescine (100mM) caused increased fluorescein flux (3.50±0.24 vs 1.44±0.04 mg/mL) and a rapid loss of transepithelial resistance compared to controls (% of Baseline TER, 36.6±8.2 vs 102.8±5.4, 3h postexposure). Ex vivo addition of putrescine caused a loss of TER (57% vs 89% of baseline by 3h) and an increased rate of 4kDa FITC-Dextran flux compared to untreated controls (1236±131 vs 562±70 ng/hr/cm2). Transcriptomic profiles show decreased expression of ornithine decarboxylase antienzyme (OAZ1, FDR=0.018) in IBS which inhibits the rate limiting enzyme for putrescine production. Increased lactulose excretion (2-24h), suggestive of increased distal colonic permeability, correlated with N-acetylputrescine concentration (p=0.07). N-acetylputrescine also correlated positively with increased stool frequency (p=0.009) and looser consistency (p=0.013). C. aerofaciens, a polyamine consumer, negatively correlated with n-acetylputrescine (FDR=0.02) suggesting a loss of specific taxa may lead to insuffcient polyamine metabolism. Conclusion: Polyamines, namely putrescine, disrupts intestinal barrier integrity, correlate with in vivo permeability, and are decreased in the colon by commensal microbiota. These observations identify putrescine as a negative effector of barrier function and suggest a novel role for the commensal microbiome in regulating barrier integrity through polyamine metabolism. Further work investigating host and microbiota mechanisms of polyamine metabolism, signaling and the specific taxa involved is needed to better understand the effects polyamines have on GI physiology. NIH DK 127998 (MG), support from Mayo Clinic Center for Individualized Medicine (CIM), and Mayo Clinic Center for Cell Signalling in Gastroenterology (C-Sig) P30DK084567. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The cryo-EM structure of ASK1 reveals an asymmetric architecture allosterically modulated by TRX1.

Apoptosis signal-regulating kinase 1 (ASK1) is a crucial stress sensor, directing cells toward apoptosis, differentiation, and senescence via the p38 and JNK signaling pathways. ASK1 dysregulation has been associated with cancer and inflammatory, cardiovascular, and neurodegenerative diseases, among others. However, our limited knowledge of the underlying structural mechanism of ASK1 regulation hampers our ability to target this member of the MAP3K protein family towards developing therapeutic interventions for these disorders. Nevertheless, as a multidomain Ser/Thr protein kinase, ASK1 is regulated by a complex mechanism involving dimerization and interactions with several other proteins, including thioredoxin 1 (TRX1). Thus, the present study aims at structurally characterizing ASK1 and its complex with TRX1 using several biophysical techniques. As shown by cryo-EM analysis, in a state close to its active form, ASK1 is a compact and asymmetric dimer, which enables extensive interdomain and interchain interactions. These interactions stabilize the active conformation of the ASK1 kinase domain. In turn, TRX1 functions as a negative allosteric effector of ASK1, modifying the structure of the TRX1-binding domain and changing its interaction with the tetratricopeptide repeats domain. Consequently, TRX1 reduces access to the activation segment of the kinase domain. Overall, our findings not only clarify the role of ASK1 dimerization and inter-domain contacts but also provide key mechanistic insights into its regulation, thereby highlighting the potential of ASK1 protein-protein interactions as targets for anti-inflammatory therapy.

The cryo-EM structure of ASK1 reveals an asymmetric architecture allosterically modulated by TRX1

Apoptosis signal-regulating kinase 1 (ASK1) is a crucial stress sensor, directing cells toward apoptosis, differentiation, and senescence via the p38 and JNK signaling pathways. ASK1 dysregulation has been associated with cancer and inflammatory, cardiovascular, and neurodegenerative diseases, among others. However, our limited knowledge of the underlying structural mechanism of ASK1 regulation hampers our ability to target this member of the MAP3K protein family towards developing therapeutic interventions for these disorders. Nevertheless, as a multidomain Ser/Thr protein kinase, ASK1 is regulated by a complex mechanism involving dimerization and interactions with several other proteins, including thioredoxin 1 (TRX1). Thus, the present study aims at structurally characterizing ASK1 and its complex with TRX1 using several biophysical techniques. As shown by cryo-EM analysis, in a state close to its active form, ASK1 is a compact and asymmetric dimer, which enables extensive interdomain and interchain interactions. These interactions stabilize the active conformation of the ASK1 kinase domain. In turn, TRX1 functions as a negative allosteric effector of ASK1, modifying the structure of the TRX1-binding domain and changing its interaction with the tetratricopeptide repeats domain. Consequently, TRX1 reduces access to the activation segment of the kinase domain. Overall, our findings not only clarify the role of ASK1 dimerization and inter-domain contacts but also provide key mechanistic insights into its regulation, thereby highlighting the potential of ASK1 protein-protein interactions as targets for anti-inflammatory therapy.

The nuclear pore protein NUP98 impedes LTR-driven basal gene expression of HIV-1, viral propagation, and infectivity.

Nucleoporins (NUPs) are cellular effectors of human immunodeficiency virus-1 (HIV-1) replication that support nucleocytoplasmic trafficking of viral components. However, these also non-canonically function as positive effectors, promoting proviral DNA integration into the host genome and viral gene transcription, or as negative effectors by associating with HIV-1 restriction factors, such as MX2, inhibiting the replication of HIV-1. Here, we investigated the regulatory role of NUP98 on HIV-1 as we observed a lowering of its endogenous levels upon HIV-1 infection in CD4+ T cells. Using complementary experiments in NUP98 overexpression and knockdown backgrounds, we deciphered that NUP98 negatively affected HIV-1 long terminal repeat (LTR) promoter activity and lowered released virus levels. The negative effect on promoter activity was independent of HIV-1 Tat, suggesting that NUP98 prevents the basal viral gene expression. ChIP-qPCR showed NUP98 to be associated with HIV-1 LTR, with the negative regulatory element (NRE) of HIV-1 LTR playing a dominant role in NUP98-mediated lowering of viral gene transcription. Truncated mutants of NUP98 showed that the attenuation of HIV-1 LTR-driven transcription is primarily contributed by its N-terminal region. Interestingly, the virus generated from the producer cells transiently expressing NUP98 showed lower infectivity, while the virus generated from NUP98 knockdown CD4+ T cells showed higher infectivity as assayed in TZM-bl cells, corroborating the anti-HIV-1 properties of NUP98. Collectively, we show a new non-canonical function of a nucleoporin adding to the list of moonlighting host factors regulating viral infections. Downregulation of NUP98 in a host cell upon HIV-1 infection supports the concept of evolutionary conflicts between viruses and host antiviral factors.

Atypical inflammatory kinase IKBKE phosphorylates and inactivates FoxA1 to promote liver tumorigenesis.

Physiologically, FoxA1 plays a key role in liver differentiation and development, and pathologically exhibits an oncogenic role in prostate and breast cancers. However, its role and upstream regulation in liver tumorigenesis remain unclear. Here, we demonstrate that FoxA1 acts as a tumor suppressor in liver cancer. Using a CRISPR-based kinome screening approach, noncanonical inflammatory kinase IKBKE has been identified to inhibit FoxA1 transcriptional activity. Notably, IKBKE directly binds to and phosphorylates FoxA1 to reduce its complex formation and DNA interaction, leading to elevated hepatocellular malignancies. Nonphosphorylated mimic Foxa1 knock-in mice markedly delay liver tumorigenesis in hydrodynamic transfection murine models, while phospho-mimic Foxa1 knock-in phenocopy Foxa1 knockout mice to exhibit developmental defects and liver inflammation. Notably, Ikbke knockout delays diethylnitrosamine (DEN)-induced mouse liver tumor development. Together, our findings not only reveal FoxA1 as a bona fide substrate and negative nuclear effector of IKBKE in hepatocellular carcinioma (HCC) but also provide a promising strategy to target IKBEK for HCC therapy.

Prognostic significance of the FSTL1-DIP2A axis in early-stage tongue cancer.

In tongue cancer, many patients already have metastasis at the time of diagnosis, and such cases are usually unresponsive to treatment, resulting in a poor prognosis. Therefore, there is an urgent need to develop more effective diagnostic and therapeutic methods to cure tongue cancer at the earliest possible stage in clinical practice. Follistatin-like 1 (FSTL1) is known as a negative effector molecule that induces and enhances the refractoriness of cancer cells directly and indirectly via suppressing anti-tumor immunity in various types of cancer. However, the molecular expression, functions, and clinical significance of FSTL1 and its receptor DIP2A in tongue cancer remains to be elucidated. In this study, we revealed that FSTL1, which is highly expressed in tongue cancer cells, plays a key role in its malignancy and is a significant risk factor for recurrence of early-stage tongue cancer. Basic study shows that FSTL1 is abundantly produced from human tongue cancer cell lines, and blocking FSTL1 with specific siRNAs or mAb significantly suppresses cellular functions. Clinical study shows that both FSTL1 and its receptor DIP2A are highly and correlatively expressed in tumor tissues of tongue cancer patients, and high expression levels of both in stage I tumors are significantly associated with shorter relapse-free survival. These suggest that targeting the FSTL1-DIP2A axis may be useful as a biomarker for early prediction of prognosis in tongue cancer patients, and as a therapeutic target for developing new drugs to treat tongue cancer more effectively. This strategy will contribute to improving clinical outcomes in tongue cancer.

Effect of proline content and histidine ligation on the dynamics of Ω-loop D and the peroxidase activity of iso-1-cytochrome c

To study how proline residues affect the dynamics of Ω-loop D (residues 70 to 85) of cytochrome c, we prepared G83P and G83A variants of yeast iso-1-cytochrome c (iso-1-Cytc) in the presence and absence of a K73H mutation. Ω-loop D is important in controlling both the electron transfer function of Cytc and the peroxidase activity of Cytc used in apoptosis because it provides the Met80 heme ligand. The G83P and G83A mutations have no effect on the global stability of iso-1-Cytc in presence or absence of the K73H mutation. However, both mutations destabilize the His73-mediated alkaline conformer relative to the native state. pH jump stopped-flow experiments show that the dynamics of the His73-mediated alkaline transition are significantly enhanced by the G83P mutation. Gated electron transfer studies show that the enhanced dynamics result from an increased rate of return to the native state, whereas the rate of loss of Met80 ligation is unchanged by the G83P mutation. Thus, the G83P substitution does not stiffen the conformation of the native state. Because bis-His heme ligation occurs when Cytc binds to cardiolipin-containing membranes, we studied the effect of His73 ligation on the peroxidase activity of Cytc, which acts as an early signal in apoptosis by causing oxygenation of cardiolipin. We find that the His73 alkaline conformer suppresses the peroxidase activity of Cytc. Thus, the bis-His ligated state of Cytc formed upon binding to cardiolipin is a negative effector for the peroxidase activity of Cytc early in apoptosis.

Cell Free Bacteriophage Synthesis from Engineered Strains Improves Yield.

Phage therapy to treat life-threatening drug-resistant infections has been hampered by technical challenges in phage production. Cell-free bacteriophage synthesis (CFBS) can overcome the limitations of standard phage production methods by manufacturing phage virions in vitro. CFBS mimics intracellular phage assembly using transcription/translation machinery (TXTL) harvested from bacterial lysates and combined with reagents to synthesize proteins encoded by a phage genomic DNA template. These systems may enable rapid phage production and engineering to accelerate phages from bench-to-bedside. TXTL harvested from wild type or commonly used bacterial strains was not optimized for bacteriophage production. Here, we demonstrate that TXTL from genetically modified E. coli BL21 can be used to enhance phage T7 yields in vitro by CFBS. Expression of 18 E. coli BL21 genes was manipulated by inducible CRISPR interference (CRISPRi) mediated by nuclease deficient Cas12a from F. novicida (dFnCas12a) to identify genes implicated in T7 propagation as positive or negative effectors. Genes shown to have a significant effect were overexpressed (positive effectors) or repressed (negative effectors) to modify the genetic background of TXTL harvested for CFBS. Phage T7 CFBS yields were improved by up to 10-fold in vitro through overexpression of translation initiation factor IF-3 (infC) and small RNAs OxyS and CyaR and by repression of RecC subunit exonuclease RecBCD. Continued improvement of CFBS will mitigate phage manufacturing bottlenecks and lower hurdles to widespread adoption of phage therapy.

ΔNp63 Regulates Homeostasis, Stemness, and Suppression of Inflammation in the Adult Epidermis

The p63 transcription factor is critical for epidermis formation in embryonic development, but its role in the adult epidermis is poorly understood. In this study, we show that acute genetic ablation of ΔNp63, the main p63 isoform, in adult epidermis disrupts keratinocyte proliferation and self-maintenance and, unexpectedly, triggers an inflammatory psoriasis-like condition. Mechanistically, single-cell RNA sequencing revealed the downregulation of cell cycle genes, upregulation of differentiation markers, and induction of several proinflammatory pathways in ΔNp63-ablated keratinocytes. Intriguingly, ΔNp63-ablated cells disappear by 3 weeks after ablation, at the expense of the remaining nonablated cells. This is not associated with active cell death and is likely due to reduced self-maintenance and enhanced differentiation. Indeed, invivo wound healing, a physiological readout of the epidermal stem cell function, is severely impaired upon ΔNp63 ablation. We found that the Wnt signaling pathway (Wnt10A, Fzd6, Fzd10) and the activator protein 1 (JunB, Fos, FosB) factors are the likely ΔNp63 effectors responsible for keratinocyte proliferation/stemness and suppression of differentiation, respectively, whereas IL-1a, IL-18, IL-24, and IL-36γ are the likely negative effectors responsible for suppression of inflammation. These data establish ΔNp63 as a critical node that coordinates epidermal homeostasis, stemness, and suppression of inflammation, upstream of known regulatory pathways.

Evidence That PbrSAUR72 Contributes to Iron Deficiency Tolerance in Pears by Facilitating Iron Absorption.

Iron is an essential trace element for plants; however, low bioactive Fe in soil continuously places plants in an Fe-deficient environment, triggering oxidative damage. To cope with this, plants make a series of alterations to increase Fe acquisition; however, this regulatory network needs further investigation. In this study, we found notably decreased indoleacetic acid (IAA) content in chlorotic pear (Pyrus bretschneideri Rehd.) leaves caused by Fe deficiency. Furthermore, IAA treatment slightly induced regreening by increasing chlorophyll synthesis and Fe2+ accumulation. At that point, we identified PbrSAUR72 as a key negative effector output of auxin signaling and established its close relationship to Fe deficiency. Furthermore, the transient PbrSAUR72 overexpression could form regreening spots with increased IAA and Fe2+ content in chlorotic pear leaves, whereas its transient silencing does the opposite in normal pear leaves. In addition, cytoplasm-localized PbrSAUR72 exhibits root expression preferences and displays high homology to AtSAUR40/72. This promotes salt tolerance in plants, indicating a putative role for PbrSAUR72 in abiotic stress responses. Indeed, transgenic plants of Solanum lycopersicum and Arabidopsis thaliana overexpressing PbrSAUR72 displayed less sensitivity to Fe deficiency, accompanied by substantially elevated expression of Fe-induced genes, such as FER/FIT, HA, and bHLH39/100. These result in higher ferric chelate reductase and root pH acidification activities, thereby hastening Fe absorption in transgenic plants under an Fe-deficient condition. Moreover, the ectopic overexpression of PbrSAUR72 inhibited reactive oxygen species production in response to Fe deficiency. These findings contribute to a new understanding of PbrSAURs and its involvement in Fe deficiency, providing new insights for the further study of the regulatory mechanisms underlying the Fe deficiency response.

Improved biosynthesis of heme in Bacillus subtilis through metabolic engineering assisted fed-batch fermentation

BackgroundHeme is an iron/porphyrin complex compound, widely used in the health care, food, and pharmaceutical industries. It is more advantageous and attractive to develop microbial cell factories to produce heme by fermentation, with lower production costs and environmentally more friendly procedures than those of the traditional extraction based on animal blood. In this study, Bacillus subtilis, a typical industrial model microorganism of food safety grade, was used for the first time as the host to synthesize heme.ResultsThe heme biosynthetic pathway was engineered as four modules, the endogenous C5 pathway, the heterologous C4 pathway, the uroporphyrinogen (urogen) III synthesis pathway, and the downstream synthesis pathway. Knockout of hemX encoding the negative effector of the concentration of HemA, overexpression of hemA encoding glutamyl-tRNA reductase, and knockout of rocG encoding the major glutamate dehydrogenase in the C5 pathway, resulted in an increase of 427% in heme production. Introduction of the heterologous C4 pathway showed a negligible effect on heme biosynthesis. Overexpression of hemCDB, which encoded hydroxymethylbilane synthase, urogen III synthase, and porphobilinogen synthase participating in the urogen III synthesis pathway, increased heme production by 39%. Knockouts of uroporphyrinogen methyltransferase gene nasF and both heme monooxygenase genes hmoA and hmoB in the downstream synthesis pathway increased heme production by 52%. The engineered B. subtilis produced 248.26 ± 6.97 mg/L of total heme with 221.83 ± 4.71 mg/L of extracellular heme during the fed-batch fermentation in 10 L fermenter.ConclusionsStrengthening endogenous C5 pathway, urogen III synthesis pathway and downstream synthesis pathway promoted the biosynthesis of heme in B. subtilis. The engineered B. subtilis strain has great potential as a microbial cell factory for efficient industrial heme production.

Glucolipotoxic Stress-Induced Mig6 Desensitizes EGFR Signaling and Promotes Pancreatic Beta Cell Death.

A loss of functional beta cell mass is a final etiological event in the development of frank type 2 diabetes (T2D). To preserve or expand beta cells and therefore treat/prevent T2D, growth factors have been considered therapeutically but have largely failed to achieve robust clinical success. The molecular mechanisms preventing the activation of mitogenic signaling pathways from maintaining functional beta cell mass during the development of T2D remain unknown. We speculated that endogenous negative effectors of mitogenic signaling cascades impede beta cell survival/expansion. Thus, we tested the hypothesis that a stress-inducible epidermal growth factor receptor (EGFR) inhibitor, mitogen-inducible gene 6 (Mig6), regulates beta cell fate in a T2D milieu. To this end, we determined that: (1) glucolipotoxicity (GLT) induces Mig6, thereby blunting EGFR signaling cascades, and (2) Mig6 mediates molecular events regulating beta cell survival/death. We discovered that GLT impairs EGFR activation, and Mig6 is elevated in human islets from T2D donors as well as GLT-treated rodent islets and 832/13 INS-1 beta cells. Mig6 is essential for GLT-induced EGFR desensitization, as Mig6 suppression rescued the GLT-impaired EGFR and ERK1/2 activation. Further, Mig6 mediated EGFR but not insulin-like growth factor-1 receptor nor hepatocyte growth factor receptor activity in beta cells. Finally, we identified that elevated Mig6 augmented beta cell apoptosis, as Mig6 suppression reduced apoptosis during GLT. In conclusion, we established that T2D and GLT induce Mig6 in beta cells; the elevated Mig6 desensitizes EGFR signaling and induces beta cell death, suggesting Mig6 could be a novel therapeutic target for T2D.

Abstract 2548: Substrate binding, not phosphorylation, activates CBL poly-ubiquitination activity

Abstract Casitas B-lineage lymphoma (CBL) is a family of RING-type E3 ligases that target proteins for degradation via the proteasomal pathway. CBL-b specifically functions as a negative effector of T cell activation by down regulating the T cell receptor, whereas c-CBL interacts with various receptor tyrosine kinases involved in cell signaling and activation, targeting them for degradation. Therefore, the CBL family regulates the biology of immune cells and targeting CBL-b or c-CBL is a potential therapeutic strategy for the treatment of cancer, infection, or autoimmune diseases. In this study, we used TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) to uncover new mechanistic aspects of CBL activation. Our assays were designed and optimized using purified components (E1/E2/c-CBL or CBL-b E3 ligase and kinase substrate Tyro3 or Axl) to recreate the polyubiquitination cascade in vitro. The assays use a Europium cryptate-labeled Ubiquitin (donor) and a Cy5-labeled Ubiquitin (acceptor) to complete the TR-FRET pairing. Since both the TR-FRET donor and acceptor are incorporated into poly-ubiquitin chains formed on the E3 ligase, the technique measures only poly-ubiquitination and not mono-ubiquitination. This FRET-based assay is homogeneous, making it especially suitable for screening applications and real-time kinetics. Validation of the assay indicated that the presence of a kinase substrate (Tyro3 or Axl) activates CBL-b and c-CBL, which are known to display a similar mechanism of activation. Importantly, our assay design allowed us to show that known CBL-B inhibitor CBL-B-IN-1 is not strictly selective toward CBL-B but also affects c-CBL E3 ligase activity to similar extend since the IC50 of the compound for c-CBL and CBL-B differ by only 2-fold. Finally, it has been proposed that CBL-b phosphorylation at Y106, Y133, and Y363 by Tyro3 kinase is required for its activation. Indeed, in our assay, we observed that CBL-b mutation at Y363F decreases TYRO3 ubiquitination drastically. Surprisingly, phospho-mimetic mutation Y363E did not restore CBL-b-mediated ubiquitination of Tyro3, indicating that substrate binding close to Y363 is the predominant requirement for CBL activation. The weak E3 ligase activity of CBL-b Y363E mutant was insensitive to CBL-B-IN-1 treatment.In conclusion, our data indicates that no significant difference in the parameters of substrate ubiquitination exists between CBL-B and c-CBL, and that ubiquitination of receptor tyrosine kinase Tyro3 by either c-CBL or CBL-B depends on the kinase binding to CBL but not on the phosphorylation of CBL. Citation Format: Kasia Zientara-Rytter, Veronique Baron, Pavel Shashkin, Henry Zhu. Substrate binding, not phosphorylation, activates CBL poly-ubiquitination activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2548.

Abstract 2184: Short Peptides derived from MIEN1 and their analogs exhibit anti-cancer activity in breast and prostate cancer cells

Abstract Migration and invasion enhancer 1 (MIEN1) is overexpressed in various cancers and plays an important role in cancer cell migration and invasion. Conserved regions of ITAM and prenylation motif in MIEN1 were used as a template to identify anti-cancer peptides. RNA-seq, qPCR analysis and western blots showed changes in the transcriptome and protein expression after peptide treatment. The two bioactive peptides inhibited genes and proteins responsible for cancer cell migration and invasion in both MDA-MB-231 breast cancer and DU-145 prostate cancer cells. The mechanism of the action of the peptides involves the inhibition of key pathways like Epithelial-Mesenchymal transition and Epidermal Growth Factor-mediated NF-κB pathway to exert their anti-cancer activity. Interestingly, the peptides targeted the same signal transduction pathways followed by parental MIEN1 to show its cancer properties. Thus, the two peptides acted as dominant negative effectors of MIEN1 activity. Additionally, Peptide-treatments induced apoptosis in mice groups bearing tumors derived from MDA-MB-231 cells as evidenced by increased levels of cleaved caspase-3 and PARP proteins in western blots. Interestingly, the MIEN1 mRNA and protein expression was lowered in the in vivo breast cancer tumor models which remained unchanged in MDA-MB-231 breast cancer cells indicating an improved therapeutic activity in living system. The peptides did not cause any toxicity in the mice group which received peptides only, at three times the dose used during in vivo assays. Citation Format: AMIT Kumar Tripathi, Jamboor K. Vishwanatha. Short Peptides derived from MIEN1 and their analogs exhibit anti-cancer activity in breast and prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2184.

Jasmonate signaling controls negative and positive effectors of salt stress tolerance in rice.

Plant responses to salt exposure involve large reconfiguration of hormonal pathways that orchestrate physiological changes towards tolerance. Jasmonate (JA) hormones are essential to withstand biotic and abiotic assaults, but their roles in salt tolerance remain unclear. Here we describe the dynamics of JA metabolism and signaling in root and leaf of rice, a plant species that is highly exposed and sensitive to salt. Roots activate the JA pathway in an early pulse, while 2 nd leaf displays a biphasic JA response with peaks at 1 hour and 3 days post-exposure. Based on higher salt tolerance of a rice JA-deficient mutant (aoc), we examined through kinetic transcriptome and physiological analysis the salt-triggered processes that are under JA control. Profound genotype-differential features emerged that could underlie observed phenotypes. ABA content and ABA-dependent water deprivation responses were impaired in aoc shoots. Moreover, aoc accumulated more Na + in roots, and less in leaf, with reduced ion translocation correlating with root derepression of the HAK4 Na + transporter. Distinct reactive oxygen species scavengers were also stronger in aoc leaf, along with reduced senescence and chlorophyll catabolism markers. Collectively, the data identify contrasted contributions of JA signaling to different sectors of the salt stress response in rice.

Hfq protein and GcvB small RNA tailoring of oppA target mRNA to levels allowing translation activation by MicF small RNA in Escherichia coli

ABSTRACT Traffic of molecules across the bacterial membrane mainly relies on porins and transporters, whose expression must adapt to environmental conditions. To ensure bacterial fitness, synthesis and assembly of functional porins and transporters are regulated through a plethora of mechanisms. Among them, small regulatory RNAs (sRNAs) are known to be powerful post-transcriptional regulators. In Escherichia coli, the MicF sRNA is known to regulate only four targets, a very narrow targetome for a sRNA responding to various stresses, such as membrane stress, osmotic shock, or thermal shock. Using an in vivo pull-down assay combined with high-throughput RNA sequencing, we sought to identify new targets of MicF to better understand its role in the maintenance of cellular homoeostasis. Here, we report the first positively regulated target of MicF, the oppA mRNA. The OppA protein is the periplasmic component of the Opp ATP-binding cassette (ABC) oligopeptide transporter and regulates the import of short peptides, some of them bactericides. Mechanistic studies suggest that oppA translation is activated by MicF through a mechanism of action involving facilitated access to a translation-enhancing region in oppA 5ʹUTR. Intriguingly, MicF activation of oppA translation depends on cross-regulation by negative trans-acting effectors, the GcvB sRNA and the RNA chaperone protein Hfq.

Invasive Meningococcal Disease and Meningococcal Serogroup B Vaccination in Adults and Their Offspring: Knowledge, Attitudes, and Practices in Italy (2019).

Despite its effectiveness in preventing invasive meningococcal disease (IMD), pediatric uptake of recombinant meningococcal vaccination for serogroup B meningitis (MenB) is low in Italy. This study aimed to investigate knowledge, attitudes, and practice (KAP) about IMD and the vaccine uptake for MenB from July to December 2019, in a sample collected from a series of local Facebook discussion groups from the provinces of Parma and Reggio Emilia (North-Eastern Italy; 337,104 registered users). A self-administered anonymous web-based questionnaire was used to collect demographics, knowledge status, perceived risk for contracting meningitis, attitude towards the utility of meningococcal vaccine, and willingness to receive/perform MenB vaccine in their offspring. In total, 541 parents returned a fully completed questionnaire (response rate of 1.6% of potential recipients), with a mean age of 39.2 years ± 6.3 (78.1% females). Meningococcal infection was identified as severe or highly severe by most participants (88.9%), while it was recognized as being frequent/highly frequent in the general population by 18.6% of respondents. The overall knowledge status was unsatisfactory (57.6% ± 33.6 of correct answers to the knowledge test). Even though 63.4% of participants were somewhat favorable to MenB/MenC vaccines, offspring's vaccination towards MenB was reported by only 38.7% of participants. In a binary logistic regression model, the male gender of respondents (adjusted odds ratio [aOR] 3.184, 95% confidence interval [95%CI] 1.772 to 5.721), living in a municipality >15,000 inhabitants (aOR 1.675, 95%CI 1.051 to 2.668), reporting a favorable attitude on meningococcus B vaccine (aOR 12.472, 95%CI 3.030 to 51.338), having been vaccinated against serogroup B (aOR 5.624, 95%CI 1.936 to 16.337) and/or serogroup C (aOR 2.652, 95%CI 1.442 to 4.872), and having previously vaccinated their offspring against serogroup C meningococcus (aOR 6.585, 95%CI 3.648 to 11.888) were characterized as positive effectors of offspring's vaccination. On the contrary, having a higher risk perception on vaccines was identified as the only negative effector (aOR 0.429, 95%CI 0.241 to 0.765). Our results hint towards extensive knowledge gaps on IMD and preventive interventions in the general population, suggesting that a positive attitude towards vaccines and vaccinations could be identified as the main effector also for MenB acceptance. Interventions in the general population aimed at improving confidence, compliance, and acknowledgment of the collective responsibility, as well as preventing actual constraints and the sharing of false beliefs on infectious diseases and their preventive measures, could therefore increase vaccination acceptance in both targeted individuals and their offspring.

Lipid environment determines the drug-stimulated ATPase activity of P-glycoprotein.

P-glycoprotein (Pgp), also known as ABCB1 or multidrug resistance protein 1 (MDR1), is expressed in the luminal membrane of the intestine, liver, and kidney where it regulates the absorption, distribution, metabolism, and excretion (ADME) of a wide range of xenobiotic compounds and metabolites, and at the blood-brain where it protects the sanctuary from uptake of toxic compounds and enhances excretion of β-amyloid and metabolites. Its expression in cancer cells has been linked to multidrug resistance against many chemotherapeutic agents leading to ineffective treatment and relapse. Besides chemotherapeutic drugs, lipids have been suggested to be transported by themselves or co-transported with drugs by Pgp. Here we tested the hypothesis that the plasma membrane lipid composition modulates how drugs interact with Pgp and may greatly affect both the affinity of the transport substrates and its maximum activity. Our general approach was to test varieties of synthetic lipid mixtures starting with the bilayer-forming phosphatidylcholine (PC) and supplementing with phospholipids carrying different head groups as well as cholesterol. We present the first results of Pgp interactions with PC, phosphatidylethanolamine (PE), sphingomyelin (SM), and cardiolipin which we found to act as positive effectors and increase the apparent affinity for substrates and increase Vmax, while others acted as negative effectors and decreased Vmax. The impact on the substrate binding and translocation mechanisms of Pgp will be discussed. The long-term goal is to establish a stable source of synthetic phospholipid mixtures supplemented with cholesterol that mimic Pgp activity in its native environment in the human body.