Endogenous Function Research Articles

Functional Food Nutrients, Redox Resilience Signaling and Neurosteroids for Brain Health

The interplay between functional food nutrients and neurosteroids has garnered significant attention for its potential to enhance stress resilience in health and/or disease. Several bioactive nutrients, including medicinal herbs, flavonoids, and bioavailable polyphenol-combined nanoparticles, as well as probiotics, vitamin D and omega-3 fatty acids, have been shown to improve blood–brain barrier (BBB) dysfunction, endogenous neurosteroid homeostasis and brain function. These nutrients can inhibit oxidative stress and neuroinflammation, which are linked to the pathogenesis of various neurological disorders. Interestingly, flavonoids exhibit dose-dependent effects, activating the nuclear factor erythroid 2–related factor 2 (Nrf2) pathway at the physiological/low dose (neurohormesis). This leads to the upregulation of antioxidant phase II genes and proteins such as heme oxygenase-1 (HO-1) and sirtuin-1 (Sirt1), which are activated by curcumin and resveratrol, respectively. These adaptive neuronal response mechanisms help protect against reactive oxygen species (ROS) and neurotoxicity. Impaired Nrf2 and neurosteroid hormone signaling in the brain can exacerbate selective vulnerability to neuroinflammatory conditions, contributing to the onset and progression of neurodegenerative and psychiatric disorders, including Alzheimer’s disease, anxiety and depression and other neurological disorders, due to the vulnerability of neurons to stress. This review focuses on functional food nutrients targeting Nrf2 antioxidant pathway and redox resilience genes to regulate the neurosteroid homeostasis and BBB damage associated with altered GABAergic neurotransmission. By exploring the underlying molecular mechanisms using innovative technologies, we aim to develop promising neuroprotective strategies and personalized nutritional and neuroregenerative therapies to prevent or attenuate oxidative stress and neuroinflammation, ultimately promoting brain health.

Attention Deficit Hyperactivity Disorder, Cannabis Use, and the Endocannabinoid System: A Scoping Review.

There is emerging evidence that the endocannabinoid system (ECS) plays a significant role in the pathophysiology of many psychiatric disorders, including attention deficit hyperactivity disorder (ADHD). Increasing evidence suggests that a number of neurobiological correlates between endogenous cannabinoid function and cognitive dysfunction are seen in ADHD, making the ECS a possible target for therapeutic interventions. Cannabis use and cannabis use disorder are more prevalent in individuals with ADHD, compared to the general population, and there is growing popular perception that cannabis is therapeutic for ADHD. However, the relationship between cannabis use and ADHD symptomology is poorly understood. Further understanding of the role of the ECS in ADHD pathophysiology and the molecular alterations that may be a target for treatment is needed. To further the science on this emerging area of research, this scoping review describes the preclinical and clinical evidence seeking to understand the relationship between the ECS and ADHD.

The impact of big fund on the operational efficiency of semiconductor companies: Corporate social responsibility as a non-economic output

This study contributes to research on the total factor operational efficiency of the semiconductor company considering corporate social responsibility (CSR) by proposing a novel integer-valued nonparametric frontier method. This method adopts an endogenous directional distance function, which first selects the benchmark and then endogenously determines the directional vector. Avoiding the issue of non-integer slack adjustment increases the convenience of formulating incentive plans and calculating efficiency. We apply this method to the input–output data of 185 listed Chinese semiconductor companies from 2010 to 2022, considering integer-valued CSR as non-economic output. Global Malmquist productivity index analysis shows that the average total factor operational efficiency of companies that received investment from the National Integrated Circuit Industry Investment Fund (Big Fund) increased by 6.9%. Using the econometric technique, we verify that the total factor operational efficiency of semiconductor companies receiving Big Fund investment increased by 0.084 compared to those without Big Fund investment. Heterogeneity analysis showed that the effect of Big Fund was more significant in improving the total factor operational efficiency of state-owned, new, and non-integrated circuit semiconductor companies, implying the necessity of purposeful investment policies for the Big Fund.

Uracil-Selective Cross-Linking in RNA and Inhibition of miRNA Function by 2-Amino-6-vinyl-7-deazapurine Deoxynucleosides.

MicroRNAs (miRNAs) regulate gene expression through RNA interference. Consequently, miRNA inhibitors, such as anti-miRNA oligonucleotides (AMOs), have attracted attention for treating miRNA overexpression. To achieve efficient inhibition, we developed 2-amino-6-vinylpurine (AVP) nucleosides that form covalent bonds with uridine counterparts in RNA. We demonstrated that mRNA cross-linked with AVP-conjugated antisense oligonucleotides with AVP were protected from gene silencing by exogenous miRNA. However, endogenous miRNA function could not be inhibited in cells, probably because of slow cross-linking kinetics. We recently developed ADpVP, an AVP derivative bearing a 7-propynyl group - which boasts faster reaction rate than the original AVP. Here, we synthesized dADpVP - a deoxy analog of ADpVP - through a simplified synthesis protocol. Evaluation of the cross-linking reaction revealed that the reaction kinetics of dADpVP were comparable to those of ADpVP. In addition, structural analysis of the cross-linked adduct discovered N3 linkage against uridine. Incorporating dADpVP into two types of miRNA inhibitors revealed a marginal impact on AMO efficacy yet improved the performance of target site blockers. These results indicate the potential of cross-linking nucleosides for indirect miRNA function inhibition.

Altered Endogenous Pain-Inhibitory Function in Older Adults With Chronic Pain Is Associated With Disruptions in Functional Connectivity During Resting State

Increasing research points to a decline in the ability to internally regulate pain as a contributing factor to the increased pain susceptibility in aging. This study investigated the connection between pain regulation and resting-state functional connectivity (rsFC) in older adults with chronic pain. We compared functional magnetic resonance imaging rsFC of 30 older adults with chronic pain (69.5 ± 6.58 years, 14 males), 29 pain-free older (70.48 ± 4.60, 15 males), and 30 younger adults (20.0 ± 1.58, 15 males). Pain inhibition and facilitatory capabilities were assessed using conditioned pain modulation (CPM) and temporal summation. Older adults with chronic pain displayed lower pain inhibition during the CPM than pain-free older and younger adults. rsFC analysis showed that older adults with chronic pain, in comparison with younger participants, displayed an abnormal hyperconnectivity between right dorsolateral prefrontal cortex and left amygdala, which was significantly correlated with lower pain inhibition during the CPM. Older adults with chronic pain displayed higher connectivity between the primary somatosensory cortex and nucleus accumbens than pain-free older adults. Finally, both older adult groups displayed reduced connectivity between brain structures involved in pain inhibition and processing in comparison with younger adults. Altogether, our results suggest that suffering from pain during aging leads to a dysfunction of pain-inhibitory processes, which significantly surpass those caused by normal aging. Furthermore, our results point to a key role of emotional and motivational brain areas, and their interaction with executive and somatosensory areas, in the reduced inhibitory capacity and likely the maintenance of chronic pain in aging. PerspectiveThis study examines the link between reduced pain-inhibition capacity and increased resting-state connectivity between affective, sensory, and executive brain structures in older adults with chronic pain. These findings could inform new pain assessment and treatment programs for this population.

Expression of VISTA regulated via IFN-γ governs endogenous T-cell function and exhibits correlation with the efficacy of CD19 CAR-T cell treated B-malignant mice

BackgroundDespite continuous improvements in the new target and construction of chimeric antigen receptor (CAR)-T, relapse remains a significant challenge following CAR-T therapy. Tumor microenvironment (TME) strongly correlates with the efficacy...

Resveralogues protect HepG2 cells against cellular senescence induced by hepatotoxic metabolites

Progressive liver disease and dysfunction cause toxic metabolites including ammonia and unconjugated bilirubin to accumulate in plasma. As the population ages alternatives to liver transplantation become increasingly important. One approach for use as a bridge to transplant or recovery is the use of bioartificial liver systems (BALS) containing primary or immortalised hepatocytes as ex-vivo replacements or supports for endogenous liver function. However, exposure to the hepatotoxic metabolites present in plasma causes the rapid failure of these cells to carry out their primary metabolic functions despite remaining viable. Hypothesizing that this loss of core hepatocyte phenotypes was caused by cell senescence we exposed HepG2 cell populations, grown in both standard two-dimensional tissue culture systems and in three dimensional cultures on novel alginate modified HEMA-MBA cryogels, to physiologically reflective concentrations of hepatotoxic metabolites and cytokines. HepG2 cells are forced into senescence by the toxic metabolites in under six hours (as measured by loss of thymidine analog incorporation or detectable Ki67 staining) which is associated with a ten to twenty-fold reduction in the capacity of the cultures to synthesise albumin or urea. This state of senescence induced by liver toxins (SILT) can be prevented by preincubation with either 2–5 µM resveratrol, its major in vivo metabolite dihydroresveratrol or a series of novel resveralogues with differential capacities to scavenge radicals and activate SIRT1 (including V29 which does not interact with the protein). SILT appears to be a previously unrecognised barrier to the development of BALS which can now be overcome using small molecules that are safe for human use at concentrations readily achievable in vivo.

Effects of perioperative low-dose naloxone on the immune system in patients undergoing laparoscopic-assisted total gastrectomy: a randomized controlled trial

BackgroundLow immune function after laparoscopic total gastrectomy puts patients at risk of infection-related complications. Low-dose naloxone (LDN) can improve the prognosis of patients suffering from chronic inflammatory diseases or autoimmune diseases. The use of LDN during perioperative procedures may reduce perioperative complications. The purpose of this study was to examine the effects of LDN on endogenous immune function in gastric cancer patients and its specific mechanisms through a randomized controlled trial.MethodsFifty-five patients who underwent laparoscopic-assisted total gastrectomy were randomly assigned to either a naloxone group (n = 23) or a nonnaloxone group (n = 22). Patients in the naloxone group received 0.05 µg/kg-1.h− 1naloxone from 3 days before surgery to 5 days after surgery via a patient-controlled intravenous injection (PCIA) pump, and patients in the nonnaloxone group did not receive special treatment. The primary outcomes were the rates of postoperative complications and immune function assessed by NK cell, CD3+ T cell, CD4+ T cell, CD8+ T cell, WBC count, neutrophil percentage, and IL-6 and calcitonin levels. The secondary outcomes were the expression levels of TLR4 (Toll-like receptor), IL-6 and TNF-α in gastric cancer tissue.ResultsCompared with the nonnaloxone group, the naloxone group exhibited a lower incidence of infection (in the incision, abdomen, and lungs) (P < 0.05). The numbers of NK cells and CD8+ T cells in the naloxone group were significantly greater than those in the nonnaloxone group at 24 h after surgery (P < 0.05) and at 96 h after surgery (P < 0.05). Compared with those in the nonnaloxone group, the CD3 + T-cell (P < 0.05) and CD4 + T-cell (P < 0.01) counts were significantly lower in the naloxone group 24 h after surgery. At 24 h and 96 h after surgery, the WBC count (P < 0.05) and neutrophil percentage (P < 0.05) were significantly greater in the nonnaloxone group. The levels of IL-6 (P < 0.05) and calcitonin in the nonnaloxone group were significantly greater at 24 h after surgery. At 24 h following surgery, the nonnaloxone group had significantly greater levels of IL-6 (P < 0.05) and calcitonin than did the naloxone group. Compared with those in the naloxone group, the expression levels of TLR4 (P < 0.05) in gastric cancer tissue in the naloxone group were greater; however, the expression levels of IL-6 (P < 0.01) and TNF-α (P < 0.01) in the naloxone group were greater than those in the nonnaloxone group.ConclusionLaparoscopic total gastrectomy patients can benefit from 0.05 ug/kg− 1. h− 1 naloxone by reducing their risk of infection. It is possible that LDN alters the number of cells in lymphocyte subpopulations, such as NK cells, CD3 + T cells, and CD4 + T cells, and the CD4+/CD8 + T-cell ratio or alters TLR4 receptor expression in immune cells, thereby altering immune cell activity.Trial registrationThe trial was registered at the Chinese Clinical Trial Registry on 24/11/2023 (ChiCTR2300077948).

Electrical and immune stimulation-based hydrogels synergistically realize scarless wound healing via amplifying endogenous electrophysiological function and promoting Macrophage Phenotype-Switching

Scars are a consequence of the wound healing process, and scarless wound healing remains a substantial clinical challenge. In this study, conductive hydrogels (named sHA/G-GM) were developed to synergistically realize scarless wound healing through electrical and immune stimulation. sHA/G-GM was prepared as high-sulfated hyaluronan (sHA) modified graphene (sHA/G) homodispersing in gelatin methacrylate, and sHA/G was released and absorbed by cytomembranes. In vitro (human scar fibroblasts and mouse primary macrophages) and in vivo (rat wound models and rabbit ear wound models) studies were conducted and demonstrated that sHA/G-GM not only improves conductivity to fibroblast activity regulation but also combines with interleukin-4 to promote macrophage phenotype switching. These results strongly suggest that sHA/G-GM synergistically amplifies endogenous electrophysiological functions and modulates the immune microenvironment to realize scarless wound healing. Therefore, the electrical and immune stimulation-based sHA/G-GM provide an effective approach to synergistically realize scarless wound healing in the future.

Carbon productivity growth in China’s transportation industry: empirical analysis using a global Malmquist-Luenberger productivity index based on an endogenous directional distance function

In China, transportation is one of the fastest-growing industries for CO2 emissions, so scientifically measuring the carbon total factor productivity growth of this industry could provide accurate guidance to achieve sustainable development. To this end, this study proposes an endogenous directional distance function (DDF) approach using a common directional vector based on satisfaction degree, and then integrates it with the global Malmquist-Luenberger productivity index (GMLPI) to measure the carbon total factor productivity growth. We employ this novel endogenous DDF-based GMLPI approach to evaluate and decompose the carbon total factor productivity growth of China’s 30 provincial-level transportation industries from 2010 to 2020. The empirical results show that the carbon total factor productivity of 14 provinces improved during 2010–2020, and 28 provinces from 2015 to 2020. Efficiency improvement and technological progress have become the joint drivers for the carbon total factor productivity growth. Targeted policy implications are provided.

In vitro and in vivo proof-of-concept studies of extracellular protein degradation using novel M6PR-targeting chimeras (MTACs).

Abstract The mannose-6-phosphate-receptor (M6PR) traffics proteins from extracellular and intracellular locales to late endosomes/lysosomes for degradation and is ubiquitously expressed. This natural endogenous function of the mannose-6-phosphate-receptor (M6PR) may be harnessed for degradation of soluble circulating proteins and cell specific degradation of membrane proteins. Here we describe the design and development of novel M6PR targeting chimeras (MTACs). MTACs are heterobifunctional molecules containing Avilar’s novel, potent, small molecule M6PR-binding ligands conjugated to ligands that bind a target protein of interest for degradation. For proof-of-concept studies, MTACs were designed to target the extracellular protein IgG, the second most abundant circulating protein with a long plasma half-life. Reduction of pathogenic IgG autoantibodies is a clinically validated strategy for treating multiple debilitating autoimmune diseases. In vitro characterization of tridentate and monodentate MTACs revealed potent biochemical binding to M6PR and IgG, and MTAC-mediated cellular uptake and degradation of IgG via the endolysosomal pathway in K562 and LNCaP cells. In vivo MTAC-mediated depletion of human IgG was demonstrated using a heterologous rat PK/PD model.

USP7-Based Deubiquitinase-Targeting Chimeras Stabilize AMPK.

Deubiquitinase-targeting chimeras (DUBTACs) have been recently developed to stabilize proteins of interest, which is in contrast to targeted protein degradation (TPD) approaches that degrade disease-causing proteins. However, to date, only the OTUB1 deubiquitinase has been utilized to develop DUBTACs via an OTUB1 covalent ligand, which could unexpectedly compromise the endogenous function of OTUB1 owing to its covalent nature. Here, we show for the first time that deubiquitinase USP7 can be harnessed for DUBTAC development. Based on a noncovalent ligand of USP7, we developed USP7-based DUBTACs that stabilized the ΔF508-CFTR mutant protein as effectively as the previously reported OTUB1-based DUBTAC. Importantly, using two different noncovalent ligands of USP7, we developed the first AMPK DUBTACs that appear to selectively stabilize different isoforms of AMPKβ, leading to elevated AMPK signaling. Overall, these results highlight that, in addition to OTUB1, USP7 can be leveraged to develop DUBTACs, thus significantly expanding the limited toolbox for targeted protein stabilization and the development of novel AMPK DUBTACs as potential therapeutics.

B-Type Natriuretic Peptide Inhibits the Expression and Function of SERCA2a in Heart Failure.

B-type natriuretic peptide (BNP) possesses protective cardiovascular properties; however, there has not been sufficient serious consideration of the side effects of BNP. As for sarcoplasmic/endoplasmic reticulum calcium ATPase 2a (SERCA2a), it was once considered a new target for the treatment of heart failure (HF). Nevertheless, clinical trials of SERCA2a gene therapy in HF have finally become unsuccessful. Research has found that elevated BNP levels and decreased SERCA2a expression are two important HF characteristics, which are always negatively correlated. We hypothesize that BNP inhibits SERCA2a expression and, therefore, exerts negative effects on SERCA2a expression and function.The effects of BNP on endogenous SERCA2a expression and function were tested in mice with HF induced by transverse aortic constriction and neonatal rat cardiomyocytes (NRCM). Furthermore, to verify the effects of BNP on exogenous SERCA2a gene transduction efficacy, BNP was added to the myocardium and cardiomyocytes infected with an adenovirus overexpressing SERCA2a.In vivo, BNP levels were increased, SERCA2a expression was reduced in both the BNP intervention and HF groups, and BNP reduced the overexpressed exogenous SERCA2a protein in the myocardium. Our in vitro data showed that BNP dose-dependently inhibited the total and exogenous SERCA2a expression in NRCM by activating the cGMP-dependent protein kinase G. BNP also inhibited the effects of SERCA2a overexpression on Ca2+ transience in NRCM.The expression and function of endogenous and exogenous SERCA2a are inhibited by BNP. The opposite relationship between BNP and SERCA2a should be given serious attention in the treatment of HF via BNP or SERCA2a gene therapy.

Functional Analyses of Four Cryptochromes From Aquatic Organisms After Heterologous Expression in Drosophila melanogaster Circadian Clock Cells.

Cryptochromes (Crys) represent a multi-facetted class of proteins closely associated with circadian clocks. They have been shown to function as photoreceptors but also to fulfill light-independent roles as transcriptional repressors within the negative feedback loop of the circadian clock. In addition, there is evidence for Crys being involved in light-dependent magneto-sensing, and regulation of neuronal activity in insects, adding to the functional diversity of this cryptic protein class. In mammals, Crys are essential components of the circadian clock, but their role in other vertebrates is less clear. In invertebrates, Crys can function as circadian photoreceptors, or as components of the circadian clock, while in some species, both light-receptive and clock factor roles coexist. In the current study, we investigate the function of Cry proteins in zebrafish (Danio rerio), a freshwater teleost expressing 6 cry genes. Zebrafish peripheral circadian clocks are intrinsically light-sensitive, suggesting the involvement of Cry in light-resetting. Echinoderms (Strongylocentrotus purpuratus) represent the only class of deuterostomes that possess an orthologue (SpuCry) of the light-sensitive Drosophila melanogaster Cry, which is an important component of the light-resetting pathway, but also works as transcriptional repressor in peripheral clocks of fruit flies. We therefore investigated the potential of different zebrafish cry genes and SpuCry to replace the light-resetting and repressor functions of Drosophila Cry by expressing them in fruit flies lacking endogenous cry function. Using various behavioral and molecular approaches, we show that most Cry proteins analyzed are able to fulfill circadian repressor functions in flies, except for one of the zebrafish Crys, encoded by cry4a. Cry4a also shows a tendency to support light-dependent Cry functions, indicating that it might act in the light-input pathway of zebrafish.

Widespread S-persulfidation in activated macrophages as a protective mechanism against oxidative-inflammatory stress

Acute inflammatory responses often involve the production of reactive oxygen and nitrogen species by innate immune cells, particularly macrophages. How activated macrophages protect themselves in the face of oxidative-inflammatory stress remains a long-standing question. Recent evidence implicates reactive sulfur species (RSS) in inflammatory responses; however, how endogenous RSS affect macrophage function and response to oxidative and inflammatory insults remains poorly understood. In this study, we investigated the endogenous pathways of RSS biogenesis and clearance in macrophages, with a particular focus on exploring how hydrogen sulfide (H2S)-mediated S-persulfidation influences macrophage responses to oxidative-inflammatory stress. We show that classical activation of mouse or human macrophages using lipopolysaccharide and interferon-γ (LPS/IFN-γ) triggers substantial production of H2S/RSS, leading to widespread protein persulfidation. Biochemical and proteomic analyses revealed that this surge in cellular S-persulfidation engaged ∼2% of total thiols and modified over 800 functionally diverse proteins. S-persulfidation was found to be largely dependent on the cystine importer xCT and the H2S-generating enzyme cystathionine γ-lyase and was independent of changes in the global proteome. We further investigated the role of the sulfide-oxidizing enzyme sulfide quinone oxidoreductase (SQOR), and found that it acts as a negative regulator of S-persulfidation. Elevated S-persulfidation following LPS/IFN-γ stimulation or SQOR inhibition was associated with increased resistance to oxidative stress. Upregulation of persulfides also inhibited the activation of the macrophage NLRP3 inflammasome and provided protection against inflammatory cell death. Collectively, our findings shed light on the metabolism and effects of RSS in macrophages and highlight the crucial role of persulfides in enabling macrophages to withstand and alleviate oxidative-inflammatory stress.

Profiling endogenous adrenal function during veno-venous ECMO support in COVID-19 ARDS: a descriptive analysis.

Prolonged critical illness is often accompanied by an impairment of adrenal function, which has been frequently related to conditions complicating patient management. The presumed connection between hypoxia and the pathogenesis of this critical- illness- related corticosteroid insufficiency (CIRCI) might play an important role in patients with severe acute respiratory distress syndrome (ARDS). Since extracorporeal membrane oxygenation (ECMO) is frequently used in ARDS, but data on CIRCI during this condition are scarce, this study reports the behaviour of adrenal function parameters during oxygenation support with veno-venous (vv)ECMO in coronavirus disease 2019 (COVID-19) ARDS. A total of 11 patients undergoing vvECMO due to COVID-19 ARDS at the Medical University of Vienna, who received no concurrent corticosteroid therapy, were retrospectively included in this study. We analysed the concentrations of cortisol, aldosterone, and angiotensin (Ang) metabolites (Ang I-IV, Ang 1-7, and Ang 1-5) in serum via liquid chromatography/tandem mass spectrometry before, after 1 day, 1 week, and 2 weeks during vvECMO support and conducted correlation analyses between cortisol and parameters of disease severity. Cortisol concentrations appeared to be lowest after initiation of ECMO and progressively increased throughout the study period. Higher concentrations were related to disease severity and correlated markedly with interleukin-6, procalcitonin, pH, base excess, and albumin during the first day of ECMO. Fair correlations during the first day could be observed with calcium, duration of critical illness, and ECMO gas flow. Angiotensin metabolite concentrations were available in a subset of patients and indicated a more homogenous aldosterone response to plasma renin activity after 1 week of ECMO support. Oxygenation support through vvECMO may lead to a partial recovery of adrenal function over time. In homogenous patient collectives, this novel approach might help to further determine the importance of adrenal stress response in ECMO and the influence of oxygenation support on CIRCI.

Structural basis of prostaglandin efflux by MRP4.

Multidrug resistance protein 4 (MRP4) is a broadly expressed ATP-binding cassette transporter that is unique among the MRP subfamily for transporting prostanoids, a group of signaling molecules derived from unsaturated fatty acids. To better understand the basis of the substrate selectivity of MRP4, we used cryogenic-electron microscopy to determine six structures of nanodisc-reconstituted MRP4 at various stages throughout its transport cycle. Substrate-bound structures of MRP4 in complex with PGE1, PGE2 and the sulfonated-sterol DHEA-S reveal a common binding site that accommodates a diverse set of organic anions and suggest an allosteric mechanism for substrate-induced enhancement of MRP4 ATPase activity. Our structure of a catalytically compromised MRP4 mutant bound to ATP-Mg2+ is outward-occluded, a conformation previously unobserved in the MRP subfamily and consistent with an alternating-access transport mechanism. Our study provides insights into the endogenous function of this versatile efflux transporter and establishes a basis for MRP4-targeted drug design.

Miniaturized CAR knocked onto CD3ε extends TCR function with CAR specificity under control of endogenous TCR signaling cascade

Immunotherapy using TCR and especially CAR transgenic T cells is a rapidly advancing field with the potential to become standard of care for the treatment of multiple diseases. While all current FDA approved CAR T cell products are generated using lentiviral gene transfer, extensive work is put into CRISPR/Cas mediated gene delivery to develop the next generation of safer and more potent cell products. One limitation of all editing systems is the size restriction of the knock-in cargo. Targeted integration under control of an endogenous promotor and/or signaling cascades opens the possibility to reduce CAR gene size to absolute minimum. Here we demonstrate that a first-generation CAR payload can be reduced to its minimum component - the antigen-binding domain - by targeted integration under control of the CD3ε promoter generating a CAR-CD3ε fusion protein that exploits the endogenous TCR signaling cascade. Miniaturizing CAR payload in this way results in potent CAR activity while simultaneously retaining the primary antigen recognition function of the TCR. Introducing CAR-specificity using a CAR binder only while maintaining endogenous TCR function may be an appealing design for future autologous CAR T cell therapies.

Perception of butenolides by Bacillus subtilis via the α/β hydrolase RsbQ

The regulation of behavioral and developmental decisions by small molecules is common to all domains of life. In plants, strigolactones and karrikins are butenolide growth regulators that influence several aspects of plant growth and development, as well as interactions with symbiotic fungi.1,2,3 DWARF14 (D14) and KARRIKIN INSENSITIVE2 (KAI2) are homologous enzyme-receptors that perceive strigolactones and karrikins, respectively, and that require hydrolase activity to effect signal transduction.4,5,6,7 RsbQ, a homolog of D14 and KAI2 from the gram-positive bacterium Bacillus subtilis, regulates growth responses to nutritional stress via the alternative transcription factor SigmaB (σB).8,9 However, the molecular function of RsbQ is unknown. Here, we show that RsbQ perceives butenolide compounds that are bioactive in plants. RsbQ is thermally destabilized by the synthetic strigolactone GR24 and its desmethyl butenolide equivalent dGR24. We show that, like D14 and KAI2, RsbQ is a functional butenolide hydrolase that undergoes covalent modification of the catalytic histidine residue. Exogenous application of both GR24 and dGR24 inhibited the endogenous signaling function of RsbQ invivo, with dGR24 being 10-fold more potent. Application of dGR24 to B.subtilis phenocopied loss-of-function rsbQ mutations and led to a significant downregulation of σB-regulated transcripts. We also discovered that exogenous butenolides promoted the transition from planktonic to biofilm growth. Our results suggest that butenolides may serve as inter-kingdom signaling compounds between plants and bacteria to help shape rhizosphere communities.

Functional interaction of Clock genes and bone morphogenetic proteins in the adrenal cortex.

The bone morphogenetic protein (BMP) system in the adrenal cortex plays modulatory roles in the control of adrenocortical steroidogenesis. BMP-6 enhances aldosterone production by modulating angiotensin (Ang) II-mitogen-activated protein kinase (MAPK) signaling, whereas activin regulates the adrenocorticotropin (ACTH)-cAMP cascade in adrenocortical cells. A peripheral clock system in the adrenal cortex was discovered and it has been shown to have functional roles in the adjustment of adrenocortical steroidogenesis by interacting with the BMP system. It was found that follistatin, a binding protein of activin, increased Clock mRNA levels, indicating an endogenous function of activin in the regulation of Clock mRNA expression. Elucidation of the interrelationships among the circadian clock system, the BMP system and adrenocortical steroidogenesis regulated by the hypothalamic-pituitary-adrenal (HPA) axis would lead to an understanding of the pathophysiology of adrenal disorders and metabolic disorders and the establishment of better medical treatment from the viewpoint of pharmacokinetics.