Negative Feedback Mechanism Research Articles

Combined in Silico and Experimental Research to Test the Possible Immunogenicity of a New Complex Biologic Medical Drug (PRS CK STORM)

Treatments with biological medicines tend to have a relative efficacy that decreases mainly for two reasons. Firstly, drugs based on the use of high doses of proteins, especially those manufactured artificially or semi-artificially by biotechnology, tend to produce immunogenicity reactions and generate Antidrug antibodies (ADAs) in a certain percentage of the patients to whom they are applied. Secondly, a therapeutic approach based on the single use of a monoclonal antibody does not cover possible positive or negative feedback mechanisms in parallel associated metabolic pathways. Our research group has developed a new biotechnological platform that allows the production of complex biological drugs for allogeneic use, based on the generation of macrophage co-culture with stromal cells or primary cells, with indirect contact. This article describes the in silico, in vitro and in vivo tests carried out with the drug PRS CK STORM, a conditioned medium of M2 macrophages produced by co-culture with indirect contact with MSCs from fat, the results of which demonstrate the absence of immunogenicity of the product applied at the doses tested, demonstrating the possible safety of the drug.

A mechanism of ACK1 activation in cancer via C‐terminal UBA domain truncation

We recently developed a machine learning (ML) based program to predict cancer hotspots. Here, we applied the ML program to 32 non‐receptor tyrosine kinases (NRTKs) and identified 36 potential cancer driver mutations, with high probability mutations in 10 genes, including ABL1, ABL2, JAK1, JAK3, and ACK1. Interestingly among all the NRTKs, ACK1 is the only kinase that, when altered, shows a significant drop in overall survival, supporting the idea that ACK1 is an oncogenic tyrosine kinase. ACK1 is a member of the poorly understood ACK family of NRTKs that also includes TNK1. Although ACK1 is an established oncogene and high‐interest therapeutic target, the exact mechanism of ACK1 regulation is largely unknown and there is still no ACK1 inhibitor in clinical use. The ACK kinase family has a unique domain arrangement with, most notably, a predicted ubiquitin association (UBA) domain at its C‐terminus. The presence of a functional UBA domain on a kinase is unique to the ACK family, but the role of the UBA domain on ACK1 is unknown. Interestingly, the ML program identified the ACK1 UBA‐truncating mutation p633fs* as a cancer hotspot. Our preliminary data suggest that the ACK1 UBA domain helps activate full‐length ACK1 through induced proximity, but also acts as a mechanism of negative feedback by tethering ACK1 to ubiquitinated cargo that is ultimately degraded. Indeed, our preliminary data suggest that truncation of the ACK1 UBA stabilizes ACK1 protein levels, which results in spontaneous ACK1 oligomerization and activation. Thus, our data provide a model to explain how human mutations in ACK1 convert the kinase into an oncogenic driver.

Importance of the Antarctic Slope Current in the Southern Ocean Response to Ice Sheet Melt and Wind Stress Change

AbstractWe use two coupled climate models, GFDL‐CM4 and GFDL‐ESM4, to investigate the physical response of the Southern Ocean to changes in surface wind stress, Antarctic meltwater, and the combined forcing of the two in a pre‐industrial control simulation. The meltwater cools the ocean surface in all regions except the Weddell Sea, where the wind stress warms the near‐surface layer. The limited sensitivity of the Weddell Sea surface layer to the meltwater is due to the spatial distribution of the meltwater fluxes, regional bathymetry, and large‐scale circulation patterns. The meltwater forcing dominates the Antarctic shelf response and the models yield strikingly different responses along West Antarctica. The disagreement is attributable to the mean‐state representation and meltwater‐driven acceleration of the Antarctic Slope Current (ASC). In CM4, the meltwater is efficiently trapped on the shelf by a well resolved, strong, and accelerating ASC which isolates the West Antarctic shelf from warm offshore waters, leading to strong subsurface cooling. In ESM4, a weaker and diffuse ASC allows more meltwater to escape to the open ocean, the West Antarctic shelf does not become isolated, and instead strong subsurface warming occurs. The CM4 results suggest a possible negative feedback mechanism that acts to limit future melting, while the ESM4 results suggest a possible positive feedback mechanism that acts to accelerate melt. Our results demonstrate the strong influence the ASC has on governing changes along the shelf, highlighting the importance of coupling interactive ice sheet models to ocean models that can resolve these dynamical processes.

Influences of Housing Environment on Functional State of the Body after Action of Ulcerogenic Stressor

Here we studied influences of housing environment on functional state of the body after action of ulcerogenic stressor. The effects of ulcerogenic stressor on the gastric mucosa, the hypothalamic‐pituitary‐adrenocortical (HPA) axis reactivity and glucocorticoid receptors in hippocampus, behavior and somatic pain sensitivity were compared in rats kept in enriched environment, social isolation, and standard laboratory conditions (control group). The rats aged 30 days were divided into groups and placed in standard cages 6 rats per cage (control) or 1 rats per cage (isolation) or in enriched cages 6 rats per cage (enriched environment). One month later, all rats (after 24 h fasting) were stressed by cold restraint (10˚C) for 3 h. In 3 h one half of rats in each group were decapitated to examine the gastric mucosa. Another half of rats were subjected to behavior tests (open field and elevated plus maze) and somatic pain sensitivity test (hot plate) as well as the HPA axis reactivity testing by plasma corticosterone levels induced by mild procedural stressor. Glucocorticoid receptor expression was estimated in hippocampus by immunohistochemistry. Body weight gradually increased in all groups during 1 month before exposure of stressor, however, in the isolated rats, the weight gain was greater than that in enriched and control groups. In all groups cold restraint caused the gastric erosion, the mean areas of which were not significantly differed. In behavior tests, the animals of isolated group were approximately equally divided into vulnerable (showing depressive‐like reactions in the open field) and resistant (not differed from the non‐stressed control) to ulcerogenic stress (active subgroup). All isolated rats showed a reduced level of exploratory activity in the elevated plus maze. Rats from enriched environment showed greater motor and exploratory activity, as well as lower anxiety level compared to those in isolated group. Isolated rats were most sensitive to action of pain, whereas rats kept in enriched environment were less sensitive to pain exposure. In the isolated group, the weakest corticosterone release among all groups was observed in response to mild procedural stress. Low HPA stress‐reactivity of isolated rats was combined with enhanced expression of glucocorticoid receptors in the hippocampus indicating a sensitization of the mechanisms of negative glucocorticoid feedback following prolonged social isolation. Thus, social isolation is a stressor leading to weight gain, a decrease in stress reactivity accompanied by an increase of glucocorticoid receptor expression in the hippocampus, a decrease of exploratory activity and increased of somatic pain sensitivity whereas enriched environment exerts beneficial action on functional state of the body: increases motor and exploratory activity, decreases anxiety and somatic pain sensitivity.

Profile of insulin resistance in adipose tissue of diet induced obese mice in response to anti‐obesity treatment

BackgroundAdipose tissue is an insulin‐responsive organ that contributes to both glucose and lipid metabolism. Insulin resistance (IR) is when insulin‐dependent tissues lose their sensitivity to insulin, resulting in high blood glucose and insulin due to impairment of the negative feedback mechanism. Insulin resistance progress to metabolic disorders like obesity and type 2 diabetes. Sulforaphane (SFN) is a potential anti‐obesity drug under investigation.AimThis study aims to understand the effect of SFN on insulin resistance pathways in adipose tissues of diet induced obese (DIO) mice.MethodsWild‐type CD1 male mice were fed a high‐fat diet (HFD) for 16 weeks to induce obesity. Mice were divided into two groups DIO‐SFN and DIO‐V. DIO mice have been injected via IP route by SFN (5 mg/kg BW) or Vehicle (25μl) for 4 weeks. Daily body weight measurements have been recorded, and glucose tolerance test (GTT) at day 21 after injection and insulin tolerance test (ITT) at day 25 after injection. Subsequently, mice were sacrificed, blood and tissue samples were collected and snap‐frozen instantly. Total RNA was extracted from epididymal white adipose tissue (eWAT). Plasma leptin, insulin, and glucose level were measured, and 84 IR genes in adipose tissues were examined using RT‐PCR array technology.ResultsSFN treatment to DIO mice causes a significant reduction in body weight by 18.4%. Furthermore, SFN causes a significant reduction in circulating leptin concentrations in DIO‐SFN compared to the vehicle‐treated group. SFN is associated with the improvement of GTT and ITT. SFN causes dysregulation of eight genes related to inflammation, adipocytokine signaling, Il‐6, and HMGB1 pathways. SFN treatment causes significant changes in functions related to adipose tissue insulin resistance such as inhibition of chemotaxsis, inflammatory response and lipid accumulation.ConclusionThis study demonstrated that SFN treatment effectively reduces body weight and can ameliorate IR in eWAT via inhibition of pathways mainly related to inflammation, such as adipocytokine signaling, Il‐6, and HMGB1. SFN can be considered a future anti‐obesity drug.

Disruption of β-catenin-mediated negative feedback reinforces cAMP-induced neuronal differentiation in glioma stem cells

Accumulating evidence supports the existence of glioma stem cells (GSCs) and their critical role in the resistance to conventional treatments for glioblastoma multiforme (GBM). Differentiation therapy represents a promising alternative strategy against GBM by forcing GSCs to exit the cell cycle and reach terminal differentiation. In this study, we demonstrated that cAMP triggered neuronal differentiation and compromised the self-renewal capacity in GSCs. In addition, cAMP induced negative feedback to antagonize the differentiation process by activating β-catenin pathway. Suppression of β-catenin signaling synergized with cAMP activators to eliminate GSCs in vitro and extended the survival of animals in vivo. The cAMP/PKA pathway stabilized β-catenin through direct phosphorylation of the molecule and inhibition of GSK-3β. The activated β-catenin translocated into the nucleus and promoted the transcription of APELA and CARD16, which were found to be responsible for the repression of cAMP-induced differentiation in GSCs. Overall, our findings identified a negative feedback mechanism for cAMP-induced differentiation in GSCs and provided potential targets for the reinforcement of differentiation therapy for GBM.

The rate‐limiting enzyme in the CDP‐choline pathway is regulated by phosphorylation‐domain charge density

Eukaryotic cell membranes are primarily composed of phospholipids, the most abundant of which is phosphatidylcholine (PC). De novo synthesis of PC by the CDP‐choline pathway is under the control of the rate‐limiting enzyme CTP:phosphocholine cytidylyltransferase (CCT). The nuclear CCTa isoform is regulated by translocation to the inner nuclear membrane (INM) and nuclear lipid droplets (nLD) in response to lipid activators, such as oleate, or deficiency in PC content. CCTa association with the INM is mediated by a positively‐charged membrane‐binding M‐domain and a phosphorylated P‐domain containing 16 serine phospho‐sites. Mutation of all P‐domain serine residues to alanine (dephosphorylated mimic) enhanced association with nLDs in oleate‐treated U2OS cells, while mutation to glutamate (phosphorylated mimic) or mutation of M‐domain lysine residues ablated nLD association. However, it is unknown if phosphorylation regulation of CCTa association with the INM and nLDs involves specific serine residues or the net charge of the P‐domain. To address this question, we mutated 4 sets of 2‐3 serine residues in the P‐domain to alanine or aspartate. CCTa cDNAs expressing different combinations of these mutations were expressed in oleate‐treated U2OS cells and the frequency of nLD association was measured. The mutants were also expressed in CHO cells with a temperature‐sensitive CCTα isoform and INM translocation in response to oleate was determined. The greatest inhibitory effect on INM and nLD association was observed when all 4 sets of serine residues were mutated to aspartate and not by any one site, suggesting that net charge density of the P‐domain regulates affinity for the NE and nLD. Additionally, CCTa serine‐to‐alanine mutants expressed in U2OS cells were unstable compared to the corresponding aspartate mutants. We conclude that CCTa association with the INM and nLD is inhibited as overall P‐domain phosphorylation increases. In its active membrane‐associated, dephosphorylated state CCTa protein has decreased stability, a potently negative feedback mechanism to control PC synthesis. These results provide novel insight into how PC synthesis is regulated that will be essential for identifying kinases and phosphatases that act on CCTα.

Open Search-Based Proteomics Reveals Widespread Tryptophan Modifications Associated with Hypoxia in Lung Cancer

The tryptophan residue has a large hydrophobic surface that plays a unique role in the folded protein conformation and functions. Tryptophan modifications are presumably to be readily detected in proteins due to the vulnerability of the indole structure to electrophilic attacks. In this study, we report a systematic identification of sequence variations at tryptophan, termed tryptophan variants, from the proteome of patients with nonsmall cell lung cancer (NSCLC). Using shotgun proteomics and a modified open search algorithm, 25 tryptophan variants on 2481 sites in over 858 proteins were identified. Among these, 6 tryptophan variants are previously identified, 15 are newly annotated, and 4 are still unknown, most of which are involved in the cascade of oxidation in the blood microparticle. Remarkably, Trp313 of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was up-oxidized whereas Trp16 and Trp38 of hemoglobin (HBB) were down-oxidized in NCSLC tissues. The results were further supported by an independent cohort of 103 lung adenocarcinoma samples, reflecting a negative feedback and potential detoxification mechanism against tumor glycolysis and hypoxia. Overall, the study reports a quick approach to explore tryptophan variants at the proteomic scale. Our findings highlight the predominant role of tryptophan oxidation in regulating the redox balance of cancer cells and its potential role as prognostic biomarker for patients with NSCLC.

Single-cell RNA Sequencing Identified Novel Nr4a1+ Ear2+ Anti-Inflammatory Macrophage Phenotype under Myeloid-TLR4 Dependent Regulation in Anti-Glomerular Basement Membrane (GBM) Crescentic Glomerulonephritis (cGN).

Previously, this study demonstrates the critical role of myeloid specific TLR4 in macrophage‐mediated progressive renal injury in anti‐glomerular basement membrane (anti‐GBM) crescentic glomerulonephritis (cGN); however, the underlying mechanism remains largely unknown. In this study, single‐cell RNA sequencing (scRNA‐seq), pseudotime trajectories reconstruction, and motif enrichment analysis are used, and macrophage diversity in anti‐GBM cGN under tight regulation of myeloid‐TLR4 is uncovered. Most significantly, a myeloid‐TLR4 deletion‐induced novel reparative macrophage phenotype (Nr4a1+ Ear2+) with significant upregulated anti‐inflammatory and tissue repair‐related signaling is discovered, thereby suppressing the M1 proinflammatory responses in anti‐GBM cGN. This is further demonstrated in vitro that deletion of TLR4 from bone marrow‐derived macrophages (BMDMs) induces the Nr4a1/Ear2‐expressing anti‐inflammatory macrophages while blocking LPS‐stimulated M1 proinflammatory responses. Mechanistically, activation of the Nr4a1/Ear2‐axis is recognized as a key mechanism through which deletion of myeloid‐TLR4 promotes the anti‐inflammatory macrophage differentiation in vivo and in vitro. This is confirmed by specifically silencing macrophage Nr4a1 or Ear2 to reverse the anti‐inflammatory effects on TLR4 deficient BMDMs upon LPS stimulation. In conclusion, the findings decode a previously unidentified role for a myeloid‐TLR4 dependent Nr4a1/Ear2 negative feedback mechanism in macrophage‐mediated progressive renal injury, implying that activation of Nr4a1‐Ear2 axis can be a novel and effective immunotherapy for anti‐GBM cGN.

Characteristics of aldosterone-producing adenomas in patients without plasma renin activity suppression.

Primary aldosteronism (PA) usually accompanies suppressed plasma renin activity (PRA) through a negative feedback mechanism. While some cases of PA with unsuppressed PRA were reported, there have been no studies about the characteristics of PA with unsuppressed PRA; thus, these characteristics were examined herein. Nine patients with unsuppressed PRA and 86 patients with suppressed PRA were examined. All patients underwent segmental adrenal venous sampling (sAVS) and adrenalectomy, and were pathologically confirmed to have cytochrome P450 11B2 (CYP11B2)-positive aldosterone-producing adenoma according to international histopathology consensus criteria. Unsuppressed and suppressed PRA were defined as PRA levels of > 1.0 and ≤ 1.0 ng/mL/hr, respectively, in multiple blood samples obtained in the resting position. The unsuppressed PRA group had higher morning cortisol levels (12.6 [8.5, 13.5] vs. 8.5 [7.1, 11.0] μg/dL, P = 0.03) and higher cortisol levels after a 1 mg dexamethasone suppression test (DST) (2.2 [1.6, 2.5] vs. 1.3 [1.0, 1.9] μ g/dL, P = 0.004) than the suppressed PRA group. The unsuppressed PRA group also showed higher aldosterone levels on the non-surgical side during sAVS (P = 0.02 before adrenocorticotropic hormone (ACTH) stimulation, P = 0.002 after ACTH stimulation), a higher intensity of CYP17 expression in the resected adrenal gland (P = 0.02), and a lower clinical complete success rate 1 year after surgery (P = 0.04) compared with those in the suppressed PRA group. These findings suggest that PA should not be ruled out by unsuppressed PRA among patients with hypertension, particularly when their cortisol levels remain unsuppressed in the 1 mg DST. Meanwhile, it should be acknowledged that patients with unsuppressed PRA have higher aldosterone levels on the non-surgical side, and a lower likelihood of postoperative complete clinical success is to be expected.

Conventional amphotericin B elicits markers of immunogenic cell death on leukemic blasts, mediates immunostimulatory effects on phagocytic cells, and synergizes with PD-L1 blockade

ABSTRACT Immunostimulatory regimens are a game changer in the fight against cancer, but still only a minority of patients achieve clinical benefit. Combination with immunomodulatory drugs and agents converting otherwise non-immunogenic forms of cell death into bona fide “immunogenic cell death” (ICD) could improve the efficacy of these novel therapies. The aim of our study was to investigate conventional Amphotericin B (AmB) as an enhancer of antitumor immune responses. In tumor cell line models, AmB induced ICD with its typical hallmarks of calreticulin (CALR) expression and release of high mobility group box 1 (HMGB1) as well as Adenosine 5’-triphosphate (ATP). Interestingly, in contrast to non-ICD inducing treatments, ICD induction led to up-regulation of PD-L1-expression by ICD experiencing cells, resulting in decreased maturation of dendritic cells (DCs). Blocking this PD-L1 expression on tumor cells could unleash full ICD effects on antigen presenting cells. Even at sub-toxic concentrations, AmB was able to enhance CALR on leukemic blasts, particularly on phagocytic monoblastic THP-1 cells, which also showed features of “M1-like” differentiation after AmB exposure. The ability of AmB to increase the immunogenicity of tumor cells was confirmed in vivo in a mouse vaccination experiment. In conclusion, we demonstrate that AmB can promote antitumor immune responses in a dose-dependent manner by ICD induction, surface translocation of CALR on leukemic blasts even at sub-toxic concentrations, and “M1-like” polarization of phagocytic cells, making it noteworthy as potential booster for cancer immunotherapy. We additionally report for the first time that PD-L1 expression may be a feature of ICD, possibly as a negative feedback mechanism regulating the maturation status of DCs and thus indirectly affecting T-cell priming.

Thyroxine Metabolism in an Avian Model of Induced Thyrotoxicosis

Craniosynostosis (CS) is the premature fusion of cranial sutures which leads to abnormal cranial structure and causes abnormal neurocognition and behavior. Maternal thyroid disorders are associated with incidence of CS in infants. However, the mechanism of TH-induced CS is still obscure. Thyroxine (T4) metabolism is regulated by the expression of deiodinases. Type I and type II deiodinases (DIO1 and DIO2) remove iodine from T4 producing active triiodothyronine (T3). Deiodinase III (DIO3) inactivates both T4 and T3. Our lab is establishing a model to study the effects of thyrotoxicosis induced CS using fertilized chicken eggs. Eggs were injected into the air cell with either saline or 25 ng T4 on days E11 and E15 of embryonic development. Heart rates were measured daily from EKG recordings and on E19, tissue samples were collected for qRT-PCR analysis. The main objective was to validate the induced state of thyrotoxicosis by evaluating negative feedback mechanisms of thyroid hormone regulation following exposure by measuring expression of DIO1 and DIO3 in embryonic livers. Since livers are the main site of metabolism, we hypothesize that levels of DIO3 will increase following thyroxine exposure. Results demonstrated a significant upregulation of DIO3 2 days after exposure and a significant downregulation 4 days following exposure. No significant differences were observed in DIO1 on any of the days after exposure. These results, along with correlating heart rates, support our model by of induced thyrotoxicosis by demonstrating the metabolism of thyroxine in response to our treatment regimen.
 
 
 Acknowledgment of NASA West Virginia Space Grant Consortium (Grant #80NSSC20M0055) and the Genomics Core Facility and WV-INBRE (NIH grant P20GM103434).

Research Progress on the Role of Mitochondrial DNA in the Pathogenesis of Chronic Liver Disease

Mitochondrial DNA is the mitochondria's own genetic material located within the mitochondrial matrix and is involved in cellular metabolism and energy supply. Mitochondrial DNA damage exacerbates oxidative stress by increasing the release of reactive oxygen species, while mitochondrial DNA release also triggers apoptosis and activates immune inflammatory responses through damage-related molecular patterns. Mitochondrial autophagy regulates mitochondrial DNA damage and release through a negative feedback mechanism to maintain intracellular homeostasis. Recent studies have shown that the occurrence and development of chronic liver disease are closely related to mitochondrial DNA-mediated immune inflammatory responses and oxidative stress.

Taste Cells of the Type III Employ CASR to Maintain Steady Serotonin Exocytosis at Variable Ca2+ in the Extracellular Medium.

Type III taste cells are the only taste bud cells which express voltage-gated (VG) Ca2+ channels and employ Ca2+-dependent exocytosis to release neurotransmitters, particularly serotonin. The taste bud is a tightly packed cell population, wherein extracellular Ca2+ is expected to fluctuate markedly due to the electrical activity of taste cells. It is currently unclear whether the Ca2+ entry-driven synapse in type III cells could be reliable enough at unsteady extracellular Ca2. Here we assayed depolarization-induced Ca2+ signals and associated serotonin release in isolated type III cells at varied extracellular Ca2+. It turned out that the same depolarizing stimulus elicited invariant Ca2+ signals in type III cells irrespective of bath Ca2+ varied within 0.5–5 mM. The serotonin release from type III cells was assayed with the biosensor approach by using HEK-293 cells co-expressing the recombinant 5-HT4 receptor and genetically encoded cAMP sensor Pink Flamindo. Consistently with the weak Ca2+ dependence of intracellular Ca2+ transients produced by VG Ca2+ entry, depolarization-triggered serotonin secretion varied negligibly with bath Ca2+. The evidence implicated the extracellular Ca2+-sensing receptor in mediating the negative feedback mechanism that regulates VG Ca2+ entry and levels off serotonin release in type III cells at deviating Ca2+ in the extracellular medium.

Alteration of Bile Acids and Omega-6 PUFAs Are Correlated With the Progression and Prognosis of Drug-Induced Liver Injury.

Background & AimsDrug-induced liver injury (DILI) is one of the leading causes of liver failure with some of the patients progressed to chronic DILI. The mechanisms underlying the severity and chronicity of DILI are poorly elucidated and the biomarkers are limited. Metabolites and gut microbiota played a crucial role in the development of various liver diseases. Herein, a systematic analysis of serum metabolites and gut microbiota was performed in DILI patients, aiming to identify metabolites correlated with the progression and clinical prognosis of DILI.MethodsVarious serum metabolites were quantitated using a metabolite array technology in this prospective study. Gut microbiome compositions and the expression profiles of liver genes were determined in patients with DILI and healthy controls.ResultsMetabolomic analysis revealed that bile acids (BAs) and polyunsaturated fatty acids (PUFAs) were closely related to DILI severity and chronicity respectively. The ratios of serum primary/secondary BAs and omega-6/omega-3 PUFAs were elevated in DILI patients. A model established by adrenic acid (AdA) and aspartic acid (Asp) exerts good performance for predicting the chronicity of DLIL. Hepatic transcriptome revealed enhanced expression of PUFA peroxidation and supressed expression of BA synthesis related genes in DILI patients. In addition, Lactic acid bacteria and BA converting bacteria were increased in gut of DILI patients. Besides, elevated serum malondialdehyde (MDA) and fibroblast growth factor 19 (FGF19) was observed in DILI patients.ConclusionBAs and PUFAs could be potent markers for the severity and chronicity of DILI respectively. The panel of AdA and Asp could be ideal predictive model for the risk of chronicity at the acute stage of DILI. Gut microbiota might act as a negative feedback mechanism to maintain the homeostasis of BAs and PUFAs via FGF19 signalling and PUFA saturation, respectively. Our study revealed novel biomarkers for severe and chronic DILI and provided new therapeutic targets for DILI.

Five million years of high atmospheric CO2 in the aftermath of the Permian-Triassic mass extinction

Abstract The end-Permian mass extinction, the largest biological crisis in Earth history, is currently understood in the context of Siberian Traps volcanism introducing large quantities of greenhouse gases to the atmosphere, culminating in the Early Triassic hothouse. In our study, the late Permian and Early Triassic atmospheric CO2 history was reconstructed by applying the paleosol pCO2 barometer. Atmospheric pCO2 shows an approximate 4× increase from mean concentrations of 412–919 ppmv in the late Permian (Changhsingian) to maximum levels between 2181 and 2610 ppmv in the Early Triassic (late Griesbachian). Mean CO2 estimates for the later Early Triassic are between 1261–1936 ppmv (Dienerian) and 1063–1757 ppmv (Spathian). Significantly lower concentrations ranging from 343 to 634 ppmv are reconstructed for the latest Early to Middle Triassic (Anisian). The 5 m.y. episode of elevated pCO2 suggests that negative feedback mechanisms such as silicate weathering were not effective enough to reduce atmospheric pCO2 to precrisis levels and that marine authigenic clay formation (i.e., reverse weathering) may have been an important component of the global carbon cycle keeping atmospheric pCO2 at elevated levels.

Inhibition of RhoA and Cdc42 by miR-133a Modulates Retinoic Acid Signalling during Early Development of Posterior Cardiac Tube Segment.

It is well known that multiple microRNAs play crucial roles in cardiovascular development, including miR-133a. Additionally, retinoic acid regulates atrial marker expression. In order to analyse the role of miR-133a as a modulator of retinoic acid signalling during the posterior segment of heart tube formation, we performed functional experiments with miR-133a and retinoic acid by means of microinjections into the posterior cardiac precursors of both primitive endocardial tubes in chick embryos. Subsequently, we subjected embryos to whole mount in situ hybridisation, immunohistochemistry and qPCR analysis. Our results demonstrate that miR-133a represses RhoA and Cdc42, as well as Raldh2/Aldh1a2, and the specific atrial markers Tbx5 and AMHC1, which play a key role during differentiation. Furthermore, we observed that miR-133a upregulates p21 and downregulates cyclin A by repressing RhoA and Cdc42, respectively, thus functioning as a cell proliferation inhibitor. Additionally, retinoic acid represses miR-133a, while it increases Raldh2, Tbx5 and AMHC1. Given that RhoA and Cdc42 are involved in Raldh2 expression and that they are modulated by miR-133a, which is influenced by retinoic acid signalling, our results suggest the presence of a negative feedback mechanism between miR-133a and retinoic acid during early development of the posterior cardiac tube segment. Despite additional unexplored factors being possible contributors to this negative feedback mechanism, miR-133a might also be considered as a potential therapeutic tool for the diagnosis, therapy and prognosis of cardiac diseases.

Effects of the Isolated and Combined Ablation of Growth Hormone and IGF-1 Receptors in Somatostatin Neurons.

Hypophysiotropic somatostatin (SST) neurons in the periventricular hypothalamic area express growth hormone (GH) receptor (GHR) and are frequently considered as the key neuronal population that mediates the negative feedback loop controlling the hypothalamic-GH axis. Additionally, insulin-like growth factor-1 (IGF-1) may also act at the hypothalamic level to control pituitary GH secretion via long-loop negative feedback. However, to the best of our knowledge, no study so far has tested whether GHR or IGF-1 receptor (IGF1R) signaling specifically in SST neurons is required for the homeostatic control of GH secretion. Here we show that GHR ablation in SST neurons did not impact the negative feedback mechanisms that control pulsatile GH secretion or body growth in male and female mice. The sex difference in hepatic gene expression profile was only mildly affected by GHR ablation in SST neurons. Similarly, IGF1R ablation in SST neurons did not affect pulsatile GH secretion, body growth, or hepatic gene expression. In contrast, simultaneous ablation of both GHR and IGF1R in SST-expressing cells increased mean GH levels and pulse amplitude in male and female mice, and partially disrupted the sex differences in hepatic gene expression. Despite the increased GH secretion in double knockout mice, no alterations in body growth and serum or liver IGF-1 levels were observed. In summary, GHR and IGF1R signaling in SST neurons play a redundant role in the control of GH secretion. Furthermore, our results reveal the importance of GH/IGF-1 negative feedback mechanisms on SST neurons for the establishment of sex differences in hepatic gene expression profile.

Proteomic Analysis of the Role of the Adenylyl Cyclase-cAMP Pathway in Red Blood Cell Mechanical Responses.

Red blood cell (RBC) deformability is modulated by the phosphorylation status of the cytoskeletal proteins that regulate the interactions of integral transmembrane complexes. Proteomic studies have revealed that receptor-related signaling molecules and regulatory proteins involved in signaling cascades are present in RBCs. In this study, we investigated the roles of the cAMP signaling mechanism in modulating shear-induced RBC deformability and examined changes in the phosphorylation of the RBC proteome. We implemented the inhibitors of adenylyl cyclase (SQ22536), protein kinase A (H89), and phosphodiesterase (PDE) (pentoxifylline) to whole blood samples, applied 5 Pa shear stress (SS) for 300 s with a capillary tubing system, and evaluated RBC deformability using a LORRCA MaxSis. The inhibition of signaling molecules significantly deteriorated shear-induced RBC deformability (p < 0.05). Capillary SS slightly increased the phosphorylation of RBC cytoskeletal proteins. Tyrosine phosphorylation was significantly elevated by the modulation of the cAMP/PKA pathway (p < 0.05), while serine phosphorylation significantly decreased as a result of the inhibition of PDE (p < 0.05). AC is the core element of this signaling pathway, and PDE works as a negative feedback mechanism that could have potential roles in SS-induced RBC deformability. The cAMP/PKA pathway could regulate RBC deformability during capillary transit by triggering significant alterations in the phosphorylation state of RBCs.

Transcriptome analysis based on dietary beta-carotene supplement reveals genes potentially involved in carotenoid metabolism in Crassostrea gigas.

Carotenoids are essential micronutrients for animals, and they can only be obtained from the diet for mollusk as well as other animals. In the body, carotenoids undergo processes including absorption, transport, deposition, and metabolic conversion; however, knowledge of the involved genes is still limited. To elucidate the molecular mechanisms of carotenoid processing and identify the related genes in Pacific oyster (Crassostrea gigas), we performed a comparative transcriptome analysis using digestive gland tissues of oysters on a beta-carotene supplemented diet or a normal diet. A total of 718 differentially expressed genes were obtained, including 505 upregulated and 213 downregulated genes in the beta-carotene supplemented group. Function Annotation and enrichment analyses revealed enrichment in genes possibly involved in carotenoid transport and storage (e.g., LOC105342035), carotenoid cleavage (e.g., LOC105341121), retinoid homeostasis (e.g., LOC105339597) and PPAR signaling pathway (e.g., LOC105323212). Notably, down-regulation of mRNA expressions of two apolipoprotein genes (LOC105342035 and LOC105342186) by RNA interference significantly decreased the carotenoid level in the digestive gland, supporting their role in carotenoid transport and storage. Based on these differentially expressed genes, we propose that there may be a negative feedback mechanism regulated by nuclear receptor transcription factors controlling carotenoid oxygenases. Our findings provide useful hints for elucidating the molecular basis of carotenoid metabolism and functions of carotenoid-related genes in the oyster.