Dependent Regulation Research Articles

Evidence of off-target effects of bosutinib that promote retinoic acid-induced differentiation of non-APL AML cells

ABSTRACT In the present study, we determined the effects of the Src family kinase (SFK) inhibitor, Bosutinib, and the engineered loss of the Lyn SFK on all-trans retinoic acid-induced leukemic cell differentiation. Retinoic acid (RA) is an embryonic morphogen and dietary factor that demonstrates chemotherapeutic efficacy in inducing differentiation of a non-APL AML cell model, the HL-60 human myeloblastic (FAB-M2) leukemia cell line, via activation of a novel signalsome containing an ensemble of signaling molecules that drive differentiation. Bosutinib is an inhibitor of SFKs used to treat myeloid leukemias where prominent high expression of SFKs, in particular Lyn, has been observed. Using either Bosutinib or loss of Lyn expression due to shRNA promoted RA-induced phenotypic differentiation, G0 arrest, and respiratory burst (functional differentiation) of HL-60 cells. Signaling events putatively seminal to RA-induced differentiation, the expression of Fgr, Cbl, Slp-76 and Vav, and the phosphorylation of c-Raf (pS259), Vav (p-tyr), and Slp76 (p-tyr) were not inhibited by Bosutinib or loss of Lyn. Nor was RA-induced upregulation of p-tyr phosphorylation of p47phox, a member of the NADPH complex that produces ROS, a putative phosphorylation dependent signaling regulator. Surprisingly, Bosutinib still works in the absence of Lyn to enhance RA-induced differentiation and neither compromised RA-induced expression, nor phosphorylation of signaling molecules that drive differentiation. These findings suggested there is a novel, off-target, Lyn-independent effect of Bosutinib that is of therapeutic significance to differentiation therapy.

RyR1-related myopathy mutations in ATP and calcium binding sites impair channel regulation

The type 1 ryanodine receptor (RyR1) is an intracellular calcium (Ca2+) release channel on the sarcoplasmic/endoplasmic reticulum that is required for skeletal muscle contraction. RyR1 channel activity is modulated by ligands, including the activators Ca2+ and ATP. Patients with inherited mutations in RyR1 may exhibit muscle weakness as part of a heterogeneous, complex disorder known as RYR1-related myopathy (RYR1-RM) or more recently termed RYR1-related disorders (RYR1-RD). Guided by high-resolution structures of skeletal muscle RyR1, obtained using cryogenic electron microscopy, we introduced mutations into putative Ca2+ and ATP binding sites and studied the function of the resulting mutant channels. These mutations confirmed the functional significance of the Ca2+ and ATP binding sites identified by structural studies based on the effects on channel regulation. Under normal conditions, Ca2+ activates RyR1 at low concentrations (µM) and inhibits it at high concentrations (mM). Mutations in the Ca2+-binding site impaired both activating and inhibitory regulation of the channel, suggesting a single site for both high and low affinity Ca2+-dependent regulation of RyR1 function. Mutation of residues that interact with the adenine ring of ATP abrogated ATP binding to the channel, whereas mutating residues that interact with the triphosphate tail only affected the degree of activation. In addition, patients with mutations at the Ca2+ or ATP binding sites suffer from muscle weakness, therefore impaired RyR1 channel regulation by either Ca2+ or ATP may contribute to the pathophysiology of RYR1-RM in some patients.

Seasonal biology of the wheat sheath miner Cerodontha australis (Diptera: Agromyzidae) in perennial ryegrass in Waikato, New Zealand

AbstractCerodontha australis Malloch (wheat sheath miner) is a leaf mining fly native to New Zealand and eastern Australia that feeds on grasses and cereals. It is thought to have limited economic impact despite being one of the most widespread and common insects in New Zealand pastures. In this study, data on C. australis were gathered over an 18‐month period from field plots of endophyte‐free Lolium perenne. Observations from pot trials on parasitism and other leaf miner species are also presented. Larval numbers were highest in late spring to early summer. One distinct peak per year of eggs and pupae indicated that this species may not be multivoltine, contradicting previous literature. Damage levels were high in spring/summer, with leaf mining in up to 53% of tillers sampled and, in mid‐summer, extending into the pseudostem in up to 41% of sampled tillers. Measurement of cephaloskeleton lengths confirmed that this species has three larval instars. Generally, only one larva per tiller survives to pupation, indicating density dependent regulation of populations, and larval/ pupal parasitism can reach high levels in summer. This study provides new information on the seasonal biology of C. australis, and its potential impact on L. perenne pastures in Waikato, New Zealand.

Calcium-Dependent Ion Channels and the Regulation of Arteriolar Myogenic Tone.

Arterioles in the peripheral microcirculation regulate blood flow to and within tissues and organs, control capillary blood pressure and microvascular fluid exchange, govern peripheral vascular resistance, and contribute to the regulation of blood pressure. These important microvessels display pressure-dependent myogenic tone, the steady state level of contractile activity of vascular smooth muscle cells (VSMCs) that sets resting arteriolar internal diameter such that arterioles can both dilate and constrict to meet the blood flow and pressure needs of the tissues and organs that they perfuse. This perspective will focus on the Ca2+-dependent ion channels in the plasma and endoplasmic reticulum membranes of arteriolar VSMCs and endothelial cells (ECs) that regulate arteriolar tone. In VSMCs, Ca2+-dependent negative feedback regulation of myogenic tone is mediated by Ca2+-activated K+ (BKCa) channels and also Ca2+-dependent inactivation of voltage-gated Ca2+ channels (VGCC). Transient receptor potential subfamily M, member 4 channels (TRPM4); Ca2+-activated Cl− channels (CaCCs; TMEM16A/ANO1), Ca2+-dependent inhibition of voltage-gated K+ (KV) and ATP-sensitive K+ (KATP) channels; and Ca2+-induced-Ca2+ release through inositol 1,4,5-trisphosphate receptors (IP3Rs) participate in Ca2+-dependent positive-feedback regulation of myogenic tone. Calcium release from VSMC ryanodine receptors (RyRs) provide negative-feedback through Ca2+-spark-mediated control of BKCa channel activity, or positive-feedback regulation in cooperation with IP3Rs or CaCCs. In some arterioles, VSMC RyRs are silent. In ECs, transient receptor potential vanilloid subfamily, member 4 (TRPV4) channels produce Ca2+ sparklets that activate IP3Rs and intermediate and small conductance Ca2+ activated K+ (IKCa and sKCa) channels causing membrane hyperpolarization that is conducted to overlying VSMCs producing endothelium-dependent hyperpolarization and vasodilation. Endothelial IP3Rs produce Ca2+ pulsars, Ca2+ wavelets, Ca2+ waves and increased global Ca2+ levels activating EC sKCa and IKCa channels and causing Ca2+-dependent production of endothelial vasodilator autacoids such as NO, prostaglandin I2 and epoxides of arachidonic acid that mediate negative-feedback regulation of myogenic tone. Thus, Ca2+-dependent ion channels importantly contribute to many aspects of the regulation of myogenic tone in arterioles in the microcirculation.

Sex-Specific MicroRNAs in Neurovascular Units in Ischemic Stroke.

Accumulating evidence pinpoints sex differences in stroke incidence, etiology and outcome. Therefore, more understanding of the sex-specific mechanisms that lead to ischemic stroke and aggravation of secondary damage after stroke is needed. Our current mechanistic understanding of cerebral ischemia states that endothelial quiescence in neurovascular units (NVUs) is a major physiological parameter affecting the cellular response to neuron, astrocyte and vascular smooth muscle cell (VSMC) injury. Although a hallmark of the response to injury in these cells is transcriptional activation, noncoding RNAs such as microRNAs exhibit cell-type and context dependent regulation of gene expression at the post-transcriptional level. This review assesses whether sex-specific microRNA expression (either derived from X-chromosome loci following incomplete X-chromosome inactivation or regulated by estrogen in their biogenesis) in these cells controls NVU quiescence, and as such, could differentiate stroke pathophysiology in women compared to men. Their adverse expression was found to decrease tight junction affinity in endothelial cells and activate VSMC proliferation, while their regulation of paracrine astrocyte signaling was shown to neutralize sex-specific apoptotic pathways in neurons. As such, these microRNAs have cell type-specific functions in astrocytes and vascular cells which act on one another, thereby affecting the cell viability of neurons. Furthermore, these microRNAs display actual and potential clinical implications as diagnostic and prognostic biomarkers in ischemic stroke and in predicting therapeutic response to antiplatelet therapy. In conclusion, this review improves the current mechanistic understanding of the molecular mechanisms leading to ischemic stroke in women and highlights the clinical promise of sex-specific microRNAs as novel diagnostic biomarkers for (silent) ischemic stroke.

Salicylic acid mediated protection of Brassica campestris sp. chinensis from saline stress via SA receptor NPR1 dependent transcriptional regulation and biosynthesis of related biochemicals

Salt stress has a significant impact on plant physiological processes and morphology, resulting in reduced plant performance. This study aimed to evaluate the hormonal regulation of Pakchoi ( Brassica campestris spp. chinensis) in response to saline stress imposition and exogenous salicylic acid application, focusing on morphological and physiological parameters. Saline stress imposition altered the concentration of endogenous hormonal levels, as demonstrated by Jasmonic acid (JA) and abscisic acid (ABA). Alterations in the level of plant hormones coincided with the enhanced reactive oxygen species (ROS) generation and proline accumulation followed by redox imbalances. Proline biosynthesis and antioxidative enzymes were significantly elevated with SA pretreatment. SA augmented stress responses are corroborated by the upregulation of SA signaling genes ( NPR1 and PR-1 ) and with a repressive effect on the expression of JA and ABA synthesis-related genes ( NCED3 , MYC2, PDF1.2 ). Moreover, the concerted action of SA-mediated genes might be associated with upregulation of flavonoid biosynthesis pathway genes ( FLS, CHS, CHI, F3H and ANS ), resulting in an enhanced level of phenolic compounds in SA pre-treated plants. A substantial scavenging property is attributed to phenolic compounds for the maintenance of cellular redox status. The results suggest that SA receptor NPR1 dependent transcriptional regulation and biosynthesis of related biochemicals are collaborative processes to counteract the adverse effects of salinity and maintain plant cellular redox homeostasis. • S.A pretreatment remediates saline stress induced symptoms. • Salicylic acid mediated modulation of endogenous hormones, as well as their signaling genes. • SA Receptor NPR1 dependent transcriptional regulation of biochemicals involves in redox control under saline stress.

Size and roughness dependent temperature effects on surface charge of silica nanoparticles

Silica nanoparticles (SNP) with different sizes and surface areas are used in numerous micro/nanofluidic applications, while their surface charge properties play a major role in their function. In many of these applications, SNPs also undergo temperature variation. We present that an increase in temperature yields a substantial increase in SNP surface charge depending on nanoparticle size and surface roughness, which cannot be estimated by existing theory. As a continuation of our earlier work characterizing the deviation of SNP surface charging from theoretical predictions due to curvature and EDL overlap effects, this study presents the differentiation from the theory in temperature dependence under various conditions. As we calculate surface chemistry as a function of local ionic conditions (Charge Regulation), temperature variation changed the equilibrium constants of protonation/deprotonation reactions of the SNP surface, in addition to changes occurring in relative permittivity and ionic mobilities. Results show that variation of SNP surface charge by temperature decreases by decreasing particle size and/or increasing roughness size, compare to theoretical flat plate calculations considering similar temperature-dependent properties and charge regulation on the surface. We characterized these deviations by obtaining an “electrokinetic similarity” between different systems of various size and roughness at various ionic conditions based on the non-dimensional groups of λ/D P and λ/D R . Based on these, we devised a phenomenological model as an extension to the flat plate theory to successfully predict the surface charge of SNPs as a function of the particle size, roughness size, and temperature. The current findings are important for the characterization of SNPs through temperature variations and can also be used to adjust the surface charge of SNPs by tuning the temperature. • Nanoparticle surface charge increases with increasing temperature. • Temperature effects on surface charge change with particle size and surface condition. • Temperature dependent ionic diffusion and surface charge regulation were considered. • Temperature dependent solution of PNP with charge regulation is presented. • A model for nanoparticle charging is extended from analytical solution.

SETD2-mediated epigenetic regulation of noncanonical Wnt5A during osteoclastogenesis

To define the role of SETD2 in the WNT5a signaling in the context of osteoclastogenesis, we exploited two different models: in vitro osteoclast differentiation, and K/BxN serum-induced arthritis model. We found that SETD2 and WNT5a were upregulated during osteoclast differentiation and after induction of arthritis. Using gain- and loss-of-function approaches in the myeloid cell, we confirmed that SETD2 regulated the osteoclast markers, and WNT5a via modulating active histone marks by enriching H3K36me3, and by reducing repressive H3K27me3 mark. Additionally, during osteoclastic differentiation, the transcription of Wnt5a was also associated with the active histone H3K9 and H4K8 acetylations. Mechanistically, SETD2 directed induction of NF-κβ expression facilitated the recruitment of H3K9Ac and H4K8Ac around the TSS region of the Wnt5a gene, thereby, assisting osteoclast differentiation. Together these findings for the first time revealed that SETD2 mediated epigenetic regulation of Wnt5a plays a critical role in osteoclastogenesis and induced arthritis.Graphic abstractModel for the Role of SETD2 dependent regulation of osteoclastic differentiation. A In monocyte cells SETD2-dependent H3K36 trimethylation help to create open chromatin region along with active enhancer mark, H3K27Ac. This chromatin state facilitated the loss of a suppressive H3K27me3 mark. B Additionally, SETD2 mediated induction of NF-κβ expression leads to the recruitment of histone acetyl transferases, P300/PCAF, to the Wnt5a gene and establish H3K9Ac and H4K8Ac marks. Along with other activation marks, these acetylation marks help in Wnt5a transcription which leads to osteoclastogenesis.

NMR and EPR-DEER Structure of a Dimeric Guanylate Cyclase Activator Protein-5 from Zebrafish Photoreceptors.

Retinal guanylate cyclases (RetGCs) are regulated by a family of guanylate cyclase-activating proteins (called GCAP1-7). GCAPs form dimers that bind to Ca2+ and confer Ca2+ sensitive activation of RetGC during visual phototransduction. The GCAP5 homologue from zebrafish contains two nonconserved cysteine residues (Cys15 and Cys17) that bind to ferrous ion, which stabilizes GCAP5 dimerization and diminishes its ability to activate RetGC. Here, we present NMR and EPR-DEER structural analysis of a GCAP5 dimer in the Mg2+-bound, Ca2+-free, Fe2+-free activator state. The NMR-derived structure of GCAP5 is similar to the crystal structure of Ca2+-bound GCAP1 (root-mean-square deviation of 2.4 Å), except that the N-terminal helix of GCAP5 is extended by two residues, which allows the sulfhydryl groups of Cys15 and Cys17 to become more solvent exposed in GCAP5 to facilitate Fe2+ binding. Nitroxide spin-label probes were covalently attached to particular cysteine residues engineered in GCAP5: C15, C17, T26C, C28, N56C, C69, C105, N139C, E152C, and S159C. The intermolecular distance of each spin-label probe in dimeric GCAP5 (measured by EPR-DEER) defined restraints for calculating the dimer structure by molecular docking. The GCAP5 dimer possesses intermolecular hydrophobic contacts involving the side chain atoms of H18, Y21, M25, F72, V76, and W93, as well as an intermolecular salt bridge between R22 and D71. The structural model of the GCAP5 dimer was validated by mutations (H18E/Y21E, H18A/Y21A, R22D, R22A, M25E, D71R, F72E, and V76E) at the dimer interface that disrupt dimerization of GCAP5 and affect the activation of RetGC. We propose that GCAP5 dimerization may play a role in the Fe2+-dependent regulation of cyclase activity in zebrafish photoreceptors.

Calmodulin acetylation: A modification to remember

The formation of new memories appears to require alterations in the shape and strength of synapses within the hippocampus, yet our knowledge of the molecular mechanisms underlying these changes remains incomplete. Zhang and colleagues provide new understanding of memory formation by uncovering the lysine acetyltransferase SRC3 as the key driver of the novel posttranslational modification of calmodulin (CaM) acetylation, which regulates CaM's activity and subsequent activation of CaMKII. This new pathway is demonstrated to be both necessary and sufficient for CA3→CA1 synapse long-term potentiation (LTP) and fear memory formation, and this approach may act as a blueprint for future investigation of the role of acetylation of other proteins in neuronal functions.

USP10 exacerbates neointima formation by stabilizing Skp2 protein in vascular smooth muscle cells

The underlying mechanism of neointima formation remains unclear. Ubiquitin-specific peptidase 10 (USP10) is a deubiquitinase that plays a major role in cancer development and progression. However, the function of USP10 in arterial restenosis is unknown. Herein, USP10 expression was detected in mouse arteries and increased after carotid ligation. The inhibition of USP10 exhibited thinner neointima in the model of mouse carotid ligation. In vitro data showed that USP10 deficiency reduced proliferation and migration of rat thoracic aorta smooth muscle cells (A7r5) and human aortic smooth muscle cells (HASMCs). Mechanically, USP10 can bind to Skp2 and stabilize its protein level by removing polyubiquitin on Skp2 in the cytoplasm. The overexpression of Skp2 abrogated cell cycle arrest induced by USP10 inhibition. Overall, the current study demonstrated that USP10 is involved in vascular remodeling by directly promoting VSMC proliferation and migration via stabilization of Skp2 protein expression.

Brain Region and Cell Compartment Dependent Regulation of Electron Transport System Components in Huntington's Disease Model Mice.

Huntington’s disease (HD) is a rare hereditary neurodegenerative disorder characterized by multiple metabolic dysfunctions including defects in mitochondrial homeostasis and functions. Although we have recently reported age-related changes in the respiratory capacities in different brain areas in HD mice, the precise mechanisms of how mitochondria become compromised in HD are still poorly understood. In this study, we investigated mRNA and protein levels of selected subunits of electron transport system (ETS) complexes and ATP-synthase in the cortex and striatum of symptomatic R6/2 mice. Our findings reveal a brain-region-specific differential expression of both nuclear and mitochondrial-encoded ETS components, indicating defects of transcription, translation and/or mitochondrial import of mitochondrial ETS components in R6/2 mouse brains.

A Novel Mechanism of Ataxia Telangiectasia Mutated Mediated Regulation of Chromatin Remodeling in Hypoxic Conditions.

The effects of genotoxic stress can be mediated by activation of the Ataxia Telangiectasia Mutated (ATM) kinase, under both DNA damage-dependent (including ionizing radiation), and independent (including hypoxic stress) conditions. ATM activation is complex, and primarily mediated by the lysine acetyltransferase Tip60. Epigenetic changes can regulate this Tip60-dependent activation of ATM, requiring the interaction of Tip60 with tri-methylated histone 3 lysine 9 (H3K9me3). Under hypoxic stress, the role of Tip60 in DNA damage-independent ATM activation is unknown. However, epigenetic changes dependent on the methyltransferase Suv39H1, which generates H3K9me3, have been implicated. Our results demonstrate severe hypoxic stress (0.1% oxygen) caused ATM auto-phosphorylation and activation (pS1981), H3K9me3, and elevated both Suv39H1 and Tip60 protein levels in FTC133 and HCT116 cell lines. Exploring the mechanism of ATM activation under these hypoxic conditions, siRNA-mediated Suv39H1 depletion prevented H3K9me3 induction, and Tip60 inhibition (by TH1834) blocked ATM auto-phosphorylation. While MDM2 (Mouse double minute 2) can target Suv39H1 for degradation, it can be blocked by sirtuin-1 (Sirt1). Under severe hypoxia MDM2 protein levels were unchanged, and Sirt1 levels depleted. SiRNA-mediated depletion of MDM2 revealed MDM2 dependent regulation of Suv39H1 protein stability under these conditions. We describe a novel molecular circuit regulating the heterochromatic state (H3K9me3 positive) under severe hypoxic conditions, showing that severe hypoxia-induced ATM activation maintains H3K9me3 levels by downregulating MDM2 and preventing MDM2-mediated degradation of Suv39H1. This novel mechanism is a potential anti-cancer therapeutic opportunity, which if exploited could target the hypoxic tumor cells known to drive both tumor progression and treatment resistance.

Polycystin-1 dependent regulation of polycystin-2 via GRP94, a member of HSP90 family that resides in the endoplasmic reticulum.

Autosomal dominant polycystic kidney disease is a common inherited renal disorder that results from mutations in either PKD1 or PKD2, encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively. Downregulation or overexpression of PKD1 or PKD2 in mouse models results in renal cyst formation, suggesting that the quantity of PC1 and PC2 needs to be maintained within a tight functional window to prevent cystogenesis. Here we show that enhanced PC2 expression is a common feature of PKD1 mutant tissues, in part due to an increase in Pkd2 mRNA. However, our data also suggest that more effective protein folding contributes to the augmented levels of PC2. We demonstrate that the unfolded protein response is activated in Pkd1 knockout kidneys and in Pkd1 mutant cells and that this is coupled with increased levels of GRP94, an endoplasmic reticulum protein that is a member of the HSP90 family of chaperones. GRP94 was found to physically interact with PC2 and depletion or chemical inhibition of GRP94 led to a decrease in PC2, suggesting that GRP94 serves as its chaperone. Moreover, GRP94 is acetylated and binds to histone deacetylase 6 (HDAC6), a known deacetylase and activator of HSP90 proteins. Inhibition of HDAC6 decreased PC2 suggesting that HDAC6 and GRP94 work together to regulate PC2 levels. Lastly, we showed that inhibition of GRP94 prevents cAMP-induced cyst formation in vitro. Taken together our data uncovered a novel HDAC6-GRP94-related axis that likely participates in maintaining elevated PC2 levels in Pkd1 mutant cells.

1807P Pan-cancer analysis of SMARCA4 genomic alterations to reveal a complex molecular landscape

SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4 (SMARCA4), encoding a subunit of the ATPase subunit of the SWI/SNF chromatin-remodeling complex, is correlated with the chromatin remodeling. Previous studies revealed that functional loss mutations of SMARCA4 result in reduced cyclin D1 expression and selective sensitivity to CDK4/6 inhibitors. The differences in mutation profiles in pan-cancer can help understand pathogenesis, prognosis, and identify targets for therapy.

Activity of the yeast vacuolar TRP channel TRPY1 is inhibited by Ca2+–calmodulin binding

Transient receptor potential (TRP) cation channels, which are conserved across mammals, flies, fish, sea squirts, worms, and fungi, essentially contribute to cellular Ca2+ signaling. The activity of the unique TRP channel in yeast, TRP yeast channel 1 (TRPY1), relies on the vacuolar and cytoplasmic Ca2+ concentration. However, the mechanism(s) of Ca2+-dependent regulation of TRPY1 and possible contribution(s) of Ca2+-binding proteins are yet not well understood. Our results demonstrate a Ca2+-dependent binding of yeast calmodulin (CaM) to TRPY1. TRPY1 activity was increased in the cmd1–6 yeast strain, carrying a non–Ca2+-binding CaM mutant, compared with the parent strain expressing wt CaM (Cmd1). Expression of Cmd1 in cmd1–6 yeast rescued the wt phenotype. In addition, in human embryonic kidney 293 cells, hypertonic shock-induced TRPY1-dependent Ca2+ influx and Ca2+ release were increased by the CaM antagonist ophiobolin A. We found that coexpression of mammalian CaM impeded the activity of TRPY1 by reinforcing effects of endogenous CaM. Finally, inhibition of TRPY1 by Ca2+–CaM required the cytoplasmic amino acid stretch E33–Y92. In summary, our results show that TRPY1 is under inhibitory control of Ca2+–CaM and that mammalian CaM can replace yeast CaM for this inhibition. These findings add TRPY1 to the innumerable cellular proteins, which include a variety of ion channels, that use CaM as a constitutive or dissociable Ca2+-sensing subunit, and contribute to a better understanding of the modulatory mechanisms of Ca2+–CaM.

Behaviorally and environmentally induced non-24-hour sleep-wake rhythm disorder in sighted patients.

To determine whether there was evidence of circadian or sleep-regulatory dysfunction in sighted individuals with non-24-hour sleep-wake rhythm disorder. Three sighted individuals with signs and/or symptoms of non-24-hour sleep-wake rhythm disorder were studied. Thirty-five- to 332-day laboratory and home-based assessments of sleep-wake and circadian timing, endogenous circadian period, photic input to the circadian pacemaker, and/or circadian and sleep-wake-dependent regulation of sleep were conducted. No evidence of circadian dysfunction was found in these individuals. Instead, sleep-wake timing appeared to dissociate from the circadian timing system, and/or self-selected sleep-wake and associated light/dark timing shifted the circadian pacemaker later, rather than the circadian pacemaker determining sleep-wake timing. These findings suggest that the etiology of this disorder may be light- and/or behaviorally induced in some sighted people, which has implications for the successful treatment of this disorder. Emens JS, St Hilaire MA, Klerman EB, etal. Behaviorally and environmentally induced non-24-hour sleep-wake rhythm disorder in sighted patients. J Clin Sleep Med. 2022;18(2):453-459.

Aldosterone suppresses cardiac mitochondria

Elevated serum aldosterone promotes arterial hypertension, cardiac hypertrophy, and diastolic dysfunction. However, the effect of elevated aldosterone levels on cardiac mitochondria remains unclear. We used primary cultures of mouse cardiomyocytes to determine whether aldosterone has direct effects on cardiomyocyte mitochondria, and aldosterone-infused mice as a preclinical model to evaluate the impact of aldosterone in vivo. We show that aldosterone suppressed mtDNA copy number and SOD2 expression via the mineralocorticoid receptor (MR)-dependent regulation of NADPH oxidase 2 (NOX2) and generation of reactive oxygen species (ROS) in primary mouse cardiomyocytes. Aldosterone suppressed cardiac mitochondria adenosine triphosphate production, which was rescued by N-acetylcysteine. Aldosterone infusion for 4 weeks in mice suppressed the number of cardiac mitochondria, mtDNA copy number, and SOD2 protein expression. MR blockade by eplerenone or the administration of N-acetylcysteine prevented aldosterone-induced cardiac mitochondrial damage in vivo. Similarly, patients with primary aldosteronism had a lower plasma leukocyte mtDNA copy number. Plasma leukocyte mtDNA copy number was positively correlated with 24-hour urinary aldosterone level and left ventricular mass index. In conclusion, aldosterone suppresses cardiac mitochondria in vivo and directly via MR activation of ROS pathways.

A simulated strategy for analysis of Short- to Long- chain fatty acids in mouse serum beyond chemical standards

Broadening coverage in fatty acid (FA) analysis benefits the understanding of metabolic regulation in biological system. However, the limited access of chemical standards makes it challenging. In this work, we introduced a simulation assisted strategy to analyze short-, medium-, long- and very-long-chain fatty acids beyond the use of chemical standards. This targeted analysis in selected reaction monitoring (SRM) mode incorporated 3-nitrophenylhydrazine derivatization and mathematical simulation of ion transitions, collision energies, RF values and retention times to identify and quantify the fatty acids without chemical standards. Serum analysis using high resolution mass spectrometry coupled with paired labeling was employed to refine the computational retention times. Based on the simulation, 116 free fatty acids from C1 to C24 were covered in a single analysis on use of 34 standard chemicals. Background interference is commonly observed in fatty acid analysis. For certain fatty acids, e.g. acetic acid or palmitic acid, reliable quantitation is largely restricted by contamination level instead of detection limit. Therefore, the background interference and quantifiable serum volume required for each fatty acid were also evaluated. At least 20 µL serum was suggested to cover most molecules. Using this approach, a total of 66 free fatty acids with various chain lengths and saturations were detected in NTCP knockout mice serum, of which 34 FAs were confirmed by chemical standards and 32 FAs were potentially assigned based on the simulation. Gender dependent fatty acid regulation was observed by NTCP knockout. This work provides a unique strategy that enables to broaden the fatty acid coverage with the absence of chemical standards and is applicable to other derivatizations.

Near-UV Light Induced ROS Production Initiates Spatial Ca2+ Spiking to Fire NFATc3 Translocation.

Ca2+-dependent gene regulation controls several functions to determine the fate of the cells. Proteins of the nuclear factor of activated T-cells (NFAT) family are Ca2+ sensitive transcription factors that control the cell growth, proliferation and insulin secretion in β-cells. Translocation of NFAT proteins to the nucleus occurs in a sequence of events that starts with activating calmodulin-dependent phosphatase calcineurin in a Ca2+-dependent manner, which dephosphorylates the NFAT proteins and leads to their translocation to the nucleus. Here, we examined the role of IP3-generating agonists and near-UV light in the induction of NFATc3 migration to the nucleus in the pancreatic β-cell line INS-1. Our results show that IP3 generation yields cytosolic Ca2+ rise and NFATc3 translocation. Moreover, near-UV light exposure generates reactive oxygen species (ROS), resulting in cytosolic Ca2+ spiking via the L-type Ca2+ channel and triggers NFATc3 translocation to the nucleus. Using the mitochondria as a Ca2+ buffering tool, we showed that ROS-induced cytosolic Ca2+ spiking, not the ROS themselves, was the triggering mechanism of nuclear import of NFATc3. Collectively, this study reveals the mechanism of near-UV light induced NFATc3 migration.