bluuebunny/crossref_metadata_2025 huggingface dataset:
Elucidating the Mechanisms of CaMKII‐CaMKAP Interactions
Authors: Quay Juliana , Gonçalves Michael , Yang Qian , Colbran Roger
Published: May 2022

Dynamic changes of intracellular calcium ion (Ca2+) concentrations are critical signaling mechanisms in all cells and organisms, and Ca2+ oscillations must be precisely decoded by intracellular signaling molecules, such as the ubiquitous Ca2+/calmodulin‐dependent protein kinase II (CaMKII). Four mammalian CaMKII genes encode diverse alternative mRNA splice variants, which are thought to be targeted to different subcellular compartments to regulate numerous cell responses (exocytosis, gene expression, cell cycle). Our lab hypothesized that the localization, activity, and biological functions of CaMKII are precisely modulated by CaMKII‐associated proteins (CaMKAPs), and we have detected over 100 CaMKAPs associated with brain CaMKII holoenzymes. Some CaMKII‐CaMKAP interactions are critical for normal physiological function. CaMKIIδ binding to the L‐type Ca2+ channel (LTCC) b2a subunit is essential for cardiac function1, and binding of neuronal CaMKII to the GluN2B subunit of the NMDA‐type glutamate receptor is necessary for synaptic plasticity2. While b2a, GluN2B and most other CaMKAPs preferentially interact with activated CaMKII, the exact molecular mechanisms for these interactions, and their physiological roles, have yet to be determined. Several residues in the catalytic domain of CaMKII have been implicated in CaMKAP binding. Previous mutagenesis studies from our lab indicated that mutation of valine 102 (V102) to glutamate disrupts CaMKII binding to LTCC α1 subunits, but not to b2a, GluN2B, or densin, and also disrupts neuronal excitation‐transcription coupling3. Additionally, a naturally‐occurring CaMKIIα Glu183Val mutation linked to autism spectrum disorder and intellectual disability disrupted interactions with all CaMKAPs tested4. To further probe the molecular basis of CaMKII‐CaMKAP interactions, we are exploiting available structural information to design additional CaMKII catalytic domain mutations, and then test their effect on CaMKII‐CaMKAP interactions, as well as CaMKII activity toward multiple substrates. We are also examining the impact of additional human CaMKII mutations. Initial studies indicate that some CaMKII mutations differentially affect binding to various CaMKAPs, CaMKII autophosphorylation, and phosphorylation of a representative substrate, the GluA1 subunit of AMPA‐type glutamate receptors. Our ongoing studies are further exploring the impact of these CaMKII mutations. Expanding our knowledge of mechanisms underlying CaMKII‐CaMKAP interactions is required to better understand CaMKII biology, the role of CaMKAPs, and the molecular basis of Ca2+signaling.

Koval et al., 2010. PNAS 107:4996.

Halt et al., 2012. EMBO J. 31:1203.

Wang et al., 2017. J Biol Chem 292:17324.

Stephenson et al., 2017. J Neurosci 37:2216.
Characterization of the Nickel‐inserting Cyclometallase LarC from Moorella thermoacetica and Identification of a CMPylated Reaction Intermediate
Authors: Turmo Aiko , Sui Dexin , Kim Kelly H. , Hu Jian , Hausinger Robert P.
Published: May 2022

The nickel‐pincer nucleotide (NPN) cofactor is required for lactate racemase (LarA) activity1, and LarB, LarE, and LarC are needed to generate this coenzyme2. LarC catalyzes the last step of the biosynthesis pathway by inserting nickel into pyridinium‐3,5‐dithiocarboxylic acid mononucleotide (P2TMN), resulting in the mature NPN cofactor3. Previously, LarC from Lactobacillus plantarum was shown to be a CTP‐dependent, single‐turnover nickel insertase or cyclometallase, but a structure was only obtained for its C‐terminal domain and its reaction mechanism was not identified. We characterized LarC from Moorella thermoacetica(LarCMt) and identified a novel reaction intermediate in which the precursor P2TMN is CMPylated. We propose that LarCMt uses CTP to generate the CMP‐P2TMN species that is properly positioned in the active site for nickel insertion, analogous to the molybdenum insertion step during molybdenum cofactor biosynthesis4. LarCMt variants were characterized to further elucidate the mechanism of the transient CMPylation process. Additionally, we generated preliminary structural data for the full‐length LarCMt through cryo‐electron microscopy. Our results highlight the role of CTP in the nickel insertion reaction and provide a better structural and mechanistic understanding of the LarC reaction.

1. Desguin, B. et al. A tethered niacin‐derived pincer complex with a nickel‐carbon bond in lactate racemase. Science. 349, 66–69 (2015).

2. Desguin, B. et al. Lactate racemase is a nickel‐dependent enzyme activated by a widespread maturation system. Nat. Commun. 5, 3615 (2014).

3. Desguin, B. et al. Biosynthesis of the nickel‐pincer nucleotide cofactor of lactate racemase requires a CTP‐dependent cyclometallase. J. Biol. Chem. 293, 12303–12317 (2018).

4. Llamas, A., Mendel, R. R. & Schwarz, G. Synthesis of adenylated molybdopterin: An essential step for molybdenum insertion. J. Biol. Chem. 279, 55241–55246 (2004).
Florid Lymphovascular Invasion of Inflammatory Breast Cancer is Due to Geometric Embolic Budding
Authors: Modi Arnav P. , Nguen Julie P. T. , Wang Justin , Ahn Jonathan S. , Libling William A. , Klein Jacob M. , Mazumder Preeanka , Barsky Sanford H.
Published: May 2022

The sina qua non of inflammatory breast cancer (IBC) is numerous tumor emboli especially within overlying dermal lymphatics. The explanation remains a mystery. In human studies comparing IBC with non‐IBC, although common tumor parameters such as Ki‐67 index, mitotic count and nuclear size show a close overlap, there is a dramatic exponential difference in embolic density in IBC v non‐IBC. Using contrasting properties of two IBC xenografts, Mary‐X and Karen‐X, this study offers a novel explanation and proposed mechanism to explain the very high embolic numbers in IBC. The explanation is geometric budding of emboli. Mary‐X exhibits florid lymphovascular invasion in vivo which give rise to high numbers of CTCs and pulmonary metastases whereas Karen‐X lacks these features. Mary‐X also gives rise to compact spheroids in vitrowhich exhibit dramatic budding whereas Karen‐X exhibits only loose non‐budding aggregates. Furthermore Mary‐X emboli also bud into daughter emboli in vivo. The mechanism that regulates the compactness of the spheroids as well as the emboli involves the generation of E‐cad/NTF1, a calpain‐mediated cleavage product of membrane E‐cadherin. Inhibiting the generation of E‐cad/NTF1 by blocking either the calpain site of cleavage (SC) or the site of binding (SB) with specific decapeptides both reduces spheroid compactness and decreases budding. Since E‐cad/NFT1 retains the p120ctn binding site but loses both the β‐catenin and α‐binding sites, promoting its 360° distribution around the cell’s membrane, the varying levels of expression of this truncated molecule may trigger budding of both the spheroids as well as the emboli. Recurrent and geometric budding of parental emboli into daughter emboli then would account for the plethora of IBC tumor emboli seen in patients.
An analysis of Course‐based Undergraduate Research Experience (CURE) efficacy at community colleges, primarily undergraduate, and research institutions
Authors: Wolyniak Michael J. , Zhang Jing , DeChenne‐Peters Sue Elen , Kuhn Misty L. , Hecht David , Koletar Courtney , Galport Nicole , Eddy Rebecca , Provost Joseph J. , Bell Jessica , Bell J. E.
Published: May 2022

Course‐based Undergraduate Research Experiences (CUREs) have emerged as a well supported high‐impact practice for improving both retention of students in science, technology, engineering, and mathematics (STEM) disciplines and overall achievement of students in STEM coursework. While considerable scholarship has been performed on the overall efficacy of CUREs as a teaching approach, comparatively little work has been done to uncover the more nuanced data on how CUREs serve students at different types of undergraduate institutions (community colleges, primarily‐undergraduate institutions (PUIs), and research‐intensive institutions) as well as students characterized as Persons Excluded due to Ethnicity or Race (PEER). In this study, we leverage the Malate Dehydrogenase CURE Community, a CURE network focused on bringing research experiences in protein biochemistry to undergraduates, to collect data on student achievement and attitudes. Using several validated assessment instruments, we considered how effective the MCC CURE experience was at each type of institution as well as for PEER students versus their White/Asian counterparts. Our data revealed that the CURE approach to instruction was especially effective at PUIs, with significantly‐higher levels of student collaboration and overall positive attitudes about STEM careers found in comparison to community colleges and research‐intensive institutions. Community colleges reported the most significant gains with respect to PEER student achievement relative to White/Asian students as well as enthusiasm among PEER students for conducting future research. Taken together, the data suggest that while all undergraduates may expect to benefit from exposure to CUREs, the specific benefits vary by the type of institution as well as the PEER status of student. The outcomes of this research will ultimately enable institutions to provide biochemistry‐focused high‐impact educational experiences for undergraduates tailored to the needs of these institutions to enable them to become a well‐equipped next generation of life scientists.
Assessing the effects of COVID‐19‐induced hypoxia and ACE2 signaling on the human blood‐brain barrier in‐vitro
Authors: Pervaiz Iqra , Varahachalam Sree Pooja , Al‐Ahmad Abraham J.
Published: May 2022

Introduction

Coronavirus disease 2019 (COVID‐19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV2). The portal of entry for the virus is peptidase ACE2, a major key player of the renin‐angiotensin‐aldosterone system (RAAS), resulting in severe lung injury. Although COVID‐19 mainly manifests as an acute respiratory distress syndrome (ARDS), there is increasing evidence of neurological symptoms in patients infected by COVID‐19. Yet, there is limited understanding of how COVID‐19 impacts the central nervous system (CNS). We speculate that such neurological symptoms maybe a consequence of a dysfunction of the blood‐brain barrier (BBB) with our central hypothesis that the neurological effects of SARS CoV‐2 are driven by chronic hypoxic stress‐impaired ACE2 at the BBB.

Methods

An in‐vitro human induced pluripotent stem cells (hiPSCs) BBB model was used in the study. Such model was exposed to various levels of hypoxia (1,5 and 10%) for up to 24 hours. In addition, normoxic cells were treated with Angiotensin II (AngII) or Angiotensin 1‐7 (degradation by product of AngII by ACE2). Changes in the barrier function was assessed using TEER, permeability to fluorescein and tight junction staining. Changes in ACE2 and MasR expression was assessed by immunofluorescence, whereas ACE2 shedding and HIF‐1 alpha expression was assessed by ELISA.

Results

Mild (10%) hypoxia was sufficient to induce the loss of barrier function. Secretion of ACE2 under hypoxia followed a biphasic pattern, with highest levels at 5% and 10%. Ang II and Ang1‐7 had little effect on the barrier function under normoxic condition. The hypoxic exposure induced shedding of the membrane bound ACE2 and molecular mechanism of hypoxic exposure in regulation of ACE2 occurs in a HIF1α‐ dependent manner.

Discussion

Our preliminary data suggest that our human model of the BBB responds to hypoxia and express critical components of RAAS. Both ACE2 and MasR negatively respond to mild hypoxia followed by a decreased barrier function with no changes in tight junction complex. Such loss was correlated with increased ACE2 shedding in HIF1α‐ dependent manner. We are currently investigating role of Ang 1‐7 in rescuing barrier function under hypoxic stress.
Lack of serum albumin improves insulin sensitivity in both male and female mice
Authors: Abdollahi Afsoun , Dowden Brianna N. , Buhman Kimberly K. , Zembroski Alyssa S. , Henderson Gregory C.
Published: May 2022

Insulin resistance is a global health problem. It has been shown that free fatty acids (FFA) play a major role in insulin resistance. Albumin acts as main fatty acid binding protein in blood. To improve understanding of the role of FFA in insulin resistance, we aimed to determine whether lack of serum albumin and the associated suppression of plasma FFA concentration would improve insulin sensitivity in albumin knockout mice compared to wild type mice. Male and female homozygous albumin knockout mice and C57BL/6J wild‐type controls, each fed a chow diet, were studied at 6‐8 weeks of age. Glucose tolerance test, insulin tolerance test, tissue analyses, and plasma proteomics were performed. In both sexes of albumin knockout mice compared to wild‐type mice, we observed lower levels of hepatic diacylglycerol content, as well as lower blood glucose during glucose tolerance test and insulin tolerance test (P < 0.05). The level of mRNA measurement showed higher expression of adiponectin, lipoprotein lipase, diacylglycerol acyltransferase‐1 and ‐2, and glucose transporter‐4 in adipose tissue, as well as lower hepatic perilipin‐2 expression (P<0.05). In agreement with adipose mRNA level analysis, plasma proteomics indicated that circulating adiponectin concentration was higher in albumin knockout compared to wild‐type mice (P<0.05). The results indicate an important role of albumin and plasma FFA concentration in lipid and glucose metabolism. We suggest that the lower plasma FFA concentration in albumin knockout mice compared to wild‐type mice leads to less hepatic diacylglycerol accumulation, and the lower level of this lipotoxic compound may contribute to the enhanced insulin sensitivity.
Revealing novel molecular pathways in exosome production and pathological effects in the context of autophagy deficiency
Authors: Liu Gang , Ma Michelle , Yin Xiao‐Ming
Published: May 2022

Extracellular vehicles (EVs) are defined as membrane‐surrounded, nanometer‐sized vesicles released by cells into the extracellular space in a highly regulated manner. Exosomes, a subclass of EVs with an average size of 40 to 160 nm in diameter, play critical roles in cell‐cell communications and in pathogenesis of many diseases, including liver diseases. However, the molecular mechanism of exosome production in specific disease scenario is not known. Here we use a mouse model of liver injury caused by autophagy deficiency to investigate the induction mechanism and the role of exosome in the pathogenesis.

We found that there was an increased serum presence of liver‐specific exosomes when hepatic autophagy function was disabled through nanoparticle tracking analysis (NTA). These EVs had the average size of 100 nm, and expressed hepatocyte‐derived TSG101 and CYP2E1. Notably we defined specific markers that had not been reported before, such as Syntenin‐2, which is a Syndecan‐binding protein. This molecule is closely related to the formation of multivesicular bodies (MVBs), which is considered as the precursor of EVs. Exosome production can be increased or decreased by Syntenin‐2 overexpression or knockdown, respectively, indicating that this molecule is crucial for the exosome production. In addition, increased expression of Syntenin‐2 and increased production of exosomes in autophagy deficient mice were reversed by co‐deletion of an anti‐oxidative gene, Nrf2. Thus, we have defined a new pathway from Nrf2 to Syntenin‐2 in controlling the production of a liver specific exosome in the context of autophagy deficiency.

To under the pathological role of these exosomes, blood EVs isolated from autophagy deficient mice were incubated with macrophages derived from the bone marrows (BMDM), which are recruited to the liver and contribute to the hepatic inflammation. We found that BMDM could effectively internalize the exosomes and are then activated for the production of a number of pro‐inflammatory genes compared to BMDM co‐cultured with exosomes isolated from wild type mice. Sequence profiling of miRNAs carried by these exosomes identified miR‐3072‐3p that can stimulate BMDM to upregulate the expression of the pro‐inflammatory genes, suggesting that these cells can be activated by the exosomes via miRNA‐mediated signaling.

In summary, our work has revealed novel molecular components in promoting the production of liver‐specific exosomes under autophagy deficiency, which in turn enhances the inflammatory response in the liver through a unique miRNA‐mediated pathway.
Impaired mechanotransduction of small cerebral arteries after traumatic brain injury. Role of arachidonic acid pathway dysfunction
Authors: Koller Akos , Szenasi Annamaria , Amrein Krisztina , Czeiter Endre , Szarka Nikolett , Toth Peter
Published: May 2022

Background

Healthy functioning of the brain requires a precise autoregulation of cerebral blood flow, which in part is achieved by the pressure and flow sensitive vasomotor mechanisms. Traumatic brain injury (TBI) frequently occurs worldwide, resulting in brain diseases with high morbidity and mortality. Here, we hypothesized that TBI impairs the autoregulatory mechanisms, namely the pressure‐ and flow‐induced constrictions of isolated rat middle cerebral arteries (MCAs).

Methods

TBI was induced in anaesthetized rats by weight drop model, and then MCAs were isolated and transferred into a pressure‐flow chamber. Changes in internal diameter in response to hemodynamic forces and vasoactive drugs were measured by a video‐microscopy.

Results

In MCAs from intact rats, increases in pressure‐, flow‐ and pressure + flow elicited constrictions (max.: ‐26 ± 1.7; ‐26 ± 1.9; and ‐52 ± 2.8 µm, p < 0.05). Flow‐induced constrictions were significantly reduced by HET0016, an inhibitor of cytochrome P450 4A (CYP450 4A) or in the presence of thromboxane‐prostanoid (TP receptor) antagonist SQ 29,548. Both responses were significantly reduced after TBI. Arachidonic acid, (AA, 10−7 M), and CYP450 4A metabolite 20‐hydroxyeicosatetraenoic acid (20‐HETE) elicited constrictions of intact MCA (‐26 ± 2.3% and ‐31 ± 3.6%), which were significantly reduced after TBI (to ‐11 ± 1.3% and ‐16 ±2.5%). The TP receptor agonist U46619 (10−7 M) elicited substantial constrictions of MCA from intact rats (‐21 ± 3.3%), which were also significantly reduced, after TBI (to ‐16 ± 2.4%).

Conclusions

Both pressure and flow‐induced constrictions of MCA were impaired by traumatic brain injury, likely due to the reduced ability of cytochrome P450 4A to convert arachidonic acid to constrictor prostaglandins (cPGs) and the mitigated sensitivity of thromboxane‐prostanoid receptors to cPGs. We propose that the impairment of autoregulatory vasomotor mechanisms contribute to the TBI‐induced human brain diseases, such as headache, disruption of blood brain barrier, brain edema, Alzheimer‐type diseases and vascular dementia.
Polyethylene glycol‐20 ameliorates tissue ischemia and improves renal function following traumatic shock
Authors: Xiang Lusha , Calderon Alfredo S. , Hill Ronnie , Ryan Kathy L. , Hinojosa‐Laborde Carmen , Connor Brian S. , Bynum James
Published: May 2022

The “Golden Hour” policy used for US Troops in Iraq and Afghanistan has contributed to the highest survival rates for combat injury in history. Anticipated future combat operations require more options to optimize combat casualty care by extending “the Golden Hour” to “the Golden Day.” Low volume resuscitation with polyethylene glycol‐20K (PEG) has been shown to decrease mortality following hemorrhagic shock, however, the effect of PEG on tissue perfusion and renal function has not been determined. We hypothesized that PEG can ameliorate ischemia and prevent renal injury during prolonged traumatic shock. Anesthetized rats received extremity trauma (soft tissue injury and fracture in one hindlimb) and pressure‐controlled hemorrhage, with mean arterial pressure (MAP) maintained at 55mmHg. MAP, heart rate, renal blood flow (RBF, perivascular ultrasonic transit time flow probe), peripheral skeletal muscle microcirculatory flow and tissue oxygen partial pressure (OxyLite needle probes), blood gases, and electrolytes were monitored throughout the experiment. Glomerular filtration rate (GFR) was recorded using a non‐invasive Fluorescein isothiocyanate‐sinistrin transcutaneous technique. After 45 minutes of shock with MAP at 55mmHg, rats were randomly treated with an intravenous bolus of either 7.5% PEG or saline (1.3 ml/kg, n = 5/group). Measurements were continued from the 45th to the 165th minute without MAP control or any other treatment. Compared with saline, PEG markedly improved MAP recovery (P < 0.001) and survival (P < 0.01) (Figures 1A and 1B). Microcirculatory perfusion and oxygen partial pressure were markedly decreased after hemorrhage and saline treatment elicited no additional effect. However, with PEG, the decreases in capillary flow and tissue oxygen levels were significantly attenuated (P = 0.04 and 0.03, respectively) (Figure 2A). Moreover, blood lactate levels continuously increased in the saline group but returned to levels comparable to baseline within 30 minutes after PEG treatment (P < 0.001) (Figure 2B). In the saline group, RBF markedly decreased concomitantly with a sustained increase in renal vascular resistance (RVR) during the shock period. PEG treatment rapidly restored RBF (P < 0.001) to levels comparable to baseline along with a normalized RVR. GFR was diminished in the saline‐treated rats but was improved in the PEG‐treated group (0.07 ± 0.05 vs. 0.67 ± 0.06 ml/min/100g, respectively; P < 0.001). These results provide strong evidence that PEG rapidly improves macro‐ and microcirculatory hemodynamics ameliorates tissue ischemia, and minimize renal injury and mortality following prolonged traumatic shock.
Estrogen Sulfotransferase is Required for the Female Mice Sensitivity to T Cell‐Mediated Hepatitis in an Estrogen‐Independent Manner
Authors: Wang Jingyuan , Guan Jibin , Zhang Ziteng , Xie Wen
Published: May 2022

Introduction

Autoimmune hepatitis (AIH) is a severe T cell‐mediated chronic liver disease with a higher incidence in females. However, the pathological mechanism of AIH remains elusive and there is no effective therapy except for nonspecific immunosuppression. Estrogen sulfotransferase (EST or SULT1E1) is a phase II enzyme best known for its function in sulfonating and deactivating estrogens. Previous work from our lab showed that EST is inducible and functional in several other inflammation diseases. However, the possible role and mechanism of EST in T‐cell mediated hepatitis remain unknown. The goal of this study is to investigate the role of EST in the female preference of T‐cell mediated hepatitis.

Methods

Concanavalin A (ConA) was used to induce T cell‐mediated hepatitis in female C57BL/6J mice. For Est loss of function models, we used the whole‐body or liver‐specific Est knockout mice, or treating WT mice with the Est inhibitor Triclosan. We also used Est whole‐body knockout mice with Est specifically reconstituted in the hepatocytes. The severity of liver injury was evaluated through the serum levels of Alanine transaminase (ALT), Aspartate transaminase (AST) and the necrosis in liver histology. Inflammatory responses were characterized by the levels of cytokines and infiltrated immune cells in the liver by real‐time PCR, ELISA, and flow cytometry. To test the role of estrogens in the regulation, we depleted estrogens by ovariectomy in female mice prior to the ConA challenging.

Results

Est was highly induced in the liver of ConA‐treated mice. Knockout or pharmacological inhibition of Est significantly protected the female mice from ConA‐induced hepatitis regardless of ovariectomy. Moreover, ESTKO mice exhibited a more robust inflammatory response with a larger spleen‐to‐body‐weight ratio and a higher level of pro‐inflammatory cytokines production. ESTKO mice had a higher frequency of CD4+ T cells, including a higher level of Treg population, and less cytotoxic CD8+ T cells. The protective phenotype of ESTKO mice was abolished when the expression of EST was reconstituted in the hepatocytes.

Conclusions

Hepatic EST is required for female sensitivity to Con A‐induced T‐cell mediated hepatitis in an estrogen‐independent manner. Pharmacological inhibition of EST might be a potential strategy for the treatment of AIH.
Antisense oligonucleotides (ASO) to increase progranulin production in frontotemporal dementia
Authors: Ebenezer Sarah , Ebenezer Joel , Nguyen Andrew
Published: May 2022

Frontotemporal Dementia (FTD) is a debilitating disease that causes the individual to slowly lose their personalities, emotions, and control over social behaviors as disease progresses. FTD is caused due to significant neuronal loss in the frontal and temporal lobes of the brain. Though there is no treatment or cure yet for FTD, research has identified that the protein progranulin may be responsible for this neuronal loss, as a lack of progranulin has been linked to the degradation of neurons in FTD patients. Therefore, an effective treatment for FTD must aim to increase the production of progranulin in the brain. Previous research papers (Jiao J et al, 2010*) found that MicroRNA 29 (miR‐29) was responsible for regulating the translation of hPGRN (human progranulin) RNA through binding to the 3’UTR and preventing translation, suggesting that preventing miR‐29 from binding to the 3’UTR of hPGRN RNA should increase the overall production of progranulin. Antisense oligonucleotides (ASO) achieve this through binding to the hPGRN RNA and disrupting the binding site of miR‐29 and therefore preventing miR‐29 from binding. In our study, neuroblastoma cells were transfected with luciferase DNA as a surrogate measure of progranulin translation and then treated with increasing concentrations of M5 ASO and evaluated for luciferase expression. The results indicated that there was an increase in luciferase activity with the increasing concentrations of ASO, specifically concentrations of 10μM and 20μM which had a significant increase in progranulin production. Further tests were done in an attempt to confirm that the ASOs are working through binding to the 3’UTR and preventing miR‐29 from binding, however these tests came back inconclusive. These findings provide a beneficial insight into further development of these ASOs as possible treatments and therapies for FTD.

*Jiao J, Herl LD, Farese RV, Gao FB. MicroRNA‐29b regulates the expression level of human progranulin, a secreted glycoprotein implicated in frontotemporal dementia. PLoS One. 2010 May 10;5(5):e10551
Differential expression T4‐5’‐deiodinase activity in corpulent rats following cold exposure
Authors: Tulp Orien L. , Outtrim Deborah , Einstein George P. , Awan Aftab R.
Published: May 2022

Multiple studies indicate that the thermic responses to diet and cold exposure may be impaired in obese rodents and are likely secondary to the combined contributions of sympathetic and thyroidal components of nonshivering thermogenesis (NST). T4 is normally deiodinated via outer ring deiodinase activity by D‐1 and/or D‐2 deiodinase activity to form hormonaly active T3 during fed and cold environments, or inner ring deiodinase activity via D‐3 to form reverse T3 (rT3) during food or energy deprivation. The effect of 14 hours of 4°C cold exposure on the obese phenotype of the LA/Ntul//‐cp rats was determined in normally reared young female animals 8 to 9 weeks of age and fed the Purina Chow #5012 ad libitum throughout. Body weights and adipose tissue mass were greater in the obese than the lean phenotype (p=<0.05). RMR and the dose related thermic responses to norepinephrine (NE, 200 ug., s.c.) were greater in lean than in obese. Cold exposure at 4°C resulted in decreases in rectal but not core temperature in obese rats, and the thermic responses to 45 minutes of 4°C cold exposure on VO2 were typical but were significantly greater in lean than obese animals at all time points measured. Circulating T4 concentrations were similar in lean and obese rats and serum T3 but not T4 concentrations increased dramatically in both phenotypes following the cold exposure, consistent with phenotype‐ and maximal IBAT‐linked changes in T4‐5’‐deiodinase activity / mg protein in kidney, liver and IBAT and in temperature linked increases in IBAT deiodinase activity / mg tissue protein in this strain. When D‐1 and D‐2 deiodinase activity was computed / tissue mass, further analysis indicated that D‐1 was the predominating deiodinase in liver, kidney, and gastrocnemius muscle, while in IBAT D‐2 > D‐1 activity / IBAT depot and was greater in obese than lean rats. Cold exposure was associated with modest increases in net kidney deiodinase activity only in kidney. Thus, the cold induced increases in circulating T3 in lean and obese rats are likely attributed at least in part to modest increases in renal T4‐5’ D‐2 deiodinase activity, in addition to maximal rates of conversion in other tissues in possible combination with decreases in hormone clearance rates during cold induced stress.
Anti‐obesity effect of Premature Citrus Extract in high‐fat diet induced obesity mice
Authors: Natraj Premkumar , Rajan Priyanka , Ranaweera Sachithra S. , Dayarathne Lakshi A. , Han Chang‐Hoon
Published: May 2022

Background

Obesity is a worldwide health threat. Premature citrus extract (PCE) are known to contain high concentrations of flavonoids, polyphenols, and organic acids with health functions. The current study aimed to investigate the anti‐obesity effect of PCE on a high‐fat diet (HFD)‐induced obesity mice.

Method

Mice were fed on chow or HFD with or without Orlistat (ORL), Hesperidin (HES), and PCE treatment for 10 weeks. Triglyceride (TG) content and total cholesterol (TC), and low‐density lipoprotein cholesterol (LDL‐c), and high‐density lipoprotein cholesterol (HDL‐c) levels were determined by TG, HDL and LDL Assay Kit (BioAssay System). The phosphorylation levels of AMP‐activated protein kinase (AMPK) and acetyl Co‐A carboxylase (ACC); the expression of sterol regulatory element binding protein‐2 (SREBP‐2), 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase (HMGCR), Acyl‐coenzyme A: cholesterol acyltransferases (ACAT‐1) and mitochondrial uncoupling proteins (UCP‐3) were observed via western blot analysis.

Results

PCE and HES administered group significantly reduced the weights of body, liver, and white adipose tissue compared to the HFD group. Supplementation with PCE and HES significantly decreased the levels of TG, TC, and (LDL‐c), and increased (HDL‐c) levels in serum compared to the HFD group. Furthermore, treatment with PCE and HES significantly decreased the levels of TG and TC in the liver whereas increased the levels of TG and TC in feces compared to the HFD group. Western blot analysis revealed that PCE and HES increased the phosphorylation levels of both AMPK and ACC and significantly decreased the expression levels of SREBP‐2, HMGCR, and ACAT‐1 in the liver compared to the HFD group. In addition, both PCE and HES increased the expression level of UCP‐3 in skeletal muscle tissue.

Conclusion

Overall results suggest that PCE and HES exerts an anti‐obesity effect via activation of AMPK and associated signaling cascades related to lipid metabolism in HFD‐induced obesity mice
SHIP164 is a Chorein Motif Lipid Transfer Protein that Controls Endosome‐Golgi Membrane Traffic
Authors: Hanna Michael G. , Suen Patreece , Wu Yumei , Reinisch Karin M. , De Camilli Pietro
Published: May 2022

Recent studies have shown that proteins with an N‐terminal chorein motif are lipid transport proteins and further suggested that they mediate lipid transfer between closely apposed membranes by a novel bridge‐like mechanism. Within these proteins, the chorein motif represents the beginning of a rod‐like structure that harbors a hydrophobic groove running along its entire length. Lipids are thought to transfer between bilayers by sliding along this groove (PMID: 34216812). VPS13 and ATG2 are founding members of this family. Another protein with a predicted N‐terminal chorein motif is SHIP164 (UHRF1BP1L), previously identified as a Syntaxin 6‐binding protein (PMID: 20163565) and as a Parkinson’s Disease (PD) susceptibility candidate gene (PMID: 28137300). Here, we first explored whether the similarity of SHIP164 to VPS13 and ATG2 extends beyond the chorein domain and determined by cryo‐EM that SHIP164, like these two proteins, has an elongated hollow rod‐like structure. We also found that SHIP164 harbors glycerolipids in an aqueous environment and transports them between membrane bilayers in a liposome‐based assay. We further investigated the localization and physiological function of SHIP164 in mammalian cells. We found that endogenous SHIP164 is localized to cation‐independent mannose‐6‐phosphate receptor (MPR)‐positive vesicular structures at the cell periphery and that its over‐expression induces the abnormal formation of dynamic clusters of small 50‐70nm vesicles enriched in these receptors and anchored to endosomes. We also found that SHIP164 interacts with proteins implicated retrograde microtubule‐based transport from endosomes to the Golgi complex: the dynein/dynactin accessory factor LC8 and dynein interactor Rab45/RASEF. Accordingly, in SHIP164 knockout (KO) RPE‐1 cells, the traffic of MPR and Sortilin positive vesicles to the Golgi complex is partially impaired. An additional defect observed in SHIP164 KO cells is the absence of the largest endosomes positive for PI3P and EEA1. In view of the lipid harboring properties of SHIP164 it is tempting to speculate that the latter phenotype may be explained by its lipid transport properties that, as in the case of VPS13 and ATG2, may allow membrane expansion. Direct evidence for such a model is missing, although, we found that SHIP164 positive vesicles are also positive for ATG9, the autophagy factor recently found to function as a scramblase in partnership with ATG2 on autophagosome membranes. An attractive possibility is that SHIP164 may participate in the regulation of endocytic flow by acting in partnership with ATG9 in the control of phospholipids/protein ratio on the membranes of endocytic vesicles.
Moringa oleifera extract protects against rhabdomyolysis‐induced acute kidney injury
Authors: Falayi Olufunke , Etim Utibe , Ogunpolu Blessing , Oyagbemi Ademola , Omobowale Temidayo , Adedapo Adeolu
Published: May 2022

Acute kidney injury (AKI) is a life‐threatening abrupt disruption in kidney function. The pathophysiological mechanisms of AKI are poorly understood. Also, there are currently no effective drugs to treat AKI. Phytochemicals from Moringa oleifera plants exhibit antihypertensive, cholesterol‐lowering, diuretic, and hepatoprotective properties. This study aims to determine if Moringa oleifera extract can ameliorate rhabdomyolysis‐induced AKI and its possible mechanisms of action.

Experimental rats were randomly divided into five groups. Rhabdomyolysis was induced by intramuscular glycerol injection. Group A received distilled water only. Group B received distilled water, followed by 50% glycerol on the 8th day. Group C and D received 50 and 100 mg/kg methanol stem extract of Moringa oleifera (orally) for seven days and glycerol on the 8th day. Group E received 100 mg/kg of methanol stem extract of Moringa oleifera alone for seven days and normal saline on the 8th day. Necropsy was performed 24 h after glycerol treatment. Blood and kidneys samples were collected for biochemical, histopathological, and immunohistochemical analyses.

Significant decreases in superoxide dismutase, glutathione peroxidase, glutathione S‐transferase, glutathione, protein thiols, and non‐protein thiols were observed in animals treated with only glycerol when compared with the control groups. Glycerol treatment also caused a significant increase in markers of AKI and inflammation, including creatinine, nitric oxide, urea, hydrogen peroxide, malondialdehyde, protein carbonyl, and advanced oxidative protein products. Immunohistochemical staining of renal NF‐ƙβ and KIM‐1 was also elevated in glycerol‐treated rats. Treatment with methanol stem bark extract of Moringa oleifera ameliorated glycerol‐induced oxidative stress, kidney damage, and inflammation.

The methanol stem extract of Moringa oleifera alleviated rhabdomyolysis‐induced AKI in rats through its antioxidant and anti‐inflammatory properties.
A novel allosteric PDE4B inhibitor T2409 improves mild cognitive impairment: involvement of cAMP related anti‐inflammatory pathway
Authors: Yan Yuqing , Zhao Yuhan , Avasthi Shivani , Zhou Sicheng , O’Donnell James M. , Xu Ying
Published: May 2022

Disorders affecting memory and cognition including Alzheimer’s disease (AD) affect millions of Americans. Although dozens of compounds have been reported for the treatment of mild cognitive deficits (MCI) in Alzheimer’s disease (AD), these drugs have not proven beneficial in ameliorating cognitive impairment in clinic. Phosphodiesterase 4B (PDE4B) plays a crucial role in the mediation of memory due to its primary role in hydrolyzing cyclic adenosine monophosphate (cAMP). However, the development of PDE4B inhibitors is challengeable due to the high conservation of the catalytic site of PDE4, across subtypes, to which classical inhibitors bind. The present study suggested that the newly synthesized allosteric inhibitor of PDE4, T2409, exhibits at least 150‐fold PDE4B selectivity. It exhibited low potency in a mouse surrogate model of emesis and nausea, suggesting its low side effects. Chronic treatment of T2409 for 14 days reversed cognitive and memory deficits in the novel object recognition and Morris water maze tests as evidenced by increased discrimination index and reduced the mean latency to platform via decreasing beta amyloid plaques both in the cortex and hippocampus in 8‐month‐old APP/PS1 mouse model of AD. Further study showed that these effects were related to decreased inflammatory factors, such as IL‐6, IL‐1β and TNF‐α, in the brain, and increased cAMP signaling dependent markers of neuroprotection associated with memory, such as p‐CREB and BDNF. These results accelerate the development of highly selective PDE4B inhibitors as novel treatments for illnesses that affect memory and cognitive function, such as AD.
Evolutionary Origins of Mammalian Axial Function Revealed through Digital Bending Experiments
Authors: Jones Katrina E. , Angielczyk Kenneth D. , Pierce Stephanie E.
Published: May 2022

The origin of mammals from non‐mammalian synapsids represents an iconic locomotor transition, often described as shift from reptile‐like sprawling limbs and extensive lateral movements of the backbone to upright limbs and sagittal backbone movements that enhance mammal specific locomotor behaviors. However, recent research in comparative anatomy has called into question the lateral‐to‐sagittal functional shift, instead suggesting that vertebral morphology in basal synapsids implies a distinct functional regime not observed in extant sprawling reptiles and represents a unique ancestral condition from which mammals evolved. To investigate this idea further, we used Autobend,a novel, experimentally validated technique for estimating vertebral osteological range of motion (oROM) from skeletons using digital modeling. We applied Autobendto seven extant mammal and reptile species and 11 exceptionally preserved non‐mammalian synapsids to estimate vertebral oROM and intervertebral joint stiffness. Results revealed a clear distinction between extant mammals and reptiles in oROM, with reptiles emphasizing lateral bending and mammals sagittal bending as expected. While most extant taxa exhibited relatively similar levels of stiffness in lateral and sagittal directions, extant lizards and salamanders displayed much more compliance in lateral bending and lower stiffness overall, in keeping with their observed axial kinematics. Conversely, most non‐mammalian synapsids displayed an intermediate condition, with stiffer intervertebral joints than extant lizards, crocodiles, and therian mammals, but similar to the pattern recovered for the modern tuatara. The mammalian condition of sagittal mobility accompanied by axial twisting in the anterior column is first observed in the crownward tritylodontid cynodont Kayentatherium. Together, these results support previous morphology‐based assertions of a distinctive ancestral vertebral function for synapsids characterized by high stiffness without the specialization for lateral bending of extant lizards or the sagittal bending and twisting observed in mammals today. This indicates a more limited role for the axial skeleton in terrestrial locomotion relative to extant groups, and a greater focus on body support than propulsion during the initial stages of synapsid evolution. This stage was followed by the evolution of mammalian‐type mobility patterns in cynodonts, and a secondary reduction in axial stiffness in certain therian clades. Given the strong association between sagittal bending and asymmetric gaits in mammals, these results provide an important first step in reconstructing the evolution of synapsid locomotor patterns.
Increased Glycolysis and ATP Production Are Not Prerequisites for Cardiomyocytes Cell Cycle Induction
Authors: Tang Ling , Zhu Wuqiang
Published: May 2022

Background

It has been reported that metabolic switch from fatty acid oxidation to glycolysis is associated with cardiomyocyte cell cycle induction. However, it is unknown if increased glycolysis and ATP production are indispensable for cardiomyocyte cell cycle induction. Here, we aim to address this question.

Methods

Cell cycle was induced by overexpression of CCND2 gene or treatment of FGF1 (100 ng/mL) and CHIR99021 (5 µM) on human induced pluripotent stem cells‐derived cardiomyocytes (hiPSC‐CMs). Contraction inhibition was induced by treatment of verapamil (0.5 μM). Cell cycle was evaluated by immunostaining using antibodies against cell cycle markers. Real‐time ATP production and mitochondrial oxidative phosphorylation were determined by Seahorse assay. Metabolic profile was assessed by liquid chromatography–mass spectrometry.

Results

Treatment of FGF1 and CHIR99021 induces cell cycle of hiPSC‐CMs, reduced carbohydrate metabolism and real‐time ATP production from both mitochondria and cytoplasm. Data from RNA Sequencing showed this treatment increased expression of CCND2 and CDK4 and decreased expression of sarcomeric contractile related genes. Overexpression of CCND2 in hiPSC‐CMs also induces cell cycle, increased aerobic glycolysis (Warburg effect) and no change of real‐time ATP production. Inhibition of contraction via treatment of verapamil resulted in reduced overall metabolism and real‐time ATP production.

Conclusion

Our data suggest that cardiomyocyte cell cycle may be induced without increased glycolysis or real‐time ATP production.
Developing a Prenatal Acute Exposure and Medical Countermeasure Evaluation Model for Organophosphate Compounds in a Genetically Modified Mouse
Authors: Herrera Eric , Barry Timothy , Sriraman Aishwarya , Brecht Karen , DeBus Sandra , Ma Ki , Mccarren Hilary , Cadieux C. L.
Published: May 2022

Organophosphate (OPs) compounds make up the most common class of pesticides as well as chemical warfare nerve agents (CWNA), all of which covalently inhibit the active site of acetylcholinesterase (AChE), resulting in intoxication via a cholinergic crisis. While the biochemical mechanism of action resulting in acute intoxication of an exposed individual is well understood, less is known about the mechanism of OP intoxication via other exposure routes, particularly prenatal exposure. The clinical effects of non‐acute prenatal OP exposure have been previously studied primarily in cases involving diet and agricultural labor, while case studies documenting the effects on conception of acute exposure during chemical warfare attacks (Japan 1995, Syria 2013 and 2017) have also been published. Such exposure events were linked to a range of serious neurodevelopmental anomalies in children, but the underlying mechanism of action as well as the potential benefits of medical countermeasures have not been clearly elucidated. A robust human‐relevant animal model to test both is therefore essential for evaluating current and future OP countermeasures. The present study used a novel humanized mouse strain expressing human AChE and an inactivated native serum carboxylesterase to create a prenatal acute exposure and emergency medical countermeasure evaluation model. Pregnant mice were acutely exposed to an OP at a critical point in cholinergic development, generally equivalent to the first trimester in humans. Dams were challenged with either sarin or the pesticide paraoxon, followed by either a saline vehicle control or 2‐PAM and an atropine/midazolam cocktail, the currently fielded emergency treatment for OP intoxication. Mice were allowed to carry pups to term, and upon reaching maturity, cortex, cerebellum, bone marrow and gonads were collected from offspring in each exposure group. Phenotypical and histopathological evaluation of tissues and other factors in dams and offspring were evaluated. Gene expression analyses conducted for each tissue generated a detailed profile associated with each OP exposure/treatment group, providing robust criteria for model development. The resulting data demonstrated a significant beneficial impact of medical countermeasures for both sarin and paraoxon exposures, significantly reducing the amount and the scope of gene expression changes induced by OP exposure. This model provides insight into the developmental pathologies resulting from prenatal acute OP exposure as well as serves as an evaluation model for future treatments.