bluuebunny/crossref_metadata_2025 huggingface dataset:
Cerebral erythropoietin increases the hippocampal mitochondrial respiration during the postnatal development
Authors: Soliz Jorge , Arias-Reyes Christian , Laouafs Sofien , Jacobs Robert A. , Schneider Gasser Edith M.
Published: April 2020

Mitochondrial respiration is the metabolic process in which ATP is formed as a result of electron transfer and substrate oxidation. However, in addition to this classic role, mitochondrial respiration, and the concomitant production of ROS, are also required to maintain ionic cell membrane gradients, develop synaptic pruning (axonal and dendritic growth), provide effective neurotransmission, modulate signaling pathways and regulate neurogenesis and differentiation. While all these processes occur during brain development, little is known about the regulation of mitochondrial respiration in the postnatal development of hippocampal tissue. In parallel, studies conducted over the past two decades demonstrated that cerebral erythropoietin (Epo) plays a crucial role in the development of the nervous system. Remarkably, Epo‐mediated neurodevelopment requires the activation of molecular mechanisms that are tightly associated with mitochondrial functions. Inline, recent published literature demonstrated that Epo modulates mitochondrial bioenergetics and control of oxidative stress in several tissues, including the brain. In this work, we evaluated the impact of Epo in the regulation of hippocampal mitochondrial respiration of mice during postnatal development. To do so, the hippocampus of wild type (WT) and transgenic mice constitutively overexpressing Epo in the brain only (Tg21) at postnatal ages 3 (P3), 7 (P7), 14 (P14), and 21 (P21) was collected. Mitochondrial oxygen consumption was measured by using our oxygraph‐2K system (O2k‐OROBOROS). Recording of the mitochondrial oxygen consumption was evaluated in 2 mg of tissue permeabilized with saponin, after the activation of complexes I (CI), I+II (C I+II), and IV (CIV). Our results showed that at P14 and P21, Epo in hippocampal tissue: 1) increases the mitochondrial respiratory activity; 2) increases the mitochondrial respiratory capacity; 3) maximizes the respiratory efficiency; and 4) augments the number mitochondria per tissue. Additional analysis, performed by using colorimetric assay kits, revealed that Epo at P14 increases the activity of superoxide dismutase (SOD). Altogether, our results suggest that Epo promotes an increased oxidative metabolism in the hippocampus at an age when this structure undergoes a full process of development and maturation.

Support or Funding Information

Jorge Soliz is supported by the “Fonds de recherche du Quebec‐Santé” (FRQ‐S; FQ121919)
Modulating the immune response and the pericardial environment with LPS or prednisolone in the axolotl does not change the regenerative capacity of cryoinjured hearts
Authors: Pedersen Kathrine , Rasmussen Rikke Kongsgaard , Dittrich Anita , Pedersen Michael , Lauridsen Henrik
Published: April 2020

Intro

Humans are incapable of regenerating after suffering from a myocardial infarction. Instead a fibrotic scar without contractile or electrically conducting properties is formed. This results in cardiac remodeling, which steers the heart towards heart failure. It is hypothesized that the time frame and composition of infiltrating immune cells play a role in the fibrotic outcome.

A popular model organism capable of cardiac regeneration is the axolotl (Ambystoma mexicanum). It is believed that the immune response to cardiac injury in the axolotl progresses with a timing and distribution of infiltrated immune cells that supports regeneration rather than extensive inflammation and scar formation. Patients suffering from cardiovascular disease display a systemic pro‐inflammatory state, which includes an upregulation of inflammatory factors, and will likely influence the immune response mounted in the incidence of a myocardial infarction. Though fully healthy mammals are not able to functionally regenerate from cardiac injury such as a myocardial infarction, it is interesting to evaluate how a strong modulation of the immune response will affect an animal model capable of regeneration. Therefore, we have chosen to modulate the immune response in a pro‐ and anti‐inflammatory direction by injecting LPS or prednisolone respectively.

Methods

In this study 24 axolotls were given a cryoinfarction injury on the ventricle, thereby mimicking a myocardial infarction. The regenerative potential was observed in three different environments; a pro‐inflammatory (10 μg LPS), an anti‐inflammatory (20 μg prednisolone) and a saline control (amphibian Ringer’s solution) by injecting 20 μL of one of the three chosen substances into the pericardium of the axolotl every five days. LPS and prednisolone were hypothesized to alter the timing and distribution of infiltrating immune cells and inhibit regeneration. Echocardiography and quantitative histology were applied to measure function (ventricular stroke volume) and anatomical regeneration (healed volumetric fraction) of injured hearts.

Results

All treatment groups showed extensive heart regeneration and no statistically significant differences in stroke volume or healed fraction between groups were observed.

Conclusion

Since neither echocardiographic nor histological data, showed any statistically significant differences in the functional or anatomical regeneration, this brings into question the importance of the immune system during regeneration in the axolotl.
Looking for LARP6 function in the Brain
Authors: Rogers Beth , Warner Lisa
Published: April 2020

LARP6, or La‐related protein 6, is known to play a role in post‐transcriptional regulation of collagen mRNA. The La Module of LARP6 is an RNA recognition module that is comprised of a La Motif and an RRM domain. The La Module binds to a conserved stem loop structure on the 5′ UTR of collagen type 1a1 and 1a2 mRNA. Interaction between LARP6 and the 5′ UTR has been shown to increase the expression of collagen type 1. Interestingly, LARP6 mRNA is highly expressed within the brain while collagen mRNA is expressed at very low levels. The purpose of this project is to investigate the functions of LARP6 within a brain system, where the cognate function of LARP6 may differ from collagen rich tissues. Here we report on characterization of several potential model cell lines, SH‐SY5Y, PC12, bEnd.3 and LX‐2 cells, using western blot analysis, confocal microscopy, and qPCR for studying LARP6 in the brain. Once an ideal system is identified we will conduct CLIP‐seq, which will allow us to identify new RNA ligands of LARP6.

Support or Funding Information

The project described was supported by Institutional Development Awards (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grants #P20GM103408 and P20GM109095. We also acknowledge the support from The Biomolecular Research Center at Boise State with funding from the National Science Foundation, Grants # 0619793 and #0923535; the MJ Murdock Charitable Trust; and the Idaho State Board of Education.
Oncogenic activity of SLC45A2‐AMACR
Authors: Luo Jianhua , Zuo Zehua , Yu Yanping
Published: April 2020

Significant number of human cancers contains gene fusion between solute carrier family 45, member 2 (SLC45A2) and α‐methylacyl‐CoA racemase (AMACR). SLC45A2 is a transporter protein known to be overexpressed in melanoma, while AMACR is an enzyme involved in metabolism of branch fatty acid, and is known for its overexpression in several human malignancies. SLC45A2‐AMACR replaces 8 transmembrane and cytosolic domains of SLC45A2 with an intact racemase domain from AMACR. At least eight types of primary human malignancies and cell lines are positive for SLC45A2‐AMACR fusion. Our analysis showed that SLC45A2‐AMACR interacts and activates MAPK kinase pathway, and promote cell growth and cancer invasion both in vitro and in vivo. Hydrodynamic tail vein injection of “sleeping beauty” SLC45A2‐AMACR in conjunction with somatic Pten knockout produced spontaneous liver cancer in 4 months. Disruption of SLC45A2‐AMACR fusion in HUH7 and H1299 cells abrogated the transformation activity of these cancer cell lines, while over‐expression of the fusion gene in H1299 cells induced higher growth rate, mitgration and resistant to serum starvation induced cell death in vitro. As a result, we conclude that SLC45A2‐AMACR is a critical driver for human cancers.
Spinal delivery of ampakine CX717 impacts phrenic motor output in adult rats
Authors: Thakre Prajwal P. , Perim Raphael R. , Sunshine Michael D. , Tadjalli Arash , Mitchell Gordon S. , Fuller David D.
Published: April 2020

Throughout the brainstem and spinal cord, α‐amino‐3‐hydroxy‐5‐methy‐4‐isoxazole propionate receptors (AMPARs) play an essential role in respiratory motor control. Ampakines are positive allosteric modulators of AMPARs which can enhance glutamatergic currents. Our laboratory recently reported that intravenous delivery of low‐dose ampakine CX717 can evoke increases in phrenic nerve inspiratory burst amplitude. However, this increased phrenic bursting could reflect an impact of the ampakine on pre‐motor brainstem circuits or a direct action on spinal neurons. In ongoing experiments, we are testing the hypothesis that delivery of ampakines to the cervical spinal cord is sufficient to evoke a sustained increase in phrenic nerve inspiratory burst amplitude. Phrenic motor output was measured in urethane‐anesthetized, mechanically ventilated, vagotomized adult male Sprague‐Dawley rats. Along with amplitude of inspiratory phrenic motor output, arterial blood pressure and end‐tidal CO2 were recorded. For intrathecal delivery of ampakine CX717 (15μl, 500μM, dissolved in 10% HPCD), the spinal cord was exposed via a dorsal approach followed by laminectomy and durotomy to enable drug administration over the mid‐cervical spinal cord. At least 15 min of stable phrenic burst amplitude was recorded as baseline after which intrathecal CX717 was applied. The initial results (n=3 sham, n=3 CX717) indicate that spinally directed CX717 causes a gradual and sustained increase in phrenic inspiratory burst amplitude lasting up to 60 min. Conversely, intrathecal delivery of 10% HPCD (sham) had no consistent impact on phrenic burst amplitude. These preliminary results are consistent with the hypothesis that allosteric modulation of spinal AMPA receptors can increase phrenic motor output. Phrenic motor neurons express AMPA receptors and are a likely candidate for the site of CX717 action.

Support or Funding Information

5 R01 HL139708 02 (DDF)
Macrophage Class A Scavenger Receptors Bind Tumor‐Associated Carbohydrate Antigens and Promote Breast Cancer Growth and Metastases in Mice
Authors: Post Steven R. , Monzavi-Karbassi Behjatolah , Simecka Christy M. , Kumarapeli Asangi , Jousheghany Fariba , Webber Jessica , He Beixiang , Wadley Taylor , Meece Trevor , Siegel Eric R. , Kelly Thomas
Published: April 2020

Tumor‐associated macrophages (TAMs) play a key role in determining the immune response in cancer. Macrophages express pattern‐recognition receptors, such as Class A Scavenger Receptors (SR‐A), that bind a variety of different ligands and regulate macrophage immune phenotype. SR‐A expression by TAMs is thought to reflect a tumor‐promoting M2 macrophage phenotype, and the accumulation of SR‐A expressing macrophages is associated with increased tumor aggressiveness and poor patient prognosis in a variety of cancers, including breast cancer. Previous studies indicate that SR‐A ligands are present in the medium of cultured tumor cells. To test the possibility that SR‐A may directly bind to tumor cells, we developed a flow cytometry based binding assay using a soluble SR‐A (sSR‐A) protein and showed that SR‐A binds directly to breast cancer cells. This binding depended on glycosylation indicating SR‐A binds to cell‐surface glycans on tumor cells, which is important because tumor‐associated carbohydrate antigens (TACAs) are associated with immune suppression, tumor progression, and poor prognosis for breast cancer patients. The ability of SR‐A to bind TACAs was tested using carbohydrate mimetic peptides (CMPs) that are structurally similar to TACAs. sSR‐A bound to CMPs, and a CMP designed to react with mannose‐rich structures blocked sSR‐A binding to tumor cells. Because SR‐A induces an immune‐suppressive macrophage phenotype, we hypothesized that SR‐A would enhance the progression of breast cancer. Using mouse models of spontaneous breast cancer, our results show that development of palpable tumors was significantly delayed and the number of lung metastases significantly decreased in mice that do not express SR‐A (SR‐A−/−). These findings demonstrate that SR‐A binds to TACAs on breast cancer cells, and that SR‐A enhances breast cancer growth and metastases. Together with previous findings, our results indicate a model in which SR‐A binds to TACAs to induce an immune‐suppressive macrophage phenotype, which contributes to immune suppression, tumor growth, and metastases.

Support or Funding Information

This research was supported by grants from the NIH (R21CA185691), the Arkansas Breast Cancer Research Program, and a DEAP award from UAMS.
Improved Vascular Function Following 7 days of L‐Citrulline Administration in Heart Failure with Preserved Ejection Fraction
Authors: Ratchford Stephen , Bunsawat Kanokwan , Alpenglow Jeremy , Richardson Russell , Wright Josephine , Ryan John , Wray D. Walter
Published: April 2020

Background

There is increasing evidence to support the presence of peripheral vascular dysfunction in patients with heart failure with preserved ejection fraction (HFpEF), which may be due, at least in part, to disease‐related changes in nitric oxide (NO) signaling. L‐Arginine (L‐Arg) is the sole substrate for NO synthesis, but is susceptible to intestinal and hepatic degradation via arginase when administered orally. L‐Citrulline (L‐Cit) is catabolized to L‐Arg at the tissue level and is not a substrate for arginase, and therefore enteral L‐Cit administration has emerged as an effective approach for increasing NO substrate bioavailability.

Purpose

The present investigation sought to determine whether oral L‐Cit administration (6g/day for 7 days) would improve vascular function in patients with HFpEF.

Methods

In 13 patients NYHA Class II (n=9) and III (n=4) with HFpEF (2M/11F, 69±10y, EF: 66±7%), flow mediated dilation (FMD) and reactive hyperemia (RH) were evaluated to assess conduit artery and microvascular function, respectively. FMD was expressed as % change in brachial artery diameter (%FMD) and % change corrected for shear rate (%FMD/shear), and RH was calculated as area‐under‐the‐curve for brachial artery blood flow. Plasma L‐Cit and L‐Arg concentrations were assessed to confirm absorption and evaluate circulating NO substrate, and the ratio of L‐Arg to plasma asymmetric dimethylarginine (ADMA) was calculated as a biomarker of NO bioavailability.

Results

Compared to baseline, L‐Cit administration improved FMD (0d: 2.5 ± 0.5%, 7d: 4.7 ± 0.9%), FMD/shear (0d: 0.07 ± 0.02, 7d: 0.12 ± 0.01AU) and RH (0d: 450 ± 46ml, 7d: 574 ± 62ml). L‐Cit (0d: 42 ± 3μM/L, 7d: 370 ± 60 μM/L), L‐Arg (0d: 65 ± 8μM/L, 7d: 257 ± 25μM/L), and L‐Arg:ADMA (0d: 136 ± 14AU, 7d: 481 ± 50AU) all increased following L‐Cit administration.

Conclusions

This study has identified the efficacy of a 7d administration of L‐Cit to improve NO substrate bioavailability, the L‐Arg/ADMA ratio, and both conduit artery and microvascular function in patients with HFpEF. These results provide new insight into the mechanisms that govern peripheral vascular dysfunction in this patient group, and demonstrate a promising plasticity in the peripheral vasculature among patients with this chronic disease.

Support or Funding Information

Support or Funding Information: This project is funded in part by the National Institutes of Health (HL118313; D.W.W., R56AG057584; R.S.R., T32HL139451 R.S.R) and the U.S. Department of Veterans Affairs (I01RX001311; D.W.W., I01RX001697; R.S.R., I01RX002323; R.S.R., I21RX001433; R.S.R., I01RX000182; R.S.R.) and the American Heart Association (18POST33960192; K.B.).
Dissecting the Cellular Control of Septin Organization in a Global Cereal Killer
Authors: Proko Rinalda , Dulal Nawaraj , Liyanage Rohana , Egan Martin
Published: April 2020

Rice blast disease, caused by the fungus Magnaporthe oryzae, destroys enough rice each year to feed 60 million people, and is a major threat to global food security. To establish disease, M. oryzae forms a specialized infection structure called an appressorium, which it uses to physically break into rice leaves. Essential for this process is the timely assembly of a septin ring structure at the base of the appressorium. Septins are a conserved family of GTP‐binding proteins, forming hetero‐oligomeric rods and filaments that are organized into higher‐order structures at the cell cortex. The M. oryzae septin ring scaffolds the formation of a donut‐shaped filamentous actin network, needed for the emergence of a polarized penetration structure from the base of the appressorium. Importantly, relatively little is understood about how higher‐order septin structures form in the right place and at the right time in appressoria to drive infection. We are using a proximity‐dependent proteomics to identify novel proteins involved in septin organization, and are functionally characterizing these using reverse genetics, and cell biological approach. We genetically tagged the septin protein Cdc11/Sep5 with TurboID, and identified a host of putatively proximal and interacting proteins. Here, we validate a number of these, and investigate their broader role in fungal biology. The outcomes of this research will provide fundamental new insight into the cellular control of septin organization in a global cereal killer.
TIFA‐NF‐κB axis modulates inflammatory microenvironment to offset sorafenib cytotoxicity in hepatocellular carcinoma
Authors: Wei Tong-You Wade , Tsai Ming-Daw
Published: April 2020

Sorafenib is the promising first‐line drug to treat advanced hepatocellular carcinoma (HCC), with the acquired resistance within 6 months in most treated patients. The resistance to sorafenib treatment has been correlated to inflammation or viral hepatitis, but the underlying molecular mechanism remains elusive. We previously reported that the TRAF‐interacting protein TIFA sustains an inflammatory feedback loop of TNFα and NF‐κB to drive a survival pathway. Here we investigated how the inflammatory microenvironment is modulated by TIFA‐NF‐κB axis to confer a sorafenib‐resistant niche for HCC recurrence. The prognostic analysis showed that severe hepatitis was highly associated with poorer clinical responses to sorafenib treatment, suggesting that the sorafenib‐resistant HCC involves chronic inflammation. In support, inflammatory cytokines TNF‐α protected HCC lines form sorafenib cytotoxicity, while inhibition of TIFA‐NF‐κB axis reversed the effect. In addition, we showed that TIFA‐NF‐κB axis offset sorafenib cytotoxicity by supporting BCL‐2‐dependent survival signaling. To further explore the therapeutic relevance, we targeted TIFA‐NF‐κB axis using a liver‐tropic adeno‐associated virus to deliver shRNAs in an orthotopic xenograft HCC model, and result showed significant repression of tumor growth and prolonged animal survival in addition to sorafenib treatment. More intriguingly, such inhibition also abrogated the liver‐infiltration of tumor‐associated macrophages, a mechanistic event proven to be triggered by the TIFA‐NF‐κB‐regulated secretion of damage‐associated molecular pattern molecules S100A8/A9 in response to sorafenib, thereby recruiting precursor monocytes. As consequences of monocyte/macrophage infiltration, the TIFA‐NF‐κB axis in HCC cells compromised sorafenib cytotoxicity in alliance with pro‐tumor M2 macrophages, and harnessed their release of IL‐7 to enhance proliferation, sorafenib resistance, and directional migration of HCC cells. Our results collectively demonstrated that TIFA‐NF‐κB axis supports inflammatory responses to offset sorafenib cytotoxicity, and that its targeting provides therapeutically intervention concomitant with sorafenib treatment in HCC.

Support or Funding Information

This study was supported by Academia Sinica and the Taiwan Protein Project funded by Ministry of Science and Technology (MOST; Grant No. AS‐KPQ‐105‐TPP), and in part by grants MOST 104‐2314‐B‐182A‐060 and Chang Gung Memorial Hospital CMRPG3G0561.
Environmental noise aggravates oxidative DNA damage, granulocyte oxidative burst and nitrate resistance in Ogg1−/− mice
Authors: Oelze Matthias Walter , Kvandova Miroslava , Filippou Konstantina , Steven Sebastian , Frenis Katie , Kalinovic Sanela , Vijacic-Mirski Ksenija , Stamm Paul , Hosseinabadi Majid Bagheri , Dovinova Ima , Epe Bernd , Münzel Thomas , Kröller-Schön Swenja , Daiber Andreas
Published: April 2020

Background

Large epidemiological studies point towards a link between the incidence of arterial hypertension, ischemic heart disease, metabolic disease and exposure to traffic noise, supporting its role as an independent cardiovascular risk factor. We characterized the underlying molecular mechanisms leading to noise‐dependent adverse effects on the vasculature in an animal model of aircraft noise exposure identifying oxidative stress and inflammation as central players in mediating vascular dysfunction. Here, we want to study the impact of noise‐induced oxidative DNA damage on vascular function in DNA‐repair deficient Ogg1−/− (8‐oxoguanine glycosylase knockout) mice.

Methods and Results

Ogg1−/− and corresponding wild type mice were exposed to aircraft noise (peak sound levels of 85 dB(A) and mean sound level of 72 dB(A)) for 4 days. We could show that 4 days noise exposure induced significant degree of endothelial dysfunction in wild type and Ogg1−/− mice. In contrast, endothelium‐independent relaxation was only impaired in noise‐exposed Ogg1−/−, indicating the presence of nitrate resistance in noise‐exposed Ogg1−/− mice. The cardioprotective, aldehyde detoxifying enzyme mitochondrial aldehyde dehydrogenase (ALDH‐2), which is necessary for nitroglycerin bioactivation, was upregulated only in the noise‐exposed Ogg1−/− mice, which may indicate the compensatory upregulation of dysfunctional ALDH‐2. Aircraft noise exposure caused oxidative DNA damage (8‐oxo‐deoxyguanosin) and elevated NOX‐2 expression in wild type mice with synergistic increases in Ogg1−/− mice (shown by immunohistochemistry). A similar pattern was found for oxidative burst of blood leukocytes and other markers of oxidative stress (4‐hydroxynonenal, 3‐nitrotyrosine) and inflammation (cyclooxygenase‐2).

Conclusion

The finding that chronic noise exposure causes oxidative DNA damage in mice is worrisome since these potential mutagenic lesions could contribute to cancer progression. Human field studies have to demonstrate whether oxidative DNA damage is also found in urban populations with high levels of noise exposure as recently shown for workers with high occupational noise exposure.

Support or Funding Information

We gratefully acknowledge the financial support by the Naturwissenschaftlich‐Medizinisches Forschungszentrum (NMFZ) of the Johannes Gutenberg University Mainz (to B.E. and A.D.), the Boehringer Ingelheim Foundation „Novel and neglected cardiovascular risk factors: molecular mechanisms and therapeutic implications” is gratefully acknowledged (to S.S., T.M. and A.D.) and a postdoctoral research stipend for a scientific stay in Mainz by the Slovak Academy of Science (to M.K.).
Anti‐Oxidant Treatment Improves Aortic Vascular Compliance In Fructose‐Induced Salt‐Sensitive Hypertension in Male Rats
Authors: Levanovich Peter Eric , Komnenov Dragana , Perecki Natalia , Wu Min , Rossi Noreen F.
Published: April 2020

High fructose diet increases renal reactive oxygen species (ROS) production which enhances renal sodium reabsorption and renin secretion, thereby increasing extracellular volume and vasoconstriction. With concurrent high sodium consumption, hypertension occurs prior to the development of frank metabolic syndrome. Prolonged hypertension causes vascular dysfunction by reducing compliance, leading to end‐organ damage to the heart and kidneys. Daily treatment with Tempol®, a ROS scavenger, prevents increases in mean arterial pressure (MAP) associated with a fructose and high salt (FHS) diet, indicating a pathologic role of ROS in this model. We hypothesized that daily administration of Tempol® ameliorates the reduction in vascular compliance and abnormalities in cardiac function associated with FHS induced hypertension. Male Sprague‐Dawley rats were fed either 20% glucose or 20% fructose in drinking water with normal (0.4%) salt chow for one week followed by 3 weeks of normal or high (4.0%) salt chow. Half of each group was supplemented with Tempol®, 15 mg/400 g body weight daily in drinking water, beginning at one week after fructose or glucose feeding. Hemodynamics were monitored by telemetry. Ultrasonography was performed under isoflurane anesthesia. The change in MAP from baseline in the FHS group was significantly elevated compared with controls (4.98 vs. 0.16 mmHg, respectively; P < 0.01), and Tempol® administration prevented this increase. Despite increased MAP, plasma renin activity in FHS rats was not suppressed. Ultrasonography revealed significantly decreased aortic compliance, measured by pulse wave velocity (P < 0.01), and significantly increased renal resistive index (RRI) in the FHS group compared to glucose controls (P < 0.05). Tempol® administration reversed the increase in pulse wave velocity but had no effect on RRI. Systolic left ventricular function by echocardiographic assessment did not change but diastolic function was impaired. This was evidenced by decreased ratio of early to late transmitral filling velocity (P < 0.01). Tempol® did not restore mitral flow patterns to control levels. These findings indicate that the decrease in vascular compliance associated with FHS diet in male rats is, at least in part, mediated by oxidative stress. Other mechanisms seem to be at play in mediating the abnormal renovascular changes.

Support or Funding Information

VA Merit Award (RX000851) awarded to Noreen F. Rossi, MD. Peter Levanovich is supported in part by the Detroit Cardiovascular Training Program (NIH T32: HL 120822)
Determining conformational dynamics of the allosteric site in HRas R97G
Authors: Ojeda Monica , Johnson Christian , Mattos Carla
Published: April 2020

HRas is a small GTPase that acts as a molecular switch for the Raf/MEK/ERK pathway that leads to cell proliferation, survival, and differentiation. Oncogenic mutations in HRas renders it unable to hydrolyze GTP to GDP, resulting in constitutive activation of HRas and cell proliferation. Additionally, HRas contains an allosteric site that binds Ca2+ and a negatively charged ligand, promoting helix 3 to shift towards helix 4 and the ordering of switch II. This conformation is associated with intrinsic hydrolysis of HRas in the presence of Raf. Experiments have been performed in our lab to study the effects of point mutations in the allosteric site of HRas on intrinsic hydrolysis constants. When compared to wild‐type, HRas R97G demonstrated increased intrinsic hydrolysis when alone and a significant decrease when in the presence of Raf. Molecular dynamics simulations were utilized to further gain insight on how the dynamics of the allosteric site differed in HRas R97G compared to the wild‐type. These simulations demonstrated increased flexibility in the upper region of helix 3, with greater accessibility to the catalytic conformation for GTP hydrolysis.

Support or Funding Information

NSF MCB 1244203
Optimization of a novel D2 dopamine receptor antagonist with unprecedented selectivity as a therapeutic to treat psychosis and other neuropsychiatric disorders
Authors: Rocereta Julia , Free R. Benjamin , Garcia Andres Dulcey , Southall Noel , Shi Lei , Lane Robert , Wetsel William , Marugan Juan J. , Sibley David R.
Published: April 2020

The D2 dopamine receptor (D2R) is one of the most validated drug targets in psychiatry and there is a strong correlation between the clinical doses of antipsychotics and their potencies for blocking D2Rs. However, current FDA‐approved antipsychotic drugs typically cross‐react with other GPCRs, leading to many deleterious side‐effects. Using high throughput screening, we have identified and characterized a lead D2R antagonist with high selectivity – ML321. ML321 shows little GPCR cross‐reactivity beyond inhibition of the D2R, and to a lesser extent the D3R, demonstrating exceptional GPCR selectivity. Behavioral assays in rodents demonstrate ML321’s efficacy in models that are predictive of antipsychotic effects in humans (e.g., attenuation of both amphetamine and PCP‐induced locomotor activity and pre‐pulse inhibition). Importantly, using doses that are maximally effective in antipsychotic‐predictive assays, ML321 promotes little to no catalepsy, suggesting that ML321 may produce fewer extrapyramidal side effects in patients (an on‐target side effect). This property may be due to unique binding kinetics of ML321 at the D2R. Importantly, no other D2R antagonist exhibits this pharmacological and behavioral profile, which supports ML321’s promise as a superior therapeutic. No concerns were noted during safety/toxicity studies including hERG channel activation, cytotoxicity, an AMES test, and a CYP inhibition study. ML321 demonstrates good brain penetrability (20% vs. plasma), but also exhibits a relatively short half‐life in vivo of about 2 hr. The latter represents an impediment to the advancement of this antipsychotic candidate into clinical studies. A metabolite study was performed using human hepatocytes, which revealed that the primary ML321 metabolites involve oxidation of the alkyl‐thiophene portion of the molecule. Thus, in an attempt to create more metabolically stable derivatives of ML321, we have systematically altered the scaffold via modification or replacement of the thiophene moiety which is likely to lead to significantly greater stability. Preliminary SAR studies (Xiao et. al., J. Med. Chem., 57:3450, 2014)) have demonstrated that replacement of the thiophene can be accomplished without significant alteration of the scaffold’s pharmacological properties. Approximately 50 such ML321 analogs have been synthesized and are being characterized pharmacologically using D2R radioligand binding and functional (b‐arrestin recruitment) assays. In parallel, these structural analogs are being assessed in vitro for ADME in order to determine their metabolic stability, solubility and predicted penetrability. Overall, our expectation is that the ML321 scaffold can be optimized into an advanced drug candidate with the potential for targeting schizophrenia and other psychotic syndromes involving the D2R.
C1orf27 controls the ER‐to‐Golgi traffic of GPCRs via direct interaction
Authors: Xu Xin , Qiu Lifen , Jiang Chunyi , Wu Guangyu
Published: April 2020

C1orf27 is a human homolog of odorant response abnormal 4 which was originally identified as an endoplasmic reticulum (ER) membrane protein to regulate the delivery of odorant G‐protein‐coupled receptors (GPCRs) to cilia in C. elegans chemosensory neurons. However, its functions in the trafficking of mammalian GPCRs remain poorly understood. Here, we used α2‐adrenergic receptors (ARs) as models to define the role of C1orf27 in the biosynthesis and anterograde trafficking of GPCRs and elucidate the underlying mechanisms in several mammalian cells. We found that siRNA‐mediated depletion of C1orf27 significantly reduced the cell surface expression of α2A‐ and α2B‐ARs and in parallel receptor‐mediated signaling as measured by MAPK activation. Confocal microscopy revealed that the receptors were restricted in the perinuclear region and extensively co‐localized with the ER marker calregulin in C1orf27 siRNA‐treated cells. In contrast, C1orf27 knockdown did not affect the ER‐Golgi‐cell surface transport of non‐GPCR plasma membrane proteins, such as vesicular stomatitis virus glycoprotein. We also found that C1orf27 directly interacted with both receptors and the interactions were ionic in nature. Interestingly, two C1orf27‐binding sites were identified in the third intracellular loop (ICL3) and the C‐terminus of α2A‐AR, whereas only one binding domain was found in the ICL3 of α2B‐AR. Furthermore, the deletion of C1orf27‐binding domains clearly impaired the cell surface transport of the receptors, suggesting an important role of the interaction in receptor forward trafficking. Altogether these data have demonstrated that C1orf27 selectively modulates the ER‐to‐Golgi traffic of GPCRs which is likely mediated through direct interaction.

Support or Funding Information

NIH R01 GM119815
Pericyte Coverage and Microvessel Caliber: Association with Smoking Status and Body‐Mass‐Index in Normal and Pre‐eclamptic Pregnancies
Authors: Bouzaher Malek H. , Lord Megan , Nguyen Hoa , Davis Justin , Chada Anisha , Durica Alison Hocine , Chappell John C.
Published: April 2020

Pre‐eclampsia (PE) is a hypertensive disorder of pregnancy that is a major cause of maternal and fetal morbidity and mortality worldwide. Current evidence suggests that PE may result from shallow implantation of the developing fetus into the uterus, leading to abnormal formation of uteroplacental blood vessels. Pericytes play critical roles in neovessel formation and are thought to also have a role in blood flow regulation in microvascular networks. Little is known however about their function in the placenta and how this may be altered in disease. Here, we sought to determine whether differences exist in placental microvessel caliber or pericyte coverage between normal and PE pregnancies. We also examined whether modifiable risk factors might impact the observed phenotypes. Human placental tissue was obtained from normal and PE pregnancies (IRB protocol approved by Carilion Clinic). Tissue sections were antibody labeled, imaged by confocal microscopy, and assessed for vascular‐specific metrics. For comparative analysis, placental tissue from WT mice was sectioned, labeled, and imaged by confocal to optimize protocols and assess for pericyte localization. Measurements were collected using ImageJ/FIJI, and statistical analysis for human samples compared results from PE vs. normal controls. Researchers were blinded to disease state until after analysis was complete. Forty patients were enrolled in the study: 20 had normal pregnancies, and 20 had pregnancies complicated by PE. A significant difference (p < 0.005) was found in gestational age at birth between groups. Pre‐eclampsia was associated with larger overall microvessel caliber, diminished capillary size for microvasculature associated with pericytes, and diminished pericyte number relative to controls. BMI and smoking status were both inversely correlated with pericyte number. The phenotypic changes observed in placental microvessels from PE pregnancies may be related to placental hypoxia and are associated with modifiable risk factors known to underlie other microvascular diseases.
An Ultrasensitive Fluorescent Biosensor for Multi‐modal Kinase Activity Detection and High‐resolution Imaging in Awake Mice
Authors: Mehta Sohum , Zhang Jin-Fan , Liu Bian , Hong Ingie , Tenner Brian , Lin Wei , Huganir Richard L. , Zhang Jin
Published: April 2020

Fluorescent protein (FP)‐based kinase activity biosensors are powerful tools for probing the spatiotemporal dynamics of signaling pathways in living cells. Kinase activity imaging is frequently used to unravel the molecular regulation of Protein Kinase A (PKA), a prototypical kinase that integrates multiple pathways to orchestrate diverse physiological processes. Yet the limited sensitivity of most kinase biosensors restricts their reliable application in physiologically relevant in vivo systems or clinically useful high‐content detection modalities. We previously developed an excitation‐ratiometric PKA activity reporter (ExRai‐AKAR) which utilizes a PKA substrate peptide and phosphoamino acid‐binding forkhead‐associated 1 (FHA1) domain to modulate the peak excitation wavelength of cpEGFP in response to phosphorylation by PKA. Here, we report the further development of a new, ultrasensitive excitation‐ratiometric PKA activity reporter (ExRai‐AKAR2) obtained via high‐throughput linker library screening. This powerful and versatile molecular tool enables sensitive and rapid monitoring of live‐cell PKA activity across multiple fluorescence detection modalities, including plate reading, cell sorting, and one‐ or two‐photon imaging. In vivo visual cortex imaging in awake mice reveals highly dynamic neuronal PKA activity rapidly recruited by forced locomotion. The performance of ExRai‐AKAR2 promises to successfully expand kinase activity imaging beyond the petri dish.

Support or Funding Information

This work was supported by Brain Initiative Grant R01 MH111516 and by R35 CA197622, R01 DK073368, and R01 GM111665 (to J.Z.).
Role of transporters and S100A8/A9 in paclitaxel‐induced peripheral neuropathy
Authors: Li Yang , Zavorka Thomas Megan E. , Leblanc Alix F. , Huang Kevin M. , Chen Mingqing , Sprowl Jason A. , Gibson Alice A. , Lustburg Maryam M. , Sparreboom Alex , Hu Shuiying
Published: April 2020

BACKGROUND

Paclitaxel (PTX) is a widely used anticancer drug that causes a debilitating, dose‐limiting acute and chronic peripheral neuropathy (PIPN) in up to 80% of patients. Although the mechanistic basis of this side effect remains largely unknown, we previously identified transport by OATP1B‐type carriers as a contributor to acute and chronic forms of peripheral neuropathy associated with PTX, and this effect can be ameliorated by the OATP1B2 inhibitor, nilotinib, without compromising anti‐tumor efficacy. Prior investigations have also shown that PTX induces the toll‐like receptor 4 (TLR4) in neurons and activation of TLR4 is associated with PIPN. In this study, we will explore potential effect of drug metabolism or transporter mediated drug‐drug interactions (DDIs) on PIPN, and novel mechanism involving S100A8/A9 proteins, as important mediators of inflammatory response in PIPN.

METHODS

In vivo toxicity studies were performed in wild‐type, OATP1B2‐deficient, P‐gp‐deficient, CYP3A4‐deficient and S100A9‐deficient mice receiving PTX. Standard assessments of mechanical‐induced allodynia with Von Frey Hairs were conducted to evaluate PIPN at timed intervals before, during, and after treatment. Relationships between OATP1B2 and S100A9 gene or protein expression with or without PTX treatment was tested in mice with a deficiency of either OATP1B2 or S100A9.

RESULTS

Since PTX is substrate of P‐gp and metabolized by CYP3A4, the influence of P‐pg or CYP3A4 on PIPN was tested in mouse models deficient of either CYP3A4 or P‐gp. Our results suggest that neither CYP3A4 nor P‐gp deficient mice showed differences in sensitivity to mechanical stimuli compared to wild‐type mice after an i.v. dose of PTX. Interestingly, while OATP1B2 and S100A9 gene or protein expression are independently regulated at baseline, expression of S100A9 was dramatically upregulated in the dorsal root ganglia (DRG) of wild‐type mice after administration of a single dose of PTX. Since PIPN is functionally dependent on OATP1B2, which is sensitive to inhibition by small molecule inhibitors such as nilotinib without negatively affecting the anti‐tumor efficacy of PTX, we next evaluated the direct role of S100A9 in acute PIPN, and found that S100A9 deficiency protects against paclitaxel‐induced mechanical allodynia.

CONCLUSION

Our preliminary results suggest that OATP1B2 inhibitor nilotinib could protect PIPN in a P‐gp and CYP3A4 independent way. Genetic knockout of S100A9 protects against acute forms of PIPN. Further studies are warranted to determine similar protective effects can be mediated by S100A8 deficiency, and to evaluate the translational utility of S100A8/A9 inhibitors as modulators of PIPN.
Many hands make light work, or too many cooks spoil the soup? Lessons from the development of the BASIL biochemistry laboratory curriculum through a diverse multi‐institutional collaboration.
Authors: Pikaart Michael
Published: April 2020

The BASIL (Biochemistry Authentic Scientific Investigation Laboratory) curriculum is driven by a consortium of biochemistry and computational instructors. BASIL is a protein biochemistry Course‐based Undergraduate Research Experience (CURE) in which students discover enzymatic function of previously uncharacterized proteins. It combines computational and wet‐lab protein science to deliver foundational concepts within the undergraduate biochemistry teaching lab. BASIL came together in 2015 as instructors sought to integrate genuine research into the biochemistry laboratory course to broaden the impact of teaching through research.

In order to maximize impact, BASIL has been intentionally developed as a collaborative effort across institutions diverse in type, size, and location nationwide. This presentation summarizes lessons learned from building this collaboration, including the following aspects.

(1) Establishing the group: Participants were identified and recruited for the most part at national meetings such as Experimental Biology/ASBMB and others.

(2) Funding: Development of the BASIL curriculum was supported by NSF IUSE as a collaborative research proposal.

(3) Communication: Group members interact through videoconferencing on a weekly basis, and at national/regional meetings.

(4) Teaching resources: The BASIL curriculum has been published as a free and open‐source resource, supplemented by online teaching suggestions.

(5) Assessment: The effectiveness of this curriculum has been assessed through student surveys shared across campuses and surveys of faculty to monitor success of (and obstacles to) implementation at each home campus.

(7) Dissemination and recruitment: Availability of the BASIL resources has been published, and additional faculty/campuses continue to join as BASIL implementors.

These are presented with the goal of sharing successful (and not‐so‐successful) strategies for collaborating across campuses as a way to stimulate effective student learning while promoting faculty development and well‐being through this team model.

Support or Funding Information

NSF IUSE 1709278
Regulation of the ER stress response by a mitochondrial microprotein
Authors: Chu Qian , Martinez Thomas F. , Novak Sammy Weiser , Donaldson Cynthia J. , Tan Dan , Vaughan Joan M. , Chang Tina , Diedrich Jolene K. , Andrade Leo , Kim Andrew , Zhang Tong , Manor Uri , Saghatelian Alan
Published: April 2020

Cellular homeostasis relies on a dedicated and coordinated response to a variety of stresses. In the endoplasmic reticulum (ER) the accumulation of unfolded proteins triggers the unfolded protein response (UPR), a conserved stress response aimed at mitigating damage and returning cells to homeostasis. Dysregulation of UPR underlies many debilitating diseases. Here, we discover that a previously uncharacterized 54‐amino acid microprotein PIGBOS regulates UPR. PIGBOS localizes to the mitochondrial outer membrane where it interacts with the ER protein CLCC1 at ER–mitochondria contact sites. Functional studies reveal that the loss of PIGBOS leads to heightened UPR and increased cell death. The characterization of PIGBOS reveals an undiscovered role for a mitochondrial protein, in this case a microprotein, in the regulation of UPR originating in the ER. This study demonstrates microproteins to be an unappreciated class of genes that are critical for inter‐organelle communication, homeostasis, and cell survival.

Support or Funding Information

This research was supported by NIH/NIGMS (R01GM102491, A.S.), the Leona M. and Harry B. Helmsley Charitable Trust grant (A.S.), Dr. Frederick Paulsen Chair/Ferring Pharmaceuticals (A.S.), the George E. Hewitt Foundation for medical research (Q.C.), the Anderson Foundation (Q.C.), NIH F32 fellowship (GM123685, T.F.M.) and the Pioneer Fellowship(D.T.). Imaging work was supported by the Waitt Advanced Biophotonics Core Facility of the Salk Institute with funding from NIH‐NCI CCSG: P30 014195, NINDS Neuroscience Core Grant: NS072031 and the Waitt Foundation. Proteomics studies were supported by the Mass Spectrometry Core of the Salk Institute with funding from the NIH with an NCI Cancer Center Support Grant P30 (CA014195).
What a catch! Climate determinant structural differences in Atlantic Cod hemoglobin
Authors: Mallin Delaney , McCleery Rachael , Khandpur Anya , Gloege Sonia , Taipale Maddy , Lane Jim
Published: April 2020

As the climate changes, we must understand the biology of our fish stocks to better support the conservation of our global fisheries. Gadus Morhau, commonly known as Atlantic Cod, differs from other tetrameric hemoglobin species in that one of the key packing contacts, Phenylalanine 122, is replaced by Leucine, changing the interaction between the key packing contacts and thus the overall structure of the hemoglobin. Additionally, due to a hemoglobin polymorphism, Atlantic Cod has multiple subpopulations, the most common of which are the homozygous genotypes Hbl‐1 and Hbl‐2. Hbl‐1 and Hbl‐2 genotypes differ due to a mutation in amino acid sequence on the β1 chain, in the Hbl‐1 genotype, at Methionine 55, Lysine 62, and Leucine 122 (as mentioned above). However, on the Hbl‐2 genotype, the amino acids in the corresponding locations are Valine 55, Alanine 62, and Methionine 122. These mutations result in a significant divergence in structure that completely changes the optimal living conditions for the fish. tThe ideal environmental temperatures for different types of Atlantic Cod, the Hbl‐2 allele has a frequency of 99% in northern regions, whereas the Hbl‐1 allele is more common in warmer areas, suggesting that the Hbl‐2 allele helps the fish adapt to colder temperatures. Furthermore, temperature seems to impact the oxygen affinity of the two genotypes. Due to its structure, Hbl‐2 has higher oxygen bonding affinity at temperatures 10 degrees Celsius and lower, whereas the Hbl‐1 allele functions more efficiently at temperatures 14 degrees Celsius or higher. Using JMol modeling software, we compared the structures of the Hbl‐1 to Hbl‐2 Atlantic Cod hemoglobins paying close attention to the relationship between substitutional packing contacts Leucine 122 in Hbl‐1 and Methionine 122 in Hbl‐2, and the commonality of contact amino acid, Arginine B12, which is located on the α1 chain of the hemoglobin. We used JMol to analyze how amino acid substitutions in the β1 chain of the Atlantic Cod cause significant structural differences that leads to variation in optimal living conditions; we infer those adaptations may prove advantageous to their survival in their extreme environments. The Mahtomedi MSOE Center for BioMolecular Modeling MAPS Team used 3‐D modeling and printing technology to examine the structure‐function relationships of the Atlantic Cod hemoglobin polymorphisms. The visual model will be a valuable tool in developing our story.