bluuebunny/crossref_metadata_2025 huggingface dataset:
Dietary adaptation in lemurs, analyzed using new approaches to describing functional properties of tooth shape
Authors: Fulwood Ethan Lucas , Shan Shan , Winchester Julia , Kirveslahti Henry , Gao Tingran , Boyer Doug , Daubechies Ingrid
Published: April 2020

The postcanine teeth of mammals are shaped by the requirements of food fracture. Tooth shape descriptors attempt to quantify those aspects of tooth form that reflect adaptation to the fracture of foods differing in material properties. Dental topography metrics, which capture features of whole occlusal surfaces using digital scans, have shown promise in classifying mammals, particularly primates, to dietary ecology. However, they suffer from potential issues arising from digital mesh processing, model overfitting, and the averaging of functional signal among tooth structures that are under potentially independent selection. Here, these issues are addressed using a recently developed dental topography metric (ariaDNE) which is less sensitive to details of mesh processing; Bayesian multinomial modelling with metrics designed to measure overfitting risk; and a tooth segmentation algorithm which allows the shapes of disaggregated tooth surface features to be quantified using dental topography metrics. ariaDNE quantifies tooth curvature using a method comparable to the originally described Dirichlet Normal Energy (DNE), but averages over local bandwidths of a tooth surface in a process that is robust to variation in tooth face count and smoothing algorithms. Combinations of dental topography metrics that included ariaDNE outperformed DNE in reclassifications of individual specimens and genera by their genus means using discriminant function analysis. Bayesian multinomial models suggest that informationally rich models including other metrics may overfit in out‐of‐sample reclassifications, however. Disaggregating teeth into regions of consistent shape did not improve predicted reclassification success, suggesting that averaging of morphological information across the tooth surface does not interfere with the ability of dental topography metrics to predict dietary adaptation. ariaDNE presents a powerful tool for the description of functional shape that can be applied to a wide range of taxa and potentially aspects of morphology across the body.

Support or Funding Information

Funding was provided by the Duke Graduate School dissertation research domestic travel grant and summer research support and by the grants NSF BCS 1552848 to Doug Boyer, NSF BCS 130405 to Doug Boyer and Elizabeth St. Clair, and NSF BCS 1825129 to Doug Boyer and Arianna Harrington.
Evaluation of the Analgesic, Anti‐Cataleptic, Muscle Relaxant and Sedative Potential of The Ethanol Extract of Lophira alata Stem bark in Rodents
Authors: Iniaghe Loretta Oghenekome , Okpakpor Emma Elohor , Idemudia Joshua , Ogbuehi Joy Ozioma , Okonna Ifunanya Lydia , Ikhile Osatohamhen Favour
Published: April 2020

Background

Lophira alata, a woody plant found in West and Central Africa is used in the treatment of several systemic disorders. This study was carried out to evaluate some neurobehavioural properties of Lophira alata in murine models of algesia, sedation and catalepsy.

Method

Stem bark of Lophira alata were obtained, identified, authenticated, dried and milled to fine powder. Extraction was done using ethanol; phytochemical screening and determination of the mean lethal dose were carried out.

Analgesia was evaluated using the hot plate and acetic‐acid induced mouse writhing tests at graded doses of 200–800 mg/kg while haloperidol and reserpine induced catalepsy were carried out to determine anti‐cataleptic potential of Lophira alata. Muscle relaxant activity and sedative property were assayed via the rotarod and diazepam induced sleep tests respectively.

Results

Reduction in number of writhes in the acetic acid induced mouse writhing and increase in latency time on the hot plate were observed at dose levels of 800 mg/kg. Lophira alata at 400 and 800 mg/kg significantly reduced mean cataleptic score while no significant difference between control groups and Lophira alata treated animals were observed in the rotarod and diazepam induced sleep tests.

Conclusion

Lophira alata possesses anti‐cataleptic property and reduces algesia possibly via peripherally and centrally mediated mechanisms.

Support or Funding Information

No honorarium received
A proteome‐wide assay for protein mistargeting in living cells
Authors: Lyu Ziqi , Sycks Melody M. , Genereux Joseph C.
Published: April 2020

Nascent chains of secretory proteins are targeted to the endoplasmic reticulum (ER) to start their journey in the secretory pathway. Yet mistargeting may not be rare, especially during stress. Mistargeted nascent proteins then accumulate in the cytosol, which can be a potential threat to the proteostasis of the cell. A comprehensive understanding of how protein gets mistargeted, which kinds of proteins are more prone to mistargeting and which factors govern the regulation of mistargeting will be important. However, those questions are hard to answer due to the lack of methodologies: the gold standard relying on in vitro transcription/translation is quantitative but tedious, low through‐put and incompatible with living cells, while the naïve proxy of measuring ER maturation events is facile but not necessarily accurate. Here, we developed a protein partitioning assay by using a cytosol‐localized engineered ascorbate peroxidase (cytAPEX) or an ER lumen‐retained horseradish peroxidase (ERHRP) to proximity‐label cytosolic and ER luminal proteomes respectively. The compartmental selectivity of this assay was validated with exogenous and endogenous proteins. We showed protein localization can be independent of maturation events, like signal sequence cleavage. We measured how an ER stressor impacts the accumulation of ER proteins in the cytosol. We also characterized differential protein targeting under the condition of translocon inhibition. Using quantitative proteomics, we calculated the basal import efficiency of some ER‐directed proteins. Interestingly, these data have good agreement with the in vitro method. With this assay, we will be able to estimate the basal import efficiency or measure mistargeting at a proteome‐wide scale in living cells. This assay will also make it possible to investigate the mechanism behind mistargeting in response to ER stress.

Support or Funding Information

University of California, Riverside
Case Report: An Unusual Variant in Kidney Arterial Supply with Two Accessory Renal Arteries from Different Origins
Authors: Quiñones-Rodriguez Jailenne I. , Jiménez-Dietsch Sofia , Villamil-Rios Catalina I.
Published: April 2020

Knowledge of the arterial supply of the human kidney is of special interest due to its variable nature as well as the clinical implications during renovascular interventions. This case report describes an unusual pattern in the arterial blood supply of the kidneys. The variation was discovered in an elderly Puerto Rican female during the course of a routine dissection on an embalmed cadaver. The left kidney received arterial supply from two accessory renal arteries. An upper accessory renal artery branched directly from the aorta and entered the kidney at the upper pole. A lower accessory renal artery branched from the main renal artery and entered the main part of the kidney at the level of the hilum. Additional branches appear to supply the left suprarenal gland both directly from the aorta and from the upper accessory renal artery. The right kidney was missing due to unknown causes occurring during the life of the individual.

This variant is uncommon, and suggests an atypical embryological development. As the kidney ascends, it gathers its blood supply from nearby arteries, and when it reaches its final destination, one artery remains while the rest degenerate. When this process fails, multiple renal arteries are present in the adult, each with an important function that is not supplemental to that of the main renal artery. However, these extra renal arteries may represent the formation of additional blood vessels rather than an embryological vestige. Understanding these types of variations may help avoid clinical complications, especially during radiological examinations and surgical approaches, such as partial nephrectomies. This is especially true of the extra hilar artery occurring at the level of the hilum, which might be confused for a typical renal artery during imaging.
Case Report: An Atypical Innervation to the Gluteus Maximus by the Sciatic Nerve and its Clinical Implications
Authors: Camacho-Quiñones Amarilis , Cárdenas-Suárez Natalia Y. , La Puerta-Resto Marizabel , Jimenez-Dietsch Sofía , Quiñones-Rodríguez Jailenne I.
Published: April 2020

Anatomical variations, concerning the gluteus maximus (GM), are uncommon and of clinical importance. Usually, the GM is innervated by Inferior Gluteal Nerve (IGN), a branch of the sacral plexus arising from the ventral rami of L5 to S2. An unusual innervation was identified in routine cadaveric dissection in an elderly Puerto Rican female. Here, both the left and right GM were innervated by their corresponding IGN, yet both had an additional innervation by the Sciatic Nerve (SN). The bilateral SN branch to the GM was located proximally, when compared to the IGN, and emerged by the inferior border of the Piriformis muscle. In literature, there have been previous studies reporting multiple forms of innervations to the GM by the SN. Nevertheless, in these cases, the IGN was absent, and this anomaly was usually unilateral.

The implications of this variation encompass both clinical and surgical correlations with respect to patient care. Regarding clinical aspects, patients with this dual variation to the GM can present with unique clinical scenarios which might be easily misdiagnosed. For instance, pain caused by inflammation of the GM in these patients might be misinterpreted and associated to pain felt in other conditions related to the SN, such as sciatica and piriformis syndrome. Likewise, women considering surgical intervention in this area might be at a higher risk of an iatrogenic nerve injury due to the anomalous SN branch to the GM.

Thus, goal of this case report is to highlight the clinical and surgical implications of this finding based on the variant anatomy of the gluteal region. Physicians need to be aware of the existence of this variation. This might clarify the puzzling clinical manifestations of patients with the latter as well as avoid iatrogenic nerve injuries during surgical interventions.
A Missing Flexor Digitorum Brevis and its Evolutionary and Clinical Implications
Authors: Mantilla Cristian , Rodriquez-Reyes Felix , Villamil-Rios Catalina , Jimenez-Dietsch Sofia , Quinones-Rodriguez Jailenne I.
Published: April 2020

The flexor digitorum brevis belongs to the superficial layer of muscles of the foot and serves to flex the digits II–V at the proximal interphalangeal joint. Its tendons typically insert on the sides of the middle phalanx of each of the lateral four digits. During routine dissection for a human gross anatomy course, we discovered a bilateral variation in the structure of the flexor digitorum brevis in the feet of an older female cadaver. The variation consists of a bilaterally missing tendon to the digiti minimi (fifth digit). Further dissections were carried out on an additional 10 cadavers in the gross anatomy lab to ascertain the presence or absence of this variation, which is frequent in some populations. Preliminary results indicate that the variation is present in 40% of the Puerto Rican population.

The flexor digitorum brevis develops embryologically from anterior condensations of abaxial muscle cell precursors starting at 6 weeks. This variation in particular is not expected to have serious functional or evolutionary consequences in humans, as flexion of the toes is not as important in human locomotor function as it would be in chimpanzees or other hominoids. A lack of functional consequences may result in greater variation than would otherwise be expected. Clinically, the tendon of the flexor digitorum brevis to digit V is often used for flexion of the proximal and intermediate phalanges. Our results suggest that because of the variation’s lack of functional adversities, the absence of this tendon might become increasingly prevalent in the population and that deeper muscles might become hypertrophied to supplement the action of the missing tendon.
Differential spinal tissue oxygen profile during moderate and severe acute intermittent hypoxia
Authors: Perim Raphael Rodrigues , Mitchell Gordon S.
Published: April 2020

Plasticity is a hallmark of the neural system controlling breathing. A frequently studied model of respiratory motor plasticity is phrenic long‐term facilitation (pLTF), a persistent increase in phrenic burst amplitude induced by acute intermittent hypoxia (AIH). Two independent mechanisms underlie pLTF, depending on details of the AIH protocol. For example, moderate AIH (3, 5 min hypoxic episodes of moderate arterial PO2 with 5 min intervals) elicits a form of pLTF that requires intact carotid bodies, raphe neuron spinal serotonin release, and phrenic motor neuron serotonin type 2 receptor activation; this mechanism is known as the Q pathway to phrenic motor facilitation. The same protocol with more severe hypoxic episodes elicits a distinct cellular mechanism known as the S pathway; severe AIH leads to spinal adenosine accumulation and phrenic motor neuron adenosine 2A receptor activation, initiating the S pathway to pLTF. Since these pathways interact via mutual cross‐talk inhibition, intermediate AIH protocols fail to elicit pLTF since the Q and S pathways cancel one another. Thus, the balance of raphe serotonergic neuron activation (fast) versus spinal tissue hypoxia (slower) is a key determinant in regulating pLTF expression. Since the specific levels of spinal tissue hypoxia tipping this balance are not known, we compared spinal tissue oxygen profiles between moderate (PaO2 40 – 50 mmHg) vs severe AIH (PaO2 25 – 30 mmHg). Ventral cervical spinal tissue oxygen pressure (PtO2) and phrenic nerve activity were recorded in anesthetized, paralyzed and ventilated rats (IFO2 = 60%). As reported before, severe AIH elicited greater short‐term hypoxic phrenic responses and pLTF vs moderate AIH. Severe AIH elicited greater decreases in PtO2, reaching a nadir below ~5 mmHg. Following each episode, a transient overshoot in PtO2 was observed, reaching 90 mmHg above baseline conditions. Following the third hypoxic episode, PtO2 remained above baseline for at least 15 minutes post AIH (~ 10 mmHg). With moderate AIH, tissue hypoxia was less severe (PtO2 nadir ~ 16 mmHg), and post‐hypoxia overshoots far less prominent. In summary, spinal tissue oxygen dynamics are complex during AIH protocols; differences in PtO2 profiles likely underlie mechanistic differences in pLTF observed with moderate vs severe AIH. Understanding these dynamics (including the post‐AIH PtO2 “memory”) may help understand how to optimize AIH protocols to maximize plasticity and, potentially, functional benefits when harnessing AIH to restore breathing deficits with neural injury and/or disease.

Support or Funding Information

NIH HL147554 and HL148030
Pro‐inflammatory Cytokines Alter Colonocyte Butyrate Metabolism via p38 Signaling Pathway
Authors: Kim Ji Yeon , Park Bohye , Crumbley Christopher , Wirthwein Kaci L. , Riffey Olivia Faith , Beaird Jacob , Smith Mary , Donohoe Dallas
Published: April 2020

In ulcerative colitis (UC) and colorectal cancer (CRC), the colonocyte undergoes a metabolic shift. This metabolic shift involves a decrease in the oxidation of the microbial‐derived short‐chain fatty acid, butyrate. The factors that promote this metabolic shift in these diseases are not fully understood. Previously, we showed interleukin‐1β (IL‐1β), a pro‐inflammatory cytokine that is elevated in UC and CRC, decreased the oxidation of butyrate in CRC cells. Experiments identified p38, a mitogen‐activated protein kinase, mediates the diminished butyrate oxidation caused by IL‐1β. We have identified tumor necrosis factor a (TNF‐a) as another pro‐inflammatory cytokine that decreases butyrate oxidation in colorectal cells. The hypothesis is that IL‐1β or TNF‐a drives metabolic shifts in colonocytes that promote disease via the p38 signaling pathway. Both IL‐1β and TNF‐a activate p38 (phosphorylation of p38) in colorectal cancer cells. Next, we utilized the Seahorse XF24 Analyzer to measure butyrate oxidation with and without IL‐1β or TNF‐a treatment. The oxidation of butyrate was lower in cells treated with IL‐1β or TNF‐a compared to untreated controls. Furthermore, IL‐1β or TNF‐a showed a shift in metabolism from oxidative phosphorylation (OXPHOS) to glycolysis. CRC cells treated with IL‐1β or TNF‐a showed increased extracellular acidification rate compared to controls, which indicates that IL‐1β or TNF‐a induces a switch toward glycolysis. Inhibition of p38 or knockdown of p38 decreased IL‐1β or TNF‐a ability to suppress butyrate oxidation. These data provide a mechanism by which cytokines, through p38, regulate metabolism in the diseased colonocyte.

Support or Funding Information

USDA R011770176
MicroRNA‐200a‐3p mediates neuroprotection in Alzheimer‐related deficits and attenuates amyloid‐beta overproduction and tau hyperphosphorylation via co‐regulating BACE1 and PRKACB
Authors: Liu Rui , Wang Lin-lin , Jiang Hai-lun
Published: April 2020

Alzheimer’s disease (AD) is characterized by two landmark pathologies, the overproduction of amyloid‐beta peptides (Aβ), predominated by the β‐amyloid protein precursor cleaving enzyme 1 (BACE1), and hyperphosphorylation of the microtubule protein, tau, due to an imbalance in a kinase / phosphatase system that involves the activation of the protein kinase A (PKA). Current evidence indicates that brain microRNAs participate in multiple aspects of AD pathology. Here, the role and underlying molecular mechanisms of microRNA‐200a‐3p (miR‐200a‐3p) in mediating neuroprotection against AD‐related deficits were investigated. The expression of miR‐200a‐3p was measured in the hippocampus of APP / PS1 and SAMP8 mice and in an AD cell model in vitro, as well as in blood plasma extracted from AD patients. The targets of miR‐200a‐3p were determined using bioinformatics and dual‐luciferase assay analyses. In addition, cell apoptosis was detected using flow cytometry and related protein levels were measured using Western blot and ELISA techniques. miR‐200a‐3p was confirmed to be depressed in microarray miRNA profile analysis in vitro and in vivo, suggesting that miR‐200a‐3p is a potential biomarker of AD. Subsequently, miR‐200a‐3p was demonstrated to inhibit cell apoptosis accompanied by the inactivation of the Bax / caspase‐3 axis and downregulation of Aβ1‐42 and tau phosphorylation levels in vitro. Further mechanistic studies revealed that miR‐200a‐3p reduced the production of Aβ1‐42 and decreased hyperphosphorylation of tau by regulating the protein translocation of BACE1 and the protein kinase cAMP‐activated catalytic subunit beta (PRKACB) associated with the three prime untranslated regions (3’‐UTRs), respectively. Importantly, the function of miR‐200a‐3p was reversed by overexpression of BACE1 or PRKACB in cultured cells. This resulted in an elevation in cell apoptosis, and an increase in Aβ1‐42, and tau hyperphosphorylation levels, involving the epitopes, threonine 205, serine 202, 214, 396, and 356, the favorable phosphorylated sites of PKA. In conclusion, our study suggests that miR‐200a‐3p is implicated in the pathology of AD, exerting neuroprotective effects against Aβ‐induced toxicity by two possible mechanisms: one involving the inhibition of Aβ overproduction via suppressing the expression of BACE1 and; two, synergistically, decreasing the hyperphosphorylation of tau via attenuating the expression of PKA.

Support or Funding Information

This study was supported by the National NaturalScience Foundation of China (No. U1803281 and 81673411), China; the Non‐profitCentral Research Institute Fund of Chinese Academy of Medical Sciences(2018RC350013), China; and Chinese Academy Medical Sciences (CAMS) InnovationFund for Medical Science (2017‐I2M‐1‐016), China.
Immunopathology in Lungs after Intranasal Challenge with Live Virus in an EHV‐1 Murine Model: Lessons Learned from Unexpected Findings
Authors: Awan Aftab , Tulp Orien Lee , Einstein George P. , Field Hugh
Published: April 2020

Equine herpes virus (EHV‐1) causes wide spread infection among equine populations worldwide. EHV‐1 causes respiratory disease, abortion, neonatal death, paresis, retinopathy and latent viremia. Horses show a transient immunity after natural or experimental EHV‐1 but infection and immune responses to EHV‐1 started to decline after a few months of infection. As a result recovered horses are prone to subsequent EHV‐1 infection. As a result of transient immune responses, effective and lasting vaccination remains a challenge. Human herpesvirus in a murine model provides solid protection to the animals, and re‐infected recovered mice were not easily reinfected after the initial infection. In this study we infected mice with EHV‐1 via intranasal route and after three months mice that had recovered were re‐infected with the same strain of EHV‐1 along with previously placebo control mice that were now infected for the first time in addition to addition of a new new placebo control group. It was proposed that mice that had recovered from a first infection when later challenged with live virus might show some measure of protection, but in fact they showed unexpectedly severe clinical signs. These mice demonstrated severe clinical signs with abdominal breathing, weight loss and a higher mortality rate compared to a same age group of mice who had been exposed to the virus for the first time or the placebo control. The unexpected findings of their worst clinical signs came from postmortem findings and histopathological study, which indicated severe consolidation and profound infiltration of inflammatory cells such that the normal parenchyma and architecture of lungs were completely lost. In this communication Immunopathological findings of the study will be discussed in detail. The results of this study suggest that an immunoreactive mechanism should be considered in designing and selecting epitopes for intranasal vaccine preparation for EHV‐1 and possibly other respiratory infections both in animals and humans.

Support or Funding Information

Supported by Institutional Resources of the University of Cambridge, UK, the University of Health and Humanities BVI, the Einstein Institute USA, and the University of Science Arts & Technology, Montserrat, BWI.
Toxoplasma gondii GRA8‐derived peptide immunotherapy improves tumor targeting of colorectal cancer
Authors: Yang Chul-Su
Published: April 2020

Targeted tumor and efficient, specific biological drug delivery in vivo has been one of the main challenges in protein‐based cancer‐targeted therapies. Mitochondria are potential therapeutic targets for various anti‐cancer drugs. We have previously reported that protein kinase Cα‐mediated phosphorylation of Toxoplasma gondii GRA8 is required for mitochondrial trafficking and regulating the interaction of the C‐terminal of GRA8 with ATP5A1/SIRT3 in mitochondria. Furthermore, SIRT3 facilitates ATP5A1 deacetylation, mitochondrial activation, and subsequent antiseptic activity in vivo. Herein we developed a recombinant acidity‐triggered rational membrane (ATRAM)‐conjugated multifunctional GRA8 peptide (rATRAM‐G8‐M/AS) comprising ATRAM as the cancer‐targeting cell‐penetrating peptide and essential, minimal residues for mitochondrial targeting or ATP5A1/SIRT3 binding. This peptide construct showed considerable improved potency compared with rGRA8 alone in HCT116 human carcinoma cells, reaching an IC50 value of up to 200‐fold lower in vitro and 500‐fold lower in vivo. Notably, rATRAM‐G8‐M/AS treatment showed significant therapeutic effects in a mouse xenograft model through mitochondrial metabolic resuscitation, and it produced negligible immunogenicity and immune responses in vivo. Thus, these results demonstrate that rATRAM‐G8‐M/AS represents a useful therapeutic strategy against tumors, particularly colon cancer. This strategy represents an urgently needed paradigm shift for therapeutic intervention.

Support or Funding Information

This work was supported by the NRF grant funded by the Korea government (MSIP) 2019R1I1A2A01064237.
Will development of human anatomy revolutionize medical education?
Authors: Singh Rajani
Published: April 2020

The growing trend of vanishing cadaveric dissection, pruning of anatomical curriculum, compression of teaching schedule of Anatomy, deployment of inappropriately qualified tutors and demonstrators and removal of expert and experienced anatomical faculty with closing of Anatomy department all over the world in general and USA, Canada, UK, Australia and New Zealand in particular due to commercialization of medical education has not only eroded standard of medical education but also accelerated the failure cases evidenced by rising medico‐legal cases. So there is worldwide clamor about inadequate knowledge of Anatomy among tomorrows’ doctors causing impediments to comprehend successful clinical practice in general and pathology, surgery, imagery and anestheology in particular. Although many anatomic and clinical stalwarts proposed ways and means to reform delivery/acquisition of Anatomy but hunger of earning chunk of profit from medical institutions never let the quality of medical education improve. Therefore, a new model of Anatomy, to strengthen delivery and acquisition of Anatomy for revolutionizing medical education, has been designed. If adopted divorcing greed of making money from medical schools to save holy and sacred quality medical education from ruining further, the medical profession in general and pathology, surgery, imagery and anestheology in particular will regain their lost glory.

The literature was reviewed and factors, continuously contributing to falling standard and growing failure cases due to insufficient knowledge of Anatomy in trainees effecting clinical practice in medical education, were analysed. The analysis evolved model for development of Anatomy as a subject to root out these deficiencies by standardizing curriculum to be taught by medically qualified and experienced faculties with strong background of beautifully blended standalone and collaborative research in variant macro/microanatomy arresting failure cases and litigations. In addition to this, the model will create an environment for easy and necessary interaction of anatomists with clinicians/trainees through providing instant anatomical solution at all levels of medical education during clinical training of medical trainees directly from well guided cadaveric dissection by experienced faculty. The model will also generate readymade records in form of table or charts containing the variant macro/microanatomical information corresponding to relevant clinical complications to help clinicians/clinical trainees during clinical practice. This model will not only provide future reservoir of competent anatomical faculty but also revolutionize clinical skill tremendously among medical trainees.
Discovery and structure‐activity relationship study of novel series of mono‐amine transporter inhibitors for the treatment of neurodegenerative diseases
Authors: Ashraf-Uz-Zaman Md , Ji Guangchen , Alvina Karina , Sajib Md Sanaullah , Mikelis Constantinos , Avila Mirla , Neugebauer Volker , German Nadezhda A.
Published: April 2020

Monoamines, such as dopamine (DA), serotonin (5‐HT) and norepinephrine (NE), have an important modulatory role in neurotransmission in the central nervous system (CNS). They are involved in numerous physiological functions and pathological conditions. Therapeutic application of selective or mixed monoamine transporter (DAT, SERT or NET) inhibitors includes a variety of neurologic disorders, such as Parkinson’s disease, Alzheimer’s disease, multiple sclerosis, epilepsy, and others. The optimal selectivity profile varies between pathological conditions; however, the exact trend is not well understood yet. Thus, for direct comparison of produced effects, it can be beneficial to identify a class of compounds that includes structurally similar ligands with different selectivity profiles.

Recently, we have serendipitously discovered a novel selective DAT inhibitor. Our compound UR118 showed strong DAT inhibition (Ki= 15 nM) with a moderate affinity to SERT (Ki= 1620nM). We design and synthesized a series of analogs of UR118 to initiate a structure‐activity relationship (SAR) study. From the first series of analogs, we obtained selective DAT inhibitors (Ki= 40), as well as DAT‐NET inhibitors (Ki= 6.5 and 167 nM, respectively). In addition to inhibition of transporters, UR118 blocked signaling of H1 (Ki= 32nM) and 5HT‐2C (Ki= 335 nM) receptors. Further, this compound did not affect the remaining 45 GPCRs in the tested panel of CNS‐related receptors. All tested analogs of UR118 have shown similar binding patterns with different selectivity profiles (data obtained from PDSP).

Further, UR118 was found to moderately permeate the BBB (Cmax = 1000nM, 10 mg/kg, i.p in mice) with the accumulation in adipose tissue, lung, and liver. UR118 was stable in rat liver microsome (63% remaining at 1 hr.). In pertussis‐induced multiple sclerosis model (mice), we observed a significant improvement of symptoms in the UR118‐treated group when compared to the vehicle‐treated group of animals.

In conclusion, we have discovered a novel series of monoamine transporter inhibitors with varying selectivity profile and favorable CNS‐drug‐like characteristics. Compound UR118 with DAT selectivity has shown definite therapeutic potential in in vivo model of multiple sclerosis.

Support or Funding Information

South Plains Foundation
The Effect of NaCl Concentrations on the NCC and ENaC Interaction
Authors: Walker Michael Austin , King-Medina Keyona , Hoover Robert S.
Published: April 2020

The kidneys play a key role in maintenance of blood pressure. Within the nephron, the smallest functional unit of the kidney, the sodium chloride cotransporter (NCC) and the epithelial sodium channel (ENaC) are co‐expressed in the second part of the Distal Convoluted Tubule (DCT2). These sodium transport proteins are critically important for regulating blood pressure. Recent studies have established that these two proteins closely associate and are likely bound together. Here, we seek to examine if changes in sodium chloride concentration alter the interaction between NCC and ENaC. We hypothesize that the change in ion concentrations will significantly influence the interaction between NCC and ENaC. After incubating 4 dishes of mouse DCT (mDCT15) cells with different sodium chloride concentrations for 30 minutes, Co‐immunoprecipitation (CO‐IP) was conducted to examine changes in interactions between NCC and ENaC. We have confirmed expression of NCC, ENaC and phospho‐NCC under experimental conditions. Experiments to determine whether the interaction between NCC and ENaC is changed are ongoing. With our results, our knowledge of how different ion concentrations influence the interaction of NCC and ENaC will be enhanced and will be used to better understand how this mechanistic relationship can lead to the development of new therapies or the better utilization of existing therapies that will serve to improve the treatment of hypertension and salt sensitivity.

Support or Funding Information

R25 DK 101390: Summer Undergraduate Program in Renal Research (SUPERR)

T32: 5T32DK007656‐28 (Dr. Keyona King‐Medina)

VA Merit Awards: I01BX002322‐01 (RSH) and I01BX000994 (HC) (Dr. Robert S. Hoover)
Extracellular Superoxide Dismutase (ecSOD) Modulates TNFα Signaling in Human Lung Endothelial Cells
Authors: Miller Michael , Choi Hyehun , Lamb Fred
Published: April 2020

Tumor necrosis factor‐α (TNFα) signaling requires superoxide (O2−•) to be produced extracellularly by NADPH oxidase, but it remains unclear how this redox signal is transduced into activation of intracellular signaling pathways. We previously demonstrated in vascular smooth muscle cells (VSMCs) that TNFα signaling is dependent upon the extracellular isoform of superoxide dismutase (ecSOD). In cultured VSMCs siRNA knockdown of ecSOD decreased downstream MAPK phosphorylation and NFκB activation. In contrast, integrin receptor activation was enhanced, inducing higher levels of focal adhesion kinase (FAK) phosphorylation. These findings hint at a role for ecSOD in cytokine signaling that is independent from an anti‐oxidant influence. Studies in mice have shown that conditional knockout of ecSOD induces a rapid and lethal ARDS phenotype. Due to the critical nature of TNFα and reactive oxygen in the development of acute respiratory distress syndrome (ARDS) we sought to determine the signaling role of extracellular O2−• and ecSOD in endothelial cells (ECs). We employed cultured human lung microvascular endothelial cells (HLMVEC) to assess the impact of ecSOD knockdown (siRNA) on TNFα signaling. ecSOD expression was reduced ~ 50% by a 72hr exposure to siRNA. In siRNA treated HLMVECs, phosphorylation of extracellular signal‐regulated kinase (ERK) following a 15 min exposure to TNFα was significantly increased (siControl vs siSOD3 – 0.82 vs 1.93 mean signal intensity; p = 0.016). This short‐term exposure to TNFα also induced a significant reduction in the abundance of ecSOD detected by Western blot (61.6% signal intensity after TNF treatment). This suggests either rapid TNFα‐induced degradation of ecSOD, or dissociation of the protein from the cell surface, where ecSOD is known to bind to either heparin sulfate or Fibulin 5, a component of the extracellular matrix that also binds to integrins. A transendothelial resistance (TEER) assay was employed to assess the role of ecSOD in formation of a tight EC monolayer. HLMVECs treated with ecSOD siRNA were significantly impaired in their ability to develop TEER compared to cells treated with scrambled siRNA. Together, these data suggest that ecSOD plays a critical role in normal endothelial cell function and in signaling in response to TNFα, including a role in regulation of endothelial monolayer integrity. Ongoing work seeks to define the contribution of ecSOD‐dependent signaling to EC inflammation in ARDS.

Support or Funding Information

This work was supported by R01HL128386 and 5T32HD060554‐10
Antioxidant Activity of Graviola Extracts Extracted with Different Solvents
Authors: Kang Myunghwa , Kim Dohee , Seo Hyungsuk , Lee Hajong , Choo Yeanhan , Kim Doungjin
Published: April 2020

The gravureola (Annona muricata L.Graviola) has long been a traditional "natural remedy" for tropical Amazonians and is called a "God's gift." Graviola is attracting attention in medicine and pharmacy because it contains a lot of antioxidants, such as tannins, saponins, flavonoids and anthraquinones, which are highly researched. Therefore, this study was performed to extract gravolae from different solvents and to compare the antioxidant activity of gravureola extract.

The extraction solvent used in this study was prepared by drying extracted with heat mentle by adding water, 30% ethanol and 80% ethanol. Antioxidant effect of each extract was measured by measuring total phenolic acid contents and flavonoids contents which measured DPPH radical scavenging activity, ABTs scavenging activity, SOD‐liked activity, reducing power and antioxidant content. As a result, the DPPH radical scavenging activity showed the highest activity at 20 minutes for the 30% ethanol extract and at 20 minutes for the 80% ethanol extract.

ABTs scavenging activity showed similar effects in all extracts. The SOD‐liked activity was very high in 80% ethanol extract compared to other extracts. Reducing power was 80% ethanol> 30% ethanol> water. Total phenolic acid and flavonoids contents were 80% ethanol> 30% ethanol> water and contained less than 0.2mg / g. As a result, the antioxidant effect of graviola was the highest in 80% ethanol extract, suggesting the possibility of development as a functional material.
Regulation of Human Organic Cation Transporter 3 (hOCT3) by the Dual‐Specifity Tyrosine‐(Y)‐Phosphorylation Regulated Kinase (DYRK1A)
Authors: Ciarimboli Giuliano , Stapel Mira , Barz Vivien , Pavenstädt Hermann , Rinschen Markus M.
Published: April 2020

hOCT3, a transmembrane protein with broad tissue distribution, is involved in the low‐affinity, high‐capacity transport of monoamine substrates across the cell membrane. In mice OCT3 is important for cerebral monoaminergic neurotransmission and for the effects of monoamines on behavior. The molecular regulation of hOCT3, however, is unknown.

In this study, we have identified the dual‐specificity tyrosine‐(Y)‐phosphorylation regulated kinase 1A (DYRK1A) as a key interaction partner of hOCT3 using a membrane‐based Split‐Ubiquitin‐Yeast‐Two‐Hybrid technique, and pull‐down experiments in hOCT3 and DYRK1A transfected human embryonic kidney (HEK) cells. In these cells, DYRK1A overexpression resulted in hOCT3 phosphorylation at the serines 3 and 537 as determined by phosphoproteomic analysis, and in a significant inhibition of the transport of 4‐4‐dimethylamino‐styryl‐N‐methylpyridinium (ASP+), a fluorescent hOCT3 substrate. DYRK1A overexpression reduced the maximum transport velocity (Vmax) of hOCT3, without changing its affinity for ASP+. The hOCT3 transporter function was rescued by inhibition of DYRK1A with 100 nM harmine. In the seminoma cell line Tcam‐2, which endogenously expresses hOCT3 and DYRK1A, harmine treatment resulted in 1) a significant increased hOCT3 expression in the plasma membrane, as determined by biotinylation experiments; 2) an increase of ASP+ uptake; and 3) a decrease of hOCT3/DYRK1A co‐localization as determined by confocal microscopy. Moreover, in Tcam2 cells, phosphatase activation by 4 h incubation with 10 μM ceramide induced a significant increase of ASP+ uptake.

Taken together, these data show that hOCT3 is regulated by DYRK1A via interaction, phosphorylation and change of intracellular distribution. The gene coding for the DYRK1A is localized in the Down syndrome (DS) critical region of chromosome 21 and is a strong candidate gene for DS‐associated mental retardation. Since hOCT3 functions as neurotransmitter reuptake transporter in the brain, we speculate that its inhibition by DYRK1A results in an impaired reuptake of neurotransmitters, and therefore an impoverishment of brain neurotransmitters content. This mechanism may be one of the molecular causes of mental retardation and susceptibility to Alzheimer diseases in DS patients.

Support or Funding Information

Deutsche Forschungsgemeinschaft CI 107/11.1
A new molecular mechanism of evasion of apoptosis revealed by novel C‐kinase activity reporter
Authors: Kajimoto Taketoshi , Caliman Alisha D. , Tobias Irene S. , Okada Taro , Pilo Caila A. , Van An-Angela N. , McCammon J. Andrew , Nakamura Shun-ichi , Newton Alexandra C.
Published: April 2020

It is well known that cells, especially cancer cells, have an ability of evasion of apoptosis by cellular stress like nutrient starvation. And the balance between apoptosis signal and apoptosis‐resistant signal will determine the fate of cells, dead or alive. Here we found new cell signaling system that plays a role in applying the brakes to cell death that in case cancer cells avoid apoptosis by cellular stress like starvation using newly developed biosensor and in silico docking simulation technique. The new cell signaling system is that second messenger, sphingosine 1‐phosphate (S1P), directly activates a key cell signaling protein, atypical protein kinase C (aPKC).

First, we found that the inhibition of aPKC induces apoptosis of cancer cell lines. Next, for making clear the molecular mechanism of the aPKC‐induced apoptosis resistance, we generated a genetically encoded reporter with the same modular structure as the original C kinase activity reporter (CKAR) but with a unique substrate sequence that allows specific visualization of atypical PKC activity in cells. Using the atypical PKC‐specific CKAR (aCKAR) we found that intracellular S1P induces the activation of atypical PKC in an S1P receptor‐independent manner. Biochemical studies revealed that S1P directly binds to the kinase domain of atypical PKC isozymes, relieving autoinhibitory constraints to activate the enzyme. In silico docking studies were used to identify potential binding sites for aPKC, one of which was validated by biochemical and aCKAR imaging techniques.

Now we got new insights about the player of evasion of apoptosis in cancer at the molecular level, and it has potential for development of new molecular‐targeted agents to release brakes against cell death.

Support or Funding Information

This study was supported by NIH to A.C.N., JSPS KAKENHI, Kobe University Grant for Japan‐US Collaboration, Nakatani Foundation Grant for Technology Development Research to T.K., NIH, HHMI, NBCR, NSF to J.A.M., JSPS KAKENHI to S.N. and T.O. A.D.C. was supported in part by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology.
Effects of Various Hypoxia vs Normoxia on Metabolic and Skeletal Muscle Oxygenation Function during Graded Exercise in Healthy Males
Authors: Seo Jongbeom , Jung Won-Sang , Park Hun-Young , Lim Kiwon
Published: April 2020

Purpose

The purpose of this study was to investigate the effect of various hypoxia vs. normoxia on metabolic and skeletal muscle oxygenation function during grated exercise and exercise performance.

Methods

Eleven healthy male participants (21.5 ± 2.3 years) performed graded exercise test (GXT) using cycle ergometer at sea‐level (760 mmHg) and under various hypobaric hypoxia (1,500 m simulated altitude; 634 mmHg, 3,000 m simulated altitude; 528 mmHg and 4,500 m simulated altitude; 433 mmHg) in a random order. GXT starts at 300 kg·m·min−1 (50 watts) and increases by 150 kg·m·min−1 (25 watts) every 2 minutes until exhaustion. The pedal frequency was set at 60 rpm. Metabolic parameters (peripheral capillary oxygen saturation; SPO2, heart rate; HR, minute ventilation; VE, oxygen consumption; VO2, carbon dioxide excretion; VCO2, respiratory exchange ratio; RER, oxygen pulse, and blood lactate) and skeletal muscle oxygen profiles (oxygenated hemoglobin and myoglobin; OxyHb, deoxygenated hemoglobin and myoglobin; DeoxyHb, and tissue oxygen saturation; StO2) were measured every 1 minute during the GXT, and it also evaluated at exercise load of 50, 75, 100, 125, and 150 watts on ergometers under various hypoxia vs. normoxia. Exercise performance was evaluated by maximal oxygen consumption, peak power, and exercise time obtained through GXT.

Results

In metabolic parameters, HR, VE, VCO2, RER, and blood lactate showed a significant increase in hypoxia compared with normoxia. Also, the increase was more pronounced as the hypoxia became more severe. However, SPO2, VO2, and O2pulse presented a significant decrease in hypoxia compared with normoxia. Also, the decrease was more pronounced as the hypoxia became more severe. In skeletal muscle oxygen profiles, OxyHb showed a significant decrease in 3,000 m and 4,500 m simulated altitude compared with normoxia. In exercise performance, VO2max and peak power were significantly decreased in 3,000 m and 4,500 m simulated altitude compared with normoxia, and exercise time decreased significantly with increasing simulated altitude.

Conclusion

These showed a decrease in exercise performance due to a decrease in metabolic parameters and skeletal muscle oxygenation in hypoxia vs. normoxia, and more pronounced as the hypoxia became more severe.

Support or Funding Information
Beneficial Effect of TMAO in Spontaneously Hypertensive Heart Failure Rats is Associated with Diuretic and Hypotensive Actions.
Authors: Ufnal Marcin , Konop Marek , Gawrys-Kopczynska Marta , Maksymiuk Klaudia , Sozanski Krzysztof , Derzsi Ladislav
Published: April 2020

There is an ongoing debate weather trimethylamine‐oxide (TMAO), a molecule present in seafood and a derivate of microbiota metabolism, is beneficial or harmful for the circulatory system. Numerous biophysical studies show that TMAO protects proteins exposed to osmotic and hydrostatic stress (HS). We hypothesized that TMAO may benefit the circulatory system by protecting cardiac proteins exposed to HS produced by the contracting heart.

Male, 6‐week‐old, Sprague‐Dawley (SD, n=40) and Spontaneously‐Hypertensive‐Heart‐Failure (SHHF, n=18) rats were randomly divided into either control (Water) or TMAO treatment. After 50 weeks, half of SD were given Isoprenaline (s.c.) to increase arterial blood pressure (BP) and produce a mild, Takotsubo‐like cardiomyopathy (ISO). In vitro, cardiac lactic dehydrogenase (LDH) with or without TMAO was exposed to HS (changes in hydrostatic pressure similar, and higher than those present in the heart) for up to 24hrs and was evaluated using fluorescence correlation spectroscopy.

After 58 weeks of the treatment survival was 100% in SD‐Water, SD‐TMAO, ISO‐TMAO and 90% in ISO‐WATER. In SHHF‐Water survival was 66% vs 100% in SHHF‐TMAO (p=0.065). In general, TMAO‐treated rats showed higher natriuresis and diuresis. In comparison to SHHF‐Water, SHHF‐TMAO showed significantly lower plasma NT‐proBNP, lower diastolic BP, lower liver angiotensinogen expression and a trend towards lower cardiac fibrosis. In vitro, exposure of LDH to HS with or without TMAO did not affect the protein structure. TMAO does not exert negative effects. In contrast, TMAO reduces mortality in HF rats that is associated with natriuretic, diuretic and hypotensive effects. HS produced by the contracting heart is neutral for cardiac LDH protein structure.

Support or Funding Information

Supported by National Science Centre, Poland grant no 2018/31/B/NZ5/00038.
Identifying biochemical binding partners of a MAP kinase‐like protein in Toxoplasma gondii
Authors: Henriquez Sarah Ana , Hu Xiaoyu , O’Shaughnessy William , Reese Michael
Published: April 2020

While the canonical mitogen‐activated protein kinase (MAPK) pathways are well‐established and almost ubiquitous across the eukaryotic kingdoms, Toxoplasma gondii expresses a family of MAPK‐like proteins that deviate from classical kinase activation. One of these MAPK‐like proteins, MAPKL2, is exclusively found in alveolates, a superphylum that includes dinoflagellates, ciliates, and apicomplexa. T. gondii is an apicomplexan intracellular parasite that can infect nearly all warm‐blooded animals, including humans. MAPKL2 is an essential kinase without which the parasite dies, and thereby recommends itself as an ideal potential drug target for T. gondii, a pathogen for which treatment is limited, expensive, and often toxic to patients. Thus, the model organism provides both the context of an informative cellular biology and a medically relevant model for studying this non‐canonical MAP kinase. We used yeast‐two‐hybrid to identify potential binding partners and have narrowed the minimum sufficient binding region of the proteins to within 15–30 residues. By determining the minimum sufficient binding regions of the three proteins of interest, a consensus sequence may be developed for use in screening for more biochemical binders in T. gondii and other alveolates. Recent experiments suggesting localization and association with specific classes of proteins will also be discussed. Narrowing the category of proteins within which the consensus sequence is used to screen will provide a more focused direction for investigation of the role of MAPKL2 in biochemical and molecular mechanisms within the parasite and related species.