bluuebunny/crossref_metadata_2025 huggingface dataset:
Clinical Implications of Sexual Dimorphism in Gonadal Vein Valves
Authors: Hasan Shaina , Thomas Olivia , Kriegshauser J Scott , Oklu Rahmi , Langley Natalie R
Published: April 2020

The clinical significance of valves within gonadal veins is controversial. In males, the absence of valves in the left testicular vein (compared to the right) has been cited as a potential mechanism for varicocele formation in the pampiniform plexus in the scrotum. In females, ovarian vein valve absence or incompetence may lead to pelvic congestion syndrome, a disputed explanation for pelvic, abdominal, and/or flank pain of unknown etiology. One study suggests that females have incompetent valves in dilated veins more frequently than males due to past pregnancies. Furthermore, inconsistencies exist in the reported incidence of gonadal vein valves, though pelvic insufficiency is more thoroughly documented and diagnosed in males than in females.

This study assesses the presence, number, and location of gonadal vein valves in male and females. The right and left gonadal veins were evaluated in 13 embalmed cadavers (7 males, 6 females; mean age = 86.3 +/− 6.2 years). Twenty‐four veins were available for dissection; two veins were lost to dissection and therefore omitted. The veins were dissected from the ovary (females) or deep inguinal ring (males) to the point of drainage into the inferior vena cava (right gonadal vein) or left renal vein (left gonadal vein). The veins were incised along the entire length of this course, and the number and location of valves was noted and photographed. Anatomical variation and pathological dilation in the vasculature was also documented. A total of 8 valves were found among the 24 dissections. Valves were present in four of the six females (67%) compared to one out of seven males (14%). The valve in the male was located in the left gonadal vein; the bilateral distribution in females was more randomly patterned. The majority of the valves (87.5%) in females were located distally, adjacent to the ovary, while a single valve was located adjacent to the left renal vein confluence. No dilation was noted in any of the specimens.

The male population in this study had a lower incidence of gonadal vein valves than in previous studies. However, the number of valves in female gonadal veins was considerably greater than in males. This finding has not been reported previously, perhaps due to the emphasis on male pelvic congestion as a risk for infertility versus attributing the condition in females to a psychosomatic etiology. The primary limitation of this research is the small sample size. Future work will explore the clinical implications of these preliminary findings in a patient population. Clinical correlation with patients who are suspected or known to suffer from pelvic congestion syndrome may shed further light on the role of gonadal vein valves in vein sufficiency and inform more effective treatment for this poorly understood condition in the female patient population.
7‐Dimethylamino‐4‐trifluormethylcoumarin (Coumarin‐152) as a fluorogenic substrate of CYP3A4 and a tool for studying functional interrelationships among human cytochromes P450
Authors: DANGI BIKASH , Davydova Nadezhda Y. , Davydov Dimitri R.
Published: April 2020

In this study we probe the use of 7‐Dimethylamino‐4‐trifluromethylcoumarin (Coumarin 152, C152) as a fluorogenic substrate for in vitro studies of drug metabolism in human liver microsomes (HLM). Examining spectral properties of the formed fluorescent products and using LC‐MS/MS for monitoring C152 metabolism we demonstrate sequential formation of two N‐demethylated products – 7‐Methylamino‐4‐trifluoromethylcoumarin (desmethyl‐Coumarin 152, DC152) and amino‐4‐trifluoromethylcoumarin (Coumarin 151, C151). The predominant product formed by HLM is DC152, which accounts for >70% of C152 metabolized. Using insect cell microsomes containing recombinant P450 enzymes (Supersomes™) we found that C152 is metabolized by CYP2B6, CYP3A4, CYP2C19, CYP1A2 and CYP2C9 listed in the order of decreasing rates of turnover. The KM values exhibited by CYP2B6, CYP3A4, CYP1A2 and CYP2C19 were of the same order of magnitude (1.8 – 4.8 μM). Importantly, the major metabolizers of C152 – CYP2B6, CYP3A4, CYP2C19 and CYP1A2 – demonstrated different ability to catalyze the second demethylation step, with CYP1A2 being the most potent catalyst in this reaction. While over half of the substrate metabolized by CYP1A2 underwent the second demethylation, the fraction of C151 formed by other enzymes did not exceed 30%, similar to what is observed in HLM. Although the rate of turnover of C152 with CYP2B6 is higher than that observed with CYP3A4, the latter appears to be the major metabolizer of C152 due to its high abundance in HLM. This is confirmed by ketoconazole (KCZ) inhibition experiments. We demonstrate that in both CYP3A4 Supersomes™ and HLM over 80% of C152 turnover is suppressed by KCZ, whereas the activities of CYP1A2, CYP2B4 and CYP2C19 with C152 remain largely unaffected. Our results demonstrate that C152 can be used for probing functional interrelationships between multiple cytochrome P450 species in HLM as an example of a substrate that is almost exclusively metabolized by CYP3A4 though having similar affinities to most major human drug‐metabolizing cytochromes P450. In addition, we establish an easy and reliable fluorimetric assay for monitoring and analyzing the metabolism of C152 that allows discriminating between the two sequential steps of its N‐demethylation.

Support or Funding Information

This research was supported by the National Institute On Alcohol Abuse And Alcoholism of NlH under Award Number R21AA024548
Re‐expression of REST in ventricular vagal neurons aggravates myocardial infarctionevoked ventricular arrhythmia in type 2 diabetes mellitus
Authors: HU WENFENG , ZHANG DONGZE , TU HUIYIN , WADMAN MICHAEL C. , LI YULONG
Published: April 2020

Diabetes mellitus, especially type 2 diabetes mellitus (T2DM, 90 to 95% of diabetic population), is a major public health problem worldwide and has become a leading cause of mortality. Myocardial infarction (MI) is the most common cause of mortality in T2DM. Comparing with non‐diabetic patients, patients with T2DM are twice to die from MI. Withdrawal of cardiac vagal activity is considered to be an important trigger for MI‐induced ventricular arrhythmias in T2DM. Our previous study has demonstrated that N‐type Ca2+ currents and cell excitability of atrioventricular ganglion (AVG) neurons were reduced in T2DM rats. We have recently demonstrated that reduced expression of Cav2.2‐α in AVG neurons contributes to cardiac vagal dysfunction and ventricular arrhythmogenesis. The repressor element 1‐silencing transcription factor (REST) is a key factor to block Cav2.2‐α expression. Normally REST is not expressed in neuronal cells. Here, we test the hypothesis that re‐expression of REST inhibits N‐type Ca2+ channel function in AVG neurons and further reduces cardiac vagal activity and promotes MI‐evoked fatal ventricular arrhythmias in T2DM rats. Using reverse‐phase protein array, we found that there was no expression of REST in the AVG from sham rats, while REST expression was significantly increased in the AVG from T2DM rats. In vivo infection of lentiviral REST shRNA (2 μl, 1×107 TU/ml) to the AVG markedly reduced REST expression in the AVG from T2DM rats. REST shRNA also increased Cav2.2‐α expression in AVG neurons from T2DM rats. The results from whole‐cell patch clamp recording showed that REST shRNA markedly restored T2DM‐reduced N‐type Ca2+ currents and cell excitability in AVG neurons. Additionally, infection of REST shRNA to AVG neurons markedly restored cardiac vagal activity, as evidenced by blunted response of LVSP to left efferent vagal stimulation. Moreover, REST shRNA also decreased the inducibility of ventricular arrhythmia in anesthetized T2DM rats (0.67±0.6 in T2DM+REST group vs. 3.6±1.03 in T2DM group). The ECG data obtained from 24‐hour radiotelemetry recording in conscious rat demonstrated that REST shRNA significantly reduced the duration of MI‐evoked ventricular arrhythmia (39.39±13.03 sec/hour in T2DM+REST group vs. 64.26±13.17 sec/hour in T2DM group). Based on above data, we conclude that re‐expression of REST decreases Cav2.2‐α expression and N‐type Ca2+ currents in AVG neurons, and subsequently reduces cardiac vagal function and increases susceptibility to ventricular arrhythmias in the T2DM.

Support or Funding Information

DHHS/NIH/NHLBI, 34‐5370‐2003‐001
Impact of Phosphorylation on MDH2 Structure, Function and Protein
Authors: Chang Nathan M. , Rementeria Gabriel J. , Berquist Blaine E. , Provost Joseph John
Published: April 2020

Mitochondrial malate dehydrogenase (MDH2) is an enzyme utilizing the NAD/NADH cofactor system in the citric acid cycle to reversibly oxidize malate to oxaloacetate, where citrate synthase (CS) uses oxaloacetate to produce citrate. Coupling of the MDH2‐CS of the two reactions is critical as the thermodynamics of MDH2 alone would not drive the cycle forward. However, shuttling of carbon through the cycle during gluconeogenesis necessitates an uncoupling of the two proteins, allowing for the reversal of the reaction. Thus, a mechanism that favors the known interaction and disrupts substrate channeling through these two enzymes must exist. We performed an analysis of putative MDH2 phosphorylation sites, identified the potential kinases, and conducted a meta‐analysis of mass spectrometry data bases of phosphorylation. We have identified 13 predicted, and 30 identified phosphorylated sites of mammalian MDH2. Further analysis of possible interaction sites and mapping functional domains with these phosphorylation sites highlight the important yet unknown role of phosphorylation to regulate MDH structure and function. In order to better understand MDH2‐CS interactions and MDH function, we synthesized four phosphomimics (S/T‐D) using site directed mutagenesis on a codon optimized human MDH2 gene in a His‐tagged, pET28a construct for bacterial expression. Using purified protein, we show impact of each mutation structure with circular dichroism and the thermal melting profile of the phosphomimics and the wildtype hMDH2. The impact of phosphorylation on MHD function was also studied by comparing kinetic values. Lastly the impact of the mutations on MDH‐CS binding was analyzed using a micro‐protein thermal shift assay.

Support or Funding Information

NSF EHR‐IUSE Project support NSF‐1726932
Investigation of isoform‐specific interactions between citrate synthase and malate dehydrogenase
Authors: Rementeria Gabriel J. , Berquist Blaine E. , Chang Nathan M. , Ghebreigziahber Daniel , Provost Joseph John
Published: April 2020

Malate dehydrogenase (MDH) and citrate synthase (CS) catalyze subsequent metabolic reactions in the Krebs cycle. MDH and CS interact to form enzyme complexes called metabolons where substrates are directly shuttled from one active site to the other, improving efficiency of the overall metabolic cycle. Prior studies have shown that the mitochondrial isoform (MDH2) has stronger interactions with CS than the cytosolic isoform (MDH1), suggesting that isoformspecific sites of interaction are involved in the formation of the enzyme complex. This study attempts to determine key residues of MDH2 that are responsible for isoform‐specific affinities to CS, allowing for the formation of metabolon complexes. Model metabolon docking complexes between MDH2 and CS were generated computationally in Cluspro, using predicted and published interactions between the two enzymes. Potential interacting residues were identified and then mapped using clustal alignments to identify possible domains of isoformspecific interactions. Previous studies by our lab have shown that CS displays an isoformspecific affinity to MDH. Specifically, CS binds with high affinity to MDH2, moderate affinity to MDH1, and displays undetectable interaction with watermelon glyoxosomal MDH (wgMDH). We identified several regions of residues unique to MDH2 containing shown & predicted interactions with CS. Using docking analysis, we identified five regions of potential isoformspecific interactions. This combined with residue alignment of MDH2, MDH1 and wgMDH isoforms helped to further define potential isoform specific sites of interaction. Mutations exchanging mitochondrial residues for unique cytosolic counterparts or deletion mutations defined by our bioinformatic analysis were designed, generated and expressed. Enzyme kinetic assays were performed to investigate enzymatic activity of mutant constructs for comparison to native MDH1 and MDH2. Secondary structure and thermal stability were investigated using circular dichroism. Interactions between mutant MDH2 constructs and CS were quantified in fluorescence‐based thermal shift assays.

Support or Funding Information

NSF EHR‐IUSE Project support NSF‐1726932
Cardiac iron overload promotes ferroptosis and cardiac dysfunction in mice with sickle cell disease
Authors: Menon Archita Venugopal , Tsai Hanting Phoebe , Kim Jonghan
Published: April 2020

Ferroptosis is a recently defined form of iron‐dependent, non‐apoptotic programmed cell death that is characterized by accumulation of lipid peroxides. Although ferroptosis was first identified in cancer cells, recent studies have demonstrated its implications in a wide range of physiological disorders such as ischemic and hemorrhagic stroke, Alzheimer’s disease, and epilepsy. Notably, there is a significant association between iron loading and heart failure. For example, cardiomyopathy is the leading cause of mortality in thalassemia and accounts for 30% of mortality in iron overload sickle cell disease (SCD). However, the underlying mechanisms of iron‐induced cardiotoxicity are not well understood. Although cell death mechanisms such as apoptosis and necrosis have been recognized in the context of heart disease, the role of ferroptosis in cardiac damage is largely unexplored. Here we hypothesized that cardiac iron overload promotes ferroptosis and cardiac dysfunction in the Townes mouse model of SCD. To test our hypothesis, we determined and compared iron levels, cardiotoxicity and ferroptotic markers between sickling HbSS (homozygous for the human βS globin gene) and their non‐sickling heterozygous HbAS littermates. First, we observed an increase in systemic and tissue iron levels in HbSS mice. We also found increased expression of the cardiotoxic markers ANP (atrial natriuretic peptide) and BNP (brain natriuretic peptide). These results were associated with an upregulation of the ferroptotic marker, Ptgs2, and increased levels of lipid peroxidation, a characteristic of ferroptosis, in the cardiac tissue of HbSS mice. However, no differences were found in caspase‐3 levels, suggesting that cardiotoxicity does not result from apoptotic mechanisms. To distinguish between the effects of the sickling gene and iron loading, we induced iron overload in non‐sickling mice by iron dextran treatment, an established model of secondary iron overload. Similar to our findings in sickling mice, we demonstrated that induced iron overload displayed increased lipid peroxidation and cardiac damage, consistent with cardiac ferroptosis. Finally, inhibition of ferroptosis, either by chelating excess iron (using an iron chelator deferoxamine) or by inhibiting lipid peroxidation (using ferrostatin‐1, a lipid radical scavenger), improved cardiac dysfunction in sickling mice. These findings suggest a novel mechanism of cardiac cell death in SCD and could provide potential protective targets against iron‐induced cardiotoxicity.
Pentagalloyl glucose (PGG) Inhibits IL‐1β‐induced pro‐inflammatory cytokine production and matrix metalloproteinase activation in human osteoarthritis synovial fibroblasts
Authors: Haque Mahamudul , Thompson Matthew , Singh Anil , Kopczynski Kelly , Ahmed Salahuddin
Published: April 2020

Osteoarthritis (OA) is a chronic degenerative joint disease in which the pro‐inflammatory cytokine, interleukin‐1β (IL‐1β), plays a central role in propagating inflammation and cartilage degradation by activating inflammatory mediators and cartilage degrading matrix metalloproteinases (MMPs). In the present study, we evaluated the efficacy of pentagalloyl glucose (PGG), a natural antioxidant, in suppressing IL‐1β‐induced mediators of inflammation and cartilage degradation in human osteoarthritis synovial fibroblasts (OASFs). PGG treatment (0.5–50 μM) for 24 hours had no significant effect on human OASF cell viability (n=3; p<0.01). Analysis of the conditioned media using ELISA showed that PGG (0.5–5 uM/ml) pretreatment significantly inhibited IL‐1β‐induced production of IL‐6 (45–90%), IL‐8 (40–95%), MCP‐1/CCL2 up to 80%, and RANTES/CCL5 by (45–90%) (n=3; p<0.05). In addition, we observed a significant dose‐dependent inhibition in MMP‐1 and MMP‐3 production by PGG in human OASFs (n=3; p<0.05). Furthermore, in‐gel zymography results using conditioned media showed that the enzymatic activity of IL‐1β‐induced gelatinases MMP‐2 and MMP‐9 were markedly reduced upon PGG treatment (n=3; p<0.05). PGG (5 μg/mL) significantly inhibited the degradation of type‐I collagen compared to IL‐1β stimulation alone in human OASFs (n=3; p<0.05). Examination of the mitogen‐activated protein kinase (MAPK) signaling pathway showed that PGG interfered with IL‐1β‐induced ERK1/2 and JNK/SAPK, but not p38 MAPK, to inhibit downstream phosphorylation and nuclear translocation of transcription factor c‐Jun. Immunofluorescence study confirmed that PGG pretreatment inhibits phospho‐c‐Jun and NF‐κB p65 nuclear translocation. Overall, our findings suggest that PGG inhibits IL‐1β‐induced mediators of inflammation and tissue destruction in human OASFs by reducing the activation of MAPK signaling pathway.

Support or Funding Information

This study was supported by the NIH/NIAMS grant R01AR072615 (S.A.), and the Washington State University College of Pharmacy (S.A.), the Summer Undergraduate Research Fellowship from ASPET (M.T.), and Special thanks to Erwin & Jennie Foisy Scholarship Fund (M.H.).
Effect of lighting conditions on thymic mast cells’s response to melatonin intake
Authors: Luzikova Elena , Shatskikh Oksana , Gordova Valentina , Smorodchenko Alina , Sergeeva Valentina
Published: April 2020

Melatonin is the main secretory hormon of the pineal gland, which plays the fundamental role in neuroendocrine‐immunological network. We have previously shown that melatonin intake at natural light conditions induced the statistically significant changes in the number and state of degranulation of thymic mast cells (MC). The dependence of MC response to melatonin intake under constant dimming conditions has not been studied.

Thymuses were isolated from white male mice (2 months old, weight 20–22 g). The animals were divided into 4 groups (each n=20): first control group were for 2 weeks in conditions of constant dimming (with cage illumination per day 0 – 0.5 LUX), mice had free access to drinking water and food); the first experimental group was kept in the same conditions for 2 weeks and received synthetic melatonin «Melaxen» (Unipharm, USA) at a concentration of 4 mg/l with drinking water; the second control group was under dimming conditions for 4 weeks; the second experimental group received melatonin at the same concentration like first group for 4 weeks. One mouse consumed approximately 6 ml of water per day, which equaled 25 mcg of melatonin per day per animal. The thymuses were isolated from the anesthetized animals and embedded in paraffin. Sections 4 microns thick were stained with toluidine blue and methylene blue (Unna), analysed under the microscope (Zeiss Primo Star) and statistically processed by StatSoft Statistica 10.0.

The result shows that the keeping of mice under conditions of constant dimming leads to an increase in degranulation of MC in the thymus of both control groups, as well as to up‐regulation of the total MC number in the fourth week of the experiment: in the connective tissue of cortical septum ‐ 1.6 times (p <0.001), in the parenchyma of the thymic lobules ‐ 1.5 times (p <0.05).

The intake of melatonin under the constant dimming conditions during 2 and 4 weeks resulted in increasing in the amount of MC in the cortical septum and in the thymus parenchyma by 1.4 and 1.3 times (p <0.05), respectively. The intake of melatonin under constant dimming conditions increase the proportion of MC without degranulation in cortical septa (from 4% to 34% and from 1% to 33%, respectively, for periods of 2 and 4 weeks, p <0.001) and in the thymus parenchyma (from 7% to 39% and from 5% to 36%, respectively, p <0.001) compared with both control groups.

Thus, under constant dimming circumstances the thymic MC are more sensitive to melatonin intake compared to the natural light conditions what we reported earlier. Perhaps, the MC prevents the destruction of the thymus and ensures the survival of T‐lymphocytes in the absence of a photoperiod.
Analysis of a novel phosophodegron‐like selectivity mechanism in stationary phase mitophagy
Authors: Abeliovich Hagai
Published: April 2020

Mitophagy, or the autophagic degradation of mitochondria, is an important housekeeping function of eukaryotic cells that prevents the accumulation of defective mitochondria due to oxidative damage and spontaneous mutations. The culling of defective mitochondria is thought to delay the onset of aging symptoms, and defects in mitophagy have been linked to late onset disorders such as Parkinson’s disease and type II diabetes. We previously demonstrated that different mitochondrial matrix proteins undergo mitophagy at different rates. We now find that dynamic mitochondrial matrix protein phosphorylation and dephosphorylation can generate a segregation principle that would couple with mitochondrial fission and fusion to selectively degrade sub‐sets of mitochondrial proteins on the basis of differential covalent modification. Our data support a model wherein differences in protein‐protein interactions between differentially phosphorylated proteins of the same species can drive a microscopic phase separation which, coupled with fusion‐fission dynamics, may account for the observed selectivity.
Optical Determination of Age‐Related Changes in Subpleural Collagen of Live Human Lungs
Authors: Islam Mohammad Naimul , Jin Guangchun , Bhattacharya Sunita , Bhattacharya Jahar
Published: April 2020

The impact of age on lung physiology is not known. Collagen is an important determinant of lung structure and function. Therefore, age‐related changes in collagen quantity, geometry and structure can lead to changes in lung function. CT scans of asymptomatic patients reveal age‐dependent increase of subpleural radiologic densities, called interstitial lung abnormalities (ILAs) that predispose to pulmonary fibrosis. However it is not known whether ILAs are collagen deposits that impair alveolar function. Since real‐time fluorescence microscopy (RFI) of the human lung has not been done, we carried out two‐photon and confocal microscopy (2P‐CF) of live, transplant‐reject lungs to determine the age‐related profile of subpleural collagen density (SCD) and whether this affects surfactant secretion. For RFI, we cannulated the bronchus and the artery of the lingular lobe to enable inflation and perfusion. Then, we held the lobe at constant alveolar pressure of 5 cmH2O. To stain alveolar epithelial cells, we subpleurally injected fluorescent dyes by means of a 31‐gauge needle. We detected subpleural collagen as the fluorescence of second harmonics generation (SHG). We quantified subpleural collagen density (SCD) as the integrated collagen fluorescence occurring in the space between the visceral pleura and the alveolar epithelium. To determine surfactant secretion, we quantified the timed appearance of lipid‐sensitive fluorescence in the alveolar space following a transient hyperinflation of the lingular lobe. Our data show that subpleural collagen was evident as well‐defined and evenly spaced cables in young lungs (median age 37 years). By contrast, subpleural collagen in aged lungs (median age 63 years) was marked by poorly formed cables surrounded by diffuse SHG fluorescence. We show that SCDs varied in different regions in all lungs and that SCD was significantly higher in aged than young lungs. Further, in high SCD regions, hyperinflation‐induced alveolar expansion and surfactant secretion were abrogated (p<0.05). Thus, SCDs increase with age and negatively regulate surfactant secretion from adjacent alveoli. SCDs may correspond to ILAs. Our findings raise the novel possibility that a risk‐factor in development of pulmonary fibrosis might be the disruption of alveolar homeostasis caused by age‐related changes in subpleural collagen (HL145547, HL36024).

Support or Funding Information

HL145547, HL36024
FXR regulates metabolic function of fat depots during obesity
Authors: Zhou Weinan , Anakk Sayeepriyadarshini
Published: April 2020

Obesity is a major epidemic and is associated with excess calorie intake leading to adipose tissue expansion and metabolic dysfunction. Farnesoid X receptor (FXR) is a nuclear receptor that transcriptionally regulates lipid and glucose metabolism. Despite several studies revealing FXR expression in the adipose tissue, its exact role in this tissue remains elusive. We determined that both primary pre‐ and post‐differentiated adipocytes from inguinal white (WAT) and brown (BAT) adipose tissue express Fxr transcripts. Notably, adipose‐specific Fxr knockout (Ad‐FxrKO) mice recently generated in our laboratory exhibit glucose intolerance, increased fat accumulation and concomitant reduction in the expression of lipolytic genes in both WAT and BAT when challenged with a high‐fat diet. These data suggest that adipose‐specific FXR may play a crucial role in regulating fat accumulation and subsequent glucose homeostasis during diet‐induced obesity. Since BAT is specialized for non‐shivering thermogenesis by generating heat through uncoupled mitochondrial respiration, we investigated the role for FXR in brown adipocyte mitochondrial function. We found that when Fxr is deleted, genes involved in BAT mitochondrial β‐oxidation, biosynthesis and respiratory chain complex are down‐regulated. Conversely, FXR activation by its endogenous bile acid ligand, chenodeoxycholic acid increases mitochondrial membrane potential of primary differentiated brown adipocytes in vitro, suggesting a key role for FXR in controlling brown adipocyte mitochondrial function and a potential role in thermogenesis. Overall, we demonstrate that FXR can modulate fat break down and regulate mitochondrial functions in the adipose tissue.

Support or Funding Information

Supported by R01 DK113080 from NIDDK, and UIUC Start Up Funds.
Mitochondrial Calcium Signaling Tunes Voltage‐Energized ATP Production
Authors: Wescott Andrew P. , Kao Joseph P. Y. , Lederer W. Jonathan , Boyman Liron
Published: April 2020

How mitochondrial ATP production dynamically responds to cellular metabolic needs is poorly understood. Here we report on quantitative investigations in heart muscle that identify the key molecular and cellular controls of ATP production. We show that Ca2+ influx into the mitochondrial matrix through the mitochondrial calcium uniporter (MCU) is small with no cytosolic Ca2+ threshold and tissue‐specific Ca2+ sensitive gating. MCU controls ATP production by its influence on matrix Ca2+ concentration ([Ca2+]m) which regulates pyruvate and oxoglutarate dehydrogenases and hence Krebs cycle production of NADH. The NADH level affects the voltage‐gradient across the inner mitochondrial membrane (Δψm) which in turn regulates ATP synthase. Our first report of the voltage‐dependence of ATP synthase in mammalian system is determined by measuring its ATP production. By this means, we show that the Δψm dependence of ATP synthase is different from the previous “gold standard”, the voltage‐dependence of bacterial ATP synthase. Additionally, we show how ATP production in cardiac mitochondria depends on both [Ca2+]m and on cytosolic [ADP]. Higher heart rates and contraction under load increase both Ca2+ sensitive and Ca2+ insensitive ATP production. These findings provide a new and different understanding of how mitochondria generate ATP in response to metabolic need, how ATP production is powered by Δψm, and how Δψm is regulated by mitochondrial Ca2+ signaling.

Support or Funding Information

This research was supported by American Heart Association grants SDG 15SDG22100002 (to L.B.) and 16PRE31030023 (to A.P.W.); by NIH R01 HL106056, R01 HL105239, U01 HL116321, 1R01HL142290, 1 R01 HL140934 and 1R01 AR071618 (to W.J.L.); by the Medical Scientist Training Program and Training Program in Integrative Membrane Biology, NIH 2T32GM092237‐06 and 5T32GM008181‐28 (to A.P.W.).
Regulation of alpha‐Arrestin Function in G Protein‐Coupled Receptor Signaling and Trafficking
Authors: Wedegaertner Helen , Pan Wen-An , Trejo JoAnn
Published: April 2020

The ubiquitously expressed β‐arrestins are known to play key roles in regulating G protein‐coupled receptor (GPCR) desensitization, internalization, and signaling. Humans also express a newly discovered family of arrestins, termed α‐arrestins, whose function remains poorly understood. While β‐ and α‐arrestins share minimal sequence homology, they are predicted to be structurally similar, except that β‐arrestins contain clathrin motifs within their C‐tails, which are known to be important in facilitating receptor internalization, and α‐arrestins contain PPxY motifs in their C‐tails, which are known to recruit WW‐domain containing proteins, such as Nedd4‐family E3 ubiquitin ligases. This suggests a separate role for α‐arrestins, although they may be subject to similar regulatory mechanisms, including phosphorylation, ubiquitination, and conformational change. Additionally, yeast, which do not express β‐arrestins, express arrestin‐related trafficking adaptors (ARTs), which are predicted to be structurally similar to human arrestins and contain PPxY motifs. ARTs have been well‐characterized as key regulators of receptor trafficking in yeast and are also regulated through post‐translational modification. Recently, we demonstrated a role for arrestin domain‐containing protein‐3 (ARRDC3), a mammalian α‐arrestin, in regulating trafficking of protease‐activated receptor‐1, a GPCR for thrombin. However, while GPCR regulation of β‐arrestin function has been well characterized, it remains entirely unknown how GPCRs regulate α‐arrestins, such as ARRDC3, or how such regulatory mechanisms might impact α‐arrestin function. Here, we examine how PAR1 signaling regulates ARRDC3 function in facilitating PAR1 trafficking. Using a variety of biochemical and microscopy‐based techniques, we show that PAR1 may regulate ARRDC3 through modulation of post‐translational modifications, and that identified ARRDC3 regulatory domains may play a role in regulating ARRDC3 function through controlling its subcellular localization and recruitment of interaction partners. We additionally will use a variety of approaches to elucidate the role of ARRDC3 regulatory mechanisms in PAR1 trafficking and signaling. Additionally, ARRDC3 has been identified to be a tumor suppressor in invasive breast cancer, functioning at least partially through its role in controlling PAR1 signaling and trafficking. Therefore, the results of these studies will not only advance our understating of how α‐arrestins are regulated but may also lead to the identification of novel therapeutic targets to prevent or treat invasive breast cancer.

Support or Funding Information

UCSD Graduate Training Program in Cellular and Molecular Pharmacology through NIH General Medical Sciences, T32 GM007752 and by National Institute of Health NIGMS R01 GM090689 and R35 GM127121 (JoAnn Trejo)
IGFBP‐3 Blocks Hyaluronan‐CD44 Signaling, Affecting Acetylcholinesterase Levels in A549 Cell Media and Cell Viability
Authors: Price Deanna , Muterspaugh Robert , Clegg Bradley , Williams Asana , Stephens Alexis , Guthrie Jeffrey , Heyl Deborah , Guy-Evans Hedeel
Published: April 2020

Insulin‐like growth factor binding protein‐3 (IGFBP‐3) belongs to a family of six IGF binding proteins. We previously found that IGFBP‐3 exerts its cytotoxic effects on A549 (p53 wild‐type) cell survival through a mechanism that depends on hyaluronan‐CD44 interactions. To shed light on the mechanism employed, we used CD44‐negative normal human lung cells (HFL1), A549, and H1299 (p53‐null) lung cancer cells. A synthetic IGFBP‐3 peptide (215‐KKGFYKKKQCRPSKGRKR‐232) but not the mutant (K228AR230A), was able to bind hyaluronan more efficiently than the analogous sequences from the other IGFBPs. In a manner comparable to that of the IGFBP‐3 protein, the peptide blocked HA‐CD44 signaling, and more effectively inhibited viability of A549 cells than viability of either H1299 or HFL1 cell lines. Treatment with the IGFBP‐3 protein or its peptide, affected acetylcholinesterase activity in the A549 cell media but not in the media of either HFL1 or H1299, an effect that correlated with changes in cell viability. These effects were diminished upon the same treatment of A549 cells transfected with either p53 siRNA or acetylcholinesterase siRNA. Taken together, our results show that IGFBP‐3 or its peptide blocks hyaluronan‐CD44 signaling via a mechanism involving p53 and acetylcholinesterase.

Support or Funding Information

Research reported in this work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R15GM131222 to Hedeel Guy Evans.
Case Report: The Fifth Liver Lobe; A Hybrid Left‐Sided Anatomical Variation of Accessory Liver Lobes
Authors: Amador Gonzalez Rafael E. , Jimenez-Dietsch Sofia , Quiñones-Rodriguez Jailenne I.
Published: April 2020

Accessory liver lobes (ALL) are supernumerary lobes in the liver. Although their origin is not completely understood, they appear to be due to a congenital anomaly caused by hyperplasia, trauma, and/or surgery. Two types of accessory lobes are documented in the literature: continuous and ectopic accessory lobes, differing in that the ectopic lobe is anatomically separate from the normal liver as opposed to the continuous subtype. Although rare, several reports exist with Riedel’s Liver Lobe being among the most well‐known. In these cases, the ALL is normally continuous with the right side of the liver and exists as either a sessile or pedunculate attachment. In our case, a rare ALL was found on the left lobe during a routine dissection of a male cadaver. However, structurally it is different from both continuous and ectopic ALL in that it is not joined to the liver via a connecting stalk nor is completely separate. Rather, this variant, a 7.5cm long ‐ 7.0cm wide‐2cm tall ALL displays a ligamentous attachment alongside the lateral edge of the left lobe. Most cases of ALL are asymptomatic and discovered incidentally. In imaging studies, ALL may be confused with tumors leading to misdiagnosis and errors in treatment. Although infrequent, there are reports of complications of ALL including infarction, hemorrhage, biliary atresia, and gallbladder torsion. Hypotheses for the embryological process of an ALL include heteroplastic embryological development as well as increased intraabdominal tension. Awareness of this variant will help decrease risk of iatrogenic injury in suspected ALL cases.
CYTOTOXIC POTENTIALS OF METHANOL EXTRACTS OF EKLONIA RADIATA AND JANIA VERUCOSA IN DIFFERENT CANCER CELLS AND NON‐CANCEROUS CELL LINE.
Authors: SAIBU GBEMISOLA MOROUNKE , Nakin Motebang Dominic , Oyedeji Adebola Omowunmi , Oyedeji Opeoluwa , Meyer Mervin
Published: April 2020

Introduction

Finding novel anticancer agents is very important for cancer treatment. Marine organisms are valuable source for developing novel agents for clinical applications. Seaweeds have been shown to have several biological activities, including anticancer activity. This study aimed at evaluating the cytotoxicity potentials of methanol extracts of Eklonia radiata (ER) and Jania verucosa (JV).

Materials and Methods

In this study, a panel of three cancer cell lines (HepG2, MDA and CaCo2) and one non‐cancerous cell line (KMST‐6) were treated with methanol extracts of Eklonia radiate (ER) and Jania verucosa (JV). Both ER and JV were found on rocky shores of the Eastern coast of South Africa, ER belongs to Kelp family while JV belongs to Corallinaceae family. The cells were treated with various concentrations of methanol extracts of ER and JV for 24 hrs, the percentage viability values of the cells were determined by the tetrazolium salt reduction assay (MTT).

Results

Morphological alteration of the cell lines after exposure with methanol extracts of Ecklonia radiata (ER) and Janica verucosa (JV) were observed under phase contrast microscope. The cytotoxicity result showed that cells treated with both methanol extracts of ER and JV had more cytotoxicity effect on MDA treated cells followed by HepG2 and Caco2. Non‐cancerous cell line (KMST‐6) however showed some selectivity in their activities.

Conclusions

Seaweeds generally change estrogen metabolism. However, in this present study, it can be established that both Eklonia radiate (ER) and Jania verucosa (JV) had potential for chemoprevention of human breast and this can be considered as potential sources of anticancer agent. Further studies are necessary for isolation and identification of biologically active substances present in these extracts.
The Emerging Role of C‐peptide on Immune Cells
Authors: Bunn Marcus , Pinger Cody , Spence Dana
Published: April 2020

Diabetes affects 30 million individuals in the U.S. today. Approximately 10% of these cases are type 1 diabetes (T1D). T1D is an auto‐immune disease that destroys the insulin‐producing cells of the pancreas (β‐cells), resulting in increased levels of blood glucose (hyperglycemia). Consequently, people with T1D suffer from many vascular health complications, such as nephropathy, neuropathy, retinopathy, and cardiovascular disease. In addition, individuals with T1D are immune deficient. It is hypothesized that hyperglycemia effects the expression of glucose transporter 1 (GLUT1), a known glucose transporter of circulating blood cells including immune cells. This could lead to important immune cells residing in an energy deficient state.

C‐peptide is a 31‐chain amino acid peptide co‐produced with insulin in the pancreatic β‐cells. Previous work from our group has shown that C‐peptide increases glucose absorption into, and ATP release from, red blood cells (RBCs). However, we have also shown that for C‐peptide to interact specifically with RBCs to elicit a biological response, it must be co‐administered with Zn2+ and albumin. Here, we measured the interaction between C‐peptide and immune cells (Tcells and neutrophils) in the presence or absence of Zn2+ and albumin. We also investigated Zn2+ binding to T‐cells and neutrophils in the presence or absence of C‐peptide and albumin. Furthermore, we studied the effect of C‐peptide on immune‐cell activation by performing functional studies to measure cell adhesion and migration. These functional studies were enabled by a novel 3D‐printed in vitro model system.

T‐cells and neutrophils were collected from whole blood samples from consenting adults and separated from other cell types by immunomagnetic separation. Immune cells were incubated with varying combinations of C‐peptide, Zn2+, and albumin. C‐peptide binding was determined using enzyme‐linked immunosorbent assay (ELISA). Zn2+ binding was determined radiometrically using 65Zn2+, and radiation was counted using a gamma counter. Cellular adhesion and migration assays will be quantified using fluorescence spectroscopy. Our results preliminarily suggest that albumin is required for delivery of C‐peptide, but not Zn2+. With albumin present we show 2.0 × 104 C‐peptide molecules bound per neutrophil. Interestingly, when albumin is absent, we observed no significant binding of C‐peptide to neutrophils. However, there appears to be additional Zn2+ added to the neutrophils when in the presence of both the albumin and C‐peptide system, suggesting a specific binding site for both on the neutrophil. Nearly identical numbers were observed for C‐peptide binding to T‐cells.

Support or Funding Information

NIH
(‐)‐Epicatechin Activates Pregnane X Receptor (PXR) in Skeletal Muscle
Authors: Ortiz Miguel , Najera Nayelli , Villarreal Francisco , Ceballos Guillermo
Published: April 2020

(‐)‐Epicatechin (EC) is a flavanol that has shown numerous biological effects such as: decrease cardiovascular dysfunction risk, metabolism regulation, induction of skeletal muscle (SkM) cells differentiation among others. The EC‐induced effects involve its interaction with acceptor/receptor like molecules and seem to be specific. The described EC acceptor/receptor molecules do not explain at all the EC’s effect on SkM. Based on structural similitude between EC and steroidal backbone, we decided to search for proteins that can recognize/interact with steroids and which activation/deactivation leads to similar effects as those induced by EC. The pregnane X receptor (PXR) can fulfill those characteristics; although the main function of this receptor is xenobiotics depuration, it is also linked to metabolism regulation and cell proliferation due to its transcription factor function. Taking as main objective the evaluation of a possible EC‐PXR interaction, docking assays were performed in order to establish if affinity exist between both molecules and if the interaction is comparable with a known agonist: pregnenolone‐16‐carbonitrile (PCN). To confirm the affinity of EC from PXR, this receptor was isolated from rat’s skeletal muscle homogenate using affinity chromatography with a column which has the flavanol attached. Similarly, the activation of PXR by (‐)‐Epicatechin was evaluated in vitro; cultured C2C12 cells (mouse myoblast) were stimulated with 0.1μM of EC or 0.1μM of PCN in order to see the expression of Cyp3a11 (a downstream gene promoted by PXR); the effect was blocked with 1μM of the antagonist Ketoconazole. The results reported here demonstrated that (‐)‐Epicatechin could interact and activate PXR.

Support or Funding Information

Conacyt 253769
Assessing Gender Disparities in Total Joint Arthroplasty from the National Inpatient Sample Database
Authors: Cheah Cheryl , Hussein Inaya Hajj , Sayeed Zain , El-Othmani Mouhanad M.
Published: April 2020

INTRODUCTION

Disparities in the health care system signify potential risks for vulnerable groups whose needs are not appropriately met. Several studies established basic inequalities between men and women with respect to health services available, including total joint arthroplasty (TJA) procedures‐. TJA has been successful in treating end‐stage arthritis, resulting in a steady increase in demand for the procedure over the last decades. This project aimed to assess and compare the differences in preoperative risk factors and postoperative outcomes between male and female patients undergoing TJA.

METHODS

Our study utilized de‐identified patient data from the National Inpatient Sample (NIS) database from 2006–2011. The NIS records ICD‐9‐CM codes to identify granular data involved in a patient’s in‐hospital stay. The data was stratified to create two cohorts of male and female patients that underwent primary TJA. We further stratified each cohort by matching for additional comorbidities using the Elixhauser Comorbidity Index. Postoperative and economic outcomes were measured for matched cohorts, respectively. Descriptive statistics were obtained, and univariate analyses using t‐tests and chi‐squared statistics were conducted for continuous and categorical data, respectively. Odds ratios with respective confidence intervals were calculated to assess the association of gender and post‐TJA complications.

RESULTS

Female TJA patients present at a later age for TJA procedures, and present with comorbidities that result in them having greater odds of experiencing in‐hospital complications compared to male TJA patients. Furthermore, female TJA patients have a longer average inpatient stay than male TJA patients, incurring more than average total charges. Male TJA patients are more likely to be discharged home as compared to female TJA patients.

CONCLUSION

Understanding these factors will help address the unmet needs and concerns of both genders regarding TJA, ensuring that qualified patients are able to access the necessary procedures for pain relief and functional improvement

Support or Funding Information

None
Knowing Real Cardiac Anatomy Enhances Understanding of Virtual Anatomy
Authors: Spicer Diane E. , Bolender David , Mori Shumpei
Published: April 2020

Present day advances in cardiac imaging, both clinically and with embryonic development, demonstrate the need for promoting attitudinally appropriate positioning of the heart in the dissection lab and with respect to the nomenclature of the cardiac structures. One of the first rules of anatomy is that all structures within the body should be described as viewed in the so‐called anatomical position. The true details of cardiac development are now being confirmed through the use of three dimensional imaging technology and can be directly correlated with the fully developed heart. In order to correlate the anatomic features depicted by actual dissection with the ever advancing imaging techniques, the anatomic details of the heart itself must be accurate. In the dissection lab, the heart is initially viewed within the thoracic cavity, but once removed it is typically assessed in the so‐called Valentine position. This no longer correlates with the clinical images or the actual position of the cardiac structures when viewed in their appropriate anatomic positon. The many nuances of cardiac anatomy can now be effectively taught using direct comparison between virtual and real dissection with both techniques allowing for adequate examination of the cardiac segments. Each of the cardiac segments and their junctions can then be assessed in their own right for their intrinsic anatomic features.

The long axis of the heart lies roughly at a 45 degree angle to the long axis of the spine. The position of the ventricles extends from right posterior to left anterior with the so‐called right‐sided structures actually lying anterior to those described as being left‐sided. The aortic root forms the center piece of the heart and this is demonstrated well with virtual dissection. Of necessity, demonstrating this by physical dissection requires the removal of those structures located anterior to the aortic root, namely, portions of the inner heart curvature, subpulmonary infundibulm and pulmonary trunk. Once removed, the structures cannot be replaced as they can with virtual dissection. The three segments of the heart are the atriums, the ventricles and the arterial trunks, with valvar structures guarding the junctions between the segments. Developmentally it has now been demonstrated that there are two ‘true’ components making up the atrial septum. These septal structures are largely surrounded by folds that are now easily appreciated with the use virtual dissection techniques. Learning the component parts of the atriums, their appendages, true septums and interatrial folds is of high clinical significance with the development of new interventional procedures. The relationships at the atrioventricular and ventriculo‐arterial junctions are easily documented and compared between the two dissection techniques. Analysis of the cardiac segments is now performed via virtual dissection just as well, if not better than in the dissection lab.