bluuebunny/crossref_metadata_2025 huggingface dataset:
Alcohol‐induced tight junction dysfunction primes airways for RSV infection (538.1)
Authors: Simet Samantha , Wyatt Todd , Pavlik Jacqueline , Sisson Joseph
Published: June 2021

Tight junctions form a belt‐like structure between adjacent cells whose degree of permeability changes according to external stimuli, physiological and pathological conditions. We have previously shown that alcohol increases tight junction permeability and disrupts tight junction proteins (ZO‐1 and claudin) in cultured airway epithelial cells through PKCα. It is well established that individuals who consume heavy and prolonged amounts of alcohol are more likely to suffer from pulmonary diseases and infections. Alcohol triggers tight junctions in airway epithelial cells to “leak”, which likely primes the airways for pulmonary infections. Thus, a second injury, such as viral or bacterial infection, is likely intensified by alcohol consumption. To date, very few studies have focused on the combination of alcohol and RSV infection. While both alcohol and RSV have been separately shown to disrupt tight junctions, little research has examined the combination of alcohol and RSV infection on tight junction function.

We hypothesize that alcohol primes the airways for RSV infection and exacerbates RSV infection significantly increasing tight junction permeability and airway epithelial injury. Preliminary cell culture data demonstrates that Gö6976, a PKCαinhibitor, and Y27632, a Rho‐associated protein kinase (ROCK) inhibitor, prevent alcohol‐induced tight junction “leak”. In addition, the combination of alcohol exposure and RSV infection increase tight junction permeability considerably more than RSV or alcohol alone. Together our preliminary data suggest that alcohol‐induced tight junction disruption primes the airways for RSV infection.

Grant Funding Source: Supported by I01BX000728 (TAW) and R01AA008769 (JHS)
Hsp90, hsp60 and hsf‐1 genes expression in muscle, heart and brain of thermal manipulated chicken (539.12)
Authors: Alzghoul Mohammad
Published: June 2021

The aim of this study was to investigate the effect of thermal manipulation (TM during embryonic days ED12 ‐ 18) on the gene expressions of Hsp90 and Hsp60 and HSF‐1 on hatchability, growth performance and thermotolerance acquisition during thermal challenge (TC) in three different thermal manipulated groups and also to investigate their ability to cope with thermal challenged (TC) during post hatching days (10 and 28) by using real time RT‐PCR and ELISA techniques. One thousand five hundred fertile chicken eggs were divided randomly into four groups: control group (37.8°C), TM1 (39°C for 9h), TM2 (39°C for 12h) and TM3 (39°C for 18h). Chicks from each treatment group were then randomly divided into 2 sub‐treatment groups (Naive and TC). Chicks in TC groups were subjected to thermal challenge at 43.0°C for 6h on days 10 and 28 of age while naïve chicks were kept under regular conditions. Chick’s response to TC was evaluated by determination of body temperature, plasma T3 and T4 levels and mRNA levels of Hsp90, Hsp60 & Hsf‐1 of muscle, heart and brain. There was a significant differences in muscle, heart and brain mRNA levels of Hsp90, Hsp60 & Hsf‐1 during embryogenesis and during TC in post‐hatch chicks that indicates tissue responds differently to heat stress. While hatchability was adversely affected, the body weight in TM3 chicks was significantly higher at the end of the study period (35 days). Results of this study indicated a long‐term enhancement of Hsp90, Hsp60 & Hsf‐1 gene expressions associated with thermotolerance acquisition in treated chicks without adversely affecting body parameters.
Satellite cell activation and populations on single muscle‐fiber cultures from adult zebrafish (Danio rerio) (539.8)
Authors: Zhang Helia , Anderson Judy
Published: June 2021

Satellite cell activation and populations on single muscle‐fiber cultures from adult zebrafish (Danio rerio) Helia Zhang and Judy E Anderson, Department of Biological Sciences, University of Manitoba, Canada Satellite‐stem cells in adult mouse skeletal muscle are activated for growth or regeneration by signaling via nitric oxide, hepatocyte growth factor (HGF) and the c‐met receptor. Here, the regulation of satellite cell activation in adult zebrafish was assessed using the model system of isolated fibers cultured at 27°C and 21°C. Satellite cells were identified c‐met+ staining; activation was determined by uptake of bromodeoxyuridine. Satellite cells were maximally activated by the NO‐donor isosorbide dinitrate (ISDN, 1mM) or HGF (10 ng/mL); levels lower and higher respectively, than in mouse fibers. HGF‐induced activation was blocked by anti‐c‐met antibody, and reduced at 21°C. Cyclical stretch (3 hr at 4 cycles per min) increased activation; NOS inhibition and culture at 21°C reduced stretch activation. Stretching rapidly increased the number of c‐met+ cells/fiber (by 3 hr). Satellite cell activation signaling on zebrafish fibers, in particular temperature‐dependent responses to HGF and stretch, give new insights into the influences of ectothermy in regulating muscle growth and suggest the single‐fiber model system can be used to explore the basis of fiber hyperplasia in teleosts.

Grant Funding Source: Supported by Natural Sciences and Engineering Research Council
Perineural invasion as an indicator of clinical outcome in vulvar carcinoma (54.2)
Authors: Holthoff Emily , Jeffus Susanne , Stone Rebecca , Gehlot Ashita , Quick Charles , Post Steven
Published: June 2021

Vulvar squamous cell carcinoma (vSCC) is a disfiguring malignancy that represents 4% of all gynecological cancers. A pathological feature that is commonly used in determining clinical prognosis and treatment is depth of tumor invasion. Perineural invasion (PNI) is another feature that is sometimes noted in pathology reports; however, the relevance of PNI to clinical outcomes in vSCC patients is not well defined. The purpose of this study was to determine the correlation between PNI and relevant clinical parameters including cancer stage and recurrence. 146 cases of vSCC were evaluated for PNI using pathology report review, H&E morphology, and IHC dual‐chromagen staining for S‐100 & AE1/3. Medical records were reviewed for clinical and follow‐up data. Statistical evaluation was performed using chi‐squared and t‐test calculations to correlate PNI with various parameters.

Patients with PNI had a 2.7‐fold higher risk of cancer recurrence (p=0.0298). Furthermore, 50% of patients with stage IV carcinoma had tumor‐associated PNI, whereas PNI was associated with <30% of stages I‐III tumors. PNI was also more closely associated with fibromyxoid stromal response (40%) than inflammatory stromal response (18%); however, this correlation was not statistically significant. Neither lymph node involvement nor lymphovascular infiltration was associated with presence of PNI. Interestingly, our data showed no significant correlation between the presence of PNI and depth of tumor invasion suggesting that PNI is an independent prognostic indicator of cancer recurrence. The significant association of PNI with recurrence, independent of tumor depth of invasion, should cause practicing pathologists to thoroughly search for and report its presence in vSCC.
Using zebrafish to identify and characterize gene‐ethanol interactions (540.10)
Authors: Lovely C. , McCarthy Neil , Sidik Alfire , Swartz Mary , Eberhart Johann
Published: June 2021

Fetal Alcohol Spectrum Disorders (FASD) is a complex set of ethanol‐induced defects with diverse phenotypes, particularly to the face. Though little is known of the genetic predisposition to FASD, this predisposition may help explain variation within FASD. The zebrafish is well suited for identifying and characterizing gene‐ethanol interactions because of its external fertilization, transparency, rapid development and genetic amenability. Across a set of small genetic screens, we have identified gene‐ethanol interactions in the Pdgf, Fgf, Bmp, and Wnt signaling pathways. Synergy is an underlying commonality among these interactions. Ethanol‐induced phenotypes are not predicted on the basis of the untreated mutant phenotype, and ethanol‐induced haploinsufficiency is observed that is not present in the untreated heterozygotes. For instance, a cell migratory defect causes facial defects in untreated pdgfra mutants with no elevation in cell death. Ethanol exposure results in a dramatic increase in cell death leading to facial defects in the majority of heterozygotes. The phenotypic spectrum for each gene‐ethanol interaction is unique, supporting the model that susceptibility loci govern some variation in FASD. Because of the success of our initial screens, we initiated a forward genetic screen to identify novel ethanol‐sensitive mutations. We have recovered and are currently characterizing 8 ethanol‐sensitive mutants. Most of these mutants display no overt phenotype in the absence of ethanol and genetic mapping of one of these places it in a region of the genome not containing a previously characterized mutant. These data demonstrate that zebrafish will be a useful model to identify and characterize gene‐ethanol interactions that may lead to variability in FASD.

Grant Funding Source: Supported by ABMRF and NIH
A new method for objective assessment of organ position during human development (540.6)
Authors: de Bakker Bernadette , Besselink Clara , Hagoort Jaco , Moorman Antoon
Published: June 2021

It is generally accepted that throughout development organs change position to one another within the body. Nonetheless, clear‐cut reports are lacking. Therefore, we aimed to unambiguously determine the position of organs compared to spinal ganglia and vertebrae.

Sections of human embryos from the Carnegie collection (CS14‐23 or 31‐60 days) were digitized. All organs were manually segmented and 3D‐reconstructions were made. To determine the position of organs, the intersection of the developing vertebrae with the notochord was used to generate planes perpendicular to the notochord, which were used to assess the position of an organ relative to the vertebrae or spinal ganglia.

For the first time a method is presented to unambiguously determine the position of an organ, relative to the vertebrae or spinal ganglia during development. We illustrate the value of the method by an example of the supposed changing position of the kidney, which, in contrast to descriptions in current textbooks, does not change significantly from stage CS17 to CS23. This implies that the proposed relationship of the development of a horseshoe kidney and the inferior mesenteric artery is questionable, because the kidney remains in place, whereas the inferior mesenteric artery descents owing to significant growth of the aorta.
Shroom3 is required for nephron development (540.9)
Authors: Khalili Hadiseh , Drysdale Tom , Sull Alexandra , Bridgewater Darren
Published: June 2021

Reduced nephron number is linked with chronic kidney disease and adult onset hypertension. Defects in the formation of nephrons are a major factor in determining nephron number. Multiple genome wide association studies have strongly linked SHROOM3 with altered kidney function and chronic kidney disease. Yet, the role of SHROOM3 in kidney function and nephron formation is not known. We utilized Shroom3‐/‐ and Shroom3+/‐ mutant mice containing a gene trap β‐galactosidase reporter to analyze Shroom3 expression. LacZ expression localized shroom3 to cap mesenchyme and podocytes throughout kidney development. LacZ expression was only observed in the collecting ducts after E15.5. LacZ expression was maintained in the podocyte and collecting ducts in mature kidneys. Immunohistochemistry using a Shroom3 antibody confirmed the dynamic spatial and temporal expression pattern. Histological analysis of kidneys from Shroom3‐/‐ mice demonstrated numerous condensed and cystic glomeruli at E13.5. Abnormal glomeruli demonstrated markedly reduced expression of the podocyte markers Wilms’ Tumor Suppressor‐1 (Wt‐1) and Nephrin in Shroom3‐/‐ mice at E13.5 and E14.5. Glomerular counting at E18.5 demonstrated a 1.6‐fold reduction in glomerular number in Shroom3‐/‐ mice when compared to wild type (n=3). Analysis of postnatal Shroom3 +/‐ mice revealed focal segmental glomerulosclerosis (FSGS), glomerular hypercellularity, and hydronephrosis. Postnatal expression of Wt‐1 was similar to wild‐type mice but nephrin expression was virtually absent. In summary Shroom3 exhibits a dynamic expression pattern and the absence of Shroom3 leads to early defects in nephrogenesis resulting in reduced glomerular number and postnatal glomerular diseases. We conclude that Shroom3 is required for nephron formation or maintenance.
CR405884, a hypothetical uncharacterized coding sequence, exhibits a peculiar expression pattern and FGF responsiveness during limb development (541.4)
Authors: Lujan Gabrielle
Published: June 2021

CR405884, a hypothetical uncharacterized coding sequence, exhibits a peculiar expression pattern and FGF responsiveness during limb development.

Gabrielle N Lujan, Christopher Armstrong, Charmaine U. Pira, Kerby C. Oberg: Division of Human Anatomy, Loma Linda University, Loma Linda, CA During limb development, the apical ectodermal ridge (AER) secretes fibroblast growth factors (FGFs) which maintain limb outgrowth, in part, by regulating Sonic Hedgehog (SHH) expression. SHH from the zone of polarizing activity (ZPA) in the distal posterior aspect of the limb bud is responsible for posterior limb growth and patterning. The molecules and pathways used by FGF to regulate SHH expression are not known. Application of ectopic FGF in the posterior limb mesoderm can induce SHH expression. Using this capacity, we performed microarray analysis on the tissue surrounding an FGF soaked bead, 24 hrs after application. Interestingly, CR405884, an uncharacterized hypothetical coding sequence, exhibited an 11‐fold increased expression in response to FGF and thus, was analyzed further.

We isolated CR405884 from chick cDNA by PCR and generated mRNA probes for whole mount in situ hybridization. We then determined the expression profile of CR405884 during limb development in stage 17‐27 embryos. To verify the microarray data, we implanted FGF‐soaked beads into stage 23 embryo limb buds, harvested the tissue after 24 hours, and evaluated CR405884 expression.

Expression of CR405884 was first evident in stage 21 limb buds as a focal, 100 um diameter region just anterior to the SHH expression domain. Over the next several stages examined, the expression expanded anteriorly and distally. Furthermore, CR405884 expression increased in response to FGF.

The focal expression of CR405884 anterior to and coincident with the expression of SHH may indicate a role for CR405884 in localizing SHH’s expression domain. This concept is further supported by its responsiveness to FGF, a known regulator of SHH expression.
Cardiac myocyte growth response to IGF1 and increased systolic load in lambs (542.2)
Authors: Wilburn Adrienne , Giraud George , Louey Samantha , Jonker Sonnet
Published: June 2021

The plasticity of the fetal heart allows a proliferative response to growth signals such as increased systolic arterial load (Load) and insulin‐like growth factor 1 (IGF1). The neonatal responses to these stimuli are unknown. OBJECTIVE To determine the in vivo growth response of neonatal cardiomyocytes to IGF1 and to Load compared to controls. METHODS Lambs were instrumented 7 days after birth with inflatable descending thoracic aortic occluders and vascular catheters. On 11‐18 days of age, lambs were given IGF1 (149 μg/kg/day, i.v.), vehicle (i.v.; control), or the occluder was inflated (final day: +35mmHg mean arterial pressure). Hearts were then enzymatically dissociated and myocyte size, maturation (nucleation), and cell cycle activity (an index of proliferation) were measured. RESULTS Hearts were heavier in Load (9.1±0.9 g/kg) and IGF1 (7.2±0.8 g/kg) groups compared to controls (6.6±0.4 g/kg). Cell cycle activity was not different between groups. In IGF1 lambs, mono and quadrinucleated right ventricular myocytes were significantly wider than controls (by 10% and 17%, respectively). In Load lambs, left ventricular mononucleated myocytes were significantly longer (by 14%), and there were 5% fewer binucleated myocytes compared to controls. CONCLUSIONS In the second week after birth, neonatal lamb cardiomyocytes respond to IGF1 and Load with cellular hypertrophy rather than proliferation.

Grant Funding Source: Supported by R01HD071068.
Cardiac innervation: what we know from gross anatomy (543.1)
Authors: Turner Benjamin , Blaak Christa , Tubbs Shane , Loukas Marios
Published: June 2021

The cardiac plexus is an extensive network of nerves located posteroinferior to the arch of the aorta at the base of the heart. The cardiac plexus receives autonomic contributions from both cervical and upper thoracic sympathetic trunks as well as contributions from vagus nerves. It is a notoriously variable and complex area that makes for difficult dissection and classification. The cardiac nerves and their branches anastomose into what are often described as two plexuses with significant interconnections between them: a superficial plexus, located just under the arch of the aorta; and a deep plexus, located anterior to and at the bifurcation of the trachea. Both plexuses send nerve fibers that follow the coronary arteries to their respective anterior and posterior locations on the heart. Sympathetic fibers dominate the plexus and carry with them those visceral sensory fibers that are considered responsible for referred cardiac pain‐‐damage to, or altered stimulation of which may lead to profound changes in innervation and cardiac function. Surgical interventions or procedures within the vicinity of the neck can interfere with the important autonomic feedback loops and regulation of the heart and even induce complex pain syndromes and altered growth. A detailed understanding of the anatomy and variability of the cardiac plexus is therefore an important component in providing treatment for many cardiac‐related conditions. In this study we will summarize our current knowledge of the anatomy and morphology of the cardiac plexus and correlate it with current diagnostic and clinical implications.
Anatomic variations of aortic arch branching pattern (543.11)
Authors: Khan Makhdoom , Rudder Nolan
Published: June 2021

The aortic arch branching pattern was examined in 39 cadaver specimens. The classic branching pattern of the aortic arch consists of the brachiocephalic trunk, left common carotid, and left subclavian arteries.The left and right vertebral arteries are normally branches of the left and right subclavian arteries, respectively.The present study demonstrates variant occurrence of the left common carotid and the left vertebral arteries. The left common carotid in one cadaver originated from the base of the brachiocephalic artery, crossed to the left side anterior to the trachea and then ascended on the left side of the cervical region. This anomaly can have various effects on the hemodynamics of the cerebral circulation and should be identified before performing any surgical procedures involving the aortic arch. In a separate cadaver, the left vertebral artery originated from the aortic arch, approximately 5mm posterior to the origin of the left subclavian artery. The anomalous left vertebral artery measured 7.34mm in diameter, while the right vertebral artery measured 7.26mm. The origin of the left vertebral artery from the aortic arch is significant as it has been associated with a higher incidence of arterial dissection than a left vertebral artery of subclavian origin. Anomalies in the aortic arch branching pattern are likely due to atypical development of the embryonic aortic arch system. A complete understanding of the normal aortic arch branching pattern, as well as its possible variations is vital to successful surgical and medical management of vascular pathologies.
Morphological variant of the posterior cerebral artery's origin and trajectory: a case report (543.13)
Authors: Daescu Ecaterina , Enache Alexandra , Baderca Flavia , Sztika Dorina , Zahoi Delia
Published: June 2021

The complexities of cerebral pathology have led to the necessity of detailed knowledge of arterial blood flow in the region. An important part of this is represented by the cerebral arterial circle.

During a routine dissection within the Department of Anatomy and Embryology’s laboratory, an atypical cerebral arterial circle was observed on the cadaver of a 58‐year‐old male. The subject’s cause of death had been unrelated.

From their origins, both posterior cerebral arteries cross the optic tracts and descend along the medial portion of the homolateral cerebral peduncle’s anterior side, down to the pontopeduncular sulcus. At this level, the left artery anastomoses with the superior cerebellar artery at its’ origin, changes direction by almost 90 degrees, crosses the optic tract yet again and continues towards the lateral side of the peduncle. The right artery also joins the superior cerebellar artery before taking an S‐shaped path. It takes a first sharp turn, crosses the anterior side of the cerebral peduncle obliquely up to the optic tract, before taking a second, wider turn and descending parallel to the lateral side of the peduncle.

The existence and variety of such morphological anomalies need to be taken into account by practitioners, both in investigations such as angiographies as well as during surgical interventions.
Anomalous drainage of the right testicular vein: a case report (543.4)
Authors: Zahoi Delia , Enache Alexandra , Sztika Dorina , Baderca Flavia , Daescu Ecaterina
Published: June 2021

Typically, the right testicular vein drains blood into the inferior vena cava, while the left testicular veins drains into the left renal vein. However, variations in the paths of the testicular veins do occur. Such variations can influence testicular drainage and even lead to varicocele, one of the causes of male infertility.

During a routine dissection conducted within the Department of Anatomy and Embryology’s laboratory, an anomalous path of the right testicular vein was observed on the cadaver of a 60‐year old. The person’s death had been the result of an unrelated cause. The anomaly discovered was that both the left and right testicular veins drained into the left renal vein. The right testicular vein ascended diagonally, crossed in front of the inferior vena cava and joined the lower side of the left renal vein, at an almost 90 degree angle and at a distance of 0.3cm from the inferior vena cava. The left testicular vein joined the left renal vein on the same side, but 4cm further away from the vena cava.

Such anatomical variants are clinically and surgically significant, given their influence on venous drainage at a testicular level, as well as the potential haemorrhagic complications during retroperitoneal surgery.
Variations in the transverse cervical artery (543.6)
Authors: Hurley Mary , Tomasik Zachary , Heller Scott , Isbrandt Robert , Stevens Todd , Olivieri Marion
Published: June 2021

The transverse cervical artery is typically seen in both the superficial back and posterior cervical triangle dissections in a human cadaver gross anatomy course. The two most commonly used references, the atlases of Grant and Netter, cite it as being a branch of the thyrocervical trunk. Also, these sources name the dorsal scapular artery as either a branch of the transverse cervical artery or a direct branch of the subclavian artery. Additionally, the suprascapular artery is typically seen as a branch of the thyrocervical trunk, but in some cases may be a branch of the transverse cervical artery. Ten cadavers were examined for source and length of the transverse cervical artery from source to the branching off of the dorsal scapular artery, and for other arteries that branched from it. In two cadavers it was a direct branch of the subclavian artery, and the suprascapular artery was a direct branch of the transverse cervical artery in two other cadavers. In this group of dissections, the transverse cervical artery was most often a branch of the thyrocervical trunk, with an average length of 5.875 cm from source to the branching off of the dorsal scapular artery.
Endothelial insulin‐like growth factor‐1 induced proliferation of vascular smooth muscle cells in response to low shear stress (544.4)
Authors: Qiu Yingxin , Wang Luis , Han Yue , Jiang Zonglai
Published: June 2021

Objective: Endothelial cells (ECs) are directly exposed to shear stress and modulate the function of vascular smooth muscle cells (VSMCs). Our previous proteomic research revealed insulin‐like growth factors (IGFs) might participate in low shear stress (LowSS) induced vascular remodeling.

Methods and Results: Using EC/VSMC co‐cultured parallel‐plate flow chamber system, our results showed, compared with normal shear stress (NSS, 15 dyn/cm2), LowSS (5 dyn/cm2) induced IGF‐1 mRNA expression in ECs, while increased phosphorylation of IGF‐1 receptor (IGF‐1R) and Akt, and expression of Sirt2 in co‐cultured VSMCs. Decreasing expression of IGF‐1 in ECs by RNA interference (RNAi) reversed the effects of LowSS on co‐cultured VSMCs. In separated VSMCs, IGF‐1 increased phosphorylation of IGF‐1R and Akt, expression of Sirt2, and cellular proliferation. PI3K/Akt specific inhibitor decreased expression of Sirt2 and VSMC proliferation, but had no effect on IGF‐1R. Whereas Sirt2 RNAi decreased VSMC proliferation, but had no effect on IGF‐1R and Akt.

Conclusion: The present results indicated that LowSS might induce the production of IGF‐1 from ECs, which subsequently paracrine controlled the phosphorylation of IGF‐1R and Akt in VSMCs, and then induced the expression of Sirt2, which finally modulated the proliferation of VSMCs. (Supported by National Natural Science Foundation of China, Nos. 11002091 and 11222223)

Grant Funding Source: Supported by National Natural Science Foundation of China, Nos. 11002091 and 11222223
Determining the subcellular localization and function of cardiac TRPV2 Ca2+ channels through fluorescent microscopy (545.4)
Authors: Slone Samuel , Onusko Evan , Koch Sheryl , Singh Umesh , Kraynik Steve , Tranter Michael , Rubinstein Jack
Published: June 2021

Transient receptor potential vanilloid 2 (TRPV2) is a ligand‐gated ion channel that is expressed in cadiomyocytes and increases contractility following stimulation with probenecid, an agonist for TRPV2. However, the subcellular location and mechanism of activation of this receptor are unkown. We used two independent approaches in cardiac‐derived HL‐1 cells to elucidate the localization and mechanism of TRPV2. To study TRPV2‐mediated Ca2+ transport into the cytosol, HL‐1 cells were first treated with a fluorescent Ca2+ dye and multiple fluorescently tagged moieties of probenecid, some permeable and others impermeable to the cytoplasmic membrane, to determine whether probenecid‐activated TRPV2 channels are located on the cytoplasmic or internal cell membranes. In addition, fluorescently labeled TRPV2 was overexpressed to directly assess its subcellular localization. Results show immediate and maximal cytosolic Ca2+ influx after TRPV2 stimulation, consistent with TRPV2‐mediated Ca2+‐induced Ca2+ release. This effect was blocked by tranilast, a TRPV2 antagonist. In conclusion, we have built upon our previous in vivo findings that TRPV2 stimulation increases cytosolic Ca2+ concentrations consistent with Ca2+‐induced Ca2+ release that may underpin the observed TRPV2‐dependent increase in cardiac contractility.
Effects of smoking on heart rate variability in students (545.6)
Authors: Saperova Elena , Dimitriev Dmitry
Published: June 2021

The purpose of the present study was to investigate cardiac autonomic function in smokers and nonsmoker controls.

Methods: Thirty nine light smokers (smoking less than 10 cigarettes a day) and thirty nine nonsmoking students with a mean age of 20.61±0.39 years take part in the study. Subjects were matched by sex, age, menstrual cycle phase and the level of anxiety. They were studied in the morning hours under standardized conditions, in a quiet room at a comfortable temperature. Time domain (SDNN, RMSSD, pNN50) and frequency domain (HF, LF and LF/HF ratio) parameters of HRV were obtained from all participants for 10 minutes while subjects were resting in a supine position. Statistical analysis was performed using Mann‐Whitney test.

Results: In this study heart rate was found to be higher in smokers than in nonsmokers (74.08±1.31 vs 64.98±1.12 beat/min, p=0.000007). Time domain parameters were found to be lower in smokers than in nonsmokers (SDNN: 52.54±3.47 vs 72.74±5.35, р=0.0009; RMSSD: 47.68±4.37 vs 71.22±7.72 ms, p=0.009 and pNN50: 26.01±3.21 vs 39.49±3.85, p=0.01). Frequency domain measures were also significantly lower in smokers than non‐smokers (TP: 3498.94±487.42 vs 6527.34±986.47, p=0.005; VLF: 787.28±82.31 vs 1934.20±214.45, p=0.0003; LF: 1147.14±257.82 vs 1895.41±346.76, p=0.007 and HF: 1184.21±245.04 vs 2274.53±487.37, p=0.026). The other HRV parameters including LF/HF ratio, pLF and pHF were not significantly different.

Conclusions: These results suggest that smoking has been associated with increased activity of the sympathetic nervous system and decrease in vagal cardiac control.

This work was supported by the Ministry of Education and Science of the Russian Federation (grant 4.4904.2011, grant 14.B37.21.0215).
Phosphatidylcholine‐specific PLC plays a central role in the development of myogenic tone in murine resistance arteries (546.4)
Authors: Mauban Joseph , Zacharia Joseph , Fairfax Seth , Wier W
Published: June 2021

The Bayliss mechanism posits that an increase in transmural pressure causes an artery to contract in response to distending pressure. Previous studies have established that PLC is critical for the myogenic response. However, the functional type/s of phospholipase C (PI‐PLC or PC‐PLC) involved and actions of PLC second messenger products (IP3, DAG) have not been clarified. This study of myogenic tone was therefore carried out to 1) identify the functional type/s of PLC involved and 2) investigate the role of IP3 and IP3‐induced Ca2+ release, in the maintenance of myogenic constriction. Pressurized mesenteric arteries showed that inhibition of phosphoinositide‐specific PLC with Edelfosine, surprisingly,increases myogenic tone at physiological pressures. In contrast, inhibition of phosphatidylcholine‐specific PLC with D609 abolishes myogenic tone. The Bayliss mechanism therefore involves a hitherto unrecognized role for the cleavage of phosphatidylcholine and formation of DAG. Ca2+ waves are virtually absent in myogenically active arteries. In addition, 1) SR Ca2+ release via IP3 and ryanodine receptors, and 2) Ca2+ store emptying, all do not eliminate myogenic constriction. In summary, the Bayliss mechanism signaling cascade in murine mesenteric resistance arteries does not require 1) sustained IP3 formation, 2) or Ca2+ release from SR stores via ryanodine and IP3 receptors. Instead, the signaling cascade critically requires PLC cleavage of phosphatidylcholine.

Grant Funding Source: Supported by NHLBI RO1091969 and NIH T32HL072751
Acute BH4 depletion leads to a loss of myocardial protection conferred by cardiac‐specific overexpression of inducible nitric oxide synthase in mice (547.11)
Authors: Boslett James , Barga Elizabeth , Kotwal Ameya , Choppara Bhavana , Zweier Jay
Published: June 2021

Many studies have shown that cardiac overexpression of NOS enzymes can lead to myocardial protection against ischemia/reperfusion (I/R) injury. However, none of these studies experiment with modulating BH4 levels. Because adequate BH4 levels lead iNOS to produce nitric oxide and inadequate BH4 levels lead iNOS to produce damaging superoxide, we hypothesized that depletion of BH4 in a model of constitutive iNOS expression would lead to a loss of iNOS function and diminished recovery of post‐ischemic myocardial contractility. The study involved two groups of iNOS TG mice: untreated mice and treated mice. Treated mice received 21 days of BH4 depletion diet using food with GTP cyclohydrolase inhibitor 2,4‐舃Diamino‐舃6‐舃hydroxypyrimidine (DAHP). The 21 day DAHP treatment led to a roughly 65% myocardial depletion of BH4 as measured by HPLC. Hearts from these two groups were used in isolated heart experiments and were subjected to 30’ of ischemia and 60’ of reperfusion. Compared to untreated mice, DAHP‐treated mice had significantly less recovery of rate pressure product, left ventricular developed pressure, and significantly more elevated end diastolic pressure at 30 and 60’ of reperfusion. This work shows the importance of adequate BH4 levels in protection against myocardial I/R, especially in the context of NOS overexpression.

Grant Funding Source: 00000784
Luteolinidin, a naturally occurring flavonoid, decreases ischemia/reperfusion injury through inhibition of CD38 and preservation of NADPH and BH4 (547.14)
Authors: Boslett James , De Pascali Francesco , Hemann Craig , Zweier Jay
Published: June 2021

CD38, a consumer of NAD+ and NADP+, is activated in I/R, leading to depletion of myocardial NADP(H) and consequently, lower eNOS function due to low levels of cofactor NADPH. Isolated hearts were subjected to two treatments: pre‐ischemic luteolinidin (25μM) infusion or vehicle control infusion (empty liposomes). In vehicle‐treated hearts at 30’ ischemia and 30’ reperfusion, NADPH and BH4 were depleted by 67% and 64% respectively. With 3’ of luteolinidin infusion (25μM), these numbers changed to 23% and 32% for NADPH and BH4, respectively, representing an impressive 44% salvage of NADPH, and a 32% salvage of BH4 compared to vehicle‐treated hearts. This NADPH and BH4 salvage was manifested physiologically as an increased loss of coronary flow (CF) in response to eNOS inhibitor L‐NAME (1mM) at 30’ reperfusion. Moreover, we found in luteolinidin‐treated hearts more post‐ischemic recovery of rate pressure product, left ventricular developed pressure and CF, and, a less elevated end diastolic pressure. This work suggests that inhibition of CD38 with luteolinidin is beneficial in I/R as measured by parameters of post‐ischemic endothelial and contractile function. The work also suggests that that post‐ischemic NADPH and BH4 levels are related, possibly through modulation of dihydrofolate reductase activity by NADPH levels.

Grant Funding Source: Supported by 00000784
Chronic exposure to low doses of HgCl2 avoids the impaired on calcium handling in the right ventricle after myocardial infarction in rat (547.3)
Authors: Stefanon Ivanita , Faria Thais , Costa Gustavo , Almenara Camila , Angeli Jhuli , Padilha Alessandra , Vassallo Dalton , Vassallo Paula
Published: June 2021

The aim of this study was to investigate if chronic exposure to low doses of mercury chloride (HgCl2) causes functional alterations in right ventricle (RV) strips after myocardial infarction (MI). Male Wistar rats were divided into four groups: Control (vehicle); HgCl2 (exposure during 4 weeks‐ 1st dose 4.6 μg/kg, subsequent dose 0.07 μg/kg/day, i.m to cover daily loss); MI surgery induced and HgCl2‐MI group. One week after MI RV strips, superfused with Krebs solution, were studied using an isometric tension system. Contractile function of HgCl2 group did not change compared to Control. On basal conditions, despite similar maximum isometric force at L‐max, relaxation time was reduced in the MI group but preserved in the HgCl2‐MI compared to Control group. The MI group presented a depression in the tetanic peak and plateau of developed force. Additionally, impaired sarcoplasmic reticulum (SR) function and a reduced sarcolemmal calcium influx were observed only in MI animals. The SERCA2a protein expression was reduced in both MI groups while PLB protein expression was increased only in MI group. These results suggest that chronic exposure to low doses of HgCl2 avoids the impaired SR function and the reduced sarcolemmal calcium influx observed in MI probably acting on PLB and SERCA2a protein expression.

Grant Funding Source: Supported by: PRONEX ‐ FAPES, CNPq, CAPES
Creatine influence on acute high and low ethanol exposure on cardiac heart muscle (547.5)
Authors: Ezeamama Precious , Haddad Gabriel
Published: June 2021

Cardiovascular disease is among the major causes for increased morbidity and mortality rates. High alcohol consumption may lead to cardiomyopathy, cardiac arrhythmias, and a suite of other disorders. Previous studies in our lab have linked the Akt/PI3K (serine/threonine kinase/ phosphoinositide 3‐kinase) and mitogen‐activated protein kinase (MAPK) pathway to cardiovascular disease. Creatine acting as an energy source in the cell can activate mTOR which is a mediator in the Akt/PI3K pathway which is involved in activating stress relief signals. In this study, since we know that alcohol is a stressor in the cardiovascular system, we hypothesize that high creatine can abrogate the stress of alcohol. We tested cardiac tissue perfused with low and high alcohol and two levels of creatine (30 µM and 100 µM). Our data shows a 133% decrease in ERK1/2 activity with high alcohol and 30 µM creatine. However, ERK1/2 activity shows a 74% increase with high alcohol and 100 µM creatine. P38 expression showed a general decrease with high alcohol and both levels of creatine (30 µM and 100 µM) as compared to low alcohol with 30 µM creatine. These results suggest that the high creatine levels (100 µM) may not be abrogating the MAPKs expression; however, it seems to have a small effect on the p38 stress signal.
Mitochondrial DNA damage and mitochondrial function during acute oxidative stress induced by H2O2 in Saccharomyces cereviseae (550.4)
Authors: Arroyo Yaria , Acevedo‐Torres Karina , Ayala‐Peña Sylvette , Torres‐Ramos Carlos
Published: June 2021

Base excision repair (BER) is the main repair mechanism responsible for the removal of mitochondrial DNA (mtDNA) damage, however, the relationship between mtDNA damage and mitochondrial function is not fully understood. We seek to determine the effect of mtDNA damage and mitochondrial function in yeast strains harboring mutations in BER genes after acute oxidative stress induced by H2O2. We observed that compared to a wild type (Wt) strain, mtDNA lesions after H2O2 treatment increased 4.3 fold in an ogg1∆ strain, 4.1 fold in an apn1∆ strain and 21.2 fold in a double mutant ogg1∆ apn1∆ strain. Similarly, mtDNA mutation rate increased in cells treated with H2O2 in the following manner: Wt strain, 7.7 fold; ogg1∆ strain, 11.9 fold; apn1∆ strain, 19.9 fold, and ogg1∆ apn1∆ strain, 30.6 fold. H2O2 treatment disrupted mitochondrial function as determined by mitochondrial membrane potential measurements: 15%, 15%, 10% and 24% decreased in Wt, ogg1∆, apn1∆, and ogg1∆ apn1∆, respectively. Finally, increased superoxide anion generation was observed after H2O2 treatment, particularly in the BER double mutant strain. We can conclude that an interaction between the OGG1 and APN1 genes is required for the maintenance of mitochondrial DNA integrity and function after an acute oxidative stress insult.

Grant Funding Source: Supported by 5SC3GM08475902, R25GM068138 and G12RR03051
Translocation of catalase and SODs protects from doxorubicin‐induced nuclear damage in cultured human ovarian cancer cells (554.2)
Authors: Best Charles
Published: June 2021

Doxorubicin is applied clinically to treat ovarian cancer, and yet the molecular mode of actions of doxorubicin is far from clear. Our most recent study has shown that antioxidant N‐acetylcysteine (NAC) potentiates doxorubicin‐induced activation of p53 and ATM, suggesting that endogenous antioxidant enzymes may play important roles in the response of ovarian cancer cells to doxorubicin treatment. In this study, we investigated the expression of catalase and SOD1, 2 and 3 in cultured ovarian cancer cells (CaOV3). Western blot data showed that CaOV3 cells express catalase and SOD1, 2, and 3. Serum starvation reduces expression of those enzymes, while EGF treatment induces EGFR and mTOR activation and the expression of those enzymes. mTOR inhibitor, Rapamycin, PI3K/AKT inhibitor LY294002, and MEK/ERK inhibitor U0126 inhibit expression. Interestingly, doxorubicin treatment induces translocation of those enzymes from cytoplasm to nucleus in a dose and time dependent manner, however, chemo drug paclitaxel has no such effect. Our data suggest that to avoid widespread oxidative stress and nuclear damage, ovarian cancer cells protect themselves via translocation of the antioxidant enzymes in response to doxorubicin treatment. Thus, inhibition of such translocation may enhance the efficacy of doxorubicin in vivo. Collectively, our data support the notion that combo treatment with inhibitors of mTOR and MEK/ERK pathways and doxorubicin may yield better clinical outcome.

Grant Funding Source: Rhode Island INBRE
Endoplasmic reticulum‐associated degradation of mutant rhodopsin disrupts protein homeostasis in photoreceptor cells during retinal degeneration (558.3)
Authors: Chiang Wei‐Chieh , Kroeger Heike , Messah Carissa , Yasumura Douglas , Matthes Michael , Sakami Sanae , Palczewski Krzysztof , LaVail Matthew , Lin Jonathan
Published: June 2021

Rhodopsin is a G‐protein coupled receptor that is expressed exclusively by rod photoreceptors of the retina and is essential for phototransduction and vision. Over 150 missense mutations in rhodopsin have been identified in the hereditary forms of retinal degeneration. Many of these missense mutations, such as P23H mutation, cause rhodopsin protein misfolding and ultimately lead to rod cell death and vision loss via poorly understood mechanisms. Here, we examined a P23H knock‐in mouse model that exactly recapitulates the gene dosage and retinal degeneration disease phenotype seen in human families carrying P23H rhodopsin alleles. In these retinas, we find that photoreceptors immediately target P23H rhodopsin for elimination as soon as it is produced. Through analysis of P23H knock‐in mice crossed with Xbp1‐Venus reporter mice, we find that photoreceptors expressing P23H rhodopsin strongly activate the IRE1 branch of the UPR. IRE1 activation leads to pronounced upregulation of ER‐associated protein degradation in photoreceptors, pronounced ubiquitination of P23H rhodopsin, and its rapid degradation. By contrast to heterologous cell culture studies of misfolded rhodopsin, virtually no misfolded rhodopsin protein accumulates within photoreceptors as ER‐retained aggregates. Consistent with the efficient elimination of P23H rhodopsin from the ER of photoreceptors, we find no increase in proapoptotic Chop, induced by persistent ER protein aggregation. Surprisingly, despite the induction of ERAD and rapid elimination of P23H rhodopsin, total cellular levels of ubiquitylated proteins markedly increase during retinal degeneration. Our findings show that the IRE1 branch of the UPR promotes robust ERAD of P23H rhodopsin in photoreceptors. We suggest that the increased buildup of damaged, ubiquitylated proteins arises as a consequence of ERAD of P23H rhodopsin, and disruption of cellular protein homeostasis leads to rod photoreceptor cell death and retinal degeneration.
U2 snRNP is required for the phosphorylation of Pol II CTD Ser2 and efficient transcription elongation (560.4)
Authors: Koga Mitsunori , Satoh Takayuki , Kaida Daisuke
Published: June 2021

Pre‐mRNA in eukaryotes is subjected to post‐transcriptional modifications, such as capping, polyadenylation and splicing. Transcription and post‐transcriptional modifications are coupled and this coupling stimulates post‐transcriptional modifications, however, the effects of post‐transcriptional modifications on transcription are not fully understood. Here, we report that inhibition of U2 snRNP by a splicing inhibitor, spliceostatin A (SSA), and an antisense morpholino oligonucleotide to U2 snRNA caused 3’ end down regulation in a gene‐specific manner. We also show that U2 snRNP inhibition resulted in the dephosphorylation of second serine residues (Ser2) within heptad repeats of the C‐terminal domain (CTD) of Pol II, which is important for transcription elongation; this dephosphorylation was correlated with splicing inactivation. Interestingly, SSA treatment caused accumulation of Pol II near the 5’ ends of genes, indicating that SSA treatment led to transcription elongation arrest. These findings suggest that a novel checkpoint mechanism prevent pre‐mRNA accumulation and the production of aberrant proteins translated from pre‐mRNA upon splicing deficiencies through the dephosphorylation of CTD Ser2.
RNA polymerase III transcription factor complexes block transcriptional interference from intergenic RNA polymerase II progression in Saccharomyces cerevisiae (561.3)
Authors: Korde Asawari , Rosselot Jessica , Donze David
Published: June 2021

Transcription of transfer RNA (tRNA) requires sequential assembly of RNA polymerase III (Pol III) transcription factors ‐ TFIIIC and TFIIIB – followed by the recruitment of the Pol III enzyme complex. Recent studies have shown that in addition to producing tRNAs, Pol III transcription complexes assembled on DNA can exert “extra‐transcriptional” effects on chromosome function. Our study provides evidence of a unique extra‐transcriptional activity of assembled Pol III transcription complexes at a tRNA gene that blocks progression of intergenic RNA polymerase II transcription. We demonstrate that the Pol III transcription complex bound to the tRNA gene upstream of the Saccharomyces cerevisiae ATG31 gene protects the ATG31 promoter against readthrough transcriptional interference from the upstream non‐coding intergenic SUT467. Chromatin immunoprecipitation confirmed that the protection is predominately mediated by binding of the TFIIIB complex. Failure to block this readthrough results in compromised ATG31 translation. Since the Atg31p is required for autophagy in yeast, strains expressing the readthrough transcript exhibit reduced fitness under autophagy‐inducing conditions. Given the recent discovery of pervasive transcription in yeast, protection of neighboring genes from intergenic transcriptional interference may be a key extra‐transcriptional function of assembled Pol III complexes.
Analysis of the transcriptional activation and post‐transcriptional regulation of transient receptor potential mucolipin‐2 gene (561.5)
Authors: Valadez Jessica , Cuajungco Math
Published: June 2021

Mucolipidosis type IV (MLIV) is a neurodegenerative disease caused by loss or dysfunction of the human transient receptor potential mucolipin 1 (TRPML1) protein. TRPML1 is a non‐selective cation channel related to two other proteins, TRPML2 and TRPML3. The high degree of sequence and functional similarities between TRPML1 and TRPML2 make TRPML2 an excellent candidate to potentially substitute for the loss of TRPML1 function in MLIV. In order to consider this possibility, we first needed to study the mechanisms that control the expression of TRPML2 gene. We cloned DNA regions of varying size upstream of the TRPML2 gene transcriptional start site (TSS) into a dual luciferase reporter assay. We identified a putative core promoter region between ‐320 and ‐287 of the TSS containing a consensus sequence for six transcription factors. Heterologous expression of the candidate TFs conferred an effect on the expression of endogenous TRPML2 transcripts compared to control. Meanwhile, we found that a specific micro‐RNA was responsible for post‐transcriptional regulation of endogenous TRPML2 transcripts as evidenced by real‐time quantitative PCR (qPCR) technique. We then screened a drug library and confirmed by real‐time qPCR that each drug candidate significantly increased the expression of endogenous TRPML2 transcripts. These results show that we could induce the expression of the TRPML2 gene through exogenous manipulation of transcriptional activators or post‐transcriptional regulators. Understanding the mechanism of TRPML2 expression is a step closer to our goal of using TRPML2 protein to substitute for the loss of TRPML1 protein function in MLIV.

Grant Funding Source: Supported by NIH R15‐NS070774 and CSUF Intramural Grants