bluuebunny/crossref_metadata_2025 huggingface dataset:
Epigenetic regulation of miR‐31 modulated apoptosis in prostate cancer (LB609)
Authors: Zhang Qunshu , Guo Bin
Published: June 2021

Prostate cancer is the second leading cause of cancer‐related mortality in the United States. However, our understanding of the cause of prostate cancer remains elusive. Epigenetics, heritable changes in gene expression caused by mechanisms other than classical genetic alterations, is an important mechanism in tumorigenesis. MicroRNAs function as epigenetic regulators to alter gene expression. Evasion of apoptosis is the hallmark of cancer, and it plays an important role in the growth and development of prostate cancer. We have recently demonstrated that miR‐31 and miR‐205 are downregulated in advanced prostate cancer. And anti‐apoptotic protein E2F6 was identified as the target of miR‐31 and contributed resistance to chemotherapy‐induced apoptosis. While we found that miR‐205 is silenced by gene promoter methylation, the mechanism of the downregulation of miR‐31 in prostate cancer remains poorly defined. In this study, we report that EZH2 (Enhancer of zeste 2), a histone methyltransferase, is responsible for miR‐31 silencing in prostate cancer. EZH2 plays an essential role in the epigenetic maintenance of the H3K27me3 repressive chromatin mark. Our results have shown that siRNA knockdown of EZH2 and DZNep (3‐deazaneplanocin) treatment (an EZH2 inhibitor) in prostate cancer cells increased miR‐31 expression. Moreover, EZH2 is responsible for histone methylation at the promoter of miR‐31 gene. In addition, siRNA knockdown of EZH2 and the treatment with DZNep sensitized prostate cancer cells to apoptosis. In contrast, EZH2 overexpression confers resistance to chemotherapy‐induced apoptosis in prostate cancer. Thus, polycomb protein EZH2 suppresses the expression of miR‐31 and contributes to apoptosis resistance in prostate cancer. The results will improve our understanding of the mechanism of drug resistance in prostate cancer cells. Novel strategies targeting EZH2 and miR‐31 can be developed to increase drug‐induced apoptosis and improve cancer response to chemotherapy.
Cardiac responses to selected cranial osteopathic manipulations in healthy young men (LB656)
Authors: Ioudina Marina , Toder Eric , Baldwin Cory , Lowe Rachel
Published: June 2021

Background and Purpose: Cranial manipulations (CM) as a part of osteopathic manipulative treatment have been used to treat patients. However, physiological responses to CM are not well defined in literature. The purpose of this study was to measure changes in heart rate (HR), heart rate variability (HRV), stroke volume index (SI), and cardiac index (CI) in response to selected CM and sham manipulation.

Method: Healthy 25‐35 year old men participated in this study. Non‐invasive impedance cardiography method was used to measure changes in cardiac parameters before and after manipulations. Data were recorded and analyzed using BIOPAC Systems, Inc. equipment and software. All manipulations were performed by the same osteopathic physician.

Results: CM increased parasympathetic input (70%) to the heart resulting in decreased HR (5%) and CI (7%) without significant effect on SI. These changes were not seen in sham manipulation.

Conclusion: Selected CM decreased CI by decreasing HR without significant effect on cardiac contractility in healthy young men. Future studies will compare gender and age differences in response to osteopathic techniques.

Grant Funding Source: This project was funded by the TUN Research Grant Award
High‐intensity interval exercise protects against postprandial endothelial dysfunction in obese adolescent males (LB662)
Authors: Ryder Justin , Hultgren Neil , Barazza Estela , Gaesser Glenn , Shaibi Gabriel
Published: June 2021

In adults, consuming a high‐fat meal can induce endothelial dysfunction while exercise may mitigate postprandial endothelial dysfunction. Whether exercise is protective against postprandial endothelial dysfunction in youth is unknown. The purpose of this study was to determine if high‐intensity interval exercise (HIIE) performed the evening prior to a high‐fat meal protects against postprandial endothelial dysfunction in obese adolescent males. Fourteen obese adolescent males (BMI%tile=98.5±0.6; 14.3±1.0yrs; VO2peak=2.6±0.5 L/min) completed a single HIIE session on a cycle ergometer (8 X 2min at 蠅90%HRmax, with 2min active recovery between bouts, 42min total) in the evening. Participants returned at 09:00 the following morning where brachial artery flow‐mediated dilation (FMD) was measured using duplex ultrasound after 20min of supine rest in the fasted state. Following baseline FMD (7.5±3.0%), a high‐fat milkshake (; 60% Fat, 35% CHO) consisting of 0.7g/kg of body weight was consumed (766±84 kcal). FMD was subsequently measured at 2hr and 4hr post‐consumption. Despite consuming a high‐fat meal, endothelial function was preserved at 2hr (8.4±3.4%) and 4hr (8.8±3.9%) post‐meal (p(time)=0.598). These data suggest that HIIE may protect against the deleterious vascular effects associated with a high‐fat diet in obese adolescent males.

Grant Funding Source: American College of Sports Medicine
Cardiac autonomic modulation on knockout mice for β3‐adrenergic receptor. (LB663)
Authors: Santos Fernando , Araujo Iara , Andrade Romario , Preite Z , Ribeiro Mirian , Irigoyen Maria , Fiorino Patricia , Farah Vera
Published: June 2021

Beta‐adrenoceptors (β‐AR) play an important role in cardiac autonomic function. There are three β‐AR subtypes (β(1), β(2), β(3)) described and identified. β(3) when stimulated, induce negative inotropic effects in order to protect the heart from catecholamine overstimulation. The aim of this study was to determine the cardiac autonomic modulation in experimental model of β3 adrenergic receptor absence. Knockout and wild type mice for β3 receptor were used (ARβ3KO and WT, respectively). The male animals with 8 weeks old were submitted to catheterization of carotid artery. This catheter was connected to a transducer and continuous signals of blood pressure (BP) and heart rate (HR) were recorded and analyzed by Windaq® software. The variability of resultant signal was evaluated in time and frequency domain by Fourier Fast Transform. Although there were no difference in BP and HR between the groups, the HR and BP variance are respectively 113% and 96% higher on ARβ3KO. Moreover, the LF component for HR (24.62 ± 3.4 vs. 7.90 ± 1.8 ms²) and BP (38.04 ± 8.1vs. 20.32 ± 2.8 mmHg²) were increased in ARβ3KO when compared with WT. The sympathetic‐vagal balance was increased in ARβ3KO (1.58 ± 0.28) when compared to WT (0.61 ± 0.09). In conclusion, our data showed that the absence of β3 receptor determined an increase in cardiac sympathetic modulation confirming the cardioprotective role this receptors.
Inhibition of Mammalian Sterile 20‐like Kinase 1 Rescues Cardiomyopathy through Protection from Myocyte Necrosis as well as Apoptosis (LB700)
Authors: Lee Grace , De Lorenzo Mariana , Vatner Stephen , Vatner Dorothy , Yan Lin
Published: June 2021

Inhibition of Mammalian Sterile 20‐like Kinase 1 (MST1) is known to have cardioprotective effects by preventing myocyte apoptosis. We examined the effects of MST1 inhibition in β1‐adrenergic receptor (β1‐AR) cardiomyopathy by mating dominant negative (DN)‐MST1 mice with β1‐AR mice. As the mice aged, the bigenic mice had higher left ventricular ejection fraction (67+12% vs 43+5%) and decreased cardiac fibrosis (8.8+1.5% vs 19.5+2.7%). It is generally believed that fibrosis in cardiomyopathy is secondary to necrosis, more than apoptosis. Accordingly, we hypothesized that MST1 may also mediate necrosis. We examined necrosis through in‐vivo labeling by Evans Blue Dye in DN‐MST1 mice after continuous isoproterenol (ISO) challenge. At 1 day after ISO the wild type hearts showed massive myocyte necrotic death (0.90+0.37%), which was significantly protected in DN‐MST1 (0.32+0.14% p<0.05), while having no discernible differences in myocyte apoptosis (0.10+0.03% vs 0.08+0.01%). These results suggest that MST1 may also have an important role in myocyte necrosis as well as apoptosis.
Dietary sodium loading increases systolic BP variability in salt resistant adults: a role for serum sodium (LB701)
Authors: Brian Michael , Wenner Megan , Evan Matthews , Ramick Meghan , Edwards David , Stocker Sean , Farquhar William
Published: June 2021

Dietary sodium loading increases systolic BP variability in salt resistant adults: a role for serum sodium Michael S. Brian1, Megan M. Wenner1, Evan Matthews1, Meghan G. Ramick1, David G. Edwards1,2, Sean D. Stocker3, William B. Farquhar1,2 1Department of Kinesiology & Applied Physiology and 2Department of Biological Sciences, University of Delaware, Newark, DE, 3Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, PA.

Increased systolic blood pressure (SBP) variability has been linked to poor cardiovascular outcomes. We examined the effects of dietary sodium‐induced increases in serum sodium (s[Na+]) on SBP variability in salt resistant adults. Twenty‐nine subjects (38±14 yrs) were randomized to low (LS: 20 mmol/d) and high (HS: 300 mmol/d) sodium diets (1‐week each). All subjects were salt resistant (mean BP change < 5 mmHg between diets). Subjects were ranked based on the increase in s[Na+] going from LS to HS diet, and a median split was utilized such that two groups were compared (s[Na+] responders: 2.6±0.8 mEq/L vs. s[Na+] non‐responders: ‐0.63±2.2 mEq/L). 24‐hr BPs were recorded during the last day of each diet (every 20‐30 min/24 hrs); the standard deviation (SD) of SBP was used as an index of variability. During the high dietary sodium condition, SBP variability was greater in the s[Na+] responders compared to the s[Na+] non‐responders (13.8±3.2 vs. 10.8±1.6, mmHg, p < 0.01). Moreover, the change in SBP variability going from LS to HS diet was greater in the s[Na+] responders compared to the s[Na+] non‐responders (Δ3.0±2.2 vs. Δ‐0.90±2.4 mmHg, p < 0.01). Linear regression analysis across all subjects supported a modest relation between s[Na+] and SBP variability (r2=0.32, p < 0.01). These data suggest that dietary sodium loading can increase SBP variability in salt resistant adults, but only in those that have a diet‐induced increase in s[Na+].

Grant Funding Source: NIH Grant R01 HL104106
Single‐unit muscle sympathetic recordings identify in human heart failure unique fibers discharging in response to both unloading and loading of cardiopulmonary receptors (LB735)
Authors: Millar Philip , Murai Hisayoshi , Morris Beverley , Floras John
Published: June 2021

In healthy men, multi‐unit muscle sympathetic nerve activity (MSNA) has been shown to integrate two populations of single‐units exhibiting directionally opposite discharge to acute selective perturbations in atrial pressure. The behaviour of these single‐units was consistent during application of lower body negative and positive pressure (LBNP and LBPP). The aim of the present study was to compare single‐unit discharge characteristics of patients with and without heart failure (HF). We measured central venous pressure and single‐unit MSNA in 6 HF patients (LVEF: 24±6%; 54±13 years [mean±SD]) and 9 healthy controls (56±8 years) before and during 7 minutes of LBNP and LBPP (±10mmHg). These interventions had no effect on stroke volume, heart rate or systemic blood pressure (All P>0.05). Twelve HF and 22 control single‐units were identified. In controls, single‐unit responses to LBNP and LBPP exhibited either ‘anticipated’ (increased with LBNP and decreased with LBPP) or ‘paradoxical’ (decreased with LBNP and increased with LBPP) firing behavior. In HF, the majority (8/12; 67%) of single‐units exhibited a unique U‐shaped discharge pattern, with ‘anticipated’ responses to LBNP but ‘paradoxical’ responses to LBPP. This novel observation provides evidence for the emergence in HF of a population of cardiopulmonary mechanoreceptors exerting consistently sympathoexcitatory efferent firing characteristics.

Grant Funding Source: Supported by the Canadian Institutes of Health Research (PJM) and Canadian Research Chairs program (JSF)
Expression of multiple adenylyl cyclase isoforms in pancreatic cancer cell line (LB740)
Authors: QUINN SIERRA , Sohail Syeda , SABBATINI MARIA
Published: June 2021

Introduction: Cyclic AMP (cAMP) is implicated in the proliferation and differentiation of several normal and malignant cell types, including pancreatic cancer. Adenylyl Cyclases (ACs) are enzymes which increase the levels of cAMP when activated. Nine transmembrane AC isoforms are known, and each one has its own pattern of expression and regulation. Objective: In this study we establish which transmembrane AC isoforms are expressed in the pancreatic adenocarcinoma epithelial cell line HPAC. Results: Using RT‐PCR we found five different isoforms: AC1, AC3, AC6, AC7 and AC9. The expression profile is similar to that in pancreatic duct cells, but AC1, which is only expressed in pancreatic cancer cells. Future goals: Study which isoform is involved in the development and progression of HPAC cells. Discussion: Overall, the pathophysiological significance of each AC isoform will provide an important insight into the cellular signaling in pancreatic cancer.
Brilliant Blue G attenuates the effects of intestinal ischemia/reperfusion injury in rats (LB747)
Authors: Palombit Kelly , Mendes Cristina , Castelucci Patricia
Published: June 2021

During pathological conditions there is an increased release ATP into the extracellular space, activating the P2X7 receptor. Several studies have shown that injury can be attenuated by the antagonist of this receptor, the Brilliant Blue G (BBG). In the present work we analyzed the effects of intestinal ischemia and reperfusion (I/R) and the effects of the BBG on rats ileum. Tissues were obtained from male Wistar rats (n=5). The ileal artery was occluded for 45 minutes with an atraumatic vascular clamp. BBG (50 and 100 mg/kg) or saline (vehicle) was given subcutaneous 1 h after injury (24 h group). The sham group was rats without the arterial occlusion. We analyzed also the I/R 0 h group (not reperfusion). Myenteric neurons were evaluated for immunoreactivity (ir) against the P2X7 receptor, nitric oxide synthase (NOS), neurofilament (NF), choline acetyl transferase (ChAT) and anti‐HuC/D (Hu). Technique histology staining to hematoxylin and eosin (HE) was performed to evaluate the integrity of the intestinal wall and myeloperoxidase technique (MPO) to verify the marking of neutrophils. Our qualitative results showed that I/R caused histological damage to the mucosa and muscle and infiltration of neutrophils into the muscle layers. Our quantitative results showed that there was a decrease in density (neurons/cm2) P2X7‐ir, NOS‐ir, NF‐ir, ChAT‐ir and Hu‐ir neurons in I/R 0 h and 24 h groups. In I/R groups where the antagonist BBG was injected, the neuronal density was less affected. Analysis in the area of the cell body profile different classes showed generally decreases in the profile area in the I/R groups and recovery profile area of the I/R BBG‐50 and BBG‐100 groups. This work indicates that deleterious effects are greater in groups subjected to I/R in rats without application BBG, suggesting that BBG has protective effects on the intestinal wall and in different neuronal classes.

Grant Funding Source: FAPESP
Maternal obesity reduces adipogenic progenitor density and DNA methylation of Zpf423 promoter in the visceral fat of offspring mice (LB770)
Authors: Liang Xingwei , Yang Qiyuan , Fu Xing , Rogers Carl , Zhu Meijun , Du Min
Published: June 2021

Maternal obesity (MO) is a worsening problem. Offspring born to obese mothers have high risks of obesity and type II diabetes, but the underlying mechanisms remain poorly defined. Zfp423 is a key transcription factor committing progenitor cells to preadipocytes, and its transcription is regulated by the DNA methylation. Our hypothesis was that MO reduces Zfp423 DNA methylation in the progenitor cells of offspring adipose tissue, which promotes Zfp423 expression and adipogenic differentiation of progenitors, resulting in a reduction in progenitor density. Diet‐induced obesity model was used. Female mice (4 week old) were subjected to either control (10% energy from fat, CN) or obesogenic (45% energy from fat, OB) diet for two months to induce obesity. Then, mice were mated and maintained in their respective diets during pregnancy and lactation. Visceral adipose tissues were sampled at weaning (21 days). OB weanlings had about 2 folds higher expression of Zfp423 compared to CN. Consistently, the methylation level analyzed by pyrosequencing was 45.1 ± 3.13% lower in OB offspring. We further used CD45‐/Sca1+ as surface markers for sorting adipogenic progenitor cells. OB visceral fat contained 27.9 ± 0.90 % less progenitor cells than CN fat. As in the fat tissue, Zfp423 expression was 35.0 ± 2.38% (p = 0.07) higher in the progenitors of OB than CN weanlings. Consistently, methylation level was 69.2 ± 14.80 % (p < 0.05) lower in OB weanlings. In conclusion, MO reduced the density and Zfp423 DNA methylation of adipogenic progenitor cells in visceral fat. Because progenitor cells are the source of new adipocytes, the reduction in their density and enhancement of adipogenic differentiation will likely impair the renewal and expansion of adipocytes, partially explaining adipose metabolic dysfunction in offspring (The work was funded by NIH R01HD067449).

Grant Funding Source: Supported by NIH R01HD067449
A Novel Pathway for Regulation of Insulin Secretion by Fractalkine and CX3CR1 System (LB772)
Authors: Lee Yun Sok , Morinaga Hidetaka , Kim Jane , Lagakos William , Taylor Susan , Keshwani Malik , Perkins Guy , Dong Hui , Kayali Ayse , Sweet Ian , Olefsky Jerrold
Published: June 2021

Fractalkine (FKN) (CX3CL1) and its receptor CX3CR1 mediate cell‐to‐cell interactions in different tissues. Here, we demonstrate that the FKN/CX3CR1 system represents a novel regulatory mechanism for pancreatic islet beta cell function and insulin secretion. CX3CR1 KO mice exhibit glucose intolerance with normal insulin sensitivity, due to a marked beta cell defect in glucose and GLP1‐stimulated insulin secretion. The defect in insulin secretion was also observed in vitro in isolated islets from CX3CR1 KO mice. In vivo administration of FKN improved glucose tolerance with an increase in insulin secretion. In vitro treatment of islets with FKN increased intracellular Ca2+ level and potentiated insulin secretion. The KO islets exhibited reduced expression of a set of genes which are necessary for the fully functional, differentiated beta cell state, whereas, treatment of WT islets with FKN leads to increased expression of these genes. Lastly, expression of FKN in islets was decreased by aging and HFD/obesity, suggesting that decreased fractalkine/CX3CR1 signaling could be a mechanism underlying beta cell dysfunction in type 2 diabetes.
Hypoxic and hypercapnic ventilatory response in rats with estradiol valerate‐induced polycystic ovaries (LB773)
Authors: Montrezor Luís , Carvalho Débora , Anselmo‐Franci Janete , Bícego Kênia , Gargaglioni Luciane
Published: June 2021

We evaluated pulmonary ventilation (VE) and body temperature (Tb) during hypercapnia (7%CO2) and hypoxia (7%O2) in a female rat model in which estradiol valerate (EV) (2mg/0.2mL/rat or oil (control) were administered intramuscularly in a single dose to induce Polycystic ovary syndrome (PCOS). The experiments were performed 30, 45 or 60 days after EV injection. The ovaries of 30, 45 and 60 days EV group displayed typical PCOS‐like changes. Plasma levels of estradiol (E2) and progesterone (P4) increased in 30 (E2: 74.5 ± 23.5 pg/mL; P4: 18.3 ±4.5 ng/mL), 45 (E2: 70.8 ± 17.5 pg/mL; P4: 21.3 ±7.6 ng/mL) and 60 (E2: 71.4 ± 4.7 pg/mL; P4: 23.7 ±4.5 ng/mL) days EV groups whereas testosterone (T) increased only in 45 (58.4±6.5 pg/mL) and 60 (79.5±18.3 pg/mL) days EV groups compared with control group (E2: 46.9±11.3 pg/mL; P4: 9.9±3.4 ng/mL; T: 22.6±1.9 pg/mL). 30, 45, 60 EV treatment did not affect resting ventilation and hypoxic ventilatory response. However, the increase in ventilation produced by hypercapnia was attenuated after 30 days EV group (1978.9 ± 85.7 mL.kg‐1.min‐1 in control group, 1651.2 ± 369.3 in 45 days group and 1617.4± 131.4 in 60 days group vs 771.0 ± 172.4 in 30 days group). Hypoxia induced Tb to reduce in all groups, a response that was amplified in 30 days EV group. Our results suggest that initial alterations promoted by EV treatment changes CO2‐drive to breathe and hypoxia‐induced drop in Tb, and these responses are compensated in 45 and 60 days EV females.
Enhanced SpO2 in Response to Intermittent Normobaric Hypoxia in a Patient (Case Study) with COPD (LB787)
Authors: Cardoso S , Samillan V , Werneck M , Pereira P , Quispe M , Romeo L , Horowitz M
Published: June 2021

Recent studies by Cardoso et al (The FASEB Journal, April 9, 2013; v27, 938.7) using hypoxic pre‐conditioning (HP) add support to its value in the management of T2D, CV and pulmonary disease. We now report enhancement of SpO2 levels (P<0.0001) for a 75 yr old patient with Stage IV COPD when we intermittently employed a set of pre and post conditioning 1 hr interruption (at rest with SpO2 recorded at 1 min intervals) of her 24/7 nasal oxygen flow from Mar 2012 through Dec 2013 (interventions were repeated at 1‐3 month intervals at the patient’s discretion). Normalization of SpO2 was recorded when in late Dec 2013 we added bicycling at 6 mph (off supplemental O2 during exercise) 24 hrs after our 1 hr hypoxic interventions. Mean SpO2 on Dec 30, 2013 = 87.1±3.4 and Jan 6, 9, 14 and 18, 2014 = 91.9±2.3; 95.1± 1.71; 95.7±1.1; 87.9±3.6, respectively (Dec 2013 vs Jan 2014, P<0.0001). All observations were re‐confirmed Feb 14, 2014. The results suggest patients with COPD respond to HP with improved quality of life, and increased tolerance to exercise that may lead, as in our patient, to significantly lower frequency of prescribed medication.

Grant Funding Source: San Pedro Univ. Peru; Federal Univ. COPPE, RJ, Brazil, Private donations
Role of endogenous hydrogen sulfide on hypoxia‐induced hypothermia in hypertensive rats (LB792)
Authors: Sabino Joao , Soriano Renato , Traslaviña Guilhermo , Branco Luiz
Published: June 2021

Role of endogenous hydrogen sulfide on hypoxia‐induced hypothermia in hypertensive rats. Joao Paulo J. Sabino1, Renato N. Soriano1, Guillermo A. Ariza Traslaviña2, Luiz G. S. Branco1. 1Dental School and 2Medical School of Ribeirão Preto, University of São Paulo.

Hypoxia causes a regulated drop in body temperature (Tb) but the mechanisms involved are not completely understood. The role of hydrogen sulfide (H2S) a gaseous molecule endogenously produced by cystathionine β‐synthase (CBS) has been recently documented during hypoxia‐induced hypothermia in normotensive rats. However, no information exists regarding the role of H2S during hypoxic hypothermia in spontaneous hypertensive rats (SHR). We then evaluated the effect of the H2S donor (Na2S 4 nmol/1µl) and the CBS inhibitor (AOAA 9 nmol/1µl) microinjections into the central nervous system (fourth ventricle) of rats exposed to 10% hypoxia. The Tb was similar in all groups [Saline Wistar group (37.46±0.18 ºC, n= 10), AOAA Wistar group (37.40±0.33 ºC, n= 5), Na2S Wistar group (37.52±0.13 ºC, n= 7), Saline SHR group (37.43±0.43 ºC, n= 8), AOAA SHR group (37.59±0.16 ºC, n= 10) and Na2S SHR group (37.72±0.15 ºC, n= 6)] during basal normoxic condition. Hypoxia induced a typical decrease in Tb in saline injected rats. The decrease in Tb in the Saline SHR group (Δ= ‐1.16±0.11 ºC) was similar to the Saline Wistar group (Δ= ‐0.81±0.20 ºC). In Wistar rats, Na2S administration prevented hypoxia‐induced hypothermia (Δ= ‐0.58±0.16 ºC), whereas AOAA administration significantly increased hypoxia‐induced hypothermia in Wistar rats (Δ= ‐1.60±0.20 ºC), indicating that H2S plays a key role on hypoxia‐induced changes in Tb. Surprisingly, neither Na2S (Δ= ‐1.33±0.20 ºC) nor AOAA (Δ= ‐1.61±0.21 ºC) caused any change in hypoxia‐induced hypothermia in SHR. In conclusion, the H2S prevents the hypoxia‐induced anapyrexia in normotensive rats, but plays no thermoregulatory role during hypoxia exposition in SHR.

Financial support: FAPESP (2011/14779‐4).
Fear conditioning depresses the intrinsic excitability of infralimbic neurons in adult rats (LB82)
Authors: Rivera‐Pérez Luis
Published: June 2021

Posttraumatic stress disorder (PTSD) is a severe anxiety disorder that may develop after a person experiences a traumatic event. To further increase our understanding of PTSD, we study fear learning in rats, which have brain structures and functions similar to humans. In this animal model, we use Pavlovian fear conditioning to imitate the exposure to a traumatic event in humans. A previous study in our lab found that fear conditioning depresses the intrinsic excitability of neurons in the infralimbic cortex of adolescent rats. Since adolescent are still developing different connections and synapses, the findings in adolescent rats may not apply to adult animals. To test whether fear conditioning also depresses the excitability of IL neurons in adult animals, we used whole‐cell patch‐clamp recordings and behavioral training to compare the intrinsic excitability in infralimbic neurons before and after fear conditioning in adult male Sprague Dawley rats. To acquire cued fear conditioning, rats received 3 tone‐shock pairings (tone: 30 sec; shock: 0.44 mA, 0.5 sec). To test for fear conditioning memory, rats were tested for fear expression to 2 tone alone presentations 2 days later. An untrained control group remained in the home cage until sacrifice. Fear was quantified during the experiment as time freezing during tone presentations. Immediately after the final tone alone presentation, rats were deeply anesthetized, perfused transcardially, and coronal slices of the medial prefrontal cortex were cut with a vibratome. As a measure of intrinsic excitability, we quantified the number of spikes evoked by depolarizing pulses. Infralimbic neurons from the fear conditioning group fired fewer spikes indicating that fear conditioning depressed the intrinsic excitability of infralimbic neurons in adult rats. Currently, we are examining whether fear extinction in adult rats reverses this depression as it does in adolescent rats. These results suggest that depression of prefrontal neuronal excitability could contribute to the development of PTSD in adults.

Grant Funding Source: This work is supported by MBRS‐RISE R25 GM096955 to LMR, MBRS‐RISE R25 GM082406 to OS, and by NIMHHD G12MD007579‐27 and R15 MH101700 to JTP.
Visual Cortex Controls the Gain, but Not the Selectivity, of Superior Collicular Responses to Looming Stimuli in Awake Mice (LB827)
Authors: Liu Mingna , Zhao Xinyu , Cang Jianhua
Published: June 2021

Neural circuits in the brain often receive inputs from multiple sources, such as the bottom‐up input from early processing stages and the top‐down input from higher‐order areas. Here, we study the function of top‐down input in the mouse Superior Colliculus (SC), which receives convergent inputs from the retina and visual cortex. Neurons in the superficial SC display robust speed tuning to looming stimuli that mimic approaching objects. The looming‐evoked responses are reduced by almost half when the visual cortex is optogenetically silenced in awake, but not in anesthetized mice. Silencing the cortex does not change the speed tuning of SC neurons, or the response time course except at the lowest tested speed. Consistent with these findings, we further show that cortical responses to the looming stimuli are speed‐insensitive and slow. The strong gain control we have revealed thus provides a neural substrate for the cortex to modulate SC‐mediated visual behaviors.

Grant Funding Source: Supported by US National Institutes of Health (NIH) grant (EY020950) to J.C. and HHMI international student fellowship to X.Z.
Aquaporin 11 deletion results in induceable proximal tubule injury in response to metabolic challenge (LB837)
Authors: Nielsen Søren , Rojek Aleksandra , Andreasen Arne
Published: June 2021

Aquaporin 11 (AQP11) is a channel protein with unknown biological function expressed intracellularly in multiple tissues, among others in kidney proximal tubule epithelium. The neonate Aqp11 gene knockout (KO) mice show injury of the proximal tubule (PT) cells, manifesting as dilatation of RER cisterns, leading to apoptosis of the PT epithelial cells (Morishita et al, Mol Cell Biol. 2005). The mice develop cortical cysts within the first weeks of life and die due to kidney failure. It is unclear if the kidney failure is caused by functional insufficiency of the PT in the absence of AQP11, or by the abnormal development of the nephron and resulting structural malformation of the kidney. We have developed an inducible Aqp11 KO mouse model, where we can temporally control the time point of Aqp11 gene disruption by injection of tamoxifen. We performed microscopic analyses of the PT morphology after induction of AQP11 mutation in unchallenged mice and after different metabolic challenges (fasting/re‐feeding, administration of free amino acids). In mice where AQP11 deletion was induced after the completion of kidney development we observe no cellular damage of PT cells and no cyst formation for up to 4 months after disruption of the Aqp11 gene. When the mice are metabolically stressed (fasting/re‐feeding, administration of free amino acids) the PT cells suffer acute injury with development of giant vacuoles, similar to those observed in neonatal total AQP11 KO mice. In conclusion, we propose that AQP11 function is necessary for proper PT cell function during states of increased protein/amino acid supply, such as during the neonatal period, when mice feed on protein‐rich milk, and in adults subject to high amino acid metabolism challenge. We hypothesize that continuous PT injury during neonatal period results in abnormal development of more distal tubule segments and cyst formation, possibly by impairment of fluid flow in the tubule.
TRPM4‐dependent contraction of retinal pericytes (LB846)
Authors: Gonzales Albert , Nelson Mark
Published: June 2021

Pericytes are an integral part of the neurovascular unit and likely contribute to the neuronal‐dependent regulation of capillary blood flow. Like vascular smooth muscle cells (VSMCs), pericytes are thought to contract through membrane depolarization and activation of voltage‐dependent calcium channels which then causes focal constrictions of capillaries. TRPM4, a melastatin transient receptor potential channel, is essential for VSMC membrane depolarization and contraction. Its role in pericyte contraction is not known. We used immunohistochemistry and confocal microscopy to examine the role of TRPM4 channels in pericyte contraction within the mouse retina. We observed TRPM4‐specific immunolabeling in the smooth muscle cells within precapillary arterioles (<10 µm) and in a sub‐population of pericytes surrounding the capillaries most proximal to the arterioles in the mouse retinal vasculature. In addition, we observed constriction in response to the thromboxane A2 analog, 9,11‐dideoxy‐9α,11α‐methanoepoxy prostaglandin F2α (U46619) in pre‐capillary arterioles and in distal and proximal pericyte‐containing capillaries. This response was blocked by the TRPM4 inhibitor 9‐phenanthrol in pre‐capillary arterioles and proximal pericyte‐containing capillaries, but not in pericyte‐containing capillaries distal to feeding arteriole. These findings suggest that TRPM4 plays a role in pericyte constriction within retinal capillary system.

Grant Funding Source: HL044455, R37 DK053832, and 1PO1HL095488
Expression of blood clot‐dissolving proteins in transgenic plant (LB852)
Authors: Wang Kevin , Tull Lauren
Published: June 2021

Introduction: The formation of a blood clot in the circulatory system can cause heart attack or stroke. Lumbrokinases, found in earthworms, and Nattokinase, found in Bacillus subtilis for soybean fermentation, can dissolve fibrin clots without causing excessive bleeding. Our goal was to clone Lumbrokinase and Nattokinase, to transiently and stably express the proteins in tobacco plants and to prove these recombinant proteins retain anti‐thrombolytic activity.

Methods: Codon‐optimized and wild‐type gene fragment of Lumbrokinases (PI293) and Nattokinase (GenBank: AF368283.1) were isolated and inserted into a single‐vector DNA replicon system for transient expression in plant and plant expression vector pCambia2300‐Phas1470‐Nos, for stable plant transformation and expression. We tested gene expression and function of the transient expression vector using fibrin dissolving assay.

Results: We optimized Lumbrokinase and Nattokinase gene codon respectively for tobacco codon usage bias. Upon codon optimization, codon adaptation index (CAI) of Lumbrokinase was increased from 0.69 to 0.86 and GC content was changed from 54.02% to 42.65%. CAI of Nattokinase was increased from 0.71 to 0.86 and GC content was changed from 46.78% to 42.61%. Higher CAI value and GC content, closer to that of tobacco plant, provide a good estimation for high expression foreign protein. Total eight vectors for transient expression and stable transformation were developed and confirmed by restriction reactions. The plant transient expression with those vectors tested positive for gene expression and function with the fibrin dissolving assay.

Conclusions: Expression Vectors of Lumbrokinase and Nattokinase for both transient and stable plant transformation were developed. The transient expression vectors were successfully expressed in the tobacco plant and tested positive for clot dissolving function.

Grant Funding Source: National Institutes of Health through Grant Number 8P20GM103447
Toxicity of Alpha‐Synuclein and Its Impact on Neurotransmitters (LB97)
Authors: Watts Jane , Lee Esther , George Megan , Arpornsuksant Celine , Chareddy Yogitha , Balaji Prerna , Chen Mickey , Link Tina , Sanders Teresa
Published: June 2021

The loss of the neurotransmitter dopamine (DA) in the substantia nigra, the part of the brain controlling motility, has been associated with Parkinsonʼs Disease (PD) but there has been little evidence to show what causes the loss of DA. New research indicates a relationship between a drop in DA and the presence of Lewy bodies, which include aggregates of a misfolded protein, alpha‐synuclein (α‐syn). Research indicates that α‐syn is a 140 amino acid protein involved in the exocytosis of DA from the synaptic vesicles of neurons. α ‐syn has an identifiable sequence (KTKEGV) that repeats six times at the N‐terminus, which folds a normal protein into amyloid fibers. Mutations, like A53T, A30P, and E46K, or exposure to environmental factors modify the normal quaternary structure of the protein, initiating the aggregation of misfolded α‐syn. The build‐up of abnormal α‐syn highly correlates with the drop in dopamine levels in the brain, which is associated with motor dysfunction in PD patients. The Walton SMART Team (Students Modeling A Research Topic) used 3D printing techniques to see how mutated α‐syn correlates with DA levels.

Grant Funding Source: Supported by grants from NIH‐CTSA and NIH‐SEPA.
A Comparison of Drill Guiding and Screw Guiding 3D-Printing Techniques for Intra- and Extrapedicular Screw Insertion
Authors: Pijpker Peter A.J. , Kuijlen Jos M.A. , Kraeima Joep , Groen Rob J.M. , Faber Chris
Published: June 2021

Study Design.

Screw randomized cadaveric study.

Objective.

To compare the accuracy of three-dimensional (3D)-printed drill guides versus additional screw guiding techniques for challenging intra- and extrapedicular screw trajectories.

Summary of Background Data.

Pedicle screw placement can be technically demanding, especially in syndromic scoliosis with limited bone stock. Recently, 3D-printing and virtual planning technology have become available as new tools to improve pedicle screw insertion. Differences in techniques exist, while some focus on guiding the drill, others also actively guide subsequent screws insertion. The accuracy of various 3D-printing-assisted techniques has been studied; however, direct comparative studies have yet to determine whether there is a benefit of additional screw guidance.

Methods.

Two cadaveric experiments were conducted to compare drill guides with two techniques that introduce additional screw guiding. The screw guiding consisted of either k-wire cannulated screws or modular guides, which were designed to guide the screw in addition to the drill bit. Screws were inserted intra- or extrapedicular using one of each methods according to a randomization scheme. Postoperative computed tomography scanning was performed and fused with the preoperative planning for detailed 3D screw deviation analysis.

Results.

For intrapedicular screw trajectories malpositioning was low (2%) and the modular guides revealed a statistically significant increase of accuracy (P 
= 0.05) compared with drill guides. All techniques showed accurate cervical screw insertion without breach. For the extrapedicular screw trajectories both additional screw guiding methods did not significantly (P = 0.09) improve accuracy and malpositioning rates remained high (24%).

Conclusions.

In this cadaveric study it was found that the additional screw-guiding techniques are not superior to the regular 3D-printed drill guides for the technically demanding extrapedicular screw technique. For intrapedicular screw insertion, modular guides can improve insertion; however, at cervical levels regular 3D-printed drill guides already demonstrated very high accuracy and therefore there is no benefit from additional screw guiding techniques.

Level of Evidence.

3
Delivery room resuscitation and short-term outcomes of extremely preterm and extremely low birth weight infants: a multicenter survey in North China
Authors: Li Shuai-Jun , Feng Qi , Tian Xiu-Ying , Zhou Ying , Ji Yong , Li Yue-Mei , Zhai Shu-Fen , Guo Wei , Zhang Fang , Zheng Rong-Xiu , He Hai-Ying , Liu Xia , Wang Jun-Yi , Mei Hua , Wang Hong-Yun , Xie Hua , Zeng Chao-Mei , Ma Li , Zhang Ping-Ping , Li Jin-Yu , Wang Xiao-Ying , Li Li-Hua , Cui Hong , Yang Shu-Lan , Chen Lu , Gu Xiao-Hong , Hu Yan-Ju , Que Sheng-Shun , Sun Li-Xia , Yang Ming , Zhao Wen-Li , Ma Qiu-Yan , Wang Hai-Juan , Guo Jiu-Ye
Published: June 2021

Background:

Delivery room resuscitation assists preterm infants, especially extremely preterm infants (EPI) and extremely low birth weight infants (ELBWI), in breathing support, while it potentially exerts a negative impact on the lungs and outcomes of preterm infants. This study aimed to assess delivery room resuscitation and discharge outcomes of EPI and ELBWI in China.

Methods:

The clinical data of EPI (gestational age [GA] <28 weeks) and ELBWI (birth weight [BW] <1000 g), admitted within 72 h of birth in 33 neonatal intensive care units from five provinces and cities in North China between 2017 and 2018, were analyzed. The primary outcomes were delivery room resuscitation and risk factors for delivery room intubation (DRI). The secondary outcomes were survival rates, incidence of bronchopulmonary dysplasia (BPD), and risk factors for BPD.

Results:

A cohort of 952 preterm infants were enrolled. The incidence of DRI, chest compressions, and administration of epinephrine was 55.9% (532/952), 12.5% (119/952), and 7.0% (67/952), respectively. Multivariate analysis revealed that the risk factors for DRI were GA <28 weeks (odds ratio [OR], 3.147; 95% confidence interval [CI], 2.082–4.755), BW <1000 g (OR, 2.240; 95% CI, 1.606–3.125), and antepartum infection (OR, 1.429; 95% CI, 1.044–1.956). The survival rate was 65.9% (627/952) and was dependent on GA. The rate of BPD was 29.3% (181/627). Multivariate analysis showed that the risk factors for BPD were male (OR, 1.603; 95% CI, 1.061–2.424), DRI (OR, 2.094; 95% CI, 1.328–3.303), respiratory distress syndrome exposed to ≥2 doses of pulmonary surfactants (PS; OR, 2.700; 95% CI, 1.679–4.343), and mechanical ventilation ≥7 days (OR, 4.358; 95% CI, 2.777–6.837). However, a larger BW (OR, 0.998; 95% CI, 0.996–0.999), antenatal steroid (OR, 0.577; 95% CI, 0.379–0.880), and PS use in the delivery room (OR, 0.273; 95% CI, 0.160–0.467) were preventive factors for BPD (all P < 0.05).

Conclusion:

Improving delivery room resuscitation and management of respiratory complications are imperative during early management of the health of EPI and ELBWI.
Isolated limb perfusion with melphalan as treatment for regionally advanced melanoma of the limbs: results of 60 patients treated in Finland during 2007–2018
Authors: Tulokas Sanni K. A. , Kohtamäki Laura M. , Mäkelä Siru P. , Juteau Susanna , Albäck Anders , Vikatmaa Pirkka J. , Mattila Kalle E. , Skyttä Tanja K. , Koivunen Jussi P. , Tyynelä-Korhonen Kristiina , Hernberg Micaela M.
Published: June 2021

Isolated limb perfusion (ILP) is widely accepted as treatment for recurrent melanoma limited to the limbs. The use of ILP has decreased in recent years with the introduction of potentially effective new systemic therapies. We evaluated retrospectively if ILP still may be a treatment option in locally advanced melanoma. In Finland, ILP is centralized to the Comprehensive Cancer Center of Helsinki University Hospital. We included all ILP patients treated at our hospital between 2007 and 2018. Clinical factors and treatment outcomes were retrospectively evaluated. Altogether 60 patients received ILP. Toxicity was mostly transient. The overall response rate was 77% with 35% complete responses and 42% partial responses. The median progression-free survival (PFS) was 6.1 months (range 0.6–116.5 months) and the median melanoma-specific survival (MSS) was 29.9 months (range 3.5–138.7 months). Patients with CR had superior median PFS (19.7 months, range 2.5–116.5 vs. 4.5 months, range 0.6–39.7 months, P = 0.00003) and median MSS (median MSS not reached vs. 25.9 months, range 3.5–98.7 months, P = 0.0005) compared to other responders. Younger patients (<69 years) had longer median MSS (47.2 months, range 3.5–138.7 vs. 25.9 months, range 8.4–125.4 months, P = 0.015) compared to patients over 69 years. Treatment outcomes of Finnish ILP patients were comparable to earlier studies and some long-term survivors were observed in the group of complete responders. Median PFS and OS were longer for patients achieving a CR. Treatment was well-tolerated also among older patients.
Seborrheic keratosis-like melanoma: a diagnostic challenge
Authors: Janowska Agata , Oranges Teresa , Iannone Michela , Romanelli Marco , Dini Valentina
Published: June 2021

This article summarizes the main epidemiologic and diagnostic features of seborrheic keratosis-like (SK-like) melanoma. We performed a review of the current literature. The real and current incidence and prevalence of SK-like melanoma are unknown. Many lesions are misdiagnosed and not excised with histopathologic confirmation, or excised without an appropriate surgical approach due to a benign clinical and dermoscopic appearance. SK-like melanoma presents both melanoma and SK features. SK-like melanoma and SK are often clinically indistinguishable even for experienced dermatologists. Clinically, it develops as a black-dark brown or light slightly elevated, papule, nodule or plaque with rapid growth and a regular or irregular shape. SK-like melanoma presents melanocytic and nonmelanocytic dermoscopic features. Irregular dark-brown dots/globules, a blue-grayish veil, streaks, irregular pigmentation or network and brown lines point to a melanoma diagnosis. Among the nonmelanocytic features, milia-like cysts and comedo-like openings have been highlighted. The association of polarized and nonpolarized dermoscopic techniques is more accurate for studying the dermoscopic features of SK-like melanoma. If the dermoscopic features are unclear, further investigations with reflectance confocal microscopy (RCM) or biopsy with histologic examination are essential. SK-like melanoma is a dermatologic challenge. Careful clinical and dermoscopic evaluation favor a correct diagnosis. In unclear cases, it is important to perform a biopsy with histologic examination to confirm the correct diagnosis.
Successful encorafenib desensitization in a patient with recurrent metastatic melanoma
Authors: Cullen Grace D. , Finnes Heidi D. , Markovic Svetomir N. , Volcheck Gerald W.
Published: June 2021

Type I hypersensitivity reactions (HSR) to dabrafenib are rare but have been previously described. We present a case where a 72-year-old woman with recurrent, metastatic BRAF-mutated melanoma developed a type I HSR to dabrafenib. We, therefore, developed a desensitization protocol with encorafenib, a similar class agent, to allow the patient to continue with treatment. Patients with a history of HSR to dabrafenib may be considered for encorafenib desensitization when other therapeutic options are limited.
A Case of Primary Myelofibrosis With Transformation to Leukemia Cutis
Authors: Saleh Jasmine , Regmi Aayushma , Speiser Jodi J. , Mudaliar Kumaran M. , Omman Reeba , Velankar Milind , Mirza Kamran M.
Published: June 2021

We report an extraordinary case of primary myelofibrosis with transformation to leukemia cutis. A 64-year-old Caucasian man with a history of JAK2-positive primary myelofibrosis presented with erythematous papulonodules on his right lower extremity. A punch biopsy revealed a normal epidermis with an underlying diffuse dermal infiltrate composed of medium-to-large-sized myeloid cells and leukocytes. Neoplastic cells were immunoreactive for LCA, CD34, CD61, CD117, and CD68 and negative for lysozyme, CD20, CD3, myeloperoxidase, and TdT. These findings were consistent with a diagnosis of leukemia cutis. A concurrent bone marrow biopsy demonstrated a markedly fibrotic, hypercellular marrow without a significant increase in blasts. With no morphologic evidence of bone marrow involvement by acute myeloid leukemia, our case suggests that the patient's primary myelofibrosis transformed to leukemia cutis. Our patient died 2 months after the onset of his skin nodules. Our case demonstrates that leukemia cutis should be included in the differential diagnosis for cutaneous nodular lesions in patients with a history of an advanced-stage hematological malignancy.
Selatogrel: A Novel Subcutaneous P2Y12 Inhibitor
Authors: Beavers Craig J. , Effoe Samuel. Aaron , Dobesh Paul P.
Published: June 2021

The use of a P2Y12 inhibitor as a component of dual antiplatelet therapy in patients with an acute coronary syndrome (ACS) is well established. However, the P2Y12 inhibitors currently available have pharmacokinetic limitations due to delayed absorption, lack of enteral access for administration with oral formulations, need for intravenous access with cangrelor, or need for metabolization to be ideal in the critical 3-hour window during an ACS. Selatogrel is a novel, potent, reversible, and selective 2-phenylprimdine-4-carboxamide administered subcutaneously under development. Results from preclinical, phase 1, and phase 2 trials have confirmed that the agent provides sustained and reversible P2Y12 platelet inhibition with an acceptable safety profile. The most commonly reported adverse effects include minor bleeding and dyspnea. Phase 3 trials are being designed to understand the critical role this agent can play in upstream management of patients with ACS including a more defined understanding of the adverse effect profile, how to transition from this agent to an oral agent, who will be administering, and does this agent allow for a safe and quick transition to coronary artery bypass graft surgery if needed. Should it obtain approval, selatogrel has the potential to provide a unique and advantageous mechanism for P2Y12 inhibition.