bluuebunny/crossref_metadata_2025 huggingface dataset:
The Effect of Sildenafil on Atrial Natriuretic Peptide Level in Rats Treated with Monocrotaline
Authors: AlJinabi Mukhallad M. , Khatib Said Y. , Mahmoud Sahar M.
Published: May 2022

Background

Pulmonary hypertension is associated with an increase in pulmonary vascular resistance leading to pulmonary vascular changes, right ventricular hypertrophy, and heart failure. Pulmonary hypertension can be induced in animal models similar to humans where the changes are comparable. Monocrotaline (MCT), an alkaloid derived from a toxic plant, can be used to induce pulmonary hypertension in animals. Sildenafil has been used to treat patients with pulmonary hypertension since it induces smooth muscle relaxation. Atrial natriuretic peptide (ANP) is a hormone secreted naturally from the atria of the heart. An increase in ANP in hypervolemia and hypertension is beneficial since it induces a significant decrease in blood volume, therefore, lowers blood pressure. Studies showed that atrial natriuretic peptide combined with sildenafil reduces pulmonary arterial hypertension significantly. Cardiac hypertrophy due to pulmonary hypertension increases expression of myosin heavy chain β in the right ventricle of the heart.

Aims

To explore the ability of sildenafil to alleviate PH induced by Monocrotaline (MCT) in rats and explore the ANP level before and after induction of PH, and the administration of sildenafil.

Methods

Sixty four male Wister rats, were divided randomly into 4 groups: Control A, (7 Rats). Group received MCT only (14 rats), at dose 60mg /kg body weight subcutaneously. Group C received a single injection of MCT subcutaneously at a dose of 60mg /kg for 3 weeks and then sildenafil at a dose of 50mg/kg/day from the beginning of the fourth week to the end of the sixth week (The reversal group) counted 23 rats. Group D received a single injection of MCT subcutaneously at a dose of 60mg /kg for 3 weeks at the same time sildenafil was given at a dose of 50mg/kg from day 1 to the end of day 21 (The prevention group) consisting of 20 rats.

Results

The survival rate of the prevention group was the highest compared to other groups. The results revealed that MCT‐ treated rats and the reversal group showed a significant increase in ANP levels compared to the control one. The prevention group rats showed a significant decrease in ANP levels compared to both the reversal and the MCT‐ treated group. Fulton index ratio shows a significant increase in MCT treated rats and the reversal group compared to the control group, also it has an increasingly significant level to the reversal group compared to the prevention group. The percent of β‐ myosin heavy chain (MHC) is decreased significantly in the reversal group compared to both the MCT treated group and the control group. Nitric oxide (NO) levels were increased significantly in the reversal group compared to both the MCT treated group and the control group.

Conclusion

Sildenafil significantly decreases ANP levels in rats treated with MCT, and it also decreases cardiac hypertrophy induced by MCT.
Acipimox Induces the Hepatic Accumulation of Long‐Chain Diacylglycerides and Triglycerides in Severely Burned Sprague Dawley Rats
Authors: Bhattarai Nisha , Vardarli Emre , Prasai Anesh , El ayadi Amina , Murton Andrew J.
Published: May 2022

Objective

Persistent lipolysis is a hallmark of severe burns, contributing to futile triglyceride cycling and the accumulation of lipid in ectopic tissues. It has been suggested that the administration of acipimox, a potent inhibitor of lipolysis, may blunt the hypermetabolic response to burns. However, what impact massive burns has on the accumulation of individual lipid species in the liver and how this is affected by acipimox treatment is unknown.

Hypothesis

We hypothesize that large burns results in the accumulation of lipotoxic species within the liver and that this can be prevented by administering acipimox.

Methods

Thirty‐two male Sprague Dawley rats were randomized to receive either a scald burn (~60% total body surface area) or sham procedure, performed under isoflurane general anesthesia. Afterwards, animals were further randomized to receive either acipimox (50 mg/kg/day) or vehicle (saline) by i.p. injection administered once a day for 7 days. On day 7, animals were euthanized and the liver snap frozen in liquid nitrogen. Liver lipids were extracted by using a chloroform:methanol (2:1) solution and diacylglycerol (DAG), triacylglycerol (TAG), free fatty acids (FFA) and cholesterol esters (CE) separated by thin‐layer chromatography. Afterwards, individual lipid classes were esterified to methyl esters, derivatized using heptane, and analyzed by gas chromatography (GC). The fatty acids C16:0, C18:0, cis/trans C18:1, cis 18:2, C20:0, C22:0, C24:0, and C22:6 were resolved by GC for each of the lipid classes examined.

Results

Liver weights were comparable between groups. Burn injury had no significant effect on the concentration of liver lipids compared to sham treated animals, irrespective of lipid class (P > 0.05). In contrast, the administration of acipimox significantly increased liver C16:0 (2.1‐fold; P < 0.05), trans C18:1 (2.5‐fold; P < 0.05), and cis C18:2 (5.5‐fold; P < 0.05) TAG content in burned animals, when compared to injured animals treated with vehicle. No effect of acipimox on liver lipids was observed in sham treated animals.

Conclusion

Contrary to expectations, acipimox causes the accumulation of long‐chain TAGs in rat liver following massive burns, but this is not observed in response to burn injury alone. The ability of acipimox to cause unwanted hepatic metabolic side effects warrants further investigation before it is used clinically to treat massive burns.
Mitochondria from Skeletal Muscle Cells Derived from Old Donors Show Reduced Energy Production and Respiratory Capacity
Authors: Arevalo José A. , Vázquez‐Medina José P. , Brooks George A.
Published: May 2022

With age, skeletal muscle mitochondria lose their oxidative capacity and their ability to respond to energy demand. These phenomena can lead to a reduction in skeletal muscle oxygen consumption, atrophy, and increased risk of developing age‐related diseases such as sarcopenia. Whether age‐derived changes in mitochondrial function correspond to morphological changes in the mitochondrial reticulum remains unknown. Our objective is to investigate age‐related changes in mitochondrial morphology and function using primary skeletal muscle cells derived from healthy young and old men. Primary skeletal muscle progenitor cells (SkM) derived from the Rectus abdominis muscle of healthy active 18‐19‐year‐old men (Young), and 66‐69‐year‐old men (Old) were obtained from Cook MyoSite Inc. (Pittsburgh, PA). Mitochondrial morphology was analyzed in live cells using confocal microscopy. Oxygen Consumption Rate (OCR) was measured in intact cells using the extracellular flux assay and a Seahorse analyzer (Agilent Technologies; Santa Clara, CA). Seahorse Cell Mito Stress Test of primary cells derived from Young revealed a higher Basal and Maximal OCR compared to Old (Basal: 23.20 ± 1.49 vs. 11.34 ± 2.31; Maximal: 41.50 ± 4.72 vs. 18.36 ± 1.65 pmol/min/protein). Young also had higher ATP production and Spare Respiratory Capacity (ATP: 19.08 ± 1.21 vs. 9.19 ± 2.10; SRC: 18.30 ± 3.47 vs. 7.03 ± 1.29 pmol/min/protein). No differences were revealed in morphology (Individuals: 37.80 ± 23.18 vs. 46.67 ± 22.43 counts; Networks: 10.20 ± 2.48 vs. 15.33 ± 5.75 counts; Mean Branches per Network: 15.52 ± 6.41 vs. 11.27 ± 3.67 counts). These preliminary results show primary skeletal muscle progenitor cells derived from old donors have lower respiration, produce less ATP, and have a reduced capacity to adapt to energy demands when compared to SkM derived from young donors. Further analyses can give us an insight into human skeletal muscle‐derived cellular physiological capacity. Technology to observe human muscle mitochondrial dysfunction in vitro helps us understand the effects of aging on skeletal muscle mitochondria and whole‐body declines of resting and exercise metabolic rates. Studies on aging individuals will be required to determine if predicted decrements in energy coupling efficiency occur with aging in vivo.
Evaluating the Impact of Non‐coding Variants in the Cardiac Transcription Factor NKX2‐5 Binding Sites
Authors: Peña‐Martínez Edwin G. , Rodríguez‐Martínez José A.
Published: May 2022

Transcription Factors (TFs) are DNA‐specific binding proteins that regulate gene expression and other biological processes like cell differentiation. For example, the cardiac TF NKX2‐5 has been proven to be a master regulator in the transcriptional network needed for heart development. Mutations in NKX2‐5 have been shown to cause congenital heart diseases (CHDs), the most common birth defect that result in structural abnormalities in the heart. However, ~95% of disease‐associated mutations occur within the non‐coding genome, such as promoters and enhancers. The molecular mechanism of these genetic variations and their role in many diseases has remained unexplored. We hypothesize that non‐coding variants within NKX2‐5 binding sites will affect DNA recognition and alter TF‐DNA binding. First, using ~84 million Single Nucleotide Polymorphisms (SNPs) reported in the 1000 Genomes Project, I identified 8,475 that are predicted to affect NKX2‐5 binding. After filtering these variants, I found 901 SNPs localized in cardiac enhancers, and 30 disease‐associated SNPs from the GWAS catalog predicted to affect NKX2‐5 binding sites. Using position weight matrix (PWM) DNA‐specificity models, binding scores for the identified mutations were calculated. The variants rs7350789 and rs7719885 were predicted to have the greatest impact in NKX2‐5 binding affinity with ΔPWM scores of 258 and ‐212 respectively. These variants were prioritized for in vitro validation through Electrophoretic Mobility Shift Assay (EMSA) using purified full‐length NKX2‐5 to evaluate TF‐DNA complex formation. After performing an EMSA, both variants resulted in an increase in TF‐DNA binding even though rs7719885 had a negative ΔPWM score which predicts a decrease in binding affinity. Although not as expected, two of the variants predicted to impact TF‐DNA binding were successfully validated.
Placental IL‐1β impairs endothelial functions by reducing VE‐cadherin expression in PE
Authors: Liu Xinlu , Si Yanru , Wang Guohui , Meng Yuhan , Li Qinghua , Yang Weiwei
Published: May 2022

Preeclampsia (PE) is a pregnancy‐specific syndrome that affects 5~8% pregnancies and is a leading cause of maternal and neonatal morbidity and mortality, especially in low‐income and middle‐income countries. Widespread systemic inflammation and endothelial dysfunction are hallmarks of injury in PE, however, the molecular mechanisms linking inflammation and endothelial dysfunction remain largely unknown. We tested the hypothesis that placental IL‐1β impairs endothelial functions by inhibiting VE‐cadherin expression in PE. We found that NLRP3 inflammasome was overactivated in preeclamptic placenta, which induced more IL‐1β release. We also observed that IL‐1β significantly increased endothelial permeability of Human Umbilical Vein Endothelial Cells (HUVECs) in a dose‐response manner, and inhibition of IL‐1β receptor significantly attenuated the increase of endothelial permeability induced by IL‐1β. We further revealed that both mRNA and protein level of VE‐cadherin was reduced by IL‐1β in HUVECs. and overexpression of VE‐cadherin in endothelial cells reversed IL‐1β‐induced increase in endothelial permeability. Taken together, our preliminary data suggested that excessive placental IL‐1β significantly impaired endothelial functions by inhibiting VE‐cadherin expression.
Allosteric Dopamine transporter modulator inhibits cocaine‐induced behavior
Authors: Xu Yibin , Lewandowski Stacia , Besada Christina
Published: May 2022

The dopamine transporter (DAT) is a transmembrane protein responsible for reuptake the neurotransmitter dopamine back into the presynaptic neurons. Dopamine (DA) is involved in driving motivation, it is increased when people expect a reward, and it is also involved in many central nervous systems (CNS) disorders. The inhibition of the reuptake process prevents dopamine in the synaptic cleft from getting back into the presynaptic neuron. Cocaine and amphetamine both inhibit DA reuptake by binding to the orthosteric site but through different mechanisms. Cocaine is a classical inhibitor that blocks the transporter through competitive inhibition. On the other hand, amphetamine is a substrate of DAT and causes competitive inhibition of DA reuptake and reversal of the transporter leading to the release of DA. As a result of the DA reuptake inhibition, the DA in the synaptic cleft increases, leading to amplified downstream dopaminergic signaling primarily in the mesolimbic pathway. Because DAT is a primary target of psychostimulants, compounds acting on the DAT in novel ways could potentially treat psychostimulant use disorders. We previously found a novel compound—KM822 and characterized it as an allosteric modulator of DAT function that significantly decreases cocaine‐induced locomotive response in planarians. To test if KM822 has similar effects in mammals, we administered KM822 and cocaine through intracranial infusions into Long Evans rats' nucleus accumbens (NAc) and measured locomotion. We targeted the NAc as it is a part of the brain that plays a crucial role in the mesolimbic dopaminergic pathway and has been recognized by its high density of DAT. Our results showed that KM822 significantly decreased hyper‐locomotion induced by cocaine and notably did not cause any increase in locomotion by itself. KM822's ability to block hyper‐locomotion induced by amphetamine, another psychostimulant with a different mechanism of action, was also examined. Finally, we examined KM822's ability to interfere with cocaine's rewarding effect using the conditioned place preference (CPP) assay. In this assay, animals are tested for their preference for a cocaine‐associated environment. Unlike locomotion assay, this assay is more relevant to behaviors associated with cocaine addiction and therefore has high translational value. Overall, these studies demonstrated KM822's ability to block DAT inhibitor‐induced behaviors in rats and provided strong evidence that the novel allosteric DAT modulator KM822 has significant potential for treating psychostimulant addiction.
Aspirin Treatment Improves Excess Activin A Associated Cardiac Dysfunction In Pregnancy
Authors: Bakrania Bhavisha A. , Palei Ana , Shahul Sajid , Hall Michael , Granger Joey P.
Published: May 2022

Preeclampsia (PE) is a hypertensive disorder prevalent in 3‐8% of pregnancies. The placenta plays a central role in PE as it releases anti‐angiogenic, pro‐inflammatory and pro‐fibrotic factors into the maternal circulation to cause endothelial dysfunction and end organ damage, including cardiac dysfunction. PE women have elevated plasma levels of the pro‐fibrotic factor, Activin A, that is associated with decreased cardiac global longitudinal strain (GLS), a sensitive measure of systolic function and fibrosis. In a retrospective study, we recently showed that PE women administered aspirin had significantly reduced Activin A and improved GLS. Whether aspirin has a direct effect in improving cardiac function and reducing Activin A is not known. We hypothesized that chronic excess levels of Activin A during pregnancy induces cardiac dysfunction, and that aspirin treatment in late pregnancy improves GLS. To address this, Vehicle and Activin A treated (6 µg/day) rats were assigned to placebo or aspirin treatment (1 mg/day) groups (n= 8‐12/group) on gestational day (GD) 14. All animals had an indwelling carotid catheter placed on GD 18, followed by a comprehensive echocardiography assessment and blood pressure measurement on GD19. Activin A infusion resulted in significant increases in circulating Activin A in the placebo group (Vehicle+Placebo, 701±45 pg/mL; Act+Placebo, 1248±121 pg/m; P<0.01). Interestingly, aspirin treatment reduced circulating Activin A, despite continual infusion (Act+Aspirin, 896±99 pg/mL, P<0.01), suggesting aspirin may degrade Activin A. Activin A infusion was associated with significantly impaired GLS (Vehicle+Placebo, ‐22.3±3.02 %; Activin+Placebo, ‐15.5±1.92 %; P<0.01) that was significantly improved with aspirin treatment (Activin+Aspirin, ‐19.2±1.47 %, P<0.01). Mean arterial pressure was not significantly different between groups, suggesting that Activin A associated cardiac dysfunction occurs independently of increases in blood pressure. These findings suggest that Activin A may induce cardiac abnormalities in women with PE, and that aspirin may be a potential treatment option.
The Relevance of the Adamkiewicz Artery Anatomical Variations for Resection of Spinal Tumors
Authors: Navedo‐Sánchez Luis R. , Pérez‐Verdejo Cheysaliz , Feliciano Caleb , Rodríguez‐Mercado Rafael , Quiñones‐Rodríguez Jailenne I.
Published: May 2022

The artery of Adamkiewickz (AKA) is the most prominent radiculomedullary artery derived between T9 to L2 vertebral segments, supplying oxygenated blood to the caudal thoracic and lumbar regions of the spinal cord. Anatomical comprehension of this artery is important to avoid serious neurological complications during surgery treating intraspinal tumors and spinal vascular malformations. This study aimed to review the published literature on iatrogenic injury to anatomical variations in the AKA, clinical significance, as well as assess diverse radiological imaging techniques used to visualize this vessel before surgery. A retrospective study was performed from 2016 to 2021 using PubMed as our primary search engine, to better focus on the recent five‐year range findings. As multiple variations related to the AKA are found in the various demographics around the world, surgical approaches can be improved to preserve the neurovascular structures. Thus, understanding the importance of neurovascular anatomical variations will help to comprehend spinal cord irrigation which is imperative for the safe and effective therapeutic planning and avoidance of complications.
Evaluation of Exercise‐Induced Changes in Lung Water Density in Heart Failure with Preserved Ejection Fraction
Authors: Zamani Sauyeh K. , Zaha Vlad G. , Jaffery Manall , Babb Tony G. , Sarma Satyam , MacNamara James , Levine Benjamin D. , Thompson Richard B. , Nelson Michael D.
Published: May 2022

The primary chronic symptom of heart failure with preserved ejection fraction (HFpEF) is severe exercise intolerance, often associated with dyspnea upon exertion. Here we examine the role of pulmonary congestion, by measuring lung water density (LWD, %) using a previously validated magnetic resonance imaging (MRI) approach. First, in 6 healthy reference controls (3 M/3 F, BMI: 23 ± 3 kg/m2, age: 24 ± 4 years) LWD was measured at rest and in response to six minutes of moderate intensity dynamic leg exercise, using an MRI compatible exercise ergometer. LWD decreased in all six healthy reference controls immediately following exercise (from 24.8 ± 3.0% to 18.9 ± 4.2%, p=0.003), while returning to baseline ten minutes after exercise cessation. Next, we assessed exercise‐induced changes in LWD in 10 individuals with HFpEF (4 M/6 F, BMI: 35 ± 6 kg/m2, age: 69 ± 5 years), following the same experimental protocol. In contrast to the reference control participants, LWD remained unchanged from baseline immediately after exercise (22.8 ± 4.1% vs. 21.9 ± 5.2%, respectively), and tended to increase above baseline ten minutes after exercise cessation (25.2 ± 7.7%). Together, the data support the use of cardiovascular MRI in evaluating LWD at rest and in response to exercise. More work is needed to determine whether changes in LWD contribute to exercise intolerance in HFpEF.
A prospective clinical study on the mechanisms underlying Critical Illness Myopathy – A time‐course approach
Authors: Larsson Lars , Cacciani Nicoola , Skärlén Åsa , Wen Ya , Wang Xiang , Addinsall Alex B. , Llano‐Diez Monica , Li Meishan , Gransberg Lennart , Hedström Yvette , Bellander Bo‐Michael , Nelson David , Bergquist Jonas
Published: May 2022

Background

Critical illness myopathy (CIM) is a consequence of modern critical care resulting in general muscle wasting and paralyses of all limb and trunk muscles, and prolonged weaning from the ventilator. CIM is associated with severe morbidity/mortality and significant negative socioeconomic consequences which has become increasingly evident during the current COVID‐19 pandemic, but underlying mechanisms remain elusive.

Methods

Ten neuro‐ICU patients exposed to long‐term controlled mechanical ventilation (CMV) were followed with repeated muscle biopsies 3times/week for up to 12 days. Single muscle fiber contractile recordings were conducted on the first and final biopsy. A multiomics approach was taken to analyze gene and protein expression in muscle and plasma at all six timepoints.

Findings

i) A progressive preferential myosin loss, the hallmark of CIM, was observed in all neuro‐ICU patients during the observation period. The myosin loss was coupled to a general transcriptional downregulation of myofibrillar proteins and activation of protein degradation pathways, resulting in significant muscle fiber atrophy and loss in force generation capacity which declined >65% during the 12‐day observation period, ii) membrane excitability was not affected as indicated by the maintained compound muscle action potential amplitude upon supramaximal stimulation of upper and lower extremity motor nerves, and iii), analyses of plasma revealed early activation of inflammatory and proinflammatory pathways, as well as a redistribution of zinc ions from plasma in parallel with the activation of zinc transporters.

Interpretation

The mechanical ventilation induced lung injury with release of cytokines/chemokines and the complete mechanical silencing uniquely observed in immobilized ICU patients affecting skeletal muscle gene/protein expression are forwarded as the dominant factors triggering CIM. It is therefore suggested that future intervention strategies should target the lung injury and the mechanical silencing in critical ill ICU patients.
Vitamin K2 (MK‐7) modulated Nrf2/NLRP3/caspase‐1 axis to protect against age‐related structural and cognitive deterioration in naturally aged rats
Authors: Aloufi Abdulrahman M. , Aldobikhi Yousef A. , Binhudayb Nujud A. , AlTorkai Roba H. A. , El‐Sherbiny Mohamed , Elkattawy Hany A. , Eladl Mohamed A. , El‐Shafey Mohamed , Hadhod Shaimaa , Elsherbiny Nehal
Published: May 2022

Ageing process is accompanied by disrupted maintenance of tissue homeostasis. Declined cognitive function is a hallmark of ageing process. Patients with severe dementia and elderly people at an early stage of Alzheimer's disease demonstrated marked reduction in serum concentration of vitamin K. Menaquinone‐7 (MK‐7) is a major form of vitamin K2 that can be found in animal products and fermented food. The present study aimed to evaluate the potential protective effect of MK‐7 against cognitive decline and structural alterations in brain tissue of naturally aged rats and to delineate the potential involved molecular mechanisms. Three months‐aged albino rats were randomly assigned into two groups; ageing control receiving vehicle and ageing group received MK‐7 (30 mg/kg, oral, once daily 5 days per week). Treatment was continued for 17 months. Additional group containing three‐month‐old rats was used as adult control. Modified T‐maze, Crawley's sociability test and modified forced swim test were used to assess cognitive level, anxious and depressive like behaviour, respectively. Dissected brain tissues were used to assess neuroprotective mechanisms of MK‐7 by histopathology and immunohistochemistry, real time polymerase chain reaction, spectrophotometry and Enzyme‐Linked Immunosorbent assay. Behavioural tests showed attenuated cognitive impairment, depression and anxiety and improved spatial memory and learning ability by MK‐7 administration. Additionally, brain expression of NLRP3 inflammasome components (NLRP3, caspase‐1) and production of downstream inflammatory factors (IL‐1β, IL‐6 and TNF‐α) were markedly decreased in MK‐7 treated rats. Further, MK‐7 triggered Nrf‐2 expression, reduced lipid peroxidation and restored antioxidant enzymes levels. Also, MK‐7 dampened microglial activation, modulated hippocampal and cerebral cortical structure alterations and restored tyrosine expression in aged brain. Our findings highlighted MK‐7 as a promising candidate for halting progression of structural and cognitive deterioration in aged population. Activation of Nrf‐2 accompanied by NLRP3/ caspase‐1 suppression participated in neuroprotective effect of MK‐7 in rat aged brain.
Machine Learning‐Guided Engineering of Cre‐lox Recombination for Comprehensive Analysis of Neural Networks
Authors: Yamauchi Yuji , Ueda Mitsuyoshi , Aoki Wataru
Published: May 2022

Background

Neural networks generate complex behaviors. However, the relationships between them have not been understood well. Thus, we have developed ‘functional cellomics’, which enables functional annotation of neural networks in a hypothesis‐free manner. The key is the stochastic labeling of opsin achieved by Cre‐mediated stochastic recombination. We designed a gene cassette, which contained two sets of lox variants, lox2272 and loxP, alternately. A transcription factor, QF2w, is interposed between loxP. Cre excises DNA with an exclusive choice of between lox2272 or loxP. When Cre excises lox2272, expressed QF2w induces opsin. Constructing a C. elegans strain carrying this gene cassette, we successfully implemented the stochastic labeling of opsin dependent on Cre induction [1]. However, the labeling rate of opsin was about 30 %. If a large subset of neurons is labeled by opsin, it becomes difficult to distinguish the function of each neuron. In this study, we demonstrated a strategy to precisely control the labeling rate of opsin by machine learning‐guided engineering of Cre‐lox recombination.

Method

In our genetic circuit, the opsin coding gene is expressed in neurons where Cre‐lox recombination occurs between lox2272 sequences. Therefore, we hypothesized that the labeling rate of opsin would be reduced by using lox2272 variants, which is difficult to be excised by Cre. Thus, we generated a library of randomized lox sequences by PCR and introduced them in Saccharomyces cerevisiae. We then induced Cre in the S. cerevisiae and extracted DNA from S. cerevisiae. We used NGS to assess the excision rate of the lox2272 variants. To develop a machine learning model to predict the excision rate of the lox2272 variants, we trained a Gaussian process (GP) model using data containing lox2272 sequences and their excision rates by Cre. Approximately 1000 lox2272 variants were randomly selected as training data and 100 variants were selected to test the prediction accuracy.

Results and Discussion

We quantified the excision rates of over 2000 lox2272 variants by NGS and successfully found lox2272 variants with excision efficiencies ranging from 0.05% to 100%. The results of the NGS analysis were confirmed by qPCR. Next, we created a GP model and observed a high correlation between the actual and predicted cleavage rates of the test data. Using the GP model, we predicted the efficiency of the unevaluated lox2272 sequence and successfully identified lox2272 variants that had various cleavage rates. These results demonstrate the feasibility of precise control of opsin labeling rates in functional cellomics [2].

[1] Aoki et al, Sci Rep, 8, 10380, 2018

[2] Yamauchi et al, submitted
Effect of Heparin on the Formation and the Healing of Kissing Gastric Ulcers in Rats
Authors: Sakhno Denis , Efimov Aleksei , Sigua Badri , Filaretova Ludmila
Published: May 2022

The incidence of gastric ulcers remains at a fairly high level: it is diagnosed annually in 7‐14% of the adult population. Despite the decrease in morbidity and the development of diagnostic methods as well as effective treatment, the number of complications such as bleeding and perforation is increasing. One of the significant pathological factors leading to the gastric injury is disturbances in blood flow through mucosal microvessels (providing oxygen and nutrients). In this regard, it becomes relevant to study drugs that affect the rheological properties of blood and microcirculation.

This study aimed to investigate the effect of heparin on the formation and the healing of the experimentally induced gastric ulcers in rat. Adult male Sprague‐Dawley rats weighing 230‐250 g were used after 24 h preliminary fasting. Gastric ulcers were produced by luminal application of 60% acetic acid to an area clamped with a pair of forceps (kissing ulcer model developed by S. Okabe) in anesthetized rats. Day 4 after the application (Day 0) was chosen as the time point for the ulcer formation, and Day 7 was chosen as the time point for incomplete ulcer healing. Heparin was injected at a dose of 1000 U/kg (s.c., once a day). While studying the effect on the ulcer formation, heparin was injected daily from Day 0 to Day 3. While studying the effect on the ulcer healing, heparin was injected daily from Day 4 to Day 6. Control rats were injected with saline. The blood hemoglobin levels were evaluated before the acid application surgery and in the end of experiments.

The heparin injection before the ulcer formation resulted in a decrease of the ulcer area. The average area of kissing ulcers in heparin‐treated group was significantly smaller than that in the control group (p <0.05). The ulcer healing was significantly accelerated in the heparin‐treated group in comparison to the control group (p < 0.05). Blood hemoglobin levels were decreased in both heparin‐treated and control groups without significant differences between the groups.

The results suggest that heparin therapy has the potential to accelerate gastric ulcer healing and prevent their formation.
Morphine Aggravates Inflammatory, Behavioral, and Hippocampal Structural Deficits in Septic Rats
Authors: Abuiessa Salwa A. , Ayieng’a Evans O. , Elblehi Samar S. , El‐Gowilly Sahar M. , Afify Elham A. , El‐Mas Mahmoud M.
Published: May 2022

Although pain and sepsis are standard comorbidities of intensive care units, reported data on whether pain control by opioid analgesics could alter inflammatory and end‐organ damage caused by sepsis remain contradictory and inconclusive. Here, we tested the hypothesis that morphine, the gold standard narcotic analgesic, modifies behavioral and hippocampal structural defects induced by sepsis in rats. Sepsis was induced with cecal ligation and puncture (CLP) and behavioral studies were undertaken 24 hr later in septic and/or morphine‐treated animals. The induction of sepsis or exposure to morphine (7 mg/kg) elicited similar: (i) falls in systolic blood pressure, (ii) alterations in spatial memory and learning tested by the Morris water maze (increases in escape latency, and decreases in time spent on quadrant plate, distance travelled, and number of crossings), and (iii) depression of exploratory behavior measured by the new object recognition test. These hemodynamic and cognitive defects were significantly exaggerated in septic rats treated with morphine compared with individual interventions. Similar patterns of amplified inflammatory (IL‐1β) and histopathological signs of hippocampal damage (neuronal degeneration, satellitosis, and neuronophagia) were noted in morphine‐treated septic rats. We also show that the presence of intact opioid receptors is mandatory for the elicitation of behavioral and hemodynamic effects of morphine because no such effects were observed after simultaneous blockade of these receptors by naloxone. Together, these findings suggest that morphine acts probably via opioid receptors to provoke sepsis manifestations of inflammation, and behavioral and hippocampal deficits.
In vitro Investigation of the Role of NCBE in Inflammation Induced Cerebrospinal Fluid Hypersecretion by the Choroid Plexus
Authors: Friis Kathrine A. , Johnsen Laura Ø. , Damkier Helle H.
Published: May 2022

Post‐hemorrhagic hydrocephalus (PHH) can be the result of inflammation or deposition of FeCl3 in the choroid plexus (CP) after intraventricular hemorrhage (IVH). The hemorrhage leads to increased CSF secretion from the CP also known as the blood‐CSF‐barrier (BCSFB). The precise molecular mechanism is unknown, but the Na+ dependent Cl‐/HCO3‐ exchanger, NCBE, located in the basolateral membrane of the CP epithelia has been suggested as a possible facilitator of CSF hypersecretion.

The aim of this study is to investigate the role of the NCBE located in the CP epithelia after inflammation or deposition of FeCl3in relation to the development of PHH. An in vitro model of the BCSFB was established and validated. The model was generated from mouse pup CP epithelial cells. The cells were treated with either TNF‐□, IL‐1□, LPS or FeCl3. NCBE protein expression of the CP epithelial cells was determined by immunoblotting.

The CP epithelial cell cultures showed typical cobblestone‐like morphology, polarization and formed an intermediate tight barrier demonstrated by TEER values of ~ 100 Ω*cm2and expression of tight junctions. Compared with controls TNF‐□ decreased NCBE expression by 23.8 % after 24 h (p=0.0048, n=6/group). IL‐1□ had same effect with 28.3 % decrease after 24 h (p=0.0018, n= 6/group) persisting with a decrease of 88.5 % after 3 and 82.4 % after 7 days (p=0.0004 and p=0.0035, n=6/group). LPS had no effect on NCBE expression(n=5/group). FeCl3 increased NCBE expression by 52 % after 24 h (p=0.024, n=7/group) followed by a decrease of 57.8 % after 7 days (p=0.043, n=3/group).

We hypothesized that IVH‐induced CP inflammation or FeCl3deposition leads to hydrocephalus by increasing the expression of the basolateral NCBE in the CP cells which leads to an increased rate of CSF secretion. However, the preliminary results suggest a decrease in expression of NCBE after inflammation whereas only an increase in NCBE expression was seen after FeCl3 deposition.

In conclusion, NCBE expression of the CP epithelium in cultures decreases under pro‐inflammatory conditions and increases after FeCl3 deposition. Further studies are needed to define the pathways that lead to regulation of NCBE following hemorrhage.
Morphometrical Description of the Left Ventricular Summit Vascular Triangle
Authors: Batko Jakub M. , Krawczyk‐Ożóg Agata , Pękala Jakub , Hołda Mateusz K.
Published: May 2022

Background

Precise anatomical description of coronary vascular topography in the epicardial area adjacent to left atrial appendage (LAA) is crucial for many procedures performed in this region. The aim of this study was to assess the mutual relations between branches of left coronary artery and great cardiac vein in the left ventricular summit region and to determine their neighborhood with the LAA.

Methods

Left coronary artery with its branches, great cardiac vein and selected cardiac structures were segmented from 200 contrast enhanced computed tomography scans and visualized using three dimensional semi‐automatic algorithms.

Results

In 97.0% of cases the area between left coronary artery branches and great cardiac vein had triangular‐like shape, with area of 253.0±160.0mm2. The length of the left anterior descending and left circumflex coronary artery sections forming the triangle sides were 24.6±8.8mm and 20.8±6.5mm, respectively. The mean length of the triangle base formed by great cardiac vein was 26.2±11.3mm. On average, 78.8±25,3% of the triangle area was covered by LAA. In 30.5% the ramus intermedius coronary artery intersected the triangle. Within the triangle, the Dg1 was present in 53.0% of hearts, the Dg2 in 4.5%, Mg1 in 29.0% and Mg2 in 3.5%.

Conclusions

This study provided detailed morphometrical description of the left ventricular summit vascular triangle. The proximal parts of the left coronary artery branches are closely adjacent to LAA in most cases. Three‐dimensional visualizations of coronary vessels and LAA may lead to increased safety of invasive procedures performed in this region.
Results of using probiotic based on Enterococcus Faecium L‐3 strain for enteritis of calves
Authors: Nikitina Anastasia , Lebedev Maxim , Kovalev Sergey , Trushkin Vyacheslav , Nikitin Georgy
Published: May 2022

Diseases of the digestive system of calves, including enteritis, are an urgent problem in dairy farming. The disease occurs under the influence of various causes and often has a mixed etiology. The use of veterinary bacterial drugs today has found its application in preventing and treating many animal diseases. At the same time, valuable microorganisms of the digestive system of animals ‐ lactic acid and bifidobacteria, play the role of an immunomodulator by synthesizing their antibiotic substances that stimulate the work of the organism's defenses. Thus, in the prevention and treatment of enteritis in calves, it is essential to use new effective probiotic drugs, taking into account their effect on the microflora of the digestive tract of calves. Two groups of black‐and‐white calves were formed: experimental (G1, n=20) and control (GC, n=20). The selection of animals was carried out according to the principle of analogs. Calves from the G1 were given a probiotic based on the Enterococcus Faecium L‐3 strainfrom birth to 45 days of age, 0.5 g with feed once a day. The probiotic was not fed to the calves of the GC. The tendency towards an increased level of bactericidal (BAS) and lysozyme activity (LAS) of serum was revealed in the calves of G1. Thus, at 14 days of age, the level of BAS in calves receiving the probiotic was significantly higher by 27% (P <0.05), and LAS by 21% (P <0.05) than in calves from the GC. This result was also noted at 30 and 45 days of age. BAS was significantly higher by the 30th day of life in the G1 (P <0.05) than in GC: 13.12 ± 1.95% and 9.68 ± 0.60%, respectively. The LAS level was significantly higher (P <0.01) in calves from G1 by 26% than in animals of the GC: 5.8 ± 0.43% and 4.3 ± 0.20%, respectively. At 45 days of age in animals of the G1, the level of BAS was significantly (P <0.001) higher by 61%, and LAS by 34% (P <0.001) than in GC. In the study of the intestinal microflora, it was noted that the calves of the GC had a significantly higher amount of total bacterial mass, Escherichia coli and Bacteroides fragilis group, which indicates the development of opportunistic microflora. Thus, regular feeding of a probiotic based on the Enterococcus Faecium L‐3 strain to calves of the early postnatal period contributes to better resistance to gastrointestinal disorders and indicators of nonspecific body defense, creating favorable conditions for the development of representatives of normal flora and limiting the growth of opportunistic microflora.
Accessory Soleus Muscle in a 79‐Year‐Old Female Cadaver: A Case Report
Authors: Ghyasi Niloofar , Acuna Kelly , Boyd Dave , Ford Avery , Jones Shiloh , Orellana Elise
Published: May 2022

In the present case report, we describe characteristics regarding a rare accessory soleus muscle (ASM) that was discovered and measured. The ASM is found in about 6% of people, and is most commonly asymptomatic (Mukish et al., 2012). The most common symptomatic presentation includes a soft mass in the posteromedial distal third of the leg. Pain is also present in around two‐thirds of symptomatic cases (Mukish et al., 2012). This unilateral anomaly was found during a routine dissection of the left lower ankle in a 79 year old female cadaver. In the present case, the ASM tendon originates from the middle of the tibia and inserts via tendinous insertion onto the medial surface of the calcaneus; this is one of four possible insertion variations already described in the literature. This particular insertion variation is described as Type A (Tubbs et al., 2016). The length of the muscle is [17cm] at the insertion point of the superior tendon to the inferior tendon. This accessory soleus variation has been shown to cause various clinical implications such as tibial nerve compression neuropathy in patients (Badr et al., 2020). Clinical symptoms experienced by patients due to presence of ASM are significant enough to require surgical removal of this muscle to relieve symptoms (Badr et al., 2020). Due to both the known and unknown clinical implications of the variation, it is pertinent to properly describe this so as to benefit future clinical and surgical outcomes for all patients.
The Role of Steroidogenic Factor‐1 Neurons in Predator Odor‐induced Muscle Thermogenesis
Authors: Watts Christina A. , Smith Jordan , Welch Emily , Malik Aalia , Novak Colleen
Published: May 2022

Obesity is one of the most prevalent diseases quietly plaguing this nation. With predictions of its prevalence nearing almost 50% of the adult US population by the end of this decade, it is paramount to identify underlying mechanisms. Characterized by its imbalance of energy intake and energy expenditure, obesity can be countered by amplifying metabolic rate, including inducing thermogenesis. Primarily research has sought to understand adipose tissue thermogenesis, leaving little attention directed toward the brain mechanisms that regulate skeletal muscle thermogenesis. Published evidence from our research program has established that exposure to predator odor (PO) evokes a robust thermogenic response in the skeletal muscle, suggesting that PO works through an unknown brain mechanism causing the thermogenic response. This metabolic response and the stimulation by predator threat each implicate the dorsomedial subregion of the ventromedial hypothalamus (dmVMH), and more specifically, the steroidogenic factor 1 (SF1) cells within it, as a region for further investigation. We hypothesize that SF1 neurons of dmVMH are a key node for predator odor‐induced muscle thermogenesis. We use designer receptors exclusively activated by designer drugs (DREADD) delivered using a viral vector to SF1‐Cre mice to assess this hypothesis. Utilizing DREADD technology, we stimulated the SF1 cells in the presence of a control odor or PO (ferret odor). Here, we analyze preliminary data looking at the muscle thermogenesis of SF1 Cre mice that have been bilaterally injected with mCherry control vector or an excitatory DREADD vector. Using clozapine‐N‐oxide (CNO) and manipulation of context (i.e., with or without the presence of PO), we have detected increases in thermogenesis with each PO exposure and CNO activation of SF1 neurons. However, these data provide little to no evidence of DREADD‐induced amplification of the PO‐induced thermogenesis. Our preliminary data supports the importance of SF1 neurons and their role in regulating PO‐induced skeletal muscle thermogenesis.
Visualizing the Gas Channel of a Monofunctional Carbon Monoxide Dehydrogenase
Authors: Biester Alison , Dementin Sébastien , Drennan Catherine L.
Published: May 2022

Carbon monoxide dehydrogenase (CODH) plays an important role in the processing of one‐carbon gases carbon monoxide and carbon dioxide. In CODH enzymes, these gases are channeled to and from the Ni‐Fe‐S active sites using hydrophobic cavities. In this work, we investigate these gas channels in a monofunctional CODH from Desulfovibrio vulgaris, which is unusual among CODHs for its oxygen‐tolerance. Based on computational predictions, we expect that monofunctional D. vulgaris CODH will have gas channels different from those of bifunctional Moorella thermoaceticacarbon monoxide dehydrogenase/acetyl‐CoA synthase (CODH/ACS). By pressurizing D. vulgaris CODH protein crystals with xenon and solving the structure to 2.04 Å resolution, we identify 12 xenon sites per CODH monomer, thereby elucidating hydrophobic channels used for CO transport. We find that D. vulgaris CODH has one CO channel that has not been experimentally validated previously in a CODH, and a second channel that is shared with Moorella thermoaceticacarbon monoxide dehydrogenase/acetyl‐CoA synthase (CODH/ACS). This experimental visualization of D. vulgaris CODH gas channels lays groundwork for further exploration of factors contributing to oxygen‐tolerance in this CODH, as well as study of channels in other CODHs.
Effects of Both Inherent Chemical Structure and Environment Conditions on the Enzyme‐Small Molecule Relationship of a Non‐Oxime Reactivator of Organophosphorus‐Inhibited Human Acetylcholinesterase
Authors: Sriraman Aishwarya , Lee Hannah , Cadieux C. L.
Published: May 2022

Organophosphorus (OP) nerve agents and pesticides inhibit the enzyme acetylcholinesterase (AChE) via a covalent interaction at the active site, resulting in a buildup of excess acetylcholine in the synaptic cleft. Current reactivators rely exclusively on oxime moieties that can reverse this inhibition via direct nucleophilic attack of the bound OP at the enzyme active site. Absolute molar concentration is a fundamental component of the mechanism of oxime‐mediated reactivation and forms the basis of established reactivation kinetics theory and descriptive mathematics. Recently, a non‐oxime reactivator (ADOC) was discovered that operates via a general acid‐base catalysis mechanism, which calls the fundamental assumptions of established reactivation kinetics theory into question. The objective of this study is to determine if the assumption that reactivation occurs in an absolute concentration‐dependent manner holds true for ADOC. An assay was developed to elucidate the effect of reactivator and enzyme concentration relationships as a component of either inherent compound structure‐activity or external environment‐activity relationships, and this was utilized to evaluate both an oxime and non‐oxime reactivator. Initial results suggest that while chemical structure and, therefore, mechanism of reactivation play a role in the reactivator‐to‐enzyme interaction, environmental factors are suspected to also impact this relationship only for ADOC, likely via activation of an alternate reactivation mechanism. In conclusion, expanding the reactivation kinetics theory to accommodate alternate mechanisms of reactivation may pave new avenues for the development of nerve agent countermeasure therapeutics.