bluuebunny/crossref_metadata_2025 huggingface dataset:
Student Use and Response to In‐House Cadaveric Dissection Videos
Authors: Vilburn Matthew J. , Simet Samantha M.
Published: May 2022

During the initial COVID‐19 shutdown, in the spring of 2020, dissection videos were created to meet the ongoing needs of all our student populations. In total, thirty‐one dissection videos were created, corresponding to each laboratory dissection. Videos were linked to an online Interactive Dissecting Guide, which students could access through a secure network. Dissection videos were initially utilized for virtual laboratory sessions and as a supplementary resource for students. Upon the return of in‐person courses, the videos were then primarily used as supplemental resources. The purpose of this study was to retroactively determine student usage and feedback.

Methods

An anonymous survey was sent to students in the Colleges of Medicine, Allied Health, and Graduate Studies who had access to dissection videos during their gross anatomy courses. Data regarding video views was extracted from Echo360 and mapped to student populations covering the specific content at that time.

Results

Initial analysis showed that students tended to watch the videos more at the beginning of a unit and then tapered off toward the end of the unit. Additionally, videos that accompanied faculty lab demonstrations were viewed the least. Survey analysis of student feedback demonstrated that 77.6% of the respondents found the videos to be “very useful” and the remaining students ranked them as “somewhat useful.” Of the students that viewed the videos, 86.2% of them indicated that they viewed them frequently.

Conclusions

Students utilized and valued high quality dissection videos created specifically for their laboratory dissection content. Students had a tendency to utilize the videos in higher numbers at the beginning of each unit and less at the end of each unit.
Impact of team‐based virtual microscopy on student learning in histology
Authors: Potaliya Pushpa
Published: May 2022

Introduction

Histology is a chief discipline in the basic medical science curriculum, and the practice of teaching it is based on theoretical didactic stratagem followed by practical training. Conventionally, students use to accomplish practical competence in this subject by learning optical microscopy. Nowadays, students can utilize latest information and communication technologies in the study of digital microscopic images. A virtual microscopy program was initiated at All India Institute of Medical Sciences, Jodhpur, India. In view of the fact that little pragmatic substantiation is there regarding the impact of virtual microscopy against optical microscopy on the acquisition of histology comprehension, this study was set up.

Objective

The purpose of this study was to:

Provide students with a core content resource that transcends the classroom.

To assess the students feedback and the impact of a set of measures deliberated to improve an experimental group's understanding of histology.

This study evaluate and compare overall laboratory averages and individual test scores along with a student appraisal to determine the effects of using virtual microscopy in place of optical microscopes in a large undergraduate human anatomy course.

Material and Method

A pretest‐post test and cross‐over design was adopted. The students' reactions to the instructional measures were assessed by course evaluation data. In the first segment, the experiment yielded two groups in an entire census of 100 students, Group 1 performing the practical sessions with Optical Microscopy versus Group 2 performing the same sessions with Virtual Microscopy. In the second phase, the research subjects switched conditions. The prior knowledge level of all research subjects was assessed with a pretest. Knowledge acquisition was measured with a post test after each phase. Analysis of covariance was done to study the differential gain in knowledge at T1 and T2, considering the possible differences in prior knowledge at the start of the study. Quantitative assessment of students was based on laboratory quizzes.

Result

Both short and long term outcome of implementing such module will be discussed at time of conference. Along with that, discussion would emphasize on its role in the medical education system.

Conclusion

Although allowing students access to high‐quality digitized materials and boosting prerequisite skills are clearly not entirely sufficient to boost final competence. Instead, the solution may lie in making students externally accountable for their learning throughout their training. More importantly, data from assessment scores and evaluation surveys indicated that use of the virtual microscope may significantly improve student performance and learning efficiency. Procedures for successfully implementing this change shall be discussed.
A Cadaveric Analysis of Morphological Variations of Pectoral Girdle Muscles: Axillary Arch and Coexisting Pectoralis Muscle Variations
Authors: Anderson Hana , Tucker Richard P. , Weil Jennifer A.
Published: May 2022

Muscles that demarcate the axilla function as critical landmarks of the region, therefore awareness of anatomical variations of these muscles allows more effective decision making in clinical procedures performed at a location within the axilla. One of the well‐known anatomical variations of this category is the axillary arch. The axillary arch, also referred to as Langer’s axillary arch and Achselbogen (among many other terms used in literature), is a muscular‐tendinous slip crossing the axilla anteriorly. Typically, the axillary arch originates from the anterior border of the latissimus dorsi near the base of the axilla and inserts onto the deep tendon of the pectoralis major, as well as to the coracoid process and the muscular and tendinous structures found in the upper part of the humerus. In itself the axillary arch is considered a common variant, as indicated by a recent meta‐analysis report describing its prevalence as 5.3%; however, occurrence of bilateral axillary arches as well as additional thoracic muscle variations accompanying the axillary arch are less common.

In the current study we report the identification of axillary arch variations in 4 out of 48 donors (8.3%) in the ethnically diverse whole‐body donation population in Northern California in 2020 and 2021. Two cases were accompanied with additional pectoral muscle variations. In one, bilateral axillary arches and a left pectoralis quartus were present. In this individual the proximal ends of the left axillary arch and pectoralis quartus formed a common aponeurosis and inserted onto the deep tendon of the pectoralis major. In the other, two supernumerary fascicles deep to the pectoralis major on both sides and a right axillary arch were present. The pectoral fascicles originated from the surface of the ribs and joined to the deep surface of the pectoralis major. The right axillary arch separately inserted onto the deep tendon of the pectoralis major.

Embryologically, the two pectoral girdle muscles associated with the variations described above (pectoralis major and latissimus dorsi) originate from the ventrolateral lip of the dermomyotome and develop through the “In‐Out” mechanism; that is, their myogenic precursors first migrate into the forelimb bud, but then return to the thorax to form these muscles. This complex migration pattern of the precursors would increase the likelihood of accessory muscles arising in this region. Since the axillary arch and pectoralis quartus in humans are considered as vestigial remnants of the panniculus carnosus (cutaneous skeletal muscle sheets found in many mammals and some primates), the migrating precursors may occasionally reactivate a limited fraction of the panniculus carnosus developmental program, leading to the development of the accessory muscles.
Bile Duct Ligation Differently Effects Plasma Osmolality but not Blood Pressure in Male vs Female Rats
Authors: Little Joel , Farmer George , Cunningham Joseph T.
Published: May 2022

Dilutional hyponatremia is known to increase morbidity in patients with advanced liver disease due to the inappropriate release of arginine vasopressin (AVP). Our previous studies show that bile duct ligation (BDL), an animal model of liver disease, increase circulating copeptin in male rats but a decrease in osmolality as compared to the shams. Female BDL rats did not demonstrate the same increase in circulating copeptin or decrease in plasma osmolality as compared to sham females. In this study, we tested if male and female BDL rats showed differences in their blood pressure responses to BDL. Previous studies indicate that male BDL rats become hypotensive while females have not been tested. Adult male and female Sprague Dawley rats had either BDL or sham ligation surgery and were implanted with radio telemetry transmitters for measuring Mean arterial pressure (MAP) and heart rates (HR). Rats were housed in a commercial metabolic cage for one week prior to euthanasian to measure water intake and urine output. No change was seen in daily water intake between the male BDL and sham groups, however the female BDL animals had a significant increase in their daily water intake (p < 0.05, n =6) when compared to the shams. Despite the increase in water intake seen only in BDL females, both the male and female BDL groups saw a large increase in daily urine output (p < 0.001, n = 4‐9) compared to the shams. Analysis of variance showed that there was a main effect of treatment (F(3, 20)= 6.7, P < 0.05) that was due to the MAP of both male and female BDL groups being significantly lower than female shams (P < 0.05). Heart rate was not different among either sexes or treatment groups. Liver weight to body weight ratio was significantly increased in both male and female BDL rats. These results show that both male and female BDL rats became hypotensive when compared to their shams, however the differences in development of hyponatremia among the male and female rats are not due to difference in blood pressure changes associated with BDL.
Gentiopicroside ameliorates acute inflammatory kidney injury in a mouse model induced by LPS through hampering NF‐κB, AP‐1 and IRF3 pathways
Authors: Shareef Saja M. , Kathem Sarmed H.
Published: May 2022

Background

Acute kidney injury (AKI) is one of the most critical care conditions with various etiologies and high mortality. LPS induces AKI by activation of renal inflammatory cascade, promotes the release of numerous pro‐inflammatory cytokines, and ultimately results in kidney damage. Gentiopicroside, a natural secoiridoid glycoside extracted from Gentiana lutea with gastroprotective and hepatoprotective effects. In this study, the renoprotective and anti‐inflammatory effects of gentiopicroside were investigated in a mouse model of LPS‐induced acute kidney injury.

Method

AKI induced in mice by LPS 10mg/kg/day intraperitoneally and euthanized 24 hrs later. Mice in the treatment groups received either 40mg or 80mg/kg/day of oral gentiopicroside for 5 consecutive days prior to LPS injection.

Results

The renoprotective effect of gentiopicroside (40mg/kg/day) resulted from the significant reduction of kidney injury marker (KIM‐1) mRNA expression level in kidney tissue (5.23±1.06 vs. 25.46±2.32) compared to nontreated mice. An interested dose dependent renoprotective effect observed with further significant reduction of KIM‐1 mRNA level when 80mg/kg/day were used (2.42±0.65 vs. 25.46±2.32) compared to nontreated mice. Pretreatment with Gentiopicroside 40 and 80 mg/kg/day revealed strong and dose dependent anti‐inflammatory effect as observed from the significant attenuation of the inflammatory markers IL‐1β (21.66±2.17 and 7.13±1.50 vs. 49.08±5.23) and iNOS (9.73±1.85 and 3.98±1.44 vs. 40.87±5.59) mRNA expression levels in kidney tissue compared to nontreated mice. Further investigation of the upstream mediators revealed that gentiopicroside exerted strong hampering effects on NF‐κB (0.42±0.07 and 0.21±0.12 vs. 7.66±1.39), AP‐1 (1.59±0.60 and 0.71±0.21 vs. 15.27±1.66) and IRF3 (4.89±0.76 and 4.16±1.015 vs. 19.21±3.06) mRNA expression level in kidney tissue compared to nontreated mice significantly.

Conclusion

This study clearly revealed that gentiopicroside exerted a dose dependent anti‐inflammatory and kidney protective effects in LPS‐induced acute kidney injury. The observed effects were resulted through hampering of NF‐κB, AP‐1 and IRF3 pathways.
Oxygen therapy limiting peripheral oxygen saturation to 89‐93% is associated with a better survival prognosis for critically ill COVID‐19 patients at high altitudes
Authors: Arias‐Reyes Christian , Viruez‐Soto Antonio , Arias Samuel , Casas‐Mamani Ronnie , Rada‐Barrera Gabriel , Merino‐Luna Alfredo , Molano‐Franco Daniel , Tinoco‐Solorzano Amilcar , Marques Danuzia A. , Zubieta‐DeUrioste Natalia , Zubieta‐Calleja Gustavo , Soliz Jorge
Published: May 2022

Patients admitted to the Intensive Care Unit (ICU) with acute hypoxemic respiratory failure automatically receive oxygen therapy to improve inspiratory oxygen fraction (FiO2). Supplemental oxygen is the most prescribed drug for critically ill patients regardless of altitude of residence. In high altitude dwellers (i.e. in La Paz [»3,400 m] and El Alto [»4,150 m] in Bolivia), a peripheral oxygen saturation (SatpO2) of 89‐95% and an arterial partial pressure of oxygen (PaO2) of 50‐67mmHg (lower as altitude rises), are considered normal values for arterial blood. Consequently, it has been suggested that limiting oxygen therapy to maintain SatpO2 around normoxia may help avoid episodes of hypoxemia, hyperoxemia, intermittent hypoxemia, and ultimately, mortality. In this study, we evaluated the impact of oxygen therapy on the mortality of critically ill COVID‐19 patients who permanently live at high altitudes. A multicenter cross‐sectional descriptive observational study was performed on 100 patients admitted to the ICU at the “Clinica Los Andes” (in La Paz city) and “Agramont” and “Del Norte” Hospitals (in El Alto city). Our results show that: 1) as expected, fatal cases were detected only in patients who required intubation and connection to invasive mechanical ventilation as a last resort to overcome their life‐threatening desaturation; 2) among intubated patients, prolonged periods in normoxia are associated with survival, prolonged periods in hypoxemia are associated with death, and time spent in hyperoxemia shows no association with survival or mortality; 3) the oxygenation limits required to effectively support the intubated patients’ survival in the ICU are between 89% and 93%; 4) among intubated patients with similar periods of normoxemic oxygenation, those with better SOFA scores survive; and 5) a lower frequency of observable reoxygenation events is not associated with survival. In conclusion, our findings indicate that high‐altitude patients entering an ICU at altitudes of 3,400 – 4,150 m should undergo oxygen therapy to maintain oxygenation levels between 89 and 93 %.
Characterization of structural features driving promiscuous ligand binding in GMCSF
Authors: Cui Jennifer , Lisi George
Published: May 2022

Granulocyte macrophage colony stimulating factor (GMCSF) is a cytokine that is involved in myelopoiesis as well as immunomodulation. GMCSF has been shown to worsen conditions such as rheumatoid arthritis (RA) and multiple sclerosis (MS) while ameliorating diseases such as Type I Diabetes. GMCSF has also been used clinically to combat neutropenia based on its immunostimulatory activity. Pathophysiological properties of GMCSF have been highlighted by the incidence of the global pandemic, COVID‐19 (caused by SARS‐CoV‐2), where elevated levels of GMCSF were identified in COVID‐19 patients in cases related to development of systemic hyperinflammation, pneumonia and acute respiratory distress syndrome (ARDS). A key feature of signalling molecules like GMCSF is the selective and nuanced way in which responses can be tuned to external stimuli. The mechanism of how these signals are modulated are unknown and of particular interest. GMCSF and heparin have been shown to interact under low pH conditions. This binding interaction is important in two ways. Firstly, this pH sensitive interaction can serve as a basis to understand how cytokines can selectively interact with binding partners under variable physiological conditions. Secondly, in clinical settings where both anticoagulatory and immunostimulatory drugs are required, clear understanding of the binding interactions between the two drugs will help inform treatment decisions. Previously we identified heparin dependent structural changes as well as a striking dynamic change in GMCSF that was dependent on heparin chain length and pH using solution NMR studies which we believe to be dependent on a histidine triad. Here we have characterized GMCSF mutants using sited‐directed mutagenesis to exchange critical histidine residues for alternate amino acid residues (including Ala, Phe, Tyr and Asp) to probe the contributions of histidine residues to the increased flexibility of GMCSF observed in acidic conditions. We find that the histidine residues are critical for stability and as little as one amino acid difference will alter flexibility and stability of the protein. In some instances, mutations of all three histidine residues can lead to partial protein unfolding. Our results highlight the critical chemical properties at the amino acid level that allow GMCSF to interact with heparin in an environmentally dependent way.
Surface Mapping and Functional Interactions of GPx4, a Cytoprotective Peripheral Membrane Protein
Authors: Labrecque Courtney L. , Nolan Aubree L. , Fuglestad Brian L.
Published: May 2022

The study of peripheral membrane proteins (PMPs) has long been fraught with difficulty due to the need for reliable and robust membrane models. Many PMPs are soluble in an inactive state and recruited to the membrane to perform their respective function. This is the source of difficulty: the small size of the proteins and their inherent dynamic interactions with membranes and specific lipids make them incredibly challenging to characterize using X‐ray crystallography or cryo‐EM. Using protein NMR and better optimized membrane mimics, a robust study of PMPs may proceed.

Glutathione peroxidase 4 (GPx4) is a PMP with the unique function of protection of membranes from lipid hydroperoxidation. This enzyme prevents cell death from ferroptosis by engaging the membrane and reducing lipid hydroperoxides. GPx4 has emerged as a high‐profile therapeutic target due to the ability of inhibitors to leverage ferroptosis against certain types of cancer cells.

We utilize a wide variety of membrane models and native substrates to map the binding interaction of the GPx4 with the membrane and substrate lipids. Electrostatic interactions of the cationic patch are also found to be important in the moonlighting function of GPx4 as a cross‐linker of DNA‐bound protamines. Finally, our experimentally determined membrane interface using DPC micelles to formulate and confirm a novel DLPC:DPC reverse micelle system that can act as a membrane model for future PMP studies. The novel DLPC:DPC formulation is the first step in developing reverse micelles as more biologically accurate membrane models that can be tuned depending on membrane chemistry.
Phosphorylation regulates E3 ligase activity of Mdm2
Authors: Mayo Lindsey , Nus Jordan
Published: May 2022

The Mdm2 oncogene is associated with the negative regulation of the tumor suppressor p53 in an autoregulatory feedback loop in response to DNA damage. It has been assumed that Mdm2 functions in this pathway as an E3 ubiquitin ligase and is Mdm2’s principal function under all conditions. However, the E3 ligase activity can function either as ubiquitin or nedd8 E3 ligase. This activity is dependent on specific tyrosine kinases that are activated in response to DNA damage signaling or growth factor signaling pathways. The E3 ligase activity can have a distinct effect on the substrate by either stabilizing or signaling destruction. In the context of growth signaling pathways, we show that Src phosphorylation of Mdm2 recruits Ubc12 (nedd8 E2 enzyme). This results in the neddylation of the tumor suppressors pVHL and p53. The neddylation of pVHL prevents the association with p53, thereby attenuating the induction of anti‐angiogenic genes. The phosphorylation of Mdm2 by Src is in the Zinc domain, and we found that this domain is a key regulatory domain for the recruitment of E2 enzymes. These findings suggest that the role of Mdm2 is more complex than just functioning as an E3 ubiquitin ligase to signal for the destruction of p53.
Acute wheel‐running increases markers of stress and aversion‐related signaling in the basolateral amygdala of male rats
Authors: Grigsby Kolter , Kerr Nathan , Kelty Taylor , Mao Xuansong , Booth Frank
Published: May 2022

Physical activity (PA) is a non‐invasive, cost‐effective means of reducing chronic disease. Most US citizens fail to meet PA guidelines, and individuals experiencing chronic stress are less likely to be physically active. To better understand the barriers to maintaining active lifestyles, we sought to determine the extent to which short‐ versus long‐term PA increases stress‐ and aversion‐related markers in wild‐type (WT) and low voluntary running (LVR) rats, a unique genetic model of low physical activity motivation. Here, we tested the effects of 1‐ and 4‐weeks of voluntary wheel‐running on physiological, behavioral, and molecular measures of stress and Hypothalamic Pituitary Adrenal (HPA)‐axis responsiveness (corticosterone levels, adrenal wet weights, and fecal boli counts). We further determined measures of aversion‐related signaling (kappa opioid receptor, dynorphin, and corticotropin releasing hormone mRNA expression) in the basolateral amygdala (BLA), brain region well characterized for its role in anxiety and aversion. Compared to sedentary values, 1‐, but not 4‐weeks of voluntary wheel‐running increased adrenal wet weights and plasma corticosterone levels, suggesting that HPA responsiveness normalizes following long‐term PA. BLA mRNA expression of Prodynorphin (Pdyn) was significantly elevated in WT and LVR rats following 1‐wk of wheel‐running compared to sedentary levels, suggesting that aversion‐related signaling is elevated following short‐, but not long‐term wheel‐running. In all, it appears that the stress effects of acute PA may increase molecular markers associated with aversion in the BLA, and that LVR rats may be more sensitive to these effects, providing a potential neural mechanism for their low PA motivation.
An Atypical Origin of the Saphenous Nerve and a Variation of the Right Iliacus Muscle: A Case Report
Authors: Bayron‐Ho Dhalma I. , Veras Wilson
Published: May 2022

During a routine cadaveric dissection of the posterior abdominal wall, variations of the bilateral lumbar plexus and a variant saphenous nerve, originating in the lower abdomen was noted and documented. Further study revealed variations at the root of the lumbar plexus and bilateral branching patterns. A variant iliacus muscle entrapping the superior portion of the femoral nerve was also observed on the right side within the abdominal cavity. To the best of our knowledge, the saphenous nerve arising from L1‐L3 above the inguinal ligament has not been reported in literature as of yet, which raises the question of how rare this variation may be. Anatomical variations should always be kept in mind in clinical and surgical settings, not only to prevent iatrogenic effects but also to determine proper diagnosis and care for the patient. Nerves in particular, are important to consider when a patient is experiencing pain or paresthesia, or when performing invasive procedures.
HuR Mediates Thermogenic Metabolism in Brown Adipose Tissue Through Control of Sarco‐Endoplasmic Calcium Cycling
Authors: Guarnieri Adrienne R. , Anthony Sarah , Tranter Michael
Published: May 2022

Obesity is classified by the World Health Organization as one of eight principle causes of preventable chronic disease and is directly associated with decreased life expectancy due to increased risk of associated comorbidities. Brown adipose tissue (BAT) is a thermogenically active, endocrine organ regulating both glucose homeostasis and heat production, and the ability to induce BAT‐mediated energy expending represents a potential therapeutic avenue to reduce obesity.

We recently showed that the RNA‐binding protein HuR (Human antigen R) plays a functional role in BAT‐mediated thermogenic metabolism. RNA sequencing analysis from mice with an adipocyte‐specific deletion of HuR (Adipo‐HuR‐/‐) revealed a BAT‐specific decrease in the expression of many genes responsible for sarco‐endoplasmic reticulum (SER) calcium cycling, including SERCA1a, RyR1, RyR2, and additional RyR complex proteins such as triadin, junctin, and CASR. Canonical thermogenesis in BAT occurs through uncoupling protein 1 (UCP1). However, several UCP1‐independent energy expending cycles, including intracellular calcium transport, have also been shown to regulate thermogenesis. SER calcium cycling has recently been established as a thermogenic mechanism in beige adipose tissue, but its functional contributions to thermogenesis in BAT have not yet been fully elucidated.

To determine if the HuR‐dependent changes in SER calcium cycles manifest as a functional change in SER loading/cycling in BAT, we show that decreased cytosolic intracellular calcium levels in BAT isolated from Adipo‐HuR‐/‐mice. Additionally, we show that HuR is activated acutely downstream of beta‐adrenergic stimulation in BAT, and deletion of HuR blunts adrenergic‐induced increases in intracellular calcium. Furthermore, HuR directly binds RyR2 mRNA in brown adipocytes and regulates RyR2 expression through RNA stabilization. Taken together, this growing body of work suggests an important role for HuR in mediating thermogenesis through modulation of SER calcium cycling in brown adipose tissue.
Low Serum Erythropoietin Levels are Associated with Fatal COVID‐19 Cases at 4,150 meters above sea level
Authors: Arias‐Reyes Christian , Viruez‐Soto Antonio , López‐Dávalos Mónica Marlene , Rada‐Barrera Gabriel , Merino‐Luna Alfredo , Molano‐Franco Daniel , Tinoco‐Solorzano Amilcar , Zubieta‐DeUrioste Natalia , Zubieta‐Calleja Gustavo , Soliz Jorge
Published: May 2022

Previous studies suggested that erythropoietin (EPO) may protect against severe COVID‐19‐induced injuries, ultimately preventing mortality. This hypothesis is based on the fact that, in addition to promoting the increase in red blood cells, EPO is an anti‐inflammatory, anti‐apoptotic and protective factor in several non‐erythropoietic tissues. Furthermore, EPO promotes nitric oxide production in the hypoxic lung and stimulates ventilation by interacting with the respiratory centers of the brainstem. Given that EPO in the blood is increased at high‐altitude, we evaluated the serum levels of EPO in critical patients with COVID‐19 at “Hospital Agramont” in the city of El Alto (4,150 masl) in Bolivia. A total of 16 patients, 15 men, one woman, with a mean age of 55.8 ± 8.49 years, admitted to the Intensive Care Unit were studied. All patients were permanent residents of El Alto, with no travel history below 3,000 masl for at least one year. Blood samples were collected upon admission to the ICU. Serum EPO concentration was assessed using an ELISA kit, and a standard technique determined hemoglobin concentration. Only half of the observed patients survived the disease. Remarkably, fatal cases showed 2.5 times lower serum EPO than survivors (2.78 ± 0.8643 mU/ml vs 7.06 ± 2.713 mU/ml; p = 0.0096), and 1.24 times lower hemoglobin levels (13.96 ± 2.56 g/dL vs 17.41 ± 1.61 g/dL; p = 0.0159). While the number of cases evaluated in this work is low, our findings strongly warrant further investigation of EPO levels in COVID‐19 patients at high and low altitudes. Our results also support the hypothesis that exogenous EPO administration could help critically ill COVID‐19 patients overcome the disease.
Locomotor Training Enhances Respiratory Plasticity in a Pre‐clinical Model of Cervical Spinal Cord Injury
Authors: Schardien Kayla A. , Randelman Margo L. , Fortino Tara A. , Wilkinson Tucker , Capello Tyler , Cusimano Marissa A. , Hall Adam A. , Zholudeva Lyandysha V. , Bezdudnaya Tatiana , Lane Michael A.
Published: May 2022

Spinal cord injury (SCI) is a devastating and irreversible injury that impacts thousands of people each year. Most injuries occur at cervical levels, compromising the phrenic motor network, resulting in life‐threatening respiratory deficits. While there is mounting clinical and pre‐clinical evidence for spontaneous plasticity, it is limited, and life‐threatening deficits persist. One strategy for therapeutically enhancing plasticity is the use of activity‐based therapies, such as respiratory and locomotor training. Clinical studies using treadmill training have shown improved respiratory function for individuals with SCI. Therefore, the present work reverse translates these promising clinical findings back to animal models of mid‐cervical SCI to assess the effects of treadmill training on anatomical and functional respiratory plasticity after injury.

Adult female Sprague‐Dawley rats (~200g) received a lateralized C3/4 contusive injury. One week post injury, the animals were blindly divided into two groups: untrained or treadmill trained (30 min, once a day, 5 days a week) for 4 weeks. Breathing behavior was measured each week using whole‐body plethysmography. After training completion, all animals were anatomically traced with retrograde and transsynaptic pseudorabies virus (PRV) to the left hemidiaphragm to label the phrenic motor network, ipsilateral to injury. Three days later, animals were anesthetized and diaphragm electromyography (dEMG) was performed to observe ipsilateral diaphragm deficits and respiratory recovery, and animals were perfused for histology. Immunohistological assessment was used to quantify interneuronal connectivity and density of descending serotonergic input to the phrenic motor network. Preliminary data demonstrated increased interneuronal connectivity with injured phrenic motor network, and increased serotonergic input to spinal networks caudal to injury. Electrophysiological assessment of diaphragm function revealed increased diaphragm activity ipsilateral to injury, after 4 weeks of training. Ongoing analyses will further elucidate the therapeutic effects of locomotor training for improving breathing post‐SCI.
Development of Novel M4 Muscarinic Acetylcholine Receptor Antagonists for the Treatment of Movement Disorders
Authors: Rook Jerri , Bender Aaron , Dickerson Jonathan , Foster Daniel , Rodriguez Alice , Niswender Colleen , Hess Ellen , Conn Jeff , Lindsley Craig
Published: May 2022

Non‐selective antagonists of muscarinic acetylcholine receptors (mAChRs) that broadly inhibit all five mAChR subtypes provide an efficacious treatment for some movement disorders, including Parkinson’s disease and dystonia. Despite their efficacy in these and other central nervous system disorders, anti‐muscarinic therapy has limited utility due to severe adverse effects that often limit their tolerability by patients. Recent advances in understanding the roles that each mAChR subtype plays in disease pathology suggest that highly selective ligands for individual subtypes may underlie the anti‐parkinsonian and anti‐dystonic efficacy observed with the use of non‐selective anti‐muscarinic therapeutics. Our recent work has indicated that the M4 muscarinic acetylcholine receptor has several important roles in opposing aberrant neurotransmitter release, intracellular signaling pathways, and brain circuits associated with movement disorders. This raises the possibility that selective antagonists of M4 may recapitulate the efficacy of non‐selective anti‐muscarinic therapeutics and may decrease or eliminate the adverse effects associated with these non‐selective drugs. However, this hypothesis has not been directly tested due to lack of selective antagonists of M4. Here we utilize genetic mAChR knockout animals in combination with non‐selective mAChR antagonists to confirm that the M4 receptor underlies the locomotor‐stimulating and anti‐parkinsonian efficacy in rodent models. We also report the synthesis, discovery, and characterization of a series of first‐in‐class selective M4 antagonists. These novel compounds have ideal pharmacokinetic properties for in vivo evaluations, and we have confirmed that these optimized compounds have anti‐parkinsonian and anti‐dystonic efficacy in pharmacological and genetic models of movement disorders. These data provide critical pre‐clinical rationale for the development of M4 antagonists and represent a potential novel treatment mechanism to meet the unmet clinical need across several movement disorders.
Bio‐Imaging quorum sensing signal molecules in a soil‐mimic gel
Authors: Jiang Nianyu , Roghair Stroud Marissa , Banerjee Soma , Vang Dua , Anisuzzaman Sharif , Shrotriya Pranav , Halverson Larry , Nilsen‐Hamilton Marit
Published: May 2022

Rhizosphere, the narrow region surrounding plant roots directly influenced by root exudates and the root associated microbiome, plays an important role in plant productivity and the rhizosphere is well known for stimulating microbial metabolic activities. How microbial communities interact to form stable, metabolically interconnected functional communities is an area of intense interest. The question remains on how microbially produced secreted molecules that function as intercellular communication signals shape the structure and function of microbial communities. Our goal is to detect and quantify signal molecules produced by microbes or plants in the root‐soil environment, spatially and temporarily. As a proof‐of‐concept, we are imaging diffusible extracellular microbial metabolites involved in a bacteria‐bacteria communication process called quorum‐sensing. Quorum‐sensing relies on the accumulation of high concentrations of signal molecules in the environment to control bacterial gene expression, influencing rhizosphere colonization and plant health. Local concentration of quorum sensing molecules are determined using aptamer based sensors. Aptamers that can specifically bind the desired signal molecules are selected through SELEX and immobilized on the surface of nano‐porous membranes. Binding of the signal molecules and aptamer covered surface results in changes in surface charge distribution and steric hinderance and thus modifying the transmembrane ionic transport. Changes in transmembrane impedance can be measured through electrochemical impedance spectroscopy methods to monitor local concentrations of signal molecules. Sensor responses were determined for different concentrations of signal molecules and results showed detection of C4‐HSL in a soil‐mimic solution with KD of 10 nM. We demonstrate that the quorum‐sensing signal molecule C4‐homoserine lactone and (C4‐HSL) can rapidly diffuse in a soil‐mimic gel, providing evidence for our use of a soil‐mimic for further aptasensor development and validation. The sensors were then inserted into soil mimic gel for monitoring C4‐HSL diffusion and the resulted impedance changes were used to determine the C4‐HSL concentration at different positions in the gel. Measurements of local C4‐HSL concentrations variation and numerical solution of diffusion equation were used to create 4D images of C4‐HSL molecule diffusion in the soil‐mimic gel.
Targeted Nanoparticles to Mitigate Radiation‐Induced Blood‐Brain Barrier Disruption and Cognitive Impairment
Authors: Lindsey Nathanial R. , Witwatchaitawee Kanawat , Leng Linette , Salem Aliasger , Grumbach Isabella M. , Ait‐Aissa Karima
Published: May 2022

Background

Up to 90% of brain cancer survivors develop radiation‐induced cognitive dysfunction following treatment. Damage to neurovascular endothelium contributes to cognitive decline through increased blood‐brain barrier (BBB) permeability. In our preliminary studies of endothelial cells (ECs) and mice, increased mitochondrial reactive oxygen species (mtROS), inducing heightened mitochondrial DNA (mtDNA) damage and release as cell‐free DNA are detected following irradiation (IR). This cell‐free mtDNA is known to activate Toll‐like receptor 9 (TLR9), triggering an immune response and further ROS. These endothelial responses are prevented with TLR9 inhibition and mtROS scavenging, suggesting a key role for TLR9 in sustained IR‐induced endothelial damage and dysfunction.

Here, we posit that specific inhibition of TLR9 in cerebral ECs during radiation treatment will prevent subsequent BBB permeability and cognitive decline. Thus, the objective of this study is to examine the role of TLR9 in IR‐induced cognitive decline using TLR‐specific loaded nanoparticles (NP).

Methods and Results

To target TLR9 in irradiated ECs, poly (lactic‐co‐glycolic acid)‐based NP coated with cyclic Arg‐Gly‐Asp (cRGD)‐peptide were designed and loaded with TLR9 inhibitor (ODN‐2088, 2.23ug/mg particle). cRGD‐peptide is a ligand for integrin αvβ3, which is highly expressed in ECs post‐IR. cRGD‐NP loaded with coumarin‐6 were used to first evaluate specificity in vitro and in vivo. ECs 24h post‐IR (4 Gy) displayed higher fluorescence than non‐IR, indicating selective uptake. Moreover, in C57Bl6 mice 24h post unilateral cerebral IR (12Gy) with cRGD‐NP, uptake accumulated in endothelium of the IR hemisphere.

Mice were administered NP‐ODN‐2088 or empty NP (3mg injection) and subjected to IR as described above. 30 days post‐IR, they were tested for memory and learning ability using the Novel Object Recognition test. IR induced a significant impairment in the memory ability of empty NP treated mice compared to NP‐ODN‐2088. BBB permeability was analyzed in the same mice 30 days post‐IR with immunohistochemistry using anti‐mouse IgG. Significant BBB permeability was detected in the IR hemisphere of empty NP mice compared to the non‐IR hemisphere. However, the IR hemisphere of NP‐ODN‐2088 mice showed attenuated permeability compared to empty NP treated mice.

Conclusion

Inhibition of TLR9 in irradiated ECs was observed to protect against BBB disruption and cognitive decline. These data support a pathway of radiation‐induced endotheliopathy through which TLR9 activation links vascular mitochondrial injury to neuropathology. Currently there are no pharmacological options for treating radiation‐induced normal tissue injury and targeting TLR9 poses a promising strategy.
RETRACTED: Bacillus Subtilis DE111 Improves Endothelial Dependent Vascular Dilation in Mice Fed a Western Diet
Authors: Risk Briana D.
Published: May 2022

Vascular dysfunction often precedes the development of cardiovascular disease in which data from our lab and others suggest a role of the gut microbiome in the development of this defect. In a pilot study of healthy humans, we observed that the spore‐based probiotic, Bacillus Subtilis DE111(DE111), improved reactive hyperemia index (RHI) scores, indicating improved vascular dilation. To further explore whether DE111 impacts cardiovascular function, the current study examined its efficacy in a diet‐induced obesity model in mice. Male C57/BJ mice were fed a standard diet (SD; n=24) or Western diet (WD; n=24) for 8 weeks to induce vascular dysfunction and were then further divided such that (n=12) half of the animals on each diet received 1billion colony forming units per day (CFU/d) of DE111. Weekly body weight was measured until termination at 6 months of age. WD and WD+DE111 mice exhibited similar increases in body weight (SD: 31.2±2.3 vs WD: 47.4±3.3 and WD+DE111: 45.7±4.0) and impairments in glucose tolerance compared to SD mice (SD: 237.1±68.0 vs WD: 410±26.9 and WD+DE111: 460±116.8, p<0.05). A 28% decrease in vessel dilation was observed with mice on a WD as compared to mice on a SD after addition of the endothelial‐dependent dilator acetylcholine (AUC, 918.3±49.25, p<0.05). However, the addition of DE111 improved endothelial mediated vessel dilation in mice fed a WD+DE111 (AUC, 991.7±47.9, p<0.05). This increase was observed independent of body weight changes and improvement in glucose tolerance. In conclusion, the addition of DE111 increased endothelial‐dependent dilation in mice fed a WD. Further analyses are underway to establish the mechanisms of this gut‐vasculature crosstalk, but our animal and human studies both suggest that the novel probiotic, DE111 may support cardiovascular health.
Studies of the 5′ Untranslated Region of sagA/Pel in Streptococcuspyogenes
Authors: Brown Alexis S. , Carroll Cameron R. , Nibar Sara G. , Perez Alvarado Gabriela C. , Lee Brian M.
Published: May 2022

Streptococcus pyogenes is a human pathogen that is also known as group A Streptococcus (GAS), which is responsible for the development of sore throats and skin infections as well as the more severe condition, necrotizing fasciitis. The streptolysin associated gene A, sagA, codes for the protein streptolysin A (SagA), which is modified and exported as the cytotoxin streptolysin S (SLS). SLS is a virulence factor that inhibits the response of neutrophils during necrotizing fasciitis. The sagA gene is contained within the pleiotropic effect locus (pel). Previous studies have identified sagA as both the gene for the streptolysin S peptide, and the small regulatory RNA (sRNA), known as Pel, which influences expression of multiple virulence genes. The 5′ UTR of sagA/Pel contains the regions necessary for both transcription initiation and ribosomal binding for translation. Focusing on the 5′ UTR of sagA, we hypothesize that the RNA structure of this region affects the transcription of sagA and the initiation of translation. Our goal is to characterize the secondary and 3D structure of the sagA/Pel mRNA/sRNA, which will allow for a better understanding for the role it plays in streptococcal pathogenesis. In this study, RNA constructs containing predicted structured regions were produced by in vitro transcription. Secondary structures were predicted using the mfold and RNAfold webserver based programs. The predicted secondary structures were used to generate three‐dimensional models with the program FARFAR through the ROSIE webserver. Differential scanning fluorimetry was also used to assess the structural stability of the RNA constructs and to screen a variety of conditions to be used in crystallization studies. These experiments provided evidence that the 5′ UTR of sagA/Pel contains significant structured regions and optimal conditions were identified for future structural studies. Potential interactions between the sagA/Pel sRNA and regulated mRNA targets were predicted using the TargetRNA2 webserver. The potential sites for recognition were used to evaluate the functional significance of the structured regions within the 5′ UTR of sagA/Pel. Identification of structural motifs necessary for regulating the expression of virulence genes will aid in the design of therapeutic strategies to inhibit the production of the virulence factors, such as SLS. In future studies, we plan to use structural techniques such as NMR spectroscopy and X‐ray crystallography to help further understand the role that sagA/Pel plays in streptococcal diseases.
Role of the Dimerization Domain of Filamin in Dictyostelium discoideum Response to Shear Flow
Authors: Buckler Sarah , Artemenko Yulia
Published: May 2022

Molecular mechanisms by which cells sense and directionally migrate in response to mechanical perturbation, which is critical in homeostasis and many diseases, are not well understood. Dictyostelium discoideum cells exposed to a brief burst of shear flow show rapid and transient activation of multiple components of the signal transduction network that participates in directed migration of these cells. Previous data from our laboratory demonstrated that actin crosslinking protein filamin is involved in the ability of cells to respond to shear flow. We also found that the actin‐binding domain is required for filamin’s function in this context. To determine if the dimerization domain (DD) is also required for filamin’s role in sensing/transmitting mechanical stimuli we generated a truncation construct of filamin lacking DD (FLNΔDD) and expressed it in wild‐type or filamin‐null cells. We found that FLNΔDDwas able to relocalize to the cortex of both wild‐type and filamin‐null cells following 2‐sec stimulation with shear flow, suggesting that dimerization between filamin molecules is not required for their recruitment to the cortex. To detect activation of the signal transduction network in the presence or absence of FLNΔDD, we used fluorescently‐tagged Ras binding domain biosensor that detects active Ras and was previously shown to relocalize to the cortex following mechanical stimulation. Surprisingly, FLNΔDDwas able to rescue the reduced response of filamin‐null cells to shear flow stimulation, suggesting that dimerization of filamin may not be needed for its ability to sense or transmit mechanical cues. However, the presence of FLNΔDD may also alter the overall organization of the cytoskeletal network, potentially explaining changes in mechanotransduction. We are currently investigating these possibilities.
Influence of Cognitive Challenges During Exercise on Post‐Exercise Cerebrovascular Function
Authors: Corkery Adam , Eisenmann Nicole , Pearson Andrew , Miller Kathleen , Howery Anna , Barnes Jill
Published: May 2022

Peripheral vascular function is acutely improved following a single session of aerobic exercise. This improvement is likely due to increased blood flow during aerobic exercise. Cognitive activity and aerobic exercise both acutely increase cerebral blood flow, and while previous studies have examined the influence of acute exercise on post‐exercise cerebrovascular function, the effects of increased cognitive activity during exercise on post‐exercise cerebrovascular function remains unclear. Therefore, the purpose of this study was to examine the influence of increased cognitive activity during aerobic exercise on post‐exercise cerebrovascular function. We hypothesized that performing cognitive challenges during exercise would result in greater post‐exercise cerebrovascular function. METHODS: Ten participants (age: 27 ± 5 y; VO2max = 53 ± 10 mL/kg/min; men = 5, women = 5) completed 2 study days, in a randomized order, in which they either walked at 30% VO2max for 30 min (WALK) or walked at 30% VO2max for 30 min while completing N‐back cognitive tasks (WALK+COG). Cerebrovascular reactivity to hypercapnia (CVR) was measured pre‐ and 90 min post‐exercise as a measure of cerebrovascular function. Middle cerebral artery velocity (MCAv), mean arterial pressure (MAP), and end‐tidal CO2 (ETCO2) were continuously monitored during the hypercapnia protocol. Cerebrovascular conductance index (CVCi) was calculated as MCAv/MAP. CVR was calculated as the slope between the changes in MCAv or CVCi and ETCO2. RESULTS: During WALK and WALK+COG, there was no difference in MCAv from pre‐ to post‐exercise (p ≥ 0.05 for both), despite a significant post‐exercise reduction in ETCO2 (WALK: pre = 43 ± 2 mmHg, post = 41 ± 3 mmHg; WALK+COG: pre = 41 ± 4 mmHg, post = 40 ± 3 mmHg; p < 0.05 for both). While WALK had no significant change in MAP from pre‐ to post‐exercise (p = 0.10), WALK+COG had a significant increase in MAP (pre = 88 ± 18 mmHg, post = 98 ± 14 mmHg; p = 0.01). Both WALK and WALK+COG had a decrease in CVCi from pre‐ to post‐exercise (WALK: pre = 0.70 ± 0.15 cm/s/mmHg, post = 0.65 ± 0.17 cm/s/mmHg; WALK+COG: pre = 0.75 ± 0.15 cm/s/mmHg, post = 0.66 ± 0.14 cm/s/mmHg; p < 0.05 for both). There was no significant change in CVR from pre‐ to post‐exercise during either study days (assessed as MCAv or CVCi; p ≥ 0.05 for both). Finally, there was no significant difference in the magnitude of change in CVR from pre‐ to post‐exercise between the study days (assessed as MCAv or CVCi; p ≥ 0.05 for both). CONCLUSION: Cognitive challenges during exercise did not influence post‐exercise cerebrovascular function. Despite this finding, cognitive challenges during walking may influence resting systemic hemodynamics, as it resulted in an increased post‐exercise MAP compared with walking alone.