bluuebunny/crossref_metadata_2025 huggingface dataset:
How Useful Was That Critical Reflection? Technology Enhanced Learning in Physiology Skill Lab
Authors: Malik Samina
Published: May 2022

Introduction

Critical reflection blended with technology is essential to gain insight into one’s action leading to enhanced learning & performance in Objectively Structured Clinical Evaluation (OSCE). However, it is still being under‐utilized by teachers and students in most second year undergraduate clinical physiology curricula of Pakistan including University College of Medicine and Dentistry.

Aims and objectives

To evaluate the usefulness of critical reflection blended with technology as a learning enhancement tool.

Subjects and methodology

Teachers and second year physiology students of University College of Medicine and Dentistry, University of Lahore mutually planned an experimental study of QUAL‐quan design, using critical reflection as a tool to enhance learning and performance in cranial nerve examination. The technological stimulus or intervention involved student engagement by video‐presentation of role‐play conducted by class‐fellows on cranial nerves examination. Each of the three randomized groups (n=50) experienced conventional practical demonstration by a standardized common demonstrator followed by hands‐on‐practice by common tutors. Group B was additionally involved in video recording of role‐play, with one student as examiner, second student as a subject, and the third as the examiner who marked the structured key. The remaining students observed the entire activity. Group C additionally watched recorded video followed by reflective group discussion analyzing examination against structured key and tutor’s performance and corrected the mistakes presented in the video through critique and collaborative feedback. Open and closed ended feedback was obtained from students and tutors. Qualitative analysis was conducted on piloted and peer‐reviewed feedback questionnaires whereas quantitative analysis was done on surprise formative assessment scores after couple of months to see the behavioral change as per Kirkpatrick’s model.

Results

Comparatively significant quantitative and qualitative responses were obtained from group C on ANOVA, including enjoyable learning environment, enhanced memory, critical thinking, clarity of concepts, time‐management & confident performance.

Conclusion

Critical reflection blended with technology may enhance learning and performance.
The synthetic non pungent capsaicin analog Arvanil displays robust anti‐tumor activity in human small cell lung cancer
Authors: Merritt Justin C. , Friedman Jamie R. , Nolan Nicholas A. , Moles Emily G. , Hopper Kelly J. , Chen Yi Charlie , Dasgupta Piyali
Published: May 2022

The nutritional compound capsaicin is the hot spicy pungent ingredient of chili peppers. Capsaicin is a pain‐relieving agent and is found in several over‐the counter pain medications. However, recent studies have shown that capsaicin has profound anti‐neoplastic effects in several types of human cancers. The applications of capsaicin as a clinically useful drug are limited by its unpleasant side effects, such as gastric irritation, stomach cramps and burning sensation. This has led to extensive research focused on the identification and rational design of second‐generation capsaicin analogs, which possess greater bioactivity than capsaicin. Our laboratory performed an SAR study using a panel of non‐pungent capsaicin analogs and identified Arvanil as the hit compound. Arvanil triggered robust apoptosis in a panel of human small cell lung cancer (SCLC). The growth‐suppressive activity of Arvanil was confirmed in chicken chorioallantoic membrane (CAM) models. Finally, the dietary administration of Arvanil potently decreased the growth rate of human SCLC tumors xenotransplanted in athymic mice. The pro‐apoptotic activity of Arvanil was mediated by the TRPV1 receptor and the calpain pathway. Non‐pungent capsaicin analogs like Arvanil may be promising drug candidates for the management and treatment of human SCLC.
Epigenetics: A Gatekeeper to DNA Amplification and Replication Control
Authors: Whetstine Johnathan R.
Published: May 2022

DNA amplification is associated with pathological states such as neurological disorders, cardiac disease and cancer. At least 50% of the amplifications in cancer are transient extrachromosomal DNA (ecDNA). The transient behavior contributes to copy number plasticity, results in heterogeneous oncogene expression and alters therapeutic response. The unanswered question remains as to whether distinct mechanisms control ecDNA copy gains within cells and how they impact copy gain events associated with disease. Our laboratory aims to define the principles regulating selective DNA amplification and the associated plasticity, so we may control these events in disease. By resolving general mechanisms governing selective DNA amplifications, we will build a framework to further understand the molecular basis by which copy gains occur in neoplasia and other diseases.

Recent studies have begun to uncover the importance of epigenetic states and histone lysine methyltransferases (KMTs) and demethylases (KDMs) in regulating extrachromosomal transient site‐specific DNA copy number gains (TSSGs). In fact, our laboratory discovered the first chromatin factor promoting TSSGs in the human genome. We have revealed a critical interplay between a myriad of KMTs and KDMs in modulating methylation balance in order to control rereplication and extrachromosomal amplification events. The methyl‐lysine modifier networks that control site‐specific DNA amplification were resolved through genetic and chemical treatments coupled to cytological approaches, epigenomic profiling and 3D genome organizational analyses. While interrogating their role in controlling DNA amplification, we have also begun to appreciate their impact on chromatin modification dynamics and global DNA replication timing. We have established that enhancers coupled to methylation changes in broad methylation domains strongly predicts replication timing changes when perturbed by specific lysine modifiers. Taken together, we have established that methyl‐lysine modifiers are key regulators promoting or preventing DNA amplification and play instrumental roles in regulating replication timing control through the combined regulation of methylation states. The most recent studies addressing these observations will be presented at the meeting.
Cellular Molecular of Osteopontin in the Pathogenesis of Psoriasis
Authors: Salleh Mohd Nazil , Hui Juan Mira Ong , Mustaffa Mohd Irfan Raimi
Published: May 2022

Psoriasis (PS) is a chronic inflammatory skin disease of complex pathogenesis with genetic predisposition, environmental factors and immunological disturbances. Osteopontin (OPN) is known to enhance Th1 responses, inhibit Th2 ones, and take part in the modulation of Th17 cell lineage. Due to its impact on Th1/Th17 immune responses, several reports have suggested that OPN may be of pathogenic importance in psoriasis. However, the involvement of this OPN in the onset of psoriasis have not been fully elucidated. We investigate the cellular localization of OPN protein expression and histopathological features of psoriasis. The study was conducted on 40 patients affected with plaque psoriasis, 10 healthy volunteers and ER+ breast cancer as a positive control. Severity of PS was assessed using the qualitative assessment and severity index score. Our results showed statistically significant differences in the expression of OPN, between lesional and non lesional skin as well as between non lesional skin and control group (P ≤ 0.05). In addition, there was a significant difference in the expression of OPN, between control and lesional group. We concluded that, OPN expressions and severity of psoriasis showed a strong significant negative correlation and also a moderate significant negative correlation. With this notion we postulate OPN expressions will stimulate the inflammatory response, by generating anti‐OPN antibodies and eventually determining OPN expression will become a useful therapeutic tool in assessing and establishing early detection of PS.
The bare bones of bifurcation: small flavoproteins as artificial electron bifurcases
Authors: Greene Brandon L.
Published: May 2022

Flavin‐based electron bifurcation (FBEB) is an essential mode of energy conservation in bacteria and archaea wherein specific flavoenzymes couple exergonic and endergonic metabolic electron flux from a single electron donor. While our understanding of the role of FBEB in metabolism continues to grow, the mechanism by which the flavin cofactor and the protein scaffold coordinate electron bifurcation remains elusive, largely due to the complexity of native bifurcases. We have leveraged the light‐oxygen‐voltage (LOV) domain flavoprotein as a robust and straight‐forward scaffold to interrogate the operant principles of electron bifurcation, including the role of the flavin binding pocket on tuning the flavin redox potential, proton‐coupled electron transfer, and potential “inversion,” as well as the role of conformational gating. Using bioinformatics and structural homology, we have remodeled the LOV flavin binding site to mimic that of native bifurcases and demonstrated key residues stabilize the anionic hydroquinone and increases potential inversion by 60 mV relative to wild‐type LOV. Furthermore, reactions with single electron donors/acceptors implicates a reactive neutral semiquinone, and lay the groundwork for kinetic electron bifurcation studies. Using cytochrome c as a model one‐electron acceptor, we can model the conformational gating mechanism by controlling donor‐acceptor concentrations, and have demonstrated the facile inter‐protein electron transfer, and photochemical crosslinking indicates a unique electron transfer interface. Kinetic modeling, including short‐circuit pathways, suggests that a bifurcation prone state should be long enough lived for direct observation, and studies are ongoing to spectroscopically identify this species. These results illustrate the utility of this reductionist approach in gaining insight into the physical principles that dictate FBEB and inform novel bifurcase design.
Screening for novel fluorescent nucleobase analogs (FBAs) using computational and experimental methods ‐ 2‐amino‐8‐vinylpurine (2A8VP), as a Case study
Authors: Russel Nadim S. , Kodali Goutham , Stanley Robert , Narayanan Madhavan
Published: May 2022

Fluorescent nucleobase analogs (FBAs) are molecular reporters that are structurally similar to naturally occurring nucleotides but that have significantly higher emission quantum yields and red‐shifted absorption and emission maxima. These reporter molecules play an important role in the study of structure and dynamics of nucleic acids and proteins by providing deeper insight into the mechanisms of various biological processes. The experimental synthesis and characterization of FBAs usually precedes their computational characterization. Here, we start from computation to screen for fluorescent emission wavelength and fluorescence intensity thus reducing the time and material needed in developing new novel fluorescent reporters. The photophysical properties of a putative fluorescent analog 2‐amino‐8‐vinylpurine (2A8VP) have been predicted using TD‐DFT theory. Based on these results, we have attempted its synthesis starting from 2‐amino‐6‐chloro‐purine nucleobase. Several blue‐emissive molecules have been obtained, one with a particularly high quantum yield. These reaction products have been characterized by NMR, LC‐MS, and HPLC to obtain their structural, absorbance, and fluorescence properties to compare them with the computed properties of the 2A8VP target molecule.
IGF2BP2 Promotes Cancer Progression by Degrading the RNA Transcript Encoding a v‐ATPase Subunit
Authors: Wang Fangyu , Latifkar Arash , Mullmann James , Panizza Elena , Fernandez Irma , Ling Lu , Miller Andrew , Fischbach Claudia , Weiss Robert , Lin Hening , Cerione Richard , Antonyak Marc
Published: May 2022

IGF2BP2 binds to a number of RNA transcripts and has been suggested to function as a tumor promoter, although little is known regarding the mechanisms that regulate its roles in RNA metabolism. Here we demonstrate that IGF2BP2 binds to the 3’ untranslated region of the transcript encoding ATP6V1A, a catalytic subunit of the v‐ATPase, and serves as a novel substrate for the NAD+‐dependent deacetylase SIRT1, which regulates how IGF2BP2 affects the stability of the ATP6V1A transcript. When sufficient levels of SIRT1 are expressed, it catalyzes the deacetylation of IGF2BP2, which can bind to the ATP6V1A transcript but does not mediate its degradation. However, when SIRT1 expression is low, the acetylated form of IGF2BP2 accumulates, and upon binding to the ATP6V1A transcript, recruits the XRN2 nuclease which catalyzes transcript degradation. Thus, the stability of the ATP6V1A transcript is significantly compromised in breast cancer cells when SIRT1 expression is low or knocked‐down. This leads to a reduction in the expression of functional v‐ATPase complexes in cancer cells and to an impairment in their lysosomal activity, resulting in the production of a cellular secretome consisting of increased numbers of exosomes enriched in ubiquitinated protein cargo and soluble hydrolases including cathepsins, that together combine to promote tumor cell survival and invasiveness. These findings describe a previously unrecognized role for IGF2BP2 in mediating the degradation of an mRNA transcript essential for lysosomal function and highlight how its sirtuin‐regulated acetylation state can have significant biological and disease consequences.
The vital landmarks of human body (Marma) according to TCAM and its application in Physical medicine and Rehabilitation
Authors: Kumar Madan , Meshram Akashdeep A.
Published: May 2022

According to Traditional Complementary and Alternative Medicine (TCAM), the universe as well as the human body, is grossly and subtly comprised of five elements. The five elements namely space, air, fire, water and earth. These are the prime factors for origin of life. Among the five elements, space is related with mind, air with body movements, fire with thermodynamics of the body, water with structural integrity of the body and earth with gross body. The Indian System of Medical science has contributed various insights into the understanding of human body using various philosophical, analytical, experimental and spiritual modalities. Marma sastra and varma sastraare two nomenclatures given for the same type of human anatomical body landmarks, which strongly works on the principles of anatomy and physiology and energy principles. Varma/marma is air (vayu). Varma is also referred to as energy. Thirteen energy locations, grouped into three categories has been explained in detail. In order to understand the varma techniques, an additional knowledge regarding the basic Indian philosophy, is also needed.

Experts have given various classifications of marma. Among them the most practical system is based on method of application. It illustrates the following: 1) Thodu Varmam (stimulated by touching), 2) Padu Varmam (activate other regions), 3) Thattu Varmam (stimulated by gentle tap), 4) Enai Varmam (unified activation of two points can relieve symptoms), 5) Pakka Varmam (action of adjacent varmas), 6) Ellidai Varmam (points related in between joints), 7) Oothu Varmam (stimulation by blowing; for children), 8) Nakku Varmam (stimulation by licking; for children), 9) Naal Varmam (associated with astrological stars), 10) Griha Varma (associated with astrological planets), 11) Patchi Varmam (points related to internal organs), 12) Vaatha Varmam, 13) Pitha Varmam, 14) Sleshma Varmam, (12, 13 and 14 areregulated by bodily humor 15) Nokku Varmam (stimulated by looking). More than fifty manuscripts have been published in the Nation Workshop on Manuscriptology, India, 1999. The present session is a representation of the major varma/marma landmarks of human body and its utility based on connecting this physical landmarks to improve the mobility standards of human body. These principles will be detailed using most recent photographs from eminent resources followed by detailed diagrammatic representation of their practical applications. Though based on ancient knowledge, the matter detailed in this session is based on current practice of these methods in India. The topic gives a new dimension and scope in using the Varma/Marma as a anatomico‐physiological landmarks for medical students as well as practitioners of physical medicine and rehabilitation (PMR).
Optimization of RNA Display Using GC‐Clamp Modifications to Improve Genetic Detection of RNA‐Protein Interactions
Authors: Nguyen Linh D. , Wang Suxuan , Schlein Silvie , Berry Katherine E.
Published: May 2022

Non‐coding small regulatory RNAs (sRNAs) have an important role in bacterial stress responses. The binding of sRNAs to their target mRNAs is facilitated by RNA‐binding proteins such as Hfq or ProQ. The Berry Lab has developed a bacterial three‐hybrid (B3H) assay to detect the binding of RNA with both of these RNA chaperones in vivo, by connecting the strength of an RNA‐protein interaction to the expression of a reporter gene. The interaction between a “prey” protein fused to the α‐subunit of RNA polymerase (RNAP) and a “bait” RNA tethered upstream of a test promoter stabilizes the binding of RNAP and increases transcription of the reporter gene lacZ. Despite the promise of the B3H system and its success in detecting many high‐affinity interactions, low signal‐to‐noise for other RNA‐protein interactions currently limits the broader utility of the assay. Computational structure predictions suggested that certain RNAs of interest could misfold when hybridized with other components of the hybrid RNA construct, e.g. an MS2 hairpin (MS2hp) or an exogenous intrinsic terminator. Such misfolding would likely disrupt a bait RNA’s interaction with the prey protein. To avoid this limitation, this study aims to optimize the hybrid RNA construct to improve the breadth of detectable interactions in the B3H assay. To this end, we designed new hybrid RNA constructs with the addition of a GC‐clamp – a short insert of guanines (G) and cytosines (C) flanking a region of interest – to promote proper folding and optimal display of RNA. Several sRNAs and 5’UTRs were cloned into GC‐clamp designs and their in vivo interactions with Hfq were tested in the B3H assay. Preliminary results demonstrate the promise of a short GC‐clamp in improving the B3H signal for many sRNA‐Hfq interactions, and we are currently working to test these GC constructs with additional RNAs. Increasing the sensitivity and generalizability of the B3H assay to study bacterial RNA‐protein interactions will help shed light on the molecular mechanisms of RNA‐chaperone proteins and the important processes in bacteria they regulate, such as adaptation to stress, biofilm formation and virulence.
Exercise Increases Mitochondrial Biogenesis in Single‐Housed Adolescent Female Rats Treated with Cocaine
Authors: Rodriguez‐Rivera Arianna K. , Pérez‐Cardona Enrique , Frontera Walter , Torres‐Ramos Carlos , Segarra Annabell
Published: May 2022

Prior studies from our laboratory showed that exercise obliterated the effects of increased anxiety induced by isolating rats during adolescence. The present study was designed to characterize the effects of treadmill running exercise on rat skeletal muscle. After weaning (day 23), male and female rats were assigned to one of the following groups: group‐housed, single‐housed, or single‐housed with exercise. Group‐housed, and isolated rats were further subdivided into saline or cocaine groups. Exercise training started the day of single housing and consisted of running on a treadmill once a day, five times a week for four weeks, and six times a week on the fifth and last week. Rats ran for 30 minutes at speed up to 10 m/min the first two weeks, 35 minutes at 12 m/min on the third week, 40 minutes at 15 m/min on the fourth week, and 45 minutes at 17 m/min on the last week. On day 59 rats were euthanized and the soleus and gastrocnemius muscles were dissected and stored at ‐80 ℃. The tissue was homogenized, DNA was isolated, and the amount of mitochondrial DNA copies was quantified using real time PCR. Our results indicate that female rats that were single‐housed showed a trend towards increased mitochondria DNA copies. However, single‐housed females that exercised and were injected with cocaine showed significantly higher copies of mitochondrial DNA in the soleus muscle than their Oil‐treated counterparts. No significant changes were observed in males.
Cellular Localization of Dbp2 and Identification of its Interaction Partners
Authors: Lubinga Rebekah P. N. , Tran Elizabeth
Published: May 2022

Dbp2 is a DEAD‐box RNA helicase in S. cerevisiae, whose main function is connected to the ribosomal RNA processing and transcription. Interestingly, the localization of Dbp2 is also highly dependent on the presence of glucose, which enables us to study how cellular stress impacts gene expression machinery. Studies from the Tran laboratory show that the rapid nuclear export of Dbp2 in the absence of glucose. Furthermore, unpublished studies from the Tran lab have linked specific phosphorylation events on Dbp2 to glucose‐dependent localization. To determine if phosphorylation affects Dbp2’s function and localization, our first step is to generate mutant Dbp2 proteins that localize to the nucleus or the cytoplasm, irrespective of the presence of glucose. To this end, site‐directed mutagenesis was used on plasmids containing wild type DBP2 genes to convert codons encoding select serine residues to codons that would encode amino acids mimicking the dephosphorylated and phosphorylated state of Dbp2. Following transformation of these mutant‐expressing plasmids and a wild type DBP2 as a control into a dbp2∆strain, a serial dilution spot assay was performed on glucose and galactose plates (mimicking – glucose). We observed that both the phosphorylation mutants (both the non‐phosphorylatable and the phosphor‐mimetic) grew substantially better than our wild type Dbp2 and empty vector. This was unexpected and will be followed up with future studies in the Tran laboratory. In addition to mutant Dbp2 generation, we also set out to identify protein interaction partners of Dbp2 in the cytoplasm. Our goal was to use proximity‐dependent biotin identification (BioID) using an AirID biotin ligase tag to identify Dbp2’s cytoplasmic interaction partners. Following unsuccessful isolation of proteins, immunofluorescence was performed to verify the localization of the ligase tag. Immunofluorescence indicates the AirID tag is more cytoplasmically localized in comparison to nuclear Dbp2, thus, the study was inconclusive. Due to the mislocalization of the AirID tag, in future studies we plan to use different versions of the biotin ligase tag such as the TurboID tag.
Effects of tooth loss on anxiety‐like behaviors in Heterocephalus glaber
Authors: Hite Natalee J. , Sarko Diana K.
Published: May 2022

Tooth loss is an ailment affecting a large percentage of the population, particularly individuals of lower socioeconomic status and the elderly. Extensive tooth loss is known to result in anatomical changes in the face, including atrophy of masticatory muscles and decreases in mandibular and maxillary bone density, as well as volumetric changes within the brain. Human studies have reported strong correlations between tooth loss and levels of both anxiety and depression. However, little has been reported to date regarding the effect of dental health on emotional states, with only sparse information available from animal models regarding the psychiatric effects of tooth loss.

Our study investigated the short‐term effects of tooth loss on anxiety‐like behaviors using Heterocephalus glaber, an animal with extensive sensory dedication to and behavioral reliance on its incisors. Adult naked mole‐rats underwent either tooth extraction or sham surgeries, followed by testing in both an open field maze and elevated plus maze in the weeks following. As there are no known studies to date that have utilized this animal model in behavioral tests of anxiety, our study provides critical information about the methodologies and modifications necessary to ascertain these important translational behavioral states so that future researchers can apply similar tests in other fields such as neurodegeneration, aging, and oncology.

We hypothesized that tooth extraction will result in greater anxiety‐like behaviors, manifesting in various ways: greater defecation in the mazes, more time spent in the outer regions of the open field maze, and less time in the open arms of the elevated plus maze. The study demonstrated a trend towards tooth extraction increasing anxiety‐like behaviors in naked mole‐rats when investigated using the open field maze paradigm. There is evidence that, with an increase in sample sizes, this research will make evident the causal relationship of tooth loss and anxiety. Future research will investigate the long‐term behavioral changes associated with extensive tooth loss and the neurocircuitry associated with these behavioral alterations. Uncovering these details will provide potential therapeutic targets for negating the mental health difficulties associated with deficits in dental health.
Antioxidant Effects of Guishe Extract from Agave lechuguilla on Kidney Mitochondria from Stz‐Induced Diabetic Male Rats
Authors: Esquivel‐Gutiérrez Edgar , Peña‐Montes Donovan , Saavedra‐Molina Alfredo , Reyes Ana G.
Published: May 2022

Guishe from Agave lechuguilla was used to test the capacity to mitigate the oxidative stress in kidney mitochondria from streptozotocin‐induced diabetic rats. Orally, a crude aqueous extract from lyophilized guishe was administered over 5 weeks at different doses (150, 300 y 600 mg/kg). Blood glucose and body weight were monitored. The activity of catalase, thiobarbituric acid reactive species, mitochondrial superoxide dismutase, glutathione and glutathione peroxidase were determined in isolated kidney mitochondria. Our results show that guishe extracts regulates the oxidative stress observed in isolated kidney mitochondria. In conclusion, we found that guishe at doses of 300 mg/kg of body weight has a beneficial effect as an antioxidant and can be used to mitigate the oxidative stress occurring during diabetes.
The ERAD system is controlled by ceramides in the endoplasmic reticulum membrane
Authors: Hwang Jiwon
Published: May 2022

Misfolded proteins in the endoplasmic reticulum (ER) are removed through a process known as endoplasmic reticulum associated degradation (ERAD). ERAD occurs through an integral membrane protein quality control system that recognizes substrates, retrotranslocates the substrates across the membrane, ubiquitinates and extracts the substrates from the membrane for degradation at the cytosolic proteasome. While ERAD systems are known to regulate lipid biosynthetic enzymes, the regulation of ERAD systems by the lipid composition of cellular membranes remains unexplored. Here, we report that the ER membrane composition controls ERAD function by incapacitating substrate extraction. Unbiased lipidomic profiling revealed that elevation of specific very‐long chain ceramides leads to a dramatic increase in the level of ubiquitinated substrates in the ER membrane, and concomitantly, reduces extracted substrates in the cytoplasm. This work reveals a previously unrecognized regulatory mechanism in which ER membrane lipid remodeling controls the ERAD system.
The Impact of Muscular Strength On Cardiovascular Disease Risk Factors
Authors: Harden Joel E. , Reynolds Leryn J. , Wilson Patrick B. , Gerstner Gena R.
Published: May 2022

The purpose of this study was to determine the associations between isokinetic leg muscular strength and cardiovascular disease (CVD) risk factor characterizations in Americans aged 50 and older. Using a publicly available dataset from the National Health and Nutrition Examination Survey (NHANES), a secondary analysis was conducted on participants (males ≥50 yrs; females ≥55 yrs; N=10,858) pooled from 1999 to 2002. CVD risk factors were determined using the American College of Sports Medicine (ACSM) cutoff values, with all nine ACSM risk factors analyzed. CVD risk factor characterization was determined by creating CVD risk factor profiles (i.e., the total number of CVD risk factors an individual possesses), then separating participants into low (0‐2 CVD risk factors), moderate (3‐5 CVD risk factors), and high (6‐8 CVD risk factors) risk groups. Muscular strength was determined by isokinetic maximal peak force (PF) of the leg extensors, both raw and normalized to body mass. Normalized, but not raw, muscular strength was shown to be significantly inversely associated with CVD risk factor characterization for both males and females (P<0.001). Muscular strength was separated into quintiles of strength (Q1 = lowest, Q5 = highest). Using ordinal regression and adjusting for all other CVD risk factors, age (males: OR: 1.13, 95% CI: 1.11‐1.15; females: OR: 1.12; 95% CI:1.10‐1.15) obesity (males: OR: 0.54; 95% CI: 0.40‐0.72; females: OR: 0.58; 95% CI: 0.41‐0.82), and smoking status (males: OR: 1.72; 95% CI: 1.31‐2.26; females: OR: 1.39; 95% CI: 1.04‐1.86) were significantly associated with isokinetic leg extensor muscular strength in both males and females, while blood glucose (OR: 1.41; 95% CI: 1.07‐1.86) was only associated with muscular strength in males. Evidence from the present study supports the notion that muscular strength may have a protective effect against CVD. However, this association must be shown independent of cardiorespiratory fitness (CRF) and in clinical trials before any causative link can be established.
Characterization of Electrodes to Record Neural Signals in the Periphery
Authors: Verma Nishant , Knudsen Bruce , Skubal Aaron , Gholston Aaron , Frank Jennifer , Ludwig Kip
Published: May 2022

Appropriately designed electrodes to make in vivo recordings of neural activity have been essential to understanding the nervous system. Recording electrode design has been extensively explored for use in measurement of naturally occurring neural activity in the brain. Recently, recordings of evoked compound action potentials (ECAPs) in the periphery have garnered interest due to their relevance in neuromodulation therapies for determining target engagement and enabling closed‐loop operation. Here, we compare three types of recording electrodes – microelectrodes, cuff electrodes, and intrafascicular electrodes – and their ability to make in vivo recordings of electrically ECAPs, naturally occurring neural activity, and sensory evoked neural activity in the peripheral nerves of a human‐relevant large animal model.

The great auricular nerve, innervating the auricle, and cervical vagus nerve of domestic pigs were instrumented with the three recording electrodes. Naturally occurring neural activity, sensory evoked neural activity induced by gentle brushing of the auricle, and electrically ECAPs initiated by non‐invasive and invasive stimulation electrodes were recorded. The study was pre‐registered (https://osf.io/y9k6j).

We showed that the recording cuff had superior performance in measuring invasive electrically ECAPs (p < 0.05) while the tungsten microelectrode, routinely used for microneurography in humans, had superior performance in measuring naturally occurring action potentials (p < 0.05). The microelectrode was still able to record electrically ECAPs. Further, we show how choice of reference electrode position may be optimized per application.

Microneurography recordings paired with the deployment of a non‐invasive neuromodulation therapy can be used to functionally map sensory innervation areas in patients, allowing individualized placement of stimulation electrodes to achieve on‐target neural fiber activation. These findings provide a basis to select recording electrode type and configuration for measurement of neural signals in the development and deployment of neuromodulation therapies.
Structure of Saccharomyces cerevisiae Mediator‐RNA polymerase II pre‐initiation complex for divergent transcription
Authors: Gorbea Colón Jose J. , Palao Leon , Kim Hee Jong , Tsai Kuang‐Lei , Chang Yi Wei , Murakami Kenji
Published: May 2022

The vast majority of eukaryotic promoter regions are divergently transcribed. While divergent transcription serves a key role in shaping the transcriptome, a structural basis for this process remains unexplored. Whether two transcription pre‐initiation complexes (PICs) can co‐assemble at divergent promotes, and if so, what structural relationships may emerge therefrom, remains unknown. To investigate the structural bases of this phenomenon, we pursued the assembly of the Saccharomyces cerevisiae Mediator‐PIC on a divergent promoter. Taking together observations from Cryo‐EM, crosslinking‐mass spectrometry, and in vitro transcription assays, we were able to capture two distinct PICs co‐assembled at divergent promoters, consistent with recent emerging model derived from in vivo observations. Overall, the structure of the Mediator‐PIC in this novel form is conducive to transcriptional activation for divergent transcription.
Diverse Morphology of Sympathetic Neuron Subpopulations in the Stellate Ganglia
Authors: Barrett Madeleine S. , Hegarty Deborah M. , Habecker Beth A. , Aicher Sue A.
Published: May 2022

Sympathetic nerves innervate the heart and changes in sympathetic efferent activity can modulate cardiac function. The post‐ganglionic sympathetic neurons that innervate the heart are located primarily in the stellate ganglia (SG), which also contains neurons that project to brown adipose tissue (BAT), among other target tissues. We aim to examine morphological changes in SG neurons after myocardial infarction, and characterize the specificity of neural plasticity within the ganglion. To interpret morphological changes induced by cardiac injury, we must first fully understand the normal morphology of distinct groups of SG neurons. To that end, we examined SG neurons following injections of retrograde tracers into cardiac tissue and/or intrascapular brown adipose tissue (BAT) in adult male and female C57BL/6J mice (8 males, 2 females, 28‐32 weeks, 24‐39 g; The Jackson Laboratory). Using cholera toxin B (CTb) tracers conjugated to two different fluorophores (1 mg/mL in 0.01 M PBS; Alexa Fluor 488 or Alexa Fluor 555), we assessed the number, dispersion, pre‐synaptic cholinergic inputs and morphology of BAT‐ and cardiac‐projecting SG neurons. We also examined the quality of the CTb tracer detection at varying times after BAT injection (7, 14 and 28 days; n=2 mice per time point). BAT‐projecting SG neuron visualization was optimal 7 days post‐injection compared to 14 and 28 days, and could be visualized directly or with antibodies against CTb. BAT‐projecting SG neurons were located in both left and right SG, predominately ipsilateral to the injection site and were widely dispersed. In contrast, cardiac‐projecting SG neurons were more localized near the base of the inferior cardiac nerve. In BAT‐ and cardiac‐injected mice (n=4), we found that SG neurons were not dual‐labeled, supporting the notion that sympathetic innervation to target tissues is specific. We found cardiac‐projecting SG neuron somas have more volume (mm3) and cardiac‐projecting cells are less abundant within the SG than BAT‐projecting neurons with our tracer‐labeling paradigm. We also discovered a positive correlation between number of primary dendrites per neuron and soma volume in cardiac‐projecting SG neurons, though not in BAT‐projecting SG neurons. Additionally, in both SG subpopulations, the number of cholinergic inputs marked with vesicular acetylcholine transporter (VAChT) on the cell body is positively correlated to soma volume. Tract tracing distinct subpopulations of neurons will allow us to characterize morphological changes after cardiac injury in specific cell groups. This approach will allow insights into whether mechanisms of plasticity are cell specific or occur globally within the SG.
PEARL mediated biosynthesis: a novel pathway to aromatic amines
Authors: Daniels Page , Lee Hyunji , Ting Chi , Zhu Lingyang , van der Donk Wilfred
Published: May 2022

Aromatic amines are common functional groups found in several metabolites. The biosynthesis of aromatic amines canonically involves amino group installation prior to aromatization. We report a fundamentally distinct pathway to aromatic amines by conversion of phenolic groups to amines. Recent studies identified the use of short, catalytic scaffold peptides for the biosynthesis of amino acid derived natural products. During this process, a peptide‐amino acyl tRNA ligase (PEARL) appends amino acids to the C‐terminus of a ribosomally synthesized peptide in an ATP‐ and tRNA‐dependent manner. We report two pathways in which the short, ribosomally synthesized peptides serve as the scaffold for downstream enzymes to modify a PEARL‐appended tryptophan into a pyrroloiminoquinone. These pathways feature previously unknown biochemical processes using a ribosomally synthesized peptide as a scaffold for non‐ribosomal peptide extension and chemical modification to generate an amino acid derived natural product. These pathways showcase a new use for PEARLs resulting in the amination of indoles.
The Effects of Ribosomal Proteins uS2, uS3, and uS4 on Transcription
Authors: Blaha Gregor M. , Diggs Stephen , Tam Timothy K. , Hakopian Sheron , Rodriguez Carlos
Published: May 2022

In E. coli, transcription and translation are coupled and ribosomes can directly contact RNA polymerase. The contacts are formed between the RNA exit site of the RNA polymerase and ribosomal proteins uS2, uS3, or uS4 on the ribosome. We assessed the effects of ribosomal proteins uS2, uS3, and uS4 on transcription. In varying degrees, they all reduced termination and increased pausing. Although uS2 bound directly to the polymerase, it affected transcription the least. uS3, which did not bind the polymerase, had the most prominent effects on transcription.

We suggest uS2 acts as an anchor for the polymerase during coupling of transcription and translation. Likewise, uS3 and uS4 may affect transcription depending on the length of RNA between the ribosome and polymerase. We speculate that ribosomal proteins may also coordinate the transcription of ribosomal RNAs with their assembly into ribosomes. Finally, we submit that the intrinsic effects of ribosomal proteins may drive the tight regulation of their expression in the cell.
Gender Differences in Perceived Stress and Biological Stress Markers are Associated with Changes in Resting Heart Rate
Authors: Chirico Erica N. , Khamisani Azmeer
Published: May 2022

Objective

Our study objective is to evaluate whether gender differences in both perceived and biological stress levels will alter resting heart rates.

Hypothesis

We hypothesize that females will experience higher levels of perceived and biological stress which will translate to a higher resting heart rate compared to males.

Methods

19 first‐year medical students participated in this study. Participants wore Fitbits to collect heart rate data throughout the course of the academic year. During exam weeks (the “stressor”), participants filled out a standard psychometric questionnaire to assess perceived stress levels and saliva samples were collected to measure cortisol and alpha‐amylase concentrations.

Results

Our study showed that females have higher resting heart rates than males (65.8±6.8 bpm vs 57.7±4.34 bpm, p<0.001). Females recorded having higher perceived stress levels than males (7.9±4.6 vs. 2.12±5.27, p<0.001), as well as having higher salivary alpha‐amylase concentrations than males (33.95±29.50 U/mL vs. 75.4±76.44 U/mL p=0.001). No difference in salivary cortisol concentrations between females and males are seen (0.44±0.25 ug/dL vs. 0.39±0.24 ug/dL, p= 0.3).

In females, there is a weak positive correlation between RHR and perceived stress level (0.22), as well as with amylase (0.26). In males, there is a moderate negative correlation between RHR and perceived stress level (‐0.48) and a weak positive correlation between RHR and cortisol (0.26). There was no relationship between RHR and amylase.

Conclusion

Females have higher levels of perceived stress levels which is in agreement with their higher levels of alpha‐amylase, a marker of psychosocial stress via the sympatho‐adrenal‐medullary system. As salivary alpha‐amylase has been suggested to reflect catecholaminergic reactivity, it seems likely that this is responsible for the increased resting heart rate. This relationship is not consistent in males where perceived stress is associated with a decrease in RHR. However, in males a relationship was seen between biological markers of stress (cortisol via the hypothalamic‐pituitary‐adrenal axis) and RHR. This study suggests that the relationship between biological measures of stress and heart rate vary with gender and that females experience an increased level of perceived stress overall.
Pigment epithelium‐derived factor induced CRX alterations in the mouse retina
Authors: Rebustini Ivan T. , Crawford Susan E. , Becerra S. P.
Published: May 2022

The gene Serpinf1 encodes for Pigment Epithelium‐Derived Factor (PEDF), which protects photoreceptors from cell death. The Serpinf1 null mouse exhibits normal retinal function; however, PEDF deficiency increases retinal degeneration susceptibility in rd10 mouse. CRX is a transcription factor that regulates expression of several photoreceptor specific genes, including opsins and phosphodiesterase. This study aims to explore whether regulation of CRX activation is one mechanism by which PEDF exerts photoreceptor survival.

Serpinf1‐/‐, Serpinf1+/‐ and Serpinf1+/+ mice at 3 months of age were used. We prepared cultures of mouse retinal explants. They were treated with recombinant human PEDF. Zaprinast (a PDE inhibitor) was added to induce photoreceptor death. We determined cell death using PSVue‐550, a visible fluorescent probe for detection of phosphatidylserine on the surface of cells. Subcellular distribution of CRX, PDE6A and pan‐acetylation was detected by immunofluorescence. The transcriptional levels of Crx, Pde6a, Opn1mw and Opn1sw were assessed using qPCR.

PEDF treatment induced the expression of Crx and its regulated genes Pde6a, Opn1mw and Opn1sw. PEDF also enhanced the CRX immunoreactivity in the nuclei of photoreceptors. In contrast, the nuclear CRX immunoreactivity was suppressed in photoreceptors from retinas of Serpinf1‐/‐ mice deficient of PEDF. PDE6A immunoreactivity also decreased in Serpinf1‐/‐ photoreceptors compared to those from Serpinf1+/+ mice. Histone acetylation was detected in discrete photoreceptors in Serpinf1+/+ but was undetectable in the Serpinf1‐/‐ retinas. Zaprinast‐induced photoreceptor death was more pronounced in Serpinf1‐/‐ retinal explants than in wild type controls. PEDF pre‐treatment prior to the zaprinast treatment improved photoreceptor survival and enhanced CRX immunoreactivity in both Serpinf1‐/‐ and Serpinf1+/+ retinal explants. Deletion in PEDF in the Serpinf1 null mice accelerated retinal degeneration induced by zaprinast, which was prevented by exogenous addition of PEDF in retinal explants.

The findings imply that PEDF activated the CRX‐associated transcription factor network. They provide a novel insight linking extracellular PEDF to nuclear CRX during photoreceptor survival.
Dissecting the Mechanisms Whereby Tryptophan Metabolites Alter Myocardial Function
Authors: Keceli Gizem , Jun Seungho , Pharoah Blaze M. , Pasaoa Ryla , Westbrook Reyhan M. , Abadir Peter M. , Paolocci Nazareno
Published: May 2022

Cardiovascular diseases (CVD) are a significant cause of morbidity and mortality, especially in the elderly population and they represent an important risk factor for frailty. The essential amino acid, tryptophan (TRP), acts as a precursor of neurotransmitters/hormones, such as serotonin and melatonin, and is central to many aspects of cellular metabolism. TRP degradation, however, can lead to breakdown products, such as kynurenine (KYN), whose impact on normal and diseased hearts remains primarily unknown. Moreover, in recent clinical reports, increased levels of KYN and/or its metabolites, formed via degradation of the essential amino acid tryptophan, are associated with heart diseases and atherosclerosis. KYN infusion significantly impaired cardiac function in Langendorff‐perfused hearts. Similarly in isolated cardiomyocytes, exposure to KYN increased oxidative stress and decreased fractional shortening. The ongoing studies include mechanistic approaches to determine the pathways involved in KYN and/or its metabolites’ impact on myocardial function.
Extended‐Synaptotagmins Modulate Diacylglycerol Dynamics at the Immunological Synapse in Human T Lymphocytes
Authors: Benavides Nathalia
Published: May 2022

Cytotoxic T‐lymphocytes (CTLs) regulate the immune response by recognizing and destroying cancerous or virus‐infected cells through the polarized release of apoptosis‐inducing proteins at the contact area known as the immunological synapse (IS). The synaptic membrane is a heterogenous region where protein complexes rearrange on the cell surface. Within the membrane are phospholipids that assemble to relay information from extracellular receptors to induce global cellular changes. Diacylglycerol (DAG) is a key secondary messenger of T‐cell receptor (TCR) activation which induces microtubule‐organizing center polarization towards the IS and signaling via MAPK and NFkB pathways. Abnormal lipid homeostasis at the synapse is linked to autoimmune disorders. However, our understanding on how cells regulate dynamic transfer of lipids from one cellular compartment to another is still scant. Evidence points to the role of lipid transport proteins (LTPs) in the regulation of lipid homeostasis without the need for membrane fusion. Extended‐Synaptotagmins (E‐Syts) are a group of LTPs tethering the endoplasmic reticulum (ER) to plasma membrane (PM), thus controlling membrane lipid compositions by transporting DAG down its concentration gradient. Herein, we tested the hypothesis that E‐Syts play a role in the modulation of diacylglycerol at the synaptic membrane, ER‐PM morphology and signaling dynamics by performing the following experiments: 1) assessing the role of E‐Syts in regulating the effector functions of human lymphocytes by measuring cellular cytotoxicity, degranulation, TCR signaling via phospho‐protein analysis and intracellular Ca2+ levels; 2) testing the role of E‐Syts in DAG distribution at the IS via stimulated emission depletion (STED) microscopy. Our results indicate that E‐Syts down‐modulate human lymphocyte activation as their knock‐down leads to enhanced TCR signaling, degranulation, and cytokine production measured by flow cytometry. Using GFP‐based bioprobes for DAG and tagged E‐Syt constructs, we observed proximal localization of E‐Syts with signaling protein LAT and CD4 via super‐resolution microscopy. Accordingly, E‐Syt1 knockout Jurkat cells at resting state express localized DAG accumulation similar to activated WT Jurkat cells. Our results provide, for the first time, an understanding of spatio‐temporal distribution as well as the role of lipid regulators at membrane junction sites during IS formation. We believe that E‐Syts play an essential role in maintenance of synaptic lipids via regulation of non‐vesicular lipid transport, whose loss might contribute to enhanced activity of CTLs at resting conditions. Collectively, our results suggest that E‐Syts are critical components to orchestrate lipid distribution during synapse formation. Future experiments will be required to shed light on additional steady state regulators of synaptic membrane lipids, highlighting the importance of membrane crosstalk in the setting of CTLs.