bluuebunny/crossref_metadata_2025 huggingface dataset:
Assaying fibroblast activation protein (FAP) expression in vivo and in vitro for possible targeting with chimeric antigen receptor (CAR) T cells
Authors: Méndez Fernández Pedro O. , Rurik Joel G. , Aghajanian Haig , Epstein Jonathan A.
Published: May 2022

Fibroblast activation protein (FAP) is a marker of activated fibroblasts in various human cardiovascular diseases. FAP is of special interest because it is expressed exclusively in activated, pathogenic fibroblasts and has been proposed as a target for chimeric antigen receptor (CAR) T cell ablation therapy. It has been shown previously that fibroblasts from patients with hypertrophic and dilated cardiomyopathy express FAP, while fibroblasts from non‐failing hearts did not. The aim of the project was to examine FAP expression in other human cardiac diseases by obtaining heart samples from patients who suffered diseases characterized by cardiac fibrosis so that we could determine which cardiac diseases might be amenable to FAP CAR T therapy. In addition, we sought to characterize an in vitro model of FAP expression to aid in the ability to assess the effectiveness of engineered T cells to kill FAP‐expressing cells. Heart tissue from patients diagnosed with various cardiac disorders, including idiopathic giant cell myocarditis, COVID‐19, and sarcoidosis were stained for FAP expression and compared to non‐failing, control hearts. The results showed significant FAP expression in all cardiac specimens with interstitial fibrosis compared to non‐failing hearts. Notably, samples from patients who died from COVID‐19 exhibited significant fibrosis and FAP expression. This suggests that there may be a large and growing population that may be amenable to anti‐fibrotic therapy. In order to develop an in vitro model of FAP expression to assay anti‐FAP CAR T cell activity, we treated IMR‐90 human fibroblasts with TGF‐B, a known activator of fibrosis, and assayed for FAP expression. TGF‐B exposure resulted in an increase in FAP expression 24‐hours after treatment. Our observation of fibroblast restricted FAP expression in multiple different cardiovascular diseases supports the potential use of FAP CAR T cell therapy as an anti‐fibrotic treatment in multiple heart diseases.
Mitragynine Reverses Paclitaxel Chemotherapy‐Induced Peripheral Neuropathy and is Mediated via Opioid Receptor Involvement
Authors: Ortiz Yuma , Mottinelli Marco , McCurdy Christopher , McMahon Lance , Wilkerson Jenny
Published: May 2022

Chemotherapy‐induced peripheral neuropathy (CIPN) is a problematic side effect in patients receiving chemotherapeutic cancer treatments. Clinical use of approved analgesic drugs often does not adequately control the pathological pain arising from CIPN and does not account for potential abuse with opioid therapeutics. Mitragyna speciosa (kratom) contains the alkaloid mitragynine, which exhibits analgesic properties. However, the underlying pharmacological mechanisms that underlie these analgesic properties are complex and not completely understood. Male and female C57bl/6 mice received 8 mg/kg intraperitoneal injections of paclitaxel, a taxane class chemotherapeutic, every other day over the course of 7 days. To confirm the development of CIPN, the von Frey assay was utilized to determine the onset mechanical allodynia, which arises when a previously non‐painful stimulus is perceived as painful. Intraperitoneal mitragynine and the prototypical opioid agonist morphine both dose‐relatedly reversed CIPN‐induced mechanical allodynia. Effective doses (ED)50 were as follows – morphine: 7.02 (6.56 – 7.51) mg/kg, mitragynine: 109.80 (104.27 – 115.62) mg/kg. Pretreatment with the opioid antagonist naltrexone 0.032 mg/kg, intraperitoneally produced a rightward shift in both morphine and mitragynine dose‐response curves. Effective doses (ED)50 were as follows – naltrexone + morphine: 27.93 (24.84 – 31.40), naltrexone + mitragynine: 245.41 (211.76 – 284.39), resulting in a 3.98 and 2.24 fold shift of dose response curves, respectively. Here we show that mitragynine reverses mechanical allodynia associated with paclitaxel CIPN. We also show that opioid receptors mediate such activity, though additional data suggest a role for adrenergic and serotonin receptors as well. Mitragynine may be an effective analgesic treatment option for patients experiencing painful CIPN.
Autonomic Responses to Acute Hyperoxia are Impaired in Patients with Peripheral Artery Disease
Authors: Cui Jian , Leuenberger Urs A. , Aziz Faisal , Luck Jonathan C. , Stavres Jonathan , Kim Danielle J‐K , Gao Zhaohui , Blaha Cheryl , Cauffman Aimee , Sinoway Lawrence I.
Published: May 2022

Prior reports suggest that oxidative stress plays an important role in the development of atherosclerotic diseases such as peripheral artery disease (PAD). The effects of acute hyperoxia, an important acute oxidative stressor, on autonomic function have not been well studied in PAD. We hypothesized that the autonomic responses to acute hyperoxia would be impaired in PAD. We studied 17 patients with PAD (12M, 5F, 67 ± 2 yrs) and 15 healthy controls (10M, 5F, 65 ± 2 yrs). The ankle‐brachial indexes in the patients were 0.57 ± 0.03 in the most affected leg and 0.77 ± 0.04 in the less affected leg, respectively. Beat‐by‐beat heart rate (HR, from ECG) and blood pressure (BP, with Finometer) were recorded for 10 min under normoxic resting conditions and during 5‐min hyperoxia (100% oxygen) as an acute oxidative stress test. HR variability (HRV) was calculated and cardiac baroreflex sensitivity (BRS) was estimated with the sequence technique. Under resting conditions, mean HR and mean arterial pressure (MAP) were not significantly different between the groups. Resting values of the root mean square of successive differences between normal heartbeats (RMSSD, a HRV index for parasympathetic activity; 14.1 ± 1.0 vs. 19.8 ± 2.2 ms, P = 0.032) and cardiac BRS (6.43 ± 0.78 vs. 14.86 ± 2.56 ms/mmHg, P = 0.001) were significantly lower in PAD than in healthy controls. Hyperoxia lowered HR in both groups and raised MAP only in patients with PAD. During hyperoxia both HR and MAP were significantly higher in PAD than in controls. Hyperoxia induced an increase in RMSSD and the high frequency (HF) component of HRV (another index for parasympathetic activity) in healthy subjects. However, during hyperoxia no increase in parasympathetic activity was observed in PAD. Both RMSSD and the HF component of the whole trial (i.e. baseline and hyperoxia) in PAD were lower than those in healthy control subjects. Hyperoxia did not induce changes in cardiac BRS in either group, while cardiac BRS of the whole trial in PAD was lower than that in healthy controls. These data suggest that resting parasympathetic activity and cardiac baroreflex function are impaired in PAD. Moreover, the autonomic response to acute oxidative stress (i.e. an increase in parasympathetic activity) is also impaired in PAD. Further studies are needed to examine if antioxidant agents can improve autonomic function in PAD.
Three anatomical variations in the arterial system of the neck of a female donor body: a case study
Authors: Bhattacharya Arunabh , Sanderson Jacob , Swail Charles , Gamez Mario , Chalk Robert
Published: May 2022

The common carotid artery (CCA) bifurcates into the external carotid artery (ECA) and internal carotid artery (ICA) at the level of the intervertebral disc between third and fourth cervical vertebra. After the bifurcation, the ICA normally lies posterolateral to the ECA. While ICA does not give off any branches in the neck, superior thyroid (STA), lingual (LA), facial artery, ascending pharyngeal, and occipital arteries arise from the ECA within the carotid triangle of the neck. During a routine dissection to demonstrate the neurovasculature of the neck, three anatomical variations in the arterial system were noted on the left side of a female donor body (Race: Caucasian, Age: 51 years). First, the CCA bifurcated into ECA and ICA below the level of the cricoid cartilage, approximately between the sixth and seventh cervical vertebra, at a distance of 5.7 cm from the arch of the aorta. Second, after the bifurcation, the ICA laid posteromedial to the ECA. Finally, despite the low bifurcation of CCA, STA and LA arose from a common thyrolingual trunk off the ECA (7.7 cm from the carotid bifurcation), between the second and third cervical vertebra. As a result, STA was 6 cm in length from its origin to its termination on the superior aspect of the thyroid gland. While each of these anatomical variations have been reported in the literature, to our knowledge, this is the first description of a ‘triple’ variation in the arterial network of the neck. Clinically, the knowledge of these anatomical variations may be important during surgical procedures involving the neck and its arterial network.
Structural Basis for Nanomolar‐Affinity Inhibition of Neutrophil Serine Protease Activity by the S. aureus EAP Domain Protein, Eap1
Authors: Gido Carson D.
Published: May 2022

Extracellular adherence domain proteins (EAP) are a novel class of innate immune evasion molecules secreted by the human pathogen, Staphylococcus aureus. EAP domains are potent and selective inhibitors of neutrophil elastase (NE) and cathepsin‐G (CG), which are the two most abundant neutrophil serine proteases (NSPs). Previous work from our group has shown that the prototypical EAP domain protein, EapH1, relies on structural plasticity of a single inhibitory site to block activity of NE and CG. However, whether other EAP domain proteins follow similar structure/function relationships remains unknown. To address this limitation, we studied herein the inhibitory properties of Eap1 and determined its structure both free and bound to either NE and CG. Eap1 displayed both dose‐dependent inhibition of NE and CG with Ki values of 0.24 nM +/‐ 0.1 and 8.5 nM +/‐ 1.2, respectively. The structure of EAP1 determined at 1.45 A limiting resolution adopted a compact, b‐grasp fold typical of ~100 residue EAP domain proteins. Whereas the structure of Eap1 bound to CG and determined at 1.95 A revealed an overall inhibitory pose similar to that seen for EapH1, the structure of Eap1 bound to NE and determined at 2.20 A surprisingly showed an inhibitory pose involving a distal region of the EAP domain. Although EAP domains share high levels of sequence and structural homology to one another, our work demonstrates that these high‐affinity inhibitors are capable for forming structurally divergent complexes with two target proteases that themselves share high levels of sequence and structural homology one another.
Extracellular Vesicles: Potential Non‐invasive Biological Tool for Predicting Response to Breast Cancer Treatment
Authors: Alvarez Folnetti A. , Kaddour Hussein , Okeoma Chioma M.
Published: May 2022

Breast cancer (BC) is the most diagnosed cancer among women and second leading cause of cancer death among women. BC patients diagnosed with locally advanced or aggressive early stage (I‐III) breast cancer receive systemic neoadjuvant chemotherapy (NAC) to downstage the breast tumor and avoid metastases to the axillary lymph node. Apart from patients with HER2+ tumors (~25%), use of Anthracycline, Cyclophosphamide and a Taxane (AC‐T) based NAC regimen has become standard of care. However, response to ACT NAC is poor with majority of patients showing partial or no response with residual or whole tumor present at resection. Thus, accurate evaluations of the response to NAC will provide important information on the effect of systemic NAC, to guide improved therapy and reduce unwarranted side effects. Accordingly, cancer‐extracellular vesicles (C‐EVs) represent an emerging liquid biopsy modality for therapeutic response monitoring and outcome prediction. In this study, C‐EVs isolated from blood plasma of BC patients prior to receiving NAC are characterized by their physicochemical properties such as size, concentration, and zeta‐potential. Global protein profiling of isolated C‐EVs was conducted using quantitative LC‐MS/MS. Proteomic analysis identified eight proteins enriched in C‐EVs from BC patients that did not respond to NAC compared to those who responded. Five out of the eight proteins were validated by western blot and classified as differentially present proteins. Functionally, the C‐EVs from BC patients that did not respond to NAC increased luminal A BC MCF7 and triple negative BC MDA‐MB‐231 cell viability. Altogether, our findings suggest that when properly isolated, blood plasma C‐EVs and the five proteins may be established as predictors of chemoresistance, and thus serve to identify patients that may not respond to treatment to spare them the toxic effects of NAC.
Antimicrobial Triclosan (TCS) Increases Production of Interleukin 1 beta (IL‐1β) in Human Immune Cells
Authors: Wilburn Wendy J. , Whalen Margaret
Published: May 2022

Triclosan (TCS) is an antimicrobial compound that is widely used in personal hygiene products such as mouthwash and toothpaste. TCS can be ingested or absorbed through the skin and has been found in human blood, breast milk, and urine. Interleukin‐1 beta (IL‐1β) is an important pro‐inflammatory cytokine produced by immune cells such as monocytes and lymphocytes, and plays a critical role in immune response regulation, tissue repair, and cellular growth. Overproduction of IL‐1β can contribute to chronic inflammation and inflammatory diseases such as rheumatoid arthritis and multiple sclerosis. IL‐1β has also been shown to stimulate tumor development. In a previous study, we showed that TCS at concentrations between 0.05‐5 µM increased the secretion of IL‐1β from immune cells within 24 h. Here we will examine whether the increase in IL‐1 β secretion stimulated by TCS is due to an increase in the cellular production of IL‐1β or only due to release of existing stores of the cytokine. Human peripheral blood mononuclear cells (PBMCs) were exposed to TCS at concentrations of 0‐5 µM. The cellular production (combination of secreted and intracellular levels) of IL‐1β was measured at 10 minutes, 30 minutes, 6 hours, and 24 hours. Secreted levels were measured in supernatants from exposed cells using enzyme‐linked immunosorbent assay (ELISA) and intracellular levels were measured by lysing the exposed cell pellet followed by Western Blot. Results indicate the production of IL‐1β was increased by exposure to one or more concentration of TCS at each length of exposure. The greatest increase in IL‐1β production was seen at 6 h, where all TCS exposures caused quite substantial increases in IL‐1β production in all donors. The magnitude of these increases varied dependent on the donor. These results indicate that TCS has the capacity to increase cellular production of the important pro‐inflammatory cytokine IL‐1β from immune cells. TCS‐induced increases in IL‐1 β production may serve to produce unwarranted inflammation and the pathologies associated with it.
Investigating the impact of 14‐3‐3 protein knockout on excitability of hippocampal CA1 pyramidal neurons
Authors: Logue Jordan , Zhou Yi
Published: May 2022

14‐3‐3 proteins are a family of regulatory proteins that are enriched at synaptic junctions. Many neurological and psychiatric disorders have also been linked to 14‐3‐3 protein dysfunctions. Our lab has previously shown that 14‐3‐3 protein functional knockout mice have reduced expression of synaptic proteins (including NMDA receptor subunits), deficits in hippocampal long‐term potentiation, and desynchronization in theta waves between hippocampus and prefrontal cortex. In addition, the 14‐3‐3 functional knockout mice exhibit behavior that correspond with the symptoms of psychiatric disorders such as schizophrenia. Recently, using cFos staining as an indicator, we identified an increase in activity in hippocampal CA1 and dentate gyrus neurons expressing the 14‐3‐3 inhibitor. However, it is not clear whether this increased neuronal activity is caused by altered synaptic activity, enhanced single cell excitability, or both. In this work, we are using slice electrophysiology and whole cell patch clamp to evaluate the impact of 14‐3‐3 inhibition on electrophysiological properties of hippocampal CA1 pyramidal neurons. To assess 14‐3‐3 knockout induced excitability changes, we measure differences in rheobase and firing rate between the 14‐3‐3 knockout mouse and their wild type control using current clamp. We are also using voltage clamp to identify changes in inward and/or outward current in these neurons. Additionally, we measure both miniature and spontaneous inhibitory and excitatory synaptic currents to assess potential alterations in synaptic input to the neurons in the 14‐3‐3 functional knockout mouse. We expect this study to provide critical insight that links the dysfunction of these regulatory proteins to the altered neuronal and/or synaptic activity, which may underline the neural circuitry and behavioral aberrations exhibited in these functional knockout mice.
Buoyancy and Hydrostatic Balance in The West Indian Coelacanth Latimeria chalumnae
Authors: Lauridsen Henrik , Pedersen Jens Mikkel H. , Ringgaard Steffen , Møller Peter R.
Published: May 2022

Buoyancy and balance are important parameters for static or slow moving low‐metabolic aquatic organisms. The extant coelacanths, a single genus (Latimeria) consisting of two species living at moderate depths (100 – 400 m) in the Western Indian Ocean and off Indonesia and representing an ancient group of lobe finned fishes dating back to the early Devonian (410 Myr), have among the lowest metabolic rates of any living vertebrate, and thus would be predicted to expend precious little energy to maintain buoyancy and balance. Previous observations on living coelacanths support the hypothesis that the coelcanth is neutrally buoyant and is in close to perfect hydrostatic balance in any body posture. Likewise, point‐by‐point measurements of tissue composition have shown a generally high lipid content of coelacanth tissue supporting neutral buoyancy without a gas filled bladder as found in other sarcopterygians (lungs) and in many actinopterygians (swim bladder). However, precise measurements of buoyancy and hydrostatic balance at different depths have never been made in the coelacanth. Here we show using non‐invasive imaging that buoyancy of the coelacanth closely matches its depth distribution and a close‐to‐perfect balance can be used by the animal to change body posture with little effort. We found that the lipid filled “fatty organ” of the coelacanth is well adapted to support neutral buoyancy and simple maneuvers of fins can cause a considerable shift in torque around the pitch axis. This allows the coelacanth to assume and remain in any body posture like horizontal orientation, headstand or tail stand with little physical effort. Our results demonstrate a close match between tissue composition in the extant coelacanth, depth range and behavior (e.g. effortless vertical drift hunting with the electrosensitive rostral organ in close contact with the substrate). We anticipate our non‐invasive technique of mapping tissue components to be a starting point for more sophisticated models of buoyancy and hydrostatic balance in aquatic vertebrates in general. Specifically, we suggest the technique can be used on the few late term and juvenile coelacanth specimens in existence to predict the most likely habitat depth of the juvenile life stage which is currently unknown.
Manipulation of Igf‐1 Expression in the Placenta and Impact on the Fetal Brain
Authors: Carver Annemarie , Taylor Robert , Kundu Sreelekha , Stevens Hanna
Published: May 2022

The placenta is an essential organ involved in mammalian development in utero. The main function of the placenta is the control of the transfer of nutrients and waste between mother and fetus, and production and delivery of hormones and growth factors. There is a lack of research that investigates the role of maternal and fetal genetics and molecular biology of the placenta’s impact on fetal neurodevelopment. Igf‐1 is a gene involved in mediating fetal development as well as growth of the brain. It is also involved in regulation of placental growth and transport. The placenta is the main source of fetal Igf‐1. Igf‐1 gene expression is altered in some individuals with autism spectrum disorder (ASD) which may reflect a role of Igf‐1 in ASD etiology. We previously found that prenatal stress, a risk factor for ASD, increased placental Igf‐1 expression and increased proliferation of striatal neural progenitors which express Igfreceptors. Developmental striatal changes are found in ASD. We therefore hypothesized that placental alterations in Igf‐1 lead to ASD‐relevant brain changes.

Methods

We examined this through the use of CRISPR Igf‐1 overexpression or control plasmids directly injected in mouse placenta on embryonic day 12, with subsequent tracking of changes in placenta, fetal body, and fetal brain. Igf‐1 overexpression in placenta had a variable trajectory. While placental weight was unaffected by Igf‐1 overexpression at E18, we found developmental and morphological changes of the embryo, including larger body and larger size and altered morphology of the head (macrocephaly) at E18. Brain volume measures demonstrate a range of effects on the primordial cortex and striatum at E14 and E18. One outcome of this work is advancements in methodology for understanding how placental genetics affect the development of the fetal brain. We also conclude that placental Igf‐1 expression does influence offspring growth and ASD‐relevant brain outcomes which suggests a role for placental genetics in neurodevelopmental disorders.
Reptile to chicken enhancer swaps to understand the molecular basis underlying morphological diversity in the vertebrate lung
Authors: Barrow Jeff , Edvalson Logan , Farmer Coleen
Published: May 2022

The lung is one of the most morphologically diverse organs among vertebrate species. Phylogenetically, much of this diversity appears to arise from the way epithelial tubes branch and change shape ranging from very saccular to highly branched structures. We are interested in understanding the genetic mechanisms that underlie this range in morphology. Fgf10 signaling has been demonstrated to play a critical role in regulating whether the lung epithelium undergoes branching morphogenesis (through low, graded levels of Fgf10) or forms sacs (high, uniform exposure to Fgf10). We hypothesize therefore that interspecies differences in Fgf10 expression may be a mechanism to underlie morphological diversity that exists in the vertebrate lung. To test this hypothesis, we have been performing enhancer swap experiments taking reptilian Fgf10 enhancers and knocking them into orthologous loci in the genome of chicken primordial germ cells (cPGCs). These targeted cPGCs are introduced into the gonad of developing chicken embryos where they will ultimately develop into germ cells in the adult chicken. These germ line chimeras can then be bred to generate fully transgenic chicks to determine if enhancer swaps are sufficient to drive a reptilian pattern of lung morphogenesis in an avian. I will discuss the gene editing approaches my laboratory is taking to knock in reptilian Fgf10 enhancers into the orthologous locus in in the chicken.
Klotho Expression in Aging and Disease
Authors: Choe David , Delery Elizabeth , MacLean Andrew
Published: May 2022

Klotho is a regulatory anti‐aging protein that is significantly expressed in the choroid plexus, plays an important role in regulating the immune response in the CNS, and diminishes with age. It is known that a decline of klotho expression has been linked to significant neuroinflammation, oxidative damage in the neural parenchyma, and decline in neurocognitive functions. As the choroid plexus is a known reservoir for latent Human Immunodeficiency Virus (HIV) infection, the relationship between HIV and klotho expression could be a cause of the neurocognitive dysfunction that accompanies HIV infections. The objective of this project was to observe levels of klotho expression in the choroid plexus in a Simian Immunodeficiency Virus (SIV) model of HIV infection, as well as to determine a pattern of age‐related decline in klotho expression. To carry out the study, Choroid plexus samples of Rhesus macaques of various ages and SIV‐infection status were studied. The tissues were stained using immunohistochemistry for polyclonal klotho, structural proteins (vimentin), and nuclei (DAPI), and imaged at varying magnifications of 4x, 10x, and 40x. The images obtained were then analyzed using the fluorescent imaging software Fiji. The epithelial cells of the choroid plexus were measured for their fluorescence intensity to measure their relative klotho expression levels. Although klotho expression was varied in the uninfected Rhesus macaques, there was an overall decreasing trend with the advancement of age. In SIV‐infected Rhesus macaques, the choroid plexus had significantly less klotho expression than the uninfected control group regardless of age (p=0.01). Analysis of the uninfected data shows that there was a decreasing pattern of klotho expression in Rhesus macaques. The variability of klotho levels in the uninfected individuals can be attributed to genetic variations between individuals. However, SIV infection significantly reduced klotho expression in the choroid plexus in comparison to uninfected individuals. This decrease in klotho expression was regardless of age. The exact mechanism in which SIV diminishes klotho expression is unknown and additional studies are warranted to examine this phenomenon.
Kub5‐Hera Deficiency Promotes R‐Loop‐Induced Genomic Instability and Carcinogenesis Following Whole‐Body Exposure to Ionizing Radiation
Authors: Motea Edward , Morales Julio , Pay S. L. , Crane Genevieve
Published: May 2022

Formation of persistent R‐loops (a structure containing an RNA:DNA hybrid and a displaced single‐stranded DNA) during transcription are now emerging as a direct threat to genomic stability with links to cancer and various neurodegenerative, autoimmune, and developmental disorders. However, the molecular mechanisms by which R‐loops are resolved or aberrantly formed to induce DNA damage and genomic instability remains unclear. Aberrant accumulation of these nucleic acid structures through compromised transcription and exposure to genotoxic agents (e.g., ionizing radiation, IR) imposes a roadblock to ongoing replication and transcription causing genomic instability by generating double‐stranded DNA breaks (DSBs). Kub5‐Hera (Ku70‐binding protein 5‐Hera, K‐H; also known as RPRD1B, Regulation of nuclear pre‐mRNA‐domain‐containing protein 1B) is a novel factor discovered in our lab that is involved in transcription termination and DNA repair. We found that loss of K‐H increases R‐loop‐induced DSBs that activates the error‐prone alternative non‐homologous end‐joining (alt‐EJ or back‐up NHEJ) due to defects in classical NHEJ (c‐NHEJ) and homologous recombination (HR). Using our transgenic mouse model, we discovered that a complete knockout of K‐H is embryonic lethal suggesting a critical role for this protein in cellular development and proliferation. Interestingly, deletion of one K‐H allele in mice caused rapid development of cancer‐related deaths over time after whole‐body exposure to IR in a dose‐dependent manner compared to their wild‐type littermates. Using targeted gene silencing, western blot, proximity ligation and immunofluorescent assays in our cell line models and mouse tissues, we showed that depletion of K‐H increases the formation of R‐loop‐induced double‐stranded breaks (DSBs) and PARP1 activity due to a compromised homologous recombination (HR). Loss of K‐H in cells increases the error‐prone activity of PARP1 in alt‐EJ to possibly repair harmful R‐loop‐induced single‐stranded breaks (SSBs) and DSBs due to HR and c‐NHEJ deficiency, which could provide the mechanism of carcinogenesis in mice after exposure to IR. Overall, our studies offer new mechanistic insights into carcinogenesis caused by K‐H genetic alterations and persistent R‐loops, as well as identify novel strategies to leverage Kub5‐Hera expression or the formation of lethal R‐loop‐induced‐DSBs for innovative therapeutic approaches.
The choline acetyltransferase inhibitor BW813U inhibits angiogenesis and tumor growth in human lung adenocarcinoma
Authors: Dasgupta Piyali , Richbart Stephen D. , Akers Austin T. , Friedman Jamie R. , Miles Sarah L. , Bow Eric W. , Rimoldi John M. , Chen Yi Charlie
Published: May 2022

Angiogenesis refers to the development of new blood vessels from preexisting blood vessels. The angiogenic pathway is essential for the growth and progression of almost every kind of human cancer. Nicotine, the addictive component of tobacco smoke is known to accelerate the growth of human lung tumors by stimulating tumor angiogenesis. The pro‐angiogenic effects of nicotine are mediated by nicotinic acetylcholine receptors (nAChRs). The endogenous ligand for nicotine is the neurotransmitter acetylcholine (ACh). Published reports show that ACh acts as growth factor for human lung cancers. Almost all lung tissues (including fetal lung) express signaling proteins involved in the synthesis, transport of the ACh‐signaling pathway. These include choline acetyl transferase enzyme (ChAT), vesicular acetylcholine transporter (VAChT), acetylcholine esterase (AChE) and choline transporter (ChT). The primary objective of our studies was to determine if disruption of ChAT could inhibit angiogenesis and suppress the growth of human LACs. We used a small molecule inhibitor of ChAT namely BW813U for our studies. BW813U robustly suppressed angiogenesis in Matrigel assays and chicken chorioallantoic membrane (CAM) assays. The administration of BW813U potenetly decreased the growth rate of H838 tumors xenotransplanted in SCID mice. Immunohistochemical staining experiments revealed that the anti‐tumor activity of BW813U was correlated with decrease of CD31 angiogenic biomarker in H813 tumor sections. The anti‐angiogenic activity of BW813U was mediated by the alpha7‐nAChR pathway and involved the nitric oxide pathway. Taken together, our studies show that ChAT antagonists like BW813U may have applications in the treatment of LAC.
Addressing persistence and retention of chemistry and biochemistry majors with engagement seminars beyond the first year
Authors: Booth Rachell E. , Garner Robert , Davis Julian
Published: May 2022

Undergraduate persistence and retention are two measures widely used by institutions to measure student success. In 2020, the national rate at which newly enrolled full‐time students persisted decreased for the first time in 7 years with the largest decrease in persistence rates for Hispanic students. The recent declines in both persistence and retention are attributed to the global pandemic. The University of the Incarnate Word (UIW), a Hispanic‐serving institution, is home to the Cardinal Chemistry Scholars (C2S) program that is supported by an NSF S‐STEM grant. The C2S program provides financial and academic support along with mentoring and career plan development to a financially disadvantaged cohort of chemistry and biochemistry majors. While over the last 20 years, most institutions have adopted first‐year seminars or experiences known to have positive impacts on both retention and persistence for first time freshman, we developed a series of engagement seminar courses that our C2S scholars take each semester until graduation. The engagements seminars include various activities that address the specific needs of students as they progress through their college years including skills development, career planning, academic engagement, and community building. Our preliminary data suggests that our continuous seminar series results in higher levels of persistence and retention among participating students compared to non‐participating students with no declines during the pandemic.
Anti‐invasive and anti‐metastatic activity of capsaicin and natural capsaicin‐like compounds in lung adenocarcinoma
Authors: Richbart Stephen D. , Nolan Nicholas A. , Akers Austin T. , Brown Kathleen C. , Denning Krista L. , Egleton Richard D. , Dasgupta Piyali
Published: May 2022

Lung adenocarcinoma (LAC) accounts for about 65% of all non‐small cell lung cancer (NSCLC) cases. A considerable proportion of LAC patients present (in the clinic) with local and distant metastasis at the time of their diagnosis. One of the earliest events of the metastatic pathway is the invasion of malignant cells through the surrounding extracellular basement membrane into the neighboring blood vessels and lymph nodes. The long‐term goal of our laboratory is to identify nutrition‐based agents which will suppress the distant metastasis of human LACs. Capsaicin is the pungent ingredient of chili peppers. Published reports have revealed that capsaicin inhibits the invasion and metastasis of several types of human cancers including melanoma, prostate cancer, and cholangiosarcoma. However, the clinical application of capsaicin as an anti‐cancer drug is limited by its unpleasant side‐effect profile. This led us to compare the anti‐metastatic activity of capsaicin with natural non‐pungent capsaicin‐like compounds, namely capsiate and capsiconiate. The structure and bioactivity of capsaicin closely resembles capsiate. There are no published reports involving the bioactivity of capsiconiate. We measured the anti‐invasive activity of these compounds by two independent invasion assays, namely the Boyden chamber assay and spherical invasion assay. We found that capsaicin and capsiate displayed anti‐invasive activity in three human LAC cell lines. In contrast, capsiconiate did not suppress the invasion of any LAC cell lines. Furthermore, we tested the anti‐metastatic activity of capsaicin in a syngeneic mouse model of metastasis. We observed that the daily dietary administration of 20mg capsaicin/kg food in AIN‐76A diet (with 5% lipid level) robustly decreased the area metastatic foci (in the lung) relative to vehicle‐treated mice. We also investigated the signaling pathway underlying the anti‐invasive activity of capsaicin. Our results show that capsaicin inhibits the invasion of human LAC cells via the TRPV6 receptor and the tyrosine phosphatase activity pathway. The data obtained from our research fosters the hope of nutrition‐based therapies in metastatic lung cancer.
Determining How Stem‐loop Structure Thermodynamic Stability Influences Frameshift Efficiency at the HTLV‐1 gag‐proFrameshift Site
Authors: Soliman Mary , Robinson Leila , Maille Madison , Egekeze Carolyn , Salamon Rebecca , Mouzakis Kathryn D.
Published: May 2022

The human T‐lymphotropic virus type 1 (HTLV‐1) RNA genome includes two programmed ‐1 ribosomal frameshift (‐1 PRF) sites. These sites allow ribosomes access to alternate reading frames encoding critical viral enzymes. The gag‐proframeshift site includes a slippery sequence, spacer, and stem‐loop structure. How the stem‐loop acts to promote frameshifting is unclear. Previous HTLV‐2 research showed that changes to the gag‐pro frameshift site stem‐loop thermodynamic stability influenced its frameshift efficiency to a modest degree. There is substantial conservation between the HTLV‐1 and HTLV‐2 gag‐pro frameshift site sequences (86%) and structures. We hypothesized that the HTLV‐1 gag‐pro frameshift efficiency would be similarly influenced by its stem‐loop thermodynamic stability. To test this hypothesis, we designed 15 stem‐loop mutants (SLMs) with varied base‐pair composition. These mutations decoupled changes in overall thermodynamic stability from those localized to the stem‐loop base. The SLM thermodynamic stabilities were calculated using nearest neighbor parameters and the in vitro frameshift efficiencies were measured with a dual‐luciferase assay. Correlations between frameshift efficiency and thermodynamic stability were subsequently assessed. Preliminarily results reveal a moderate correlation between the SLM stem‐loop overall thermodynamic stability and frameshifting efficiency. No correlation was observed between the thermodynamic stability of the stem‐loop base and frameshifting efficiency. While the overall thermodynamic stability does impact the frameshift efficiency, it cannot be used exclusively to predict it. This reflects a complex interplay between the frameshift site elements. Overall, our preliminary results suggest a conserved function for the gag‐pro frameshift site stem‐loop between the HTLV‐1 and HTLV‐2 retroviruses.
Neurons in the Caudate Nucleus of the Camel and Human: A Comparative Study
Authors: Bataineh Ziad M.
Published: May 2022

The caudate nucleus is described as a huge, subcortical grey mass in the forebrain known for its role in motor function and involved in a number of clinical syndromes. During ontogenesis, the caudate nucleus follows the curvature of the telencephalic vesicle, hence its curved shape. Its rostral portion referred to as the head, is far more voluminous than the body.

Aims

This study aimed to describe the different types and characteristics of neurons in the caudate nucleus of the brain of camel and human and compare these neurons with their counterparts in other species.

Method

4mm thick blocks containing caudate nucleus was cut, dry and processed according to the rapid Golgi and or the Golgi‐Kopsch methods (double impregnation). Well‐impregnated neurons studied under light microscope.

Results

According to the present study, neurons were classified according to the density and distribution of the spines along their axons and dendrites. Neurons in this study classified into three main types in the caudate nucleus of camel and human. According to the somal size each group includes one or more of the following cell size: large, medium and small. Rich‐spiny neurons (Type I): include medium and small subtypes. Hairy‐like, mushroom‐like and stubby spines were present in the secondary dendrite and their branching. Sparsely‐spiny neurons (Type II): they have relatively larger cell bodies and thicker primary dendrites than (Type I). Characterized by their very low density of spines and poorer branching of their dendrites. Aspiny‐neurons (Type III): are large, medium and small size. May have varicose, appendages with various types and short axon which gives collaterals mostly within the dendritic domain.

Conclusion

The neuronal structure of the caudate nucleus in the camel is differ from that of the human. The human neurons appear slightly larger than those of the camel and they have more dendritic trunks that break up more often than the neurons of the camel. Although the types of spines are similar in both species, the density of spines appears to be greater in the camel than in the human. The structural differences of the caudate neurons between two species may result from the fact that the homo sapeneis belongs to a phylogenetically younger order (Anthropoidea) than the camel. (Artiodactyla) which support the fact that the number and diversity of the types of neurons go to increase throughout phylogenesis.
Nitric Oxide Inhibits TRPV4‐mediated Hemichannel Opening in Porcine Nonpigmented Ciliary Epithelium
Authors: Shahidullah Mohammad , Delamere Nicholas
Published: May 2022

Previous studies showed the presence of connexin 43 (Cx43), connexin 50 (Cx50), and pannexin 1 (Panx1) in the nonpigmented ciliary epithelium (NPE) of the eye, as well as evidence of hemichannel (HC) activity. In the lens, mechanosensitive Transient Receptor Potential Vanilloid 4 (TRPV4)‐mediated calcium entry stimulates hemichannel activity. The aim here is to test for TRPV4 expression in the NPE, determine whether TRPV4 activation causes HC opening, and study the influence of nitric oxide.

Immunohistochemistry was used to probe for TRPV4 in porcine ciliary body. Propidium iodide (PI) uptake and ATP release by primary cultured NPE was used to assess HC opening. PI and ATP are large molecules (MW 668 and 507, respectively) that pass through open hemichannels but not the plasma membrane. ATP in the bathing medium was measured by luciferase luminometry and PI uptake by fluorimetry.

Abundant expression of TRPV4 was detected in the NPE cell layer of the ciliary body. Cultured NPE cells displayed an increase in PI uptake when exposed to a TRPV4 agonist GSK 1016790A (10 nM) or to hypoosmotic solution (200 mOsm) (Control 4.65 ± 0.24 vs GSK 10.26 ± 0.32 and Hypo 8.26 ± 0.42 fluorescence units per mg protein; n=6, p<0.001). PI uptake also was increased in NPE cells cultured on a flexible membrane and subjected to cyclic stretch for 1‐10 min (10%, 0.5 Hz), the peak response was at at 2 min (Control 1.5 ± 0.03 vs stretch 2.5 ± 0.10; n=6, p<0.001). The increase in PI uptake caused by hypoosmotic solution or by cyclic stretch was abolished by a selective TRPV4 antagonist HC 067047 (10 μM). Hyposmotic solution increased ATP release from 1.12 ± 0.24 to 2.81± 0.27 (n=12, p<0.001) while cyclic stretch increased ATP release from 0.20 ± 0.03 to 0.40 ± 0.03 pmoles/mg protein (n=6, p<0.001). ATP release responses caused by hypoosmotic solution and cyclic stretch were inhibited by HC 067047. Hypoosmotic solution‐induced ATP release was also inhibited by connexin inhibitory peptide, Gap‐27 (200 μM) and nitric oxide (NO) donors, sodium nitroprusside (SNP) and S‐nitroso‐N‐acetylpenicillamine (SNAP).

TRPV4 channels are expressed by the NPE. The ability of HC 067047 and Gap‐27 to prevent ATP release and PI uptake points to TRPV4‐dependent opening of connexin HCs in NPE cells. Suppression of ATP release by SNP and SNAP indicates the hemichannel opening mechanism is inhibited by NO.
High‐Performance Computational Molecular Docking for Potential Inhibitors of an Essential Enzyme of Burkholderia pseudomallei
Authors: Vincent Annabelle G. , Beckham Josh T.
Published: May 2022

Melioidosis, caused by the rod‐shaped bacteria Burkholderia pseudomallei, is thought to be an understudied and neglected disease. This life‐threatening infection, endemic across tropical areas such as northern Australia and southeast Asia, is estimated to kill at least 89,000 people worldwide annually, though this number is thought to be a serious underestimation. The symptoms of this disease are non‐specific, resembling other common illnesses, as the bacterium is highly adaptable and able to produce a range of clinical manifestations depending on the infected tissue. Melioidosis, with its difficulty in diagnosis, high‐ lethality, increasing antibiotic resistance, and airborne potential has led many governments including the U.S. to consider the disease a bioterror threat, as with other pathogens such as anthrax and Ebola. Novel drug treatments are needed, in the face of increasing antibiotic resistance. In this research, we targeted one of the essential enzymes ‐ GmhA ‐ of Burkholderia pseudomallei for small molecule drug discovery The GmhA gene is necessary for the synthesis of heptoses which are utilized in virulence and antibiotic resistance. Through high‐performance computing, virtual libraries of small molecules were docked against the x‐ray crystallography model of the active site of the enzyme. The virtual screening programs, GOLD and VINA, predicted the strength of the binding interaction with higher scores reflecting a stronger interaction and the potential to inhibit the enzyme. In order to validate the programs for this protein, control ligands with similar physiochemical properties to the known substrate M7P were found. Screening these controls resulted in similar ranges of results for both the GOLD and VINA, with M7P scoring consistently with a GOLD score of roughly 55. After validation, thousands of small molecules from four libraries were docked to the protein structure, resulting in a consistent interquartile range of scores roughly 40‐60. Significantly departing from this range, goserelin acetate scored the highest overall, with a GOLD score of 101.9. Wet lab work is being conducted on the BpGmhA target, with successful protein synthesis in E. coli from a pET expression vector resulting in affinity‐purified protein elutions of the expected molecular weight of BpGmhA of 85.56 kDa. Elutions will be further tested with top virtually scoring ligands to verify inhibition properties predicted from the computational dockings. Promising ligands will be obtained and then evaluated through binding and enzymatic assays to verify their ability to inhibit the enzyme. If confirmed, these novel ligands may be potential candidates for further drug development to treat melioidosis.
The small RNA universe of Capitella teleta
Authors: Khanal Sweta , Zancanela Beatriz , Peter Jacob , Flynt Alex
Published: May 2022

RNAi is an evolutionarily fluid mechanism with dramatically different activities across animal phyla. One major group where there has been little investigation is annelid worms. Here the small RNAs of the polychaete developmental model, Capitella teleta, are profiled across development. As is seen with nearly all animals, nearly 200 hundred microRNAs were found with 58 high confidence novel species. Greater miRNA diversity was associated with later stages consistent with differentiation of tissues. Outside miRNA, a distinct composition of other small RNA pathways was found. Unlike many invertebrates, an endogenous siRNA pathway was not observed, indicating pathway loss relative to basal planarians. No processively generated siRNA‐class RNAs could be found arising from dsRNA precursors. This has significant impact on RNAi technology development for this group of animals. Unlike the apparent absence of siRNAs, a significant population of piRNAs were observed. For many piRNAs phasing and ping pong biogenesis pathways were identified. Interestingly, piRNAs were found to be highly expressed during early development, suggesting a potential role in regulation in metamorphosis. Critically, the configuration of RNAi factors in C. teletais found in other annelids and mollusks, suggesting that similar biology is likely present in the wider clade. This study is the first providing comprehensive analysis of small RNAs in annelids.
Inhibition of vitamin C transport impairs neuronal differentiation of hiPSCs
Authors: Rashid Mohammad A. , Marchant Jonathan S. , Subramanian Veedamali S.
Published: May 2022

Vitamin C is an antioxidant that acts as a free radical scavenger and cofactor for many important biological reactions. It plays an important role in stem cell homeostasis, proliferation and differentiation. However, the role of vitamin C transporters in the neuronal differentiation of human induced pluripotent stem cells (hiPSCs) has not yet been investigated. Therefore, we investigated if vitamin C transporter knockdown impairs neuronal differentiation in hiPSCs. We differentiated hiPSCs into neurons from undifferentiated hiPSCs using established protocols and employed siRNA‐mediated knockdown experiments. Validation of results was performed by RT‐qPCR, Western blot analysis, RNA‐Sequencing (RNA‐Seq) and confocal imaging. First, we determined the expression of the human sodium‐dependent vitamin C transporters, hSVCT1 and hSVCT2 (products of the SLC23A1 and SLC23A2 genes, respectively) using RNA‐Seq analysis. RNA‐Seq analysis showed that both SLC23A1 and SLC23A2 were expressed during differentiation of neuronal hiPSCs. As expected, the immunofluorescence results showed that the hSVCT2 is predominantly expressed on the plasma membrane during neuronal hiPSCs differentiation. We validated the effectiveness of SLC23A1, SLC23A2and SCAMP2 (a vitamin C transporter interacting protein) knockdown by gene specific siRNA using RT‐qPCR analysis. RT‐qPCR analysis found that the expression of all three genes were significantly reduced. We also demonstrated that knockdown of SLC23A1, SLC23A2 and SCAMP2 was accompanied by a significant decrease in the number of Tuj‐1 (early neuron marker)‐positive cells during neuronal hiPSCs differentiation. Reciprocally, we demonstrated vitamin C supplementation dose‐dependently increases Tuj‐1 positive cells during neuronal hiPSCs differentiation. In conclusion, we demonstrate that knockdown of vitamin C transporter impairs neuronal differentiation of hiPSCs. These findings suggest that vitamin C and its transporters play an important role in neuronal differentiation, development, and function in hiPSCs.