bluuebunny/crossref_metadata_2025 huggingface dataset:
Profiling Oncogenic Ras Mutant Drugs with Homogeneous Bioluminescent Immunoassays
Authors: Zegzouti Hicham , Engel Laurie , Swiatnicki Matthew , Alves Juliano , Goueli Said
Published: May 2022

Activating KRAS mutations present in many cancers are difficult to target with drugs until recently with the FDA approval of sotorasib (AMG‐510), a KRAS G12C selective inhibitor. In an effort to study Ras inhibitors in cells and identify new compounds that inhibit Ras and its downstream signaling, assays such as western or ELISA are commonly used. These traditional immunoassays can be tedious, require multiple washing steps, and are not easily adaptable to a high throughput screening (HTS) format. To overcome these limitations, we developed Lumit, a novel immunoassay approach that combines bioluminescent enzyme subunit complementation technology and immunodetection. We applied this bioluminescent and homogeneous “Add and Read” assay to analyze Ras signaling pathway activation and inhibition through multiple nodes of the pathway including detection of phosphorylated ERK. The assay was used to profile different allele specific inhibitors with cell lines harboring different activating KRAS mutations and obtained the predicted pharmacology and selectivity. The potency of PROTAC compound targeting selective degradation of KRAS G12C was also tested using Lumit and the result showed that maximal decrease in RAS signaling was achieved. Unlike current assays that require lengthy sample preparation and multiple washing steps, the Lumit immunoassay provides an alternative platform because it is homogeneous and allows to decipher signaling pathways activities in a fast and simple manner. Our results demonstrate that this bioluminescent technology can be adapted to any signaling pathway node, allowing scientists to streamline the analysis of signaling pathways of interest such as Ras, and identify much needed inhibitors of its mutants.
A low salt diet increases blood pressure in mice with selective deletion of the dopamine D2 receptor in the renal proximal tubule
Authors: Moore Shaun C. , Asico Laureano D. , Jose Pedro A. , Armando Ines
Published: May 2022

The renal dopamine D2 receptor D2 (D2R) contributes in maintaining normal blood pressure (BP) and prevents inflammation and tissue injury. In mice, global deletion of the D2R gene (Drd2) or renal‐selective downregulation of the receptor increases BP and results in renal inflammation, tubular injury, and fibrosis. We studied the function of D2R in the renal proximal tubule, by generating Drd2fl/fl,PSGLT2::Cre+mice (D2R PT‐/‐) that lack D2R only in the renal proximal tubule and Drd2fl/fl,PSGLT2::Cre‐(D2R PT+/+) mice that do not have the deletion. Mice were genotyped for Drd2fl/fland a smaller amplicon representing the Cre deletion mutant. We studied male mice on normal salt (NS; 0.8% NaCl), high (HS; 4% NaCl), and low (LS;<0.05% NaCl) diets. On NS diet, male D2RPT‐/‐ had slightly higher BP, measured under anesthesia, than male D2RPT+/+mice (108±2 vs 102±2 mmHg, n=17‐21/group; P<0.05). On HS diet D2RPT‐/‐ had lower BP than D2RPT+/+ (100±3 vs 108±1 mmHg; P<0.04; n=12) but on LS diet D2RPT‐/‐ had higher BP than D2RPT+/+ (130±5 vs 103±4 mmHg; P<0.01; n=8). BP measured by telemetry in conscious mice confirmed the decrease in BP on HS diet and the increase in BP on LS diet, relative to BP on NS diet. The data indicated that the normal response to changes in salt intake is impaired by the deletion of the D2R only in the renal proximal tubule. On NS diet, the renal protein expressions of NHE3, Na+/K+ATPase, and NCC were similar in both groups of mice but NKCC2 was higher in D2RPT‐/‐ (190 ±5% vs 100±20; P<0.05) than in D2RPT+/+. On HS diet, Na+/K+ATPase was lower (110±5% vs 220±3%; P<0.05) but NKCC2 was higher (290±9% vs 200±5%,P<0.05) in D2RPT‐/‐ while NHE3 and NCC were similar in both groups. On LS diet, NHE3, Na+/K+ATPase, and NCC expression were similar in D2RPT‐/‐ and D2RPT+/+ but NKCC2 was lower (100±20 vs 710±10; P<0.05) in D2RPT‐/‐ than in D2RPT+/+. However, the increased BP in D2RPT‐/‐ on LS cannot be explained by changes in renal expression of sodium exchanger/transporter/pump but may be related to the increase in renal inflammation and fibrosis. These results may have significant clinical implications since in humans, the presence of D2R variants, DRD2rs6276 and rs6277, which decrease D2R protein/function, is associated with an increase in BP on LS diet, a case of inverse salt sensitivity.
vFLIP SIM Promotes Viral Latency of KSHV
Authors: Constantine Mathew A.
Published: May 2022

Kaposi’s Sarcoma‐Associated Herpesvirus (KSHV) is the etiological agent of Kaposi’s Sarcoma (KS), Primary Effusion Lymphoma (PEL), and Multicentric Castleman’s Disease (MCD). Upon primary infection, the KSHV genome primarily exists as a latent infection, tethered to the host chromatin as an episome. It is widely accepted that NFκB signaling promotes latency and that NFκB signaling is activated through expression of the viral FLICE Inhibitory Protein (vFLIP). Our objective is to reveal new insights into the regulation of KSHV latency. We have identified a putative SUMO Interaction Motif (SIM) in vFLIP and we hypothesize that the vFLIP SIM is required for maintaining latency. Mutation of this SIM abolishes the ability of vFLIP to activate NFκB signaling. We also observed reduced vFLIP degradation by the master lytic switch RTA, as well as increased lytic reactivation. Our data present a novel means by which KSHV vFLIP functions to maintain latency.
Assessment of Mitochondrial Respiratory Function in Isolated Nephron Segments of Dahl SS Rats in Salt Induced Hypertension
Authors: Yang Chun , Zheleznova Nadezhda N. , Dash Ranjan K. , Cowley Allen W.
Published: May 2022

Rationale

Kidney nephrons actively reabsorb 99% of the filtrate to maintain body fluid and solute homeostasis. A large amount of ATP synthesis and O2 consumption must work coordinal to support this demand. We hypothesized that mitochondrial function of proximal tubules (PT) and medullary thick ascending limbs (mTAL) is altered during the development of salt induced hypertension in the Dahl salt‐sensitive (SS) rat.

Method

The mitochondrial oxygen consumption rates (OCR) were determined in freshly bulk isolated PT and mTAL segments from SS rats fed 0.4% NaCl diet (low salt; LS) or 4% NaCl diet (high salt; HS) for 7 days. A protocol was developed to determine basal levels of OCR and the effects of various key circulating substrates (glucose, lactate, pyruvate, glutamine, and palmitate) upon mitochondrial respiration (OCR) using the Agilent Seahorse XF96 Extracellular Flux Analyzer combined with the Agilent Seahorse XF Cell Mito Stress Test protocol.

Results

Analysis of the renal tubular mitochondrial OCR data for five different circulating substrates confirmed that there were clear differences in the substrate preference between the PT and mTAL which were altered by HS feeding. It was found that PT preferred lactate and glutamine while mTAL preferred glucose and pyruvate and that both segments could effectively utilize palmitate as a mitochondrial substrate. PT isolated from SS rats fed HS displayed higher OCR levels compared to PT from LS fed rats, but HS diet did not result in significant differences in individual substrate effects. In contrast, isolated mTAL from HS fed rats exhibited increased OCR in response to glutamine and palmitate compared to mTAL from LS fed rats although no basal differences in OCR were observed.

Conclusion

These observations demonstrated that renal tubular mitochondrial respiratory function is altered in SS rats during the development of salt‐induced hypertension and that both PT and mTAL exhibit an increased respiratory capacity in response to HS feeding. These changes in metabolism appear necessary to meet the increased tubular workloads and need for greater ATP production and O2 utilization in both PT and mTAL. We conclude, however, that different metabolic strategies are utilized by each of these tubular segments to meet these increased metabolic requirements.
Conducted Capillary Signaling Enables Oxygen Responses in Skeletal Muscle Independent of Metabolite Production
Authors: Kowalewska Paulina M. , Milkovich Stephanie L. , Goldman Daniel , Sandow Shaun L. , Ellis Christopher G. , Welsh Donald G.
Published: May 2022

Red blood cell (RBC) flow through the microvasculature is closely matched to tissue O2 requirements. At a fundamental level, O2 demand‐supply coupling entails the sensing of PO2, and the generation of a stimulus that alters upstream arteriolar tone. However, where and how O2 needs are sensed in the microcirculation is presently contested. One idea centers on hypoxia triggering the release of K+, activating endothelial KIR2.1 channels and initiating a hyperpolarization that conducts upstream via gap junctions, comprised of connexins (Cx). We tested this idea in skeletal muscle where we controlled the local tissue O2 environment using live animal imaging in Cx40‐/‐and endothelial KIR2.1‐/‐ mice. The extensor digitorum longus muscle positioned overtop a gas control chamber allowed second‐by‐second monitoring of capillary RBC flow responses as O2 was altered around its physiological set point of 53 mmHg. A stepwise drop in PO2 at the muscle surface (53 to 15 or 0 mmHg) increased RBC supply rate in control capillaries while elevated chamber O2 elicited the opposite response; these capillaries robustly expressed Cx40. The RBC flow responses were rapid and tightly coupled to O2 levels as readily observed when chamber O2 was oscillated in a sinusoidal manner. In contrast, this blood flow response was significantly diminished in Cx40‐/‐ mice and translated into lower capillary RBC O2 saturation. KIR2.1‐/‐mice, on the other hand, had normal resting RBC O2 saturation and reacted normally to O2 changes, albeit an oscillation or a sustained stepwise decrease. Furthermore, we confirmed that RBC flow responses are conserved in endothelial KIR2.1‐/‐mice even when the low O2 challenge is applied to a restricted number of surface capillaries in the muscle; interestingly, these responses were dominated by capillary hematocrit changes in both control and endothelial KIR2.1‐/‐mice. Modelling this phenomenon supported the idea that the number of capillaries stimulated is indeed tied to conduction distance and tissue RBC distribution. In conclusion, we demonstrate that microvascular O2 responses depend on coordinated electrical signaling via gap junctions comprised of Cx40 and that endothelial KIR2.1channels do not drive the initiating electrical event. These findings reconceptualize our understanding of blood flow regulation and how O2 initiates this process at the capillary level independent of metabolite production.
Role of beetroots in oxidative stress and vascular relaxation in ovariectomized rats
Authors: Wang Hui Di , Nguyen David H. , Deng Arlinda , Longo Amanda , MacPherson Rebecca , Ward Wendy
Published: May 2022

The onset of hypertension in women is known to peak dramatically after menopause, partly due to decreased estrogen levels. It is well known that postmenopausal estrogen therapy can cause many adverse effects, and there is growing interest in using dietary products as potential treatment options. Dietary nitrates in beetroot may raise blood nitric oxide levels. We tested the hypothesis that beetroot can improve the oxidative stress and vascular relaxation responses in a model of an ovariectomized rodent.

Female ovariectomized Sprague‐Dawley rats were divided into four treatment groups: beetroot juice (nitrate content 18.75mg), nitroglycerin (0.2 mg/kg), estradiol replacement (1.5mg) or no treatment for 12 weeks (n=4 per group). The isometric contractile activities were measured in isolated aortic rings mounted in tissue baths. Phenylephrine (10‐6 M) was used to precontract the aortic rings. The contractile activities to various concentrations of acetylcholine and sodium nitroprusside were evaluated. The superoxide concentration was measured using chemiluminescence.

The beetroot treatment significantly reduced superoxide production (P<0.05). However, it did not affect acetylcholine or sodium nitroprusside‐induced vascular relaxation at any of the concentrations used in the experiments (P>0.05). There were no significant differences in acetylcholine‐induced vascular relaxation curves among the different treatment groups (P>0.05).

In conclusion, these experiments indicate that dietary beetroot does not improve acetylcholine‐mediated vascular relaxation while decreasing oxidative stress, contrary to expectations.
Virtual Screening Approach to Repurpose FDA‐approved Drugs for Targeting Acid Ceramidase‐1 in Pancreatic Cancer
Authors: Raval Nipam , Henrichs Grant
Published: May 2022

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer‐related deaths in the US. Despite extensive research and advances in molecular pathogenesis, PDAC remains a major problem in the US. It is also projected to be the second leading cause of cancer‐related deaths in the US by 2030. PDAC is an aggressive disease and characterized by poor prognosis, rapid progression, drug resistance and recurrence. The current chemotherapy treatment only improved survival to <10%, hence there is a need to identify a novel target for drug discovery.

Recently, altered sphingolipid metabolism was identified in PDAC patients. Sphingolipids are critical constituents of the cell membrane that regulate several signaling pathways related to cell survival. The major sphingolipid metabolites are ceramide and sphingosine which are interconvertible, while ceramide is pro‐apoptotic and sphingosine is anti‐apoptotic. Ceramidases are a heterogeneous enzyme family that convert ceramides into sphingosine and fatty acids. The five human ceramidases are identified are the acid ceramidase (ASAH1); neutral ceramidase (ASAH2); alkaline ceramidase 1 (ACER1); alkaline ceramidase 2 (ACER2); and alkaline ceramidase 3 (ACER3). To understand the role of ceramides in PDAC, we first interrogated the cancer genome atlas database and found that all ceramides are expressed in lower levels in PDAC except ASAH1 which is uniquely overexpressed in PDAC patients compared to normal individuals. Further, immunocytochemistry of PDAC patient tissue microarray showed higher expression of ASAH1 in PDAC tumors compared to normal pancreas. Western blot analysis also showed overexpression of ASAH1 protein in PDAC cell lines. Studies indicate that ASAH1 plays an important role in PDAC progression. Our data indicate that ASAH1 is a potential target for the treatment of PDAC. For faster translation of this target to the clinic, we used virtual screening and molecular docking approach to identify FDA‐approved drugs for targeting ASAH1. We have downloaded the 3D‐protein structure of ASAH1 from the protein data bank and currently screening a library of small molecular drugs against ASAH1 protein using multiple software programs such as Autodock vina and PyRx. We will select the top 10 drug candidates from virtual screening based on their binding energies and the ability to form hydrogen bonds with key amino acids. We will confirm these findings using a cellular thermal shift assay to analyze the binding of these drugs to ASAH1 in PDAC cell lines and test their anticancer activities in cell culture system and orthotopic PDAC mouse model.
Genetic profiling of the hippocampus during peripheral chronic inflammatory pain
Authors: Garman Adam
Published: May 2022

Brain mechanisms linking chronic pain conditions and development of comorbid clinical depression are still largely unknown. Here, we used a genome‐wide microarray analysis to examine the genetic profile of the hippocampus, a limbic region that regulates mood and stress responses, from male rats exposed to 21 days of inflammatory pain. Bioinformatic gene network/canonical pathways analyses have identified significantly dysregulated genes with known roles in either neuroinflammation or neurodegenerative processes. Akt (protein kinase B) was identified as the main network hub gene. Altered activity of Akt‐related signaling pathways has been linked to both the development of depressive state and antidepressant treatment. Furthermore, lipocalin‐2 (Lcn2) or NGAL was identified as one of the highest upregulated genes (~ 2‐fold) within the hippocampus during chronic pain state. Lcn2/NGAL is an iron‐related protein with roles in innate immune response and cell differentiation/maturation that was recently implicated in regulation of emotional behaviors and cognitive function through regulation of neuronal excitability and dendritic spine formation/maturation. Besides the hippocampus, robust increases in Lcn2/NGAL mRNA were also observed within the prefrontal cortex (PFC) and anterior cingulate cortex (ACC), as well as in the brains of female rats exposed to the same pain paradigm. Overall, the results of this study continue to strengthen the idea that dysregulation of genes involved in neuroinflammatory and neurodegenerative processes in the hippocampus and other limbic brain areas may be involved in the development of mood disorders during the chronic pain state.
Summing up 10 years in a new medical school: Per aspera ad astra
Authors: Barash Alon , Toledano Nadav , Maoz Eyla , Blumberg Yair , Dickman Nomy , Karasik David
Published: May 2022

Opening a new medical school and introducing an anatomy course is a rare opportunity for medical anatomy educators. Here we sum our experience in an ongoing 10‐year anatomy program, emphasizing the challenges we endured, the hardships and successes in the hope that others can learn from our experience.

In February 2010, Bar Ilan University was chosen by the Israeli Council for Higher Education to open a new, fifth, medical faculty. A mere eight months later, the school opened its gates to a class of 70 students in the northern city of Safed. This new faculty, dedicated to establishing connections to the local community, enabled us to develop and implement a new anatomy curriculum.

The basic course incorporated frontal lectures, dissection labs, basic systematic physiology, clinical correlation lectures and imaging labs. The initial curriculum included trunk and Head & neck regions spanning 5 weeks. After the first couple of years, the length and contents of the course started to change and adapt according to the needs of our faculty, and by input from physicians as well as students. Over the years student number rose to 100, and the course expanded to 10 weeks to incorporate the entire body aside from neuroanatomy, which is taught in a separate course. Histology and embryology were included; ultrasound hands‐on labs were added; and virtual anatomy sessions were integrated to the program. Clinical and physiological correlate lectures were cut down, with the understating, backed by students’ evaluations, that this was too early for them, and focus should remain on basic normal anatomy. On the other hand, more time was added to imaging sessions and lectures, as most modern physicians use these techniques in their everyday clinical practice. While originally relaying on retired physicians to serve as dissection instructors, we initialed a near‐peer students dissection instructors’ program that was met with great success.

In the last couple of years our program and indeed the entire medical school was heavily impacted by the ongoing Covid‐19 pandemic. While lectures were taken to virtual platform, we decided to continue dissection sessions as normally as possible, with strict mandatory regulations including personal protective gear and keeping students’ number within the lab to a minimum of 15, 7‐8 students per cadaver. Students were highly appreciative of our efforts, and course scoring was exceptionally high.

The last 10 years has shown us that anatomy, a basic and important tradition, could be taught in many exceptional ways, and we should always strive to improve. But perhaps even more important is the understating that we are educators to whom our students look up to, and endurance, perseverance, resourcefulness, and professionalism are just as important as scholastic knowledge.
Anatomical and Molecular Phenotypes of Fast and Slow Vagal Targets in the Intrinsic Cardiac Nervous System
Authors: Srivastava Ankita , Robbins Shaina , Achanta Sirisha , Schwaber James S. , Vadigepalli Rajanikanth
Published: May 2022

The vagus nerve coordinates the communication between the brain and most of the visceral organs. Vagus nerve activity is critical to promote cardiac health and cardioprotective response in heart failure. The vagal motor outflow involves two nuclei, the nucleus ambiguous (NA) and the dorsal motor nucleus of the vagus (DMV). NA acts via nicotinic cholinergic processes to elicit fast kinetics of the intrinsic cardiac nervous system (ICN) response at the heart. By contrast, the DMV influences the ICN neurons via muscarinic cholinergic processes yielding slower kinetics of response. Interestingly, NA and DMV project to distinct neuronal populations in the ICN, providing an anatomical basis for the fast versus slow vagal action on the heart. We aim to delineate the ICN neuronal network mediating the two lanes of vagal inputs for regulating distinct cardiac functions with lane‐specific transcriptomic identities of ICN neuron targets. We performed immunohistochemistry (IHC) on the rat heart tissue sections embedded in the optimal cutting temperature (OCT) followed by confocal microscopy imaging. We found that while the ICN neurons express muscarinic and nicotinic acetylcholine receptors (AChRs) broadly across the ICN, with several neurons predominantly expressing either muscarinic or nicotinic AChRs. We used Laser Capture Microscopy (LCM) to acquire neurons that predominantly express muscarinic or nicotinic AChRs. We are profiling the transcriptomic identities of these LCM‐captured neuron classes. Our results suggest that mAChR‐only and nAChR‐only neurons differentially express neuromodulatory processes, including receptors, neurotransmitter synthesis enzymes, and neuropeptides. Further, we incorporate a systems approach combining spatial single‐cell transcriptomics, neuronal network modeling, and physiological control systems modeling to identify selective targets for novel neuromodulatory interventions that improve heart health.
Facial dysmorphologies in genetic disorders: exploring the ancestry component in a Colombian population
Authors: Martínez Abadías Neus , Echeverry Luis Miguel , Gómez Eidith , Solís Paula , Candelo Estephania , Ramírez‐Montaño Diana , Ortiz Diana , González Alejandro , Sevillano Xavier , Cuéllar Juan Carlos , Pachajoa Harry
Published: May 2022

Currently there are more than 10,000 rare diseases that affect about 7% of the world's population. Up to 40% of rare genetic disorders present craniofacial dysmorphologies, which can vary from subtle facial anomalies to severe malformations. Visual assessment of facial dysmorphology is commonly used for clinical diagnosis, patient management and treatment monitoring. However, qualitative descriptions are usually vague and quantitative approaches using craniofacial phenotypes for the diagnosis of rare diseases are based on North American and European populations, disregarding the influence of population ancestry on facial variation, as in Latin‐America. In this study, we assessed facial dysmorphologies associated to three different genetic disorders (Down (DS), Morquio (MS) and Noonan syndrome (NS)) in a Latin‐American population from Cali (Colombia). We recorded the coordinates of 18 facial landmarks in 2D images from 34 pediatric patients (19 DS, 8 MS, 6 NS) and 75 controls and quantified facial differences between patients and control groups using Euclidean Distance Matrix Analysis (EDMA).

Comparisons between control and syndromic phenotypes indicated that individuals diagnosed with DS and MS presented the largest percentage of dysmorphologies, with respectively 56.2% and 54.9% of significantly different facial traits. In NS, the percentage decreased to 12.4%. Each syndrome presented a characteristic facial pattern. In DS, all facial structures were affected, with a 6% increase of relative distance between the eyes and a 7‐10% reduction in facial height. The eyes and nose were most affected in MS, but not the mouth, with higher hypertelorism and facial reduction than in DS (9% and 8‐17%). In NS, facial differences were more subtle and affected mainly the eyes and mouth, reducing the relative distance between them from 1 to 4% in patients as compared to controls. Our study provides a precise quantitative comparison of facial dysmorphologies in three genetic disorders that in the future can be compared with other world‐wide populations to test whether facial traits associated to disease are altered by different evolutionary and adaptive histories of human populations.
Modulation of lysosomal function as a therapeutic approach for coronaviral infections
Authors: Lear Travis , Larsen Mads , Lin Bo , Cao Qing , Alfaras Irene , Kennerdell Jason , Salminen Laura , Camarco Daniel , Lockwood Karina C. , Boudreau Áine , Ma Jing , Liu Jie , Tan Jay X. , Myerburg Michael M. , Chen Yanwen , St Croix Claudette , Sekine Yusuke , Evankovich John , Finkel Toren , Chen Bill B. , Liu Yuan
Published: May 2022

The endo‐lysosomal pathway plays an important role in pathogen clearance and both bacteria and viruses have evolved complex mechanisms to evade this host system. Here, we describe a novel aspect of coronaviral infection, whereby the master transcriptional regulator of lysosome biogenesis – TFEB – is targeted for proteasomal‐mediated degradation upon viral infection. Through mass spectrometry analysis and an unbiased siRNA screen, we identify that TFEB protein stability is coordinately regulated by the E3 ubiquitin ligase subunit DCAF7 and the PAK2 kinase. In particular, viral infection triggers marked PAK2 activation, which in turn, phosphorylates and primes TFEB for ubiquitin‐mediated protein degradation. Deletion of either DCAF7 or PAK2 blocks viral‐mediated TFEB degradation and protects against viral‐induced cytopathic effects. We further derive a series of small molecules that interfere with the DCAF7‐TFEB interaction. These agents inhibit viral‐triggered TFEB degradation and demonstrate broad anti‐viral activities including attenuating in vivo SARS‐CoV‐2 infection. Together, these results delineate a viral‐triggered pathway that disables the endogenous cellular system that maintains lysosomal function and suggest that small molecule inhibitors of the E3 ubiquitin ligase DCAF7 represent a novel class of endo‐lysosomal, host‐directed, anti‐viral therapies.
Analysis of Swelling Capability and Efficacy of Gelatin‐Based Antibiotic Eluting Hemostatic Agents
Authors: Lu Brandon , Kelleher Justin , Shelby Shameka
Published: May 2022

During surgical procedures, hemostatic agents are applied within incisions to accelerate the clotting process and mitigate excessive blood loss. These hemostats are typically gelatin based and remain within the surgical site after the incision is closed, which can serve as an ideal environment for microbial growth. Therefore, we developed a gelatin‐based hemostatic agent capable of sustained release of antibiotics to serve as a prophylactic measure against surgical site infections, while avoiding the disadvantages associated with systemic antibiotic administration. Gelatin matrices containing trapped/covalently bonded antibiotics were generated using type B gelatin, the cross‐linking agent 1‐ethyl‐3‐(‐3‐dimethylaminopropyl)carbodiimide, and either vancomycin, ciprofloxacin, or ceftazidime. Gelatin matrices were allowed to elute antibiotics over time in phosphate‐buffered saline solution to simulate release under native conditions, and elution samples were analyzed via infrared spectroscopy, Kirby‐Bauer, microdilution, and cell viability assays. Temporal swelling assays were also performed to assess the swelling capacity of the antibiotic‐eluting matrices. Eluted antibiotics retained structural integrity as demonstrated with infrared spectroscopy. Vancomycin and ciprofloxacin‐containing matrices inhibited microbial growth, while ceftazidime matrices exhibited no significant difference in inhibition when compared to controls. The antibiotic‐eluting matrices maintained their biocompatibility, as evaluated by cell viability assays. In addition, temporal swelling assays affirmed the ability of the matrices to perform as viable hemostats. Thus, adoption of these antibiotic‐eluting hemostatic agents in surgical practice could result in a novel method of combating surgical site infections while avoiding drawbacks associated with systemic antibiotic administration.
CRY‐BAR: A versatile light‐gated tool for the remodeling of membrane architectures
Authors: Wurz Anna I. , Bunner Wyatt P. , Szatmari Erzsebet M. , Hughes Robert M.
Published: May 2022

BAR (Bin, Amphiphysin and Rvs) protein domains are responsible for the generation of membrane curvature and represent a critical mechanical component of cellular function. Thus, BAR domains have great potential as components of membrane‐remodeling tools for cell biologists. In this work, we describe the design and implementation of a versatile light‐gated I‐BAR domain containing tool (‘CRY‐BAR’) with applications in the remodeling of membrane architectures and the control of cellular dynamics. By taking advantage of the intrinsic membrane association of the I‐BAR domain, CRY‐BAR can be used for spatial and temporal control of cellular dynamics and the initiation of cellular protrusions. CRY‐BAR function is demonstrated in immortalized and primary cell lines; Ezrin, which acts as a relay between the plasma membrane and the actin cytoskeleton, is implicated as an important mediator of switch function. Overall, CRY‐BAR holds promise as a widely applicable addition to the optogenetic toolkit.
in vitro reconstitution of RhoGEF positioning reveals that centralspindlin transports and retains RhoGEF at the plus‐end tips of microtubules
Authors: Tao Li , Warecki Brandt
Published: May 2022

Cytokinesis, the physical division of one cell into two, requires accurate positioning of the cleavage furrow between segregating chromosomes. Failure to properly position the cleavage furrow can result in aneuploidy and is a hallmark of cancer cells. However, key aspects of the molecular mechanisms that govern where the cleavage furrow forms remain poorly understood. Furrow formation is specified by localization of the conserved Rho‐activator RhoGEF, which leads to local membrane ingression. RhoGEF binds to the conserved motor protein complex centralspindlin, which performs numerous essential functions during cytokinesis. While in vivo experimentation has resulted in multiple proposed models for how the centralspindlin‐RhoGEF interaction mediates furrow formation, detailed molecular analysis is limited by the predominance of different and/or redundant pathways in various cell types. Here, we directly address this issue by performing in vitro reconstitutions of RhoGEF positioning using full‐length, recombinant proteins. We find that purified centralspindlin transports RhoGEF along single microtubules as cargo. Following transport, centralspindlin then retains RhoGEF at the plus‐end tips for extended periods of time, leading to local RhoGEF accumulation. Transport and retention are inherent behaviors of centralspindlin. Additionally, we find that RhoGEF binding to centralspindlin may stimulate centralspindlin motor activity, suggesting a possible positive feedback loop. Collectively, these results demonstrate centralspindlin’s motor activity and strong association with microtubules rapidly generate a high concentration of RhoGEF at areas where plus‐end tips are abundant, such as at overlapping aster microtubules at the cell equator, to specify the position of cleavage furrow formation.
Evans Blue Dye Interferes with Gating for Certain Fluorochrome‐Conjugated Antibodies Using Flow Cytometry in the Ischemic Rat Heart
Authors: Teng Jiajing , De Jeronimo Diaz Cesar , Flynn Ryan A. , Lin Matthew , Rogers Connie J. , Korzick Donna H.
Published: May 2022

Background

Acute myocardial infarction (AMI) is the leading cause of death in the U.S. Understanding the immune response to AMI has potential therapeutic impact. In particular, the differentiation of monocytes into macrophages post‐AMI is of interest given their role in inflammatory cytokine production and tissue repair. Recent studies suggest that impaired macrophage (Mϕ) polarization may promote a proinflammatory phenotype in the heart post‐AMI. Flow cytometry is a gold standard technique to assess immune cell populations including M1 and M2 Mϕ subtypes, using a wide range of fluorochrome‐conjugated antibodies (FC‐Abs). Evans Blue (EB) dye is widely used to assess the size of AMI, but the feasibility of the combining approaches due to spectral overlap is uncertain. Thus, the objective of this study was to determine whether the cardiac viability dye EB can be used simultaneously with FC‐Abs to quantify M1 and M2 Mϕ in cardiac tissue post‐AMI.

Methods

Immune cells were isolated from ischemic rat hearts (31 min ischemia/48 hr reperfusion) with or without EB perfusion and stained with FC‐Abs (1 μg/1 × 106 cells) to the following cell surface markers: FITC‐CD45 (experiment 1) or AF750‐CD45 (experiment 2) to detect leukocytes; Pacific Blue‐CD11b, AF700‐CD68 and PE‐CD206 (experiment 1) or Pacific Blue‐CD11b, AF488‐CD68 and PE‐CD206 (experiment 2) to quantify M1 and M2 Mϕ. A total of 500,000‐1,000,000 events were acquired using a BD LSR‐Fortessa and were analyzed and plotted using FlowJo software v10.

Results

Overlap of EB (emission peak at 680 nm) was detected in the AF700‐CD68 channel (emission peak at 720nm) and the AF750‐CD45 channel (emission peak at 775nm), interfering with gating within these two channels. In experiment 1, the M1 (CD45+CD11b+CD68+) population differed between samples with EB (2.5% of CD45+CD11b+ population, sample size n=2) vs without EB (68.5%, n=2). However, in experiment 2, the M1 population was similar between samples with EB (54.3% of CD45+CD11b+ population, n=2) and without EB (48.7%, n=1). The M2 (CD45+CD11b+CD206+) population did not differ in experiment 1 or 2.

Conclusion

Our data suggest that the EB perfusion interferes with fluorochromes with emission peaks close to 680nm. Thus, caution must be taken when selecting FC‐Abs for flow cytometry when combined with EB perfusion or other related dyes. Our next steps will be to confirm these observations in a larger sample size.
PROTECTION OF TELOMERES 1b Modulates Cellular ROS and Chromatin Structure in Arabidopsis thaliana
Authors: Castillo‐González Claudia , Barbero Barcenilla Borja , Min Ji‐Hee , Shippen Dorothy E.
Published: May 2022

Reactive oxygen species (ROS) are natural by‐products of cellular respiration, essential for cell signaling. They are also a major threat to telomere stability and can trigger senescence. Emerging studies reveal a broad role for telomere‐associated factors in modulating the response to oxidative stress. PROTECTION OF TELOMERES 1 (POT1) is one of the most conserved telomeric proteins, critical for both chromosome‐end protection and replication. POT1 is a single‐copy gene in most species. However, the flowering plant Arabidopsis thaliana encodes two highly divergent POT1 paralogs, AtPOT1a and AtPOT1b. AtPOT1a is an essential telomerase‐associated processivity factor. In contrast, AtPOT1b is not required for telomere replication or end protection, and its overexpression cannot complement the loss of AtPOT1a. Here we explore the natural separation‐of‐function within Arabidopsis POT1 paralogs and uncover a novel role for AtPOT1b in redox homeostasis. AtPOT1b expression is highly regulated, and restricted to seeds, root tips, and gametophytes, organs whose development is controlled by endogenous ROS. Strikingly, ectopic over‐expression of AtPOT1b decreases natural ROS levels throughout the plant, while a null mutation results in high levels of cellular and genomic oxidation, telomere length changes under multiple environmental insults, decreased chromatin compaction, decreased DNA methylation and increased expression of stress‐response genes. Consequently, atpot1b mutants have reduced fitness and are hypersensitive to chemical and environmental stresses. Finally, in response to oxidative stress, AtPOT1b expression increases and the protein accumulates at telomeres, suggesting that AtPOT1b may play a direct role in mitigating telomere oxidation. We postulate that AtPOT1b functions in a molecular rheostat that both senses and regulates cellular oxidation, thereby promoting genome stability in tissues where higher ROS levels are required for development.
SPARC: Intersectional labeling of vagal afferent nerve subsets using Cre and FLP dependent dual reporter strain
Authors: Kim Seol‐Hee , Hadley Stephen , Taylor‐Clark Thomas
Published: May 2022

Respiratory reflexes are tightly controlled by distinct types of vagal sensory afferent nerves. Afferent nerves can be categorized by their conduction velocity, axon diameter, myelination, responses to stimuli, ion channels/receptor expression, and embryological origin. Some airway vagal afferent nerves are sensitive to noxious stimuli (i.e. nociceptive). TRPV1, the capsaicin receptor, is selectively expressed on most vagal nociceptors. Vagal ganglia are comprised of jugular ganglia (neural crest origin) and nodose ganglia (placodal origin). Tac1 encodes Tachykinin1, the substance P precursor protein, and is mainly expressed in the jugular ganglia. P2X2, a purinergic receptor, is exclusively expressed in nodose ganglia. Previous data suggests that activation of jugular nociceptors and nodose nociceptors causes distinct reflexes. Here, we used intersectional genetics to simultaneously visualize nodose and jugular nociceptors. We generated a transgenic mouse model with dual recombinase responsive allele using Cre‐Lox and Flp‐FRT (Flippase‐Flp recognition target) systems. With the ROSA26‐RC::FLTG allele, cells with only Flp show tdTomato expression (by excising of FRT‐flanked STOP cassette) while cells with both Flp and Cre have GFP expression (by excising of both FRT‐flanked STOP cassette and loxP‐flanked tdTomato). We generated TRPV1‐FLP:Tac1‐Cre:ROSA and TRPV1‐FLP:P2X2‐Cre:ROSA strains. Offspring (6 to 8 weeks old) were used for characterization. Vagal ganglia, lung and brainstem were collected and fixed with 4% paraformaldehyde. Tissue were cryosectioned for immunohistochemistry. In TRPV1‐FLP:Tac1‐Cre:ROSA, the majority of tdTomato expressing cells were in nodose ganglia and GFP expressing neurons were in jugular ganglia. In TRPV1‐FLP:P2X2‐Cre:ROSA, as expected, GFP expressing neurons were only observed in nodose ganglia while tdTomato expressing neurons were observed in both nodose and jugular ganglia. Reporter expressing neurons in both strains overlapped with anti‐TRPV1 antibody staining indicating they were all nociceptive. In the brainstem, reporter expression was mainly in the nucleus of solitary tract (nTS) in both strains. In TRPV1‐FLP:Tac1‐Cre:ROSA strain, GFP expression (jugular nociceptors) was also observed in the paratrigeminal nucleus (Pa5). In the lung, most TRPV1+ fibers expressing Tac1 were found in close proximity to the epithelial layer of bronchi and bronchioles. Whereas TRPV1+ fibers expressing P2X2 often projected away from the conducting airways into the alveolar tissue. Taken together, we have developed cell type specific‐dual reporter mouse models using a combinatorial/intersectional Cre and FLP strategy to successfully identify distinct nociceptive subtypes of afferent nerves in vagal ganglia, brainstem, and lung.
Reduced Angiotensin II Type 2 Receptor Expression Is Associated with Gastric Cancer Progression and Enhanced Gastric Cancer Cell Migration/Invasion
Authors: Sandoval‐Borquez Alejandra M. , Wichman Ignacio , Carrasco‐Avino Gonzalo , Corvalan Alejandro , Lavandero Sergio , Quest Andrew F. G.
Published: May 2022

Introduction

Gastric cancer (GC) is one of the leading causes of death due to cancer worldwide. Most cases have a poor prognosis due to non‐curable surgical resection and the limited efficacy of chemotherapy. Angiotensin II Type 2 Receptor (AT2R), an effector protein of the renin angiotensin system (RAS), is reportedly deregulated in some human cancers and implicated in the regulation of several cancer‐related cellular processes. However, our current understanding of the role of this protein in GC is very limited. The objective of this study was to evaluate the expression of AT2R during GC progression and determine the effect of receptor activation and overexpression on GC cell migration and invasion.

Hypothesis

AT2R acts as a tumor suppressor in GC by reducing cell migration and invasion.

Methods

We used the data published in the The Cancer Genome Atlas (TCGA) to evaluate in GC the expression of AGTR2, the gene that encodes AT2R. In addition, we determined the expression of AT2R in precancerous gastric lesions, advanced GC and non‐tumor adjacent tissue samples by immunohistochemistry. Association analysis of the expression of AGTR2 and AT2R with clinicopathological features and survival curves was also performed. Besides, the effects of AT2R on cell migration, transmigration, invasion and colony formation were evaluated using human GC cells. Finally, we determined the effect of AT2R activation in an intraperitoneal carcinomatosis xenograft mouse model.

Results

We observed that AGTR2 and AT2R expression was downregulated in GC patients (Fig. 1A), and this decreased expression correlated with depth of tumor invasion, metastasis and poor overall survival (Fig 1B). Moreover, loss of AT2R expression was associated with disease progression from pre‐neoplasic lesions to GC (Fig, 1C). On the other hand, AT2R activation and overexpression reduced migration (Fig. 2A, B), transendothelial migration, invasion and colony formation of AGS and Hs 746T GC cells. Finally, AT2R activation increased mouse survival and decreased tumor formation in an in vivo model (not shown).

Conclusions

AT2R was downregulated to a significant extent in GC patients and shown to prevent GC cell migration and invasion, suggesting a role in preventing the progression of GC lesions.
Targeted Osmotic Lysis of Advanced Carcinoma in Companion Animals
Authors: Paul Dennis , Edenfield Samantha , Miller Paige R. , Sherman Kelly J. , Del Piero Fabio , Gould Harry J.
Published: May 2022

Targeted osmotic lysis (TOL) is a novel technology that involves the concomitant stimulation of voltage‐gated sodium channels (VGSCs) and pharmacological blockade of Na+, K+‐ATPase (sodium pumps) causing selective osmotic lysis of highly malignant cancer cells that may offer an option for treating advanced carcinomas in companion animals. We hypothesized, that as in experimental animals, TOL would be able to increase survival beyond that anticipated for animals with advance carcinoma without adversely affecting quality of life. Accordingly, we treated 2 feline and 3 canine companion animals in which TOL was used to safely treat advanced carcinoma. Based on available pre‐clinical evidence for safety, efficacy and lack of off‐target damage to normal tissue, companion animals diagnosed with advanced carcinomas that were experiencing impaired behavior and functionality were offered trial treatment with TOL. When practical, biopsy samples were obtained to determine the level of VGSC expression. Digoxin was administered to achieve steady‐state, therapeutic tissue levels. The animals were then exposed to pulsed electric field stimulation. Post‐treatment measurements of tumor size and assessments of behavior and functionality were compared to pre‐treatment observations made by the owners and by the treating veterinarian. The frequency of treatments and survival varied, based on the patient’s level of pre‐morbid functioning and response to treatment. In all five cases, TOL the animals survived beyond the prognosis. In 3 cases, owners reported behavioral improvement. The results support the hypothesis that TOL may provide a therapeutic option with broad application in veterinary practice as a safe, well‐tolerated and effective option for treating patients with advanced stage carcinoma.
Derivation of Actin‐Binding Peptides from a Cofilin Scaffold: A Rational Approach
Authors: O'Bryant Collin T. , Hardeman Jordan M. , Bessetti Riley N. , Litwa Karen A. , Hughes Robert M.
Published: May 2022

Actin‐binding peptides and proteins have numerous applications in the control and monitoring of cellular dynamics. This is effectively demonstrated by the wide usage of protein fusions containing the actin‐binding peptide LifeAct and fluorescent proteins for live cell imaging. Despite their popularity, there remains a need for a new generation of actin‐binding probes that are largely orthogonal to cellular processes. We propose that this need could be met by a rational deconstructive approach to well‐characterized actin‐binding peptides such as cofilin. In this work, we report on such an effort that uses structural information about the cofilin‐actin binding interface as a guide for the selection and design of cofilin‐derived peptide sequences (COFPEP). We describe the ability of these peptides to label cytoskeletal structures in live and fixed cells, and further detail methods for their improvement via rational design methods. Finally, we investigate whether these cofilin‐derived fragments can inhibit the formation of cofilin‐actin clusters and rods formed in cells undergoing energetic stress. We postulate that peptides with the ability to disrupt stress‐induced cofilin‐actin structures may have applications in studies of brain ischemia and neurodegenerative disease.