bluuebunny/crossref_metadata_2025 huggingface dataset:
Getting Better Images Easier for Single Particle Cryo‐EM
Authors: Cheng Anchi
Published: June 2021

Single‐particle Cryo‐EM is a structural biology technique that has gained increasing popularity in recent years. An increasing number protein complex structures can now be reconstructed to resolutions sufficient to build atomic models similar to those obtained with x‐ray crystallography. The single particle cryo‐EM method relies on averaging millions of projection images of the same protein complex particles preserved in a thin‐layer of vitreous ice. These projection images also need to be distributed across a large number of orientational views so that they can be combined to reconstruct the three‐dimensional volume correctly. At National Resource for Automated Molecular Microscopy (NRAMM), methods are being developed to characterize cryo‐specimen preparation and improve its reproducibility. Efforts are also being made to increase the throughput and quality of automated data acquisition. Tools are made to efficiently support EM managers at large facilities such as the Simons Electron Microscopy Center (SEMC) and the recently established National Center for CryoEM Access and Training (NCCAT). For example, we developed a blotless grid preparation instrument, Spotiton, which by decreasing the time between sample application and cryo‐plunging, serves to amerliorate effects such as preferred orientation. By compensating for beam tilt and astigmatism, we also increased the data collection rate by at least 2‐fold without compromising data quality. In this presentation, I will describe some of these problems and illustrate our solutions for getting better data easier.

Support or Funding Information

NIH National Institute of General Medical Sciences (GM103310 and U24GM129539) Simons Foundation (349247)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Sequenced and Team Approach to Surface Anatomy Education in Physical Therapy in Preparation for Clinical Practice and Collaborative Care
Authors: Topp Kimberly
Published: June 2021

A deep understanding of anatomy is foundational in the clinical practice of physical therapy, and is key to discovery and scholarly work in movement sciences. Similar to several other health science professions, education of the physical therapist begins with prerequisite coursework in an undergraduate program, continues with doctoral‐level courses in a graduate program, and often post‐licensure residency or fellowship training. Anatomy and physiology are prerequisites for many DPT programs, and anatomy is included in the required elements for accreditation of DPT programs, residencies and fellowships. Surface anatomy is foundational for clinical work in physical therapy and is honed over years of practice.

In the DPT curriculum, surface anatomy is incorporated into early courses in gross anatomy and skills acquisition, including training in goniometry and assessment of muscle strength and vital signs. Surface anatomy continues into advanced gross anatomy courses and education in physical examination, neuromusculoskeletal assessment, and movement science. Early on, students identify bony landmarks, arterial pulses and muscle bulk, similar to early learners in medical curricula. As they progress, DPT students learn to observe subtle contours and shadows, identify muscle weaknesses and joint restrictions through observation of movement, assess muscle contraction and tendon motion using ultrasound imaging, and palpate delicate superficial nerves, millimeter differences in joint motion, and directional restrictions in layers of fascia.

Licensed physical therapists gain additional knowledge of anatomy through continuing education courses, residency and fellowship training. Courses may include advanced training in such areas as pelvic health, oncological care, prosthetics, or vestibular rehabilitation. Residencies and fellowships enable clinical specialization, such as in pediatrics, orthopedics or cardiopulmonary care. Each of these courses and specialty certifications includes advanced training in surface observation of underlying anatomy.

In this talk, Dr. Topp will describe a sequenced approach to surface anatomy education in physical therapy, and how surface anatomy is used in clinical physical therapy. As budding experts in anatomy, students in physical therapy may provide guidance in surface anatomy for physicians in training, and the talk will include examples of near‐peer learning of surface anatomy and case‐based musculoskeletal examinations.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anthropometric evaluation of humerus with and without the septal foramen
Authors: Soares Rosiely Olímpio , Fazan Frederico Sassoli , Fazan Valéria Paula Sassoli
Published: June 2021

The humerus is the longest and heaviest of the bones of the upper limb. In the elbow region, the humerus condyle is composed of the trochlea, the capitulum, and related fossae. The coronoid fossa, located on the anterior surface, receives the coronoid process of the ulna when the forearm is flexed. The olecranon fossa, located on the posterior surface, is large and deep and receives the olecranon of the ulna, when the forearm is extended. The two fossae are separated by a thin bony lamina that usually allows the passage of light. In some cases, the two fossae are separated by a non‐calcified membrane which, in dry bones, gives rise to an opening or perforation, called the supratrochlear foramen or septal foramen. Individuals with this anatomical variation are able to hyper‐extend the elbow joint. In the present study, 30 left humerus and 25 right humerus were evaluated and 16.7% of the left humerus and 15% of the right humerus presented the septal opening. With the aid of a digital caliper, the length, diameter and circumference of the middle portion of the diaphysis and the inter‐epicondylar distance of the humerus with and without the septal foramen were obtained. With the aid of an analytical scale, the weight of the bones was obtained and, later, the weight/length ratio was calculated for each humerus. Statistical analysis showed no differences between the right and left normal humerus, as well as between those with septal formamen, in none of the analyzed parameters. When we compared the humerus with and without the septal foramem, the diameter and circumference of the middle portion of the diaphysis, and the inter‐epicondylar distance were significantly smaller in the humerus with the septal foramen. Our data suggest that the presence of the septal foramen may be related to a reduction of the standard anthropometric measurements of the humerus.

Support or Funding Information

FAPESP, CNPq and FAEPA

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Can Dentition Aid in Estimation of Handedness?
Authors: Jelaca Maria
Published: June 2021

Handedness is challenging to estimate on skeletonized human remains when muscular markers of it are not available. This preliminary study anthropometrically explores if human teeth could be used to aid in identification of handedness. The dental data for this research is collected from a total of fifty (N=50) living adult volunteers with good dental health of which thirty‐nine (N=39) were right handed and eleven (N=11) left‐handed. Non‐invasive anthropometric data collected via digital dental caliper consists of the Maxillary and the Mandibular quadrants in which permanent incisors and canines were analyzed. The study reconfirms that males on average have larger teeth than females. The lateral incisors were not taken into consideration as they show minimal variations across the quadrants. The left‐handed males measure slightly larger central Maxillary incisors and canines at the left and the right quadrant than the right‐handed males. The left‐handed females have smaller central incisors than the right‐handed females on the Maxillary quadrants, but slightly larger central incisors on the Mandibular quadrants. Mandibular right quadrant central incisors are slightly larger among the left‐handed than they are among the right‐handed individuals of both sex.

Support or Funding Information

SWC

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Estrogen Non‐Genomic Signaling as a Potential Therapeutic Target in Inflammatory Breast Cancer
Authors: Rodriguez Martir Keishla M , Jimenez Iancarlos , Guzman Jeisac , Montañez Katixa M , Bittman Xavier , Aviles Sila M , Negron Dalissa , Peterson Esther A
Published: June 2021

Breast cancer is the second leading cause of death in females in the United States2, being Inflammatory Breast Cancer (IBC) one of the most aggressive subtypes. The poor prognosis for patients with IBC emphasizes the need to better understand the molecular signature of this disease with the goal of developing effective targeted therapeutics. To this date there is no effective targeted therapeutics, especially for those patients that account for approximately 20–40% of IBC cases with triple‐negative breast cancer (TNBC) classification3. Importantly, several studies have shown that estrogen can exert non‐genomic effects in IBC and non‐IBC TNBC, mediated by the expression of an alternate isoform of estrogen receptor alpha, estrogen receptor alpha‐36, and GPR305,7,9. In this context, estrogen can activate non‐genomic signaling pathways involved in the acquisition of oncogenic phenotypes such as increased motility and invasion in IBC cells. Phytoestrogens, like coumestrol (Cou), are natural compounds found in plants, specifically soybeans. Also, it is a polyphenolic compound that is structurally similar to estradiol (E2) and several studies have shown that coumestrol inhibits cell proliferation and migration in TNBC. We hypothesize (1) that estrogen non‐genomic signaling has an active role in the aggressive metastatic phenotype of IBC, and (2) that Cou has anticancer activity by inhibiting estrogen non‐genomic signaling in IBC. To test this hypothesis, these cells were treated with E2 and activation of downstream kinases (ERK and AKT) was analyzed by Western blot. Immunofluorescence in IBC SUM149 cell line and Western Blot of breast cancer cell lines showed expression of estrogen receptors. In addition, preliminary observations demonstrated that upon E2 treatment there was an activation of ERK and AKT kinase in both IBC and TNBC cell line. Finally, treatment using previously reported IC50 (50μM)10–12 of Cou caused a decrease in cell viability of breast cancer cell lines. In summary, IBC cells are responsive to E2 treatment making this signaling pathway attractive for the development of innovative therapeutic strategies. Importantly, these Cou preliminary studies shows a potential anticancer activity in IBC cell lines.

Support or Funding Information

This project is supported by UPR Fondos Institucionales para la Investigación (FIPI to E. Peterson ) and RISE program (5R25GM061151‐17 to I. Jiménez).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Resveratrol on diabetes‐induced up‐regulation of apoptosis and MAPK signaling in retinal pigment epithelium of dark Agouti rats
Authors: Kittaneh Rawan , Al‐Hussaini Heba , Narayana Kilarkaje
Published: June 2021

Although diabetic retinopathy (DRT) is a treacherous outcome of uncontrolled hyperglycemia, the mechanisms which initiate the onset of DRT are not yet clear. The current study investigated modulatory effects of Resveratrol on type 1 diabetes‐induced changes in apoptosis and mitogen‐activated protein kinase (MAPK) signaling in retinal pigmented epithelium (RPE) of dark Agouti rats. Adult male rats (12–14 weeks, n=15) were segregated in duplicates into normal control, Resveratrol‐treated, streptozotocin‐induced diabetic, and Resveratrol‐treated (5mg/kg/d, from the day of confirmation of diabetes to the sample collection day) diabetic groups. The RPE was collected on days 30 and 90. For the latter sampling time, another Resveratrol‐treated (from 45d after the confirmation of diabetes to sampling day 90) diabetic group was included to investigate the effects of different treating protocol of Resveratrol. Resveratrol when administered to normal rats, increased gene expressions (RT‐PCR analysis) of caspases‐3, 8and 9, Bax, Bcl2,p38MAPK, JNK, and p53on 30d (P<0.05). On the other hand, diabetes decreased gene expressions of caspase‐3and 8, Erk, p38MAPK, and JNK(P<0.05). Resveratrol reversed the inhibited gene expressions of caspase‐8,Erk, JNKand p38MAPK to normal control levels in diabetic rats (P<0.05). Resveratrol normalized diabetes‐induced upregulation of proteins (Western blotting analysis) caspase‐3 and 9, cytochrome‐c, Bcl2 and ERK (P<0.05) on 30d. On 90d, Resveratrol recovered diabetes‐induced decreases in caspases‐3 and 8 (18 kDa fragment), cytochrome‐c, and Bcl2 proteins to the control level. Similar effects of Resveratrol were observed when given to diabetic rats only for 45d instead of 90d. In conclusion, Resveratrol when given to normal rats upregulates transcription of apoptosis and MAPK genes, however, their protein levels do not increase suggesting the lack of induction of apoptosis and MAPK‐mediated cell signaling. Resveratrol imparts its protective effects by normalizing apoptosis and MAPK signaling. These results are suggestive of beneficial effects of Resveratrol on the RPE in diabetic rats.

Support or Funding Information

College of Graduate studies and Research sector, Kuwait University

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Elongated Styloid Processes and a Triticeal Cartilage in the Stylohyoid Ligament: Clinical and Comparative Perspectives
Authors: Bender Emily Marie , Nwafor Divine C. , Petrone Ashley B. , Lambert H. Wayne , Zdilla Matthew J.
Published: June 2021

Elongation of the styloid process of the temporal bone may manifest in numerous vascular and neurological signs and symptoms known as Eagle syndrome. Though this syndrome is well‐known, oftentimes elongated styloid processes do not manifest the typical signs and symptoms of Eagle syndrome. Occasionally, elongated styloids may be identified incidentally, in which case they are important to recognize. This report details a case of bilaterally elongated styloid processes. However, unlike most reports of elongated styloids, in this case, a triticeal cartilage is located within the stylohyoid ligament. This report highlights the significance of the elongated styloids with special attention on the triticeal cartilage. The report highlights both the clinical implications and also emphasizes a comparative anatomical perspective.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Publicly Guided Effort to Rapidly Produce and Disseminate 100 Intersectional Genetic Alleles for the Study of Nervous System Circuit Development and Function in the Mouse
Authors: Martinez Vena , Sun Jenny , McKinney Andrew , Jiang Xiaolong , Ray Russell
Published: June 2021

The goal of this project is to build and distribute a community library of more than 100 mouse intersectional genetic alleles to facilitate the developmental, anatomical, molecular, and functional characterization of neural circuit organization in behavior and physiology based on public input. Even within narrowly defined cell types, significant diversity is found at multiple levels including genetic and molecular signatures, activity patterns, and synaptic connectivity. Current challenges now center on tools to identify, access, and study cell populations with increasing precision. Intersectional genetics offers exceptionally high resolution to consistently delineate distinct cell types in the embryo and adult mouse for functional, molecular and anatomical studies. Intersectional genetics utilizes a ubiquitously expressed conditional allele that is activated by Cre and Flp site specific recombinases. Upon activation, the intersectional genetic alleles may express any number of Genetically Encodable Effector Molecules (GEEMS), such as channelrhodopsins for neural activity perturbations or an L10‐GFP ribotrap fusion to affinity purify translating mRNAs. The intersectional allele is activated by overlapping expression of both Flp and Cre recombinases in the same cell. Traditionally, these recombinases have been deployed as gene knock‐in or transgenic alleles that are designed to express in a cell or gene specific fashion. The use of two selectors (Cre and Flp) to define a cellular population enables high specificity and modularity in combining any set of Cre, Flp and intersectional alleles to fit an experiment. The number of Cre and Flp recombinase mouse lines are constantly growing, giving greater access to increasing numbers of cell types. Additionally, these recombinases are the focus of multiple efforts to deploy them in ways that select cells based on other unique properties such as neuronal activity and synaptic connectivity. To make this powerful genetic approach more accessible to as many research laboratories as possible, we are building a production pipeline to create a suite of resources for anyone to easily make their own intersectional mouse line, to produce over 100 targeted ES cell lines that can be developed into mouse lines, as well as 10–15 high demand mouse lines all for public distribution. Priority for the GEEMS to be intersectionally deployed will be based on a public input collected at mouseintersectionalgenetics.org. All plasmids, cell lines and mouse lines developed under this effort will be distributed without restriction to non‐profit research organizations.

Support or Funding Information

McNair Medical Institute

R01HL130249

R01HL130249‐02S1

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Neuroinflammatory responses in a mouse model of acute organophosphate intoxication
Authors: Calsbeek Jonas , Guignet Michelle , Dawson Mallory , Bruun Donald , Lein Pamela
Published: June 2021

Organophosphates are a class of synthetic, neurotoxic compounds that include nerve agents and pesticides that can cause convulsions, status epilepticus (SE), and death following acute intoxication. The current standard of care for OP intoxication can prevent mortality; however, survivors often exhibit persistent neuropathologic changes and cognitive deficits. These observations underscore the need for improved therapies. Currently available preclinical models of acute OP intoxication almost exclusively use rats or guinea pigs, two species for which there currently are limited numbers of available transgenic lines. To overcome this limitation, we have developed a mouse model of acute OP intoxication using the OP pesticide diisopropylfluorophosphate (DFP). Within minutes after a single injection of DFP, adult male C57BL/6 mice displayed robust behavioral seizures consistent with the induction SE. At 1, 7, 14, and 28 days post‐intoxication, animals were euthanized, and the brains were removed for FluoroJade C labeling to quantify neurodegeneration, and GFAP/S100β or IBA1/CD68 immunohistochemistry to quantify reactive astrogliosis and microglial activation, respectively. A significant increase in neurodegeneration, reactive astrogliosis, and activation of microglia was observed in the somatosensory cortex, hippocampus, and piriform cortex of DFP mice relative to vehicle controls. These findings identify mice as a sensitive platform for the investigation of DFP‐induced neurological consequences that recapitulate the patterns of injury observed in widely used preclinical models. The availability of genetically modified mouse strains will provide unique opportunities to elucidate the molecular mechanisms contributing to the pathophysiology of acute OP intoxication, which will provide insight regarding more effective targets for therapeutic intervention.

Support or Funding Information

This work supported by the NINDS CounterACT Program (grant # U54 NS079202).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Stress hormones induce DNA damage in ovarian cancer cells
Authors: de Lis Lamboy Caraballo Rocio , Armaiz Guillermo
Published: June 2021

Activation of the sympathetic nervous system (SNS) is associated with cancer progression. Recent data suggest that adrenergic signaling alters the genetic integrity of cancer cells by damaging the DNA and/or influencing the DNA‐maintenance machinery composed of the DNA Damage Response Pathway and DNA Repair Pathways. Consequently, accumulation of genomic alterations can lead to genomic instability and the acquisition of other cancer hallmarks that drive tumor progression. Still, the exact mechanisms of how sustained adrenergic signaling influences genomic stability is an area that needs to be further studied. We hypothesize that increased adrenergic stimuli will induce significant levels of DNA damage. To assess the extent of DNA damage after catecholamine exposure, we determined p‐H2AX (sensitive marker for double‐stranded DNA damage) expression by western blot and immunofluorescence analyses in SKOV3 and other high‐grade serous ovarian cancer cell lines. Neutral Comet Assays were performed to assess double‐stranded DNA damage after catecholamine exposure. SKOV3 cells that were exposed to epinephrine (Epi) and norepinephrine (Ne) at 1h, 3h, 6h, 12h, and 24h displayed increased expression of p‐H2AX. Moreover, immunofluorescence analyses for p‐H2AX in SKOV3 and OV90 showed increased foci formation (reflecting 1:1 foci/double‐strand break) when compared to the control group. Lastly, Neutral Comet Assay analyses in catecholamine‐treated SKOV3 and COV362 cells displayed higher levels of double‐stranded DNA breaks in comparison to untreated cells. Lastly, to determine if DNA damage is due to adrenergic stimuli, cells were pre‐treated with propranolol (non‐specific β‐blocker) and results showed a significant reduction in the levels of DNA damage. These results suggest that catecholamines can induce double‐stranded DNA damage in ovarian cancer cells.

Support or Funding Information

NIMHHD (G12MD007579), NIGMS‐RISE R25GM082406, NCI (U54CA163071 and U54CA163068), U54CA163071‐06, and Ponce Research Institute.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synergistic and Global Effect of C‐terminus of Hfq in Small RNA Regulation
Authors: Kavita Kumari , Zhang Aixia , Tai Chin‐Hsien , Storz Gisela , Gottesman Susan
Published: June 2021

Hfq, a homohexameric RNA chaperone, is known for stabilizing small noncoding RNAs (sRNAs) and facilitating riboregulation by promoting sRNA base pairing with target mRNAs. The proteins encoded by these target mRNAs are often involved in virulence and stress responses. Hfq has a conserved N‐terminal region sufficient for most of its function and a poorly conserved disordered C‐terminus (CT). The length of the C‐terminus varies among bacteria; it is 37 aa in E. coli. Since the role of C‐terminus of Hfq has been poorly understood we have investigated its role in vivo in small RNA stability and function. Mutant derivatives of Hfq were expressed from the chromosomal locus, allowing in vivo comparisons with the wild‐type (WT) Hfq, expressed under similar conditions and at physiological levels. While deletion of the CT (Hfq65) had modest phenotypes, combinations of this deletion with a weak mutation (K31A) in the distal face of the protein was fully defective for function in different assays. This strong synergistic phenotype of CT deletion with K31A allowed us to further dissect the CT. Loss of the region between 65–72aa, the best conserved part of the CT, was critical for the synergistic defect. These results suggest that the initial region of the CT supports the function of the distal face, where many mRNAs and a subset of sRNAs are known to bind. To understand the global effect of CT on interacting RNAs, we compared RNA seq data of Hfq immunoprecipitation for WT and Hfq65. Overall, Hfq binding RNA patterns were not much affected. However, consistent with previous findings, and with the cooperation of the CT with the distal face of Hfq, Hfq65 was most defective in the accumulation of the sRNAs (Class II) that bind the proximal and distal faces of Hfq. Some class I sRNAs, which bind the proximal and rim face of Hfq, were also modestly depleted in Hfq65, suggesting the possibility of a broader role for the CT. The mechanistic aspects of CT collaboration with the Hfq core are under investigation. Our results suggest that the C‐terminus collaborates with the Hfq distal face, reinforcing the specificity and strength of RNA binding to Hfq.

Support or Funding Information

This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Point Mutation of Aquaporin 4 Impairs its Binding in Neuromyelitis Optica
Authors: Replogle Jessica Beth Sakash , Calderon Irene , Cutforth Siena , Devine Erin , Fraley James , Lamarre Thomas , Moran Elizabeth , Valencia Luis , Suder Karen Cruse
Published: June 2021

Aquaporin 4 (AQP4) is the principal aquaporin expressed in astrocytes throughout the central nervous system. An autoantibody, IgG, that binds to AQP4 leads to a rare disease called neuromyelitis optica (NMO), which is characterized by paralysis and loss of vision. Current treatments for NMO are few and involve non‐specific immunosuppression. The Summit Country Day School MSOE Center for BioMolecular Modeling MAPS Team used 3D modeling and printing technology to examine structure‐function relationships of the AQP4 epitope for NMO‐IgG. Understanding the molecular structure of the AQP4 epitope is an important step in explaining its binding to NMO‐IgG. NMO‐IgG primarily interacts with the accessible AQP4 extracellular loops A, C, and E. These loops change their conformation when AQP4 arrange into orthogonal arrays of particles, thus enabling attachment of NMO‐IgG and facilitating complement binding. Studies on the point mutation of aspartate in position 69 (D69) revealed conformational changes such as 1) reorientation of threonine in position 62, 2) weakened hydrogen bond interaction between leucine in position 53 and threonine in position 56, 3) increased mobility of loop A, and 4) disruption of epitope reorganization of AQP4. Overall, D69 substitution and the subsequent conformational changes in loop A leads to impairment of binding between AQP4 and the NMO‐IgG. Determining the role of extracellular loop A between and within AQP4 monomers will aid in understanding how it contributes to antigen recognition. Prevention of the binding of AQP4 by NMO‐IgG is an important strategy for developing targeted therapy in NMO.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Pre‐N Domain is a Distinct Feature of Grp94 that is Essential for Client Maturation and Regulation
Authors: Huck Jack D. , Que Nanette L.S. , Hong Feng , Li Zihai , Gewirth Daniel T.
Published: June 2021

The chaperone cycle of hsp90s involves a series of large conformational shifts between open and closed dimer states that are driven by the binding of ATP to the N‐terminal domain (NTD). Unlike its cytosolic counterpart, the ER hsp90 paralog, Grp94, does not possess a network of co‐chaperones to regulate dimer closure and modulate the rate of ATP hydrolysis. Although hsp90s are highly homologous with each other, regions of divergence may lead to the functional differences that are key to Grp94's adaptation to the ER. One such region, the Pre‐N domain, precedes the NTD and varies substantially in length and sequence among hsp90s, with the Grp94 Pre‐N domain (50 a.a.) dwarfing the Pre‐N domains of the other paralogs (Trap‐1: 24 aa; Hsp90a: 16 aa; Hsp90b: 11 aa). For Grp94, the Pre‐N domain slows the rate of ATP hydrolysis but the mechanism by which it does so and its role in client maturation are not understood. Here we describe crystal structures of Grp94 containing the Pre‐N domain in open and closed dimer states. Surprisingly for a significant regulatory element, the Pre‐N domain makes only minimal stabilizing contacts with the opposite NTD in the closed dimer state. In the open dimer state, however, the Pre‐N domain adopts a novel trajectory and secondary structure that may regulate the ATP‐bound conformation through allosteric auto‐inhibition of the NTD. Our functional analysis further revealed that both the length and sequence composition of the Pre‐N domain are required for client proteins to reach the cell surface. We are currently investigating the interplay between the Pre‐N domain and other ER folding factors, such as post‐translational modifications, to understand the role of the Pre‐N domain in client maturation. Taken together, these results imply a functional role for the Grp94 Pre‐N domain that mimics that of the cytoplasmic Hsp90 co‐chaperones, which are missing in the ER.

Support or Funding Information

Research in the Gewirth lab is supported by NIH grants R01‐CA095130 and P01‐CA186866, a grant from the Richard and Mae Stone Goode Foundation of Buffalo, and grant 02‐2016‐079 from the Avon Foundation.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modulation of gap junctional intercellular communication in HaCaT cells by xenobiotics
Authors: Shipman Richard David , Jost Jannika , Pollet Dieter , Waldmann Petra
Published: June 2021

Gap junctions are clusters of transmembrane proteins which enable direct communication between adjacent cells. Gap junctions allow intercellular diffusion of small molecules like metabolites (such as glucose), second messengers (such as cAMP, ATP) and ions (such as Mg2+, K+). That process, called gap junctional intercellular communication (GJIC), allows coordination of many essential functions in multicellular organisms, such as directed cell differentiation, cell growth, cellular and tissue homeostasis. Moreover, dysfunctional GJIC has been linked to a variety of human diseases.

The final aim of this project is to elucidate whether estrogenic acting chemicals play a role in GJIC. As a first step, two industrial chemicals, bisphenol A (BPA) and 4‐nonylphenol (4‐NP), were selected and assessed as modulators of GJIC in the human epidermal keratinocyte line HaCaT.

To study GJIC of HaCaT cells, the scrape loading/dye transfer (SL/DT) assay was applied to HaCaT cultures grown at standard conditions with serial dilution treatments of either BPA or 4‐NP versus ‐a vehicle treatment (< 1% ethanol) for 24 hours. The SL/DT assay entails cutting a cell monolayer with a scalpel to allow for the incorporation of a fluorescent dye (Lucifer Yellow) into the ‘scraped’ cells. The dye transfer via opened gap junctions into adjacent cells is then investigated by fluorescence microscopy and image analysis. The number of Lucifer Yellow positive cells is a quantitative descriptor of GJIC activity in treated cells versus untreated cells. In addition to the results provided by the SL/DT functional assay, quantitative PCR was performed in parallel to assess the mRNA levels for GJIC protein connexins (Cx26, Cx36, and Cx43).

The results of the SL/DT assays showed that both BPA and 4‐NP reduce GJIC in the human epidermal keratinocyte line in a dose range of 10 down to 0.1 μM. Currently qPCR experiments are being conducted to elucidate whether this is due to closed gap junctions or to modulation in expression of connexin encoding genes.

With this research, these properties alongside qPCR may be relevant to answer the question of whether the observed modulation of GJIC in skin cells is due to an estrogenic activity. Furthermore, a better understanding of GJIC modulation may clarify the role of GJIC in disease and make it a future target for pharmacological, and particularly, dermatopharmacological approaches.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Super‐SILAC based quantitative phosphoproteomics reveals the potential biomarkers in lung cancer
Authors: Hsiao Ting‐Feng , Wang Chih‐Liang , Wu Yi‐Cheng , Wu Chia‐Chun , Yu Chia‐Jung
Published: June 2021

Lung adenocarcinoma (ADC) is the most common histological subtype of lung cancer, the leading cause of cancer‐related deaths worldwide. Epidermal growth factor receptor (EGFR) mutations strikingly target lung ADC in East Asia. EGFR tyrosine kinase inhibitor (EGFR‐TKI) has been developed as a targeted therapy for treatment of advanced lung ADC with activating EGFR mutations. Unfortunately, neither druggable targets nor useful biomarkers can be used to benefit ADC patients with wild‐type (WT) EGFR. We herein aimed to identify the differentially expressed phosphoproteins and kinases in ADC tissues with EGFR‐WT. We applied super‐SILAC combined with TiO2 phosphopeptide enrichment and liquid chromatography‐tandem mass spectrometry to establish the stage‐related phosphoproteome with 20244 phosphopeptides in ADC tissues. The bioinformatics analyses revealed the stage‐dependent phosphorylation profile, upstream signaling and the kinase cascade in ADC tissues. We also observed that the impact of EGF, PTK2B, JAK2, ICAM1, or VEGF on pathogenesis of the early‐ and late‐stage cancers was indeed different. To explore the clinical applications of these dysregulated phosphoproteins in lung ADC, the potential markers were selected for further verifications/validations via western blotting and immunohistochemistry staining.

Support or Funding Information

Ministry of Science and Technology, Taiwan, R.O.C. (105‐2320‐B‐182‐035‐MY3) and Chang Gung Medical Research Fund (CMRPD1H0081, CMRPD1H0082 and BMRP894).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
mTOR inhibition promotes LPS‐induced acute lung injury by inducing endothelial hyperpermeability
Authors: Chen Xiaolin , Fan Xing , Sun Chongxiu
Published: June 2021

Although mTOR inhibitors are widely used to treat various disorders in clinic, a considerable number of side effects have been described, including pneumonitis. The intrinsic mechanisms are not completely understood. This study aimed to elucidate the role of endothelial barrier dysfunction in the pathogenesis of mTOR inhibition‐induced lung injury.

Measurement of TER (transendothelial electrical resistance) and transendothelial BSA flux indicated that inhibition of mTOR either through transfection with siRNA, or treatment with inhibitors rapamycin or torin 1, increased endothelial permeability, accompanied by F‐actin remodeling as visualized by immunofluorescent. Consistently, mice with specific knockout of Mtor in endothelial cells exhibited significantly aggravated lung injury, neutrophil infiltration and lung edema in response to LPS exposure.

Studies with Rho‐associated kinase (ROCK) inhibitor (Y‐27632) and myosin light chain (MLC) kinase inhibitor (ML‐7) indicated that the hyperpermeability response was dependent on MLC phosphorylation, which is regulated by PKC activity. PKC contributed to mTOR inhibition‐induced MLC phosphorylation and endothelial hyperpermeability through P38/NF‐κB pathway to upregulate MLC kinase expression and ROCK/MYPT1 pathway to negatively control MLC phosphatase activity.

Taken together, our results demonstrate that inhibition of mTOR promotes LPS‐induced acute lung injury, at least partly through inducing endothelial barrier dysfunction.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dietary Complexation of Peanut Protein to Polyphenolic Extracts Reduces Peanut Specific Plasma IgE levels in Peanut Sensitized C3H/HeJ mice
Authors: Randolph Priscilla D , Bansode Rishipal D , Williams Leonard L , Plundrich Nathalie , Lila Mary Ann
Published: June 2021

Peanut Allergy is a common food allergy that can cause severe anaphylaxis reactions in children and adults. Treatment strategies that are utilized to combat peanut allergy includes but not limited to strict avoidance and rescue medication upon exposure to peanut. Oral immunotherapy is another strategies used to increase the threshold of peanut sensitivity in individuals who are allergic to peanut. Cross‐linking of dietary proteins by polyphenols can change their structural and immunological properties and can affect the protein's, thermal and morphological properties. Thus the purpose of this study was to determine whether the peanut proteins complexes to polyphenols extract would reduce allergic response in C3H/HeJ mice. Five‐week‐old C3H/HeJ mice were purchased. Before exposing mice to protein‐polyphenols aggregates of either low (15%: w/w) or high (40%; w/w) complexation ratios of blueberry (BB‐Low and BB‐High) and cranberry (CB‐Low and CB‐High) extract, mice were sensitized to peanut flour. Peanut sensitivity was also established by measuring the increase in peanut specific plasma IgE and IgG levels before treatment. Once the final peanut challenge, animals were scored for anaphylactic reactions, sacrificed, and blood samples were collected. Tissues samples aliquot from the liver, adipose, pancreas, spleen, and small intestine were collected and frozen at −80oC. Western blot analysis of spleen lysates revealed CD63/ant‐IgE protein expression was reduced by 35% and 44% in CB‐low and CB‐high diet groups, respectively, compared to the control. The reduction in CD63/anti‐IgE protein expression was 22% and 53% in BB‐low and BB‐high diet, respectively when compared to the control group. Therefore, our results demonstrate that the complexation for polyphenols to peanut flour can potentially lower plasma IgE of peanut‐sensitized C3H/HeJ mice.

Support or Funding Information

USDA NIFA: 240792

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analysis of Photobiomodulation Effects in Cardiac Hypertrophy in Experimental Model of Myocardial Infarction
Authors: Costa Leandro Oliveira D'Alcantara , Atum Allan , Serra Andrey , Manchini Martha , Feliciano Regiane S. , Silva José Antônio A.
Published: June 2021

Introduction

The process of cardiac remodeling is characterized by changes in geometry, volume, mass and constitution of the heart in response to tissue aggression. Myocardial hypertrophy is an important component of cardiac remodeling, which allows the heart to maintain its basic functions under increasing load conditions. The renin‐angiotensin system plays an important role in the pathophysiology of myocardial infarction and cardiac hypertrophy and contributes to the progression of heart failure.

Objective

To evaluate the effect of photobiomodulation in genes participating in the cardiac hypertrophy process, by the analysis of Edn1, Ace, Agtr1a and Ace2 mRNA.

Materials and Methods

Fifteen female Wistar rats were divided into three groups: Sham (5), IM (5) and IM + Laser (5) (Ethics Committee: 0015–2012). The infarction induction was made by occlusion of the descending left coronary artery, the laser being applied for 60 seconds at the occlusion site immediately after the infarction. Left ventricle samples from the experimental animals were collected 72h after infarction. Modifications of the gene expression of the genes: Edn1, Ace, Agtr1a and Ace2 were analyzed by Real‐Time PCR.

Results

The IM group determined a significant increase in expression of the genes: Edn1, Ace and Agtr1a when compared to the Sham group 3 days after the infarction. Laser therapy significantly reduced the expression of these genes when compared to the IM group: Edn1, Ace and Agtr1a when compared to the IM group, suggesting a reduction in hypertrophic processes after laser therapy.

Conclusion

Increased ACE expression in experimental models of myocardial infarction, as well as increased endothelin, are related to hypertrophic processes after myocardial infarction. Our studies suggest the action of the laser in the infarcted myocardium through the participation of the renin angiotensin system with the reduction of the expression of the genes involved in these processes.

Support or Funding Information

FAPESP, CNPq, CAPES, UNINOVE

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of Gαi protein selective μ opioid receptor analgesics to limit side‐effects
Authors: Liu Jianxi , Kenny Paul J.
Published: June 2021

The clinical utility of opioids as analgesics is limited by their serious side effects, which include respiratory inhibition, gastrointestinal motility disorders, tolerance and addiction. Opioids act by binding to μ opioid receptors (MORs) to trigger G‐protein and β‐arrestin‐mediated signaling cascades. The Gαi class of G‐protein is thought to play an important role in transducing the analgesic actions of opioids. Gαi subunits include Gi1, Gi2, Gi3, Go and Gz. In addition, Gβγ subunits also contribute to the actions of opioids, and the many subtypes of Gβγ subunits adding yet more complexity to MOR‐mediated signaling.

Gαi proteins show discrete tissue and brain distributions, each subunit has discrete enzymatic kinetics, and these subunits are differentially regulated by GTPase‐activating proteins (GAPs), Regulator of G protein (RGS) proteins, and other effectors. Moreover, findings from genetically modified mice suggest that disruption of specific Gαi proteins results in discrete patterns of altered responses to opioids. Based on these observations, we hypothesize that compounds can be developed to modulate specific Gi protein coupling to μ receptors, which will result in analgesic actions with less pronounced side‐effects.

Currently, novel opioids are developed by structure‐based or Gqi5‐mediated calcium readout. Consequently, Gqi5 biased opioids are likely to be preferred. To identify functionally novel opioids, we constructed MOR/Gqi15, i35, o5 and z5 in HEK 293 cells. We profiled ten reference compounds: DAMGO, β‐endorphin, met‐enkephalin, morphine, hydrocodone, oxycodone, TRV130, PZM21, SR17018 and fentanyl in these cell lines. We found that the majority of opioids signal through all Gi proteins with similar potency and efficacy. However, a minority showed strong Gi protein bias. Finally, because neuronal excitability is mediated by Gβγ subunits acting at N‐type CaV and G protein gated inwardly‐rectifying potassium (GIRK) channels, we generated MOR/GIRK1/2 and MOR/CaV2.2 α1/α2δ1/β1 in HEK 293 cells. These lines will be used to identify molecules that influence Gβγ actions on the activity of these effector channels.

These preliminary data demonstrate that opioids are likely signal in manner that can be biased to specific Gi proteins. This offers an opportunity to differentiate/discover compounds with unique pharmacodynamic profiles. Novel opioids that act through discrete Gi protein, or on Gβγ proteins, will facilitate greater understanding about the relationship between selective G‐protein coupling and opioid‐related side‐effects. We aim to leverage this new information to develop novel opioid analgesics with limited side‐effects.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
RB and CDKN2A cause Rapamycin resistance in cancer cells
Authors: Chakraborty Sohag , Utter Matthew , Foster David
Published: June 2021

mTOR‐the mammalian target of Rapamycin acts as the central regulator of multiple cellular processes like cell growth, proliferation and survival by integrating signals via nutrients, growth factors, hormones and energy sensing. In cancer cells, the mTOR pathway is highly dysregulated providing survival signals to the cells for their uncontrolled growth. Hence, mTOR has been a potential therapeutic target for cancer treatment for the past two decades. Application of high micro‐molar doses of Rapamycin has been found to successfully inhibit mTORC1 by blocking the phosphorylation of both downstream substrates ‐ S6K and 4E‐BP1. Our lab previously reported that Rapamycin acts as a cytostatic drug and causes G1 cell cycle arrest in the presence of TGF‐β signal. However, in the absence of TGF‐β signal, the drug induces apoptosis. The rationale behind the apoptotic effect of Rapamycin in absence of TGF‐β is that without TGF‐β signals, cells do not arrest in G1 and progress into S phase where Rapamycin is cytotoxic rather than cytostatic. Of significance, we found that cancer cells with nonfunctional RB and mutated CDKN2A do not undergo apoptosis upon Rapamycin treatment in the absence of TGF‐β signal. Proteins encoded by RB (pRb) and CDKN2A (p16INK4A and p14ARF) are upstream effectors of E2F family of transcription factors that are critical for G1 to S phase progression.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.