bluuebunny/crossref_metadata_2025 huggingface dataset:
The Micro‐RNA let‐7f Regulates Expression of the Voltage‐gated Mechanosensitive Ion Channel NaV1.5 in Human Gastrointestinal Smooth Muscle
Authors: Mazzone Amelia , Strege Peter R , Bernard Cheryl E , Cima Robert R , Larson David W , Dozois Eric J , Hayashi Yujiro , Ordog Tamas , Gibbons Simon J , Beyder Arthur , Farrugia Gianrico
Published: November 2023

Background

SCN5A‐encoded NaV1.5 is a mechanosensitive voltage‐gated sodium channel expressed in human gastrointestinal smooth muscle cells. In the gastrointestinal tract, alterations in NaV1.5 current density by naturally occurring mutations or by pharmaceutical block of channel function are associated with constipation. In the heart loss of NaV1.5 current is associated with cardiac contractility disorders such as dilated cardiomyopathy. Ion channel expression level is regulated by miRNAs, and NaV1.5 is known to be regulated by miRNA in heart. However, it is not known if miRNAs regulate NaV1.5 expression in GI smooth muscle.

Aim

To identify miRNAs that regulate NaV1.5 expression in gastrointestinal smooth muscle in health and disease.

Methods

A qPCR array was used to screen for differential expression of 372 of the most common miRNAs in the miRBase database to test the differential expression of miRNA between control tissue and tissues from patients with slow transit constipation. qRT‐PCR was done to determine expression levels of SCN5A mRNA. A cell line (HuJej) was derived from human jejunum smooth muscle cells by immortalization using the SV40 large T antigen. Whole cell voltage clamp experiments were done by standard patch clamp techniques.

Results

SCN5A mRNA expression was reduced by 66±0.08% in patients with slow transit constipation (57.5±1.8 years) compared to age matched controls (53±1.7 years). qPCR array screening showed that 12 miRNAs were significantly upregulated in colonic muscularis externa from patients with STC compared to controls. Let‐7f miRNA was increased 6.46‐fold in slow transit constipation compared to controls and was the only miRNA with strong negative correlation to SCN5A mRNA expression in the same tissues. HuJej cells express NaV1.5; therefore, we investigated the effect of let‐7f on NaV1.5 currents by transfection with a stable miRNA mimic. 48 hours after transfection with let‐7f mimic, NaV1.5 currents were significantly decreased compared to untransfected cells or to cells transfected with non‐targeting miRNA (let‐7f, −0.6±0.1 pA/pF versus untransfected, −3.9±0.6 pA/pF or non‐targeting, −2.3±0.6 pA/pF).

Summary and Conclusions

SCN5A mRNA is decreased in slow transit constipation. Expression levels of let‐7f and NaV1.5 RNA are negatively correlated in human colonic smooth muscle. miRNA let‐7f regulates NaV1.5 current density. Regulation of NaV1.5 current density may have relevance in health and disease.

Support or Funding Information

This work was supported by grant DK52766.
miR429 is Upregulated in Inflammatory Monocytes and Regulates Monocyte Migration
Authors: Barry Zachary R , Kynast Ross W , Stinson W Alexander , Fox David A , Amin M Asif , Rabquer Bradley James
Published: November 2023

Purpose

Rheumatoid Arthritis (RA) is an autoimmune disease characterized by chronic inflammation and joint destruction. Inflammation is characteristic of an immune response, brought about by increased migration of leukocytes to the damaged area. Monocytes migrate from the blood into the tissue to exert their effects. A multitude of genes involved in inflammatory pathways regulate monocyte migration. miRNAs are post transcriptional regulators that effect gene expression in a variety of cell types and in response to various stimuli. We hypothesized that inflammation regulates miRNA expression, including miR429, which then plays a specific role in regulating the expression of monocyte genes involved in monocyte migration.

Methods and Results

Monocytes were isolated from the blood of normal subjects (n=5) and patients with RA (n=8). miRNA was isolated and cDNA prepared. qPCR was performed using primers specific for miR429. There was a 2.6 fold increase in the expression of miR429 in the RA patients compared to the normal subjects. The miRNA databases, miRanda and MiRwalk, were used to identify gene targets for miR429 that are involved in monocyte and inflammation pathways. This analysis identified 38 gene targets with possible relevance to monocyte inflammatory processes, including MAP4K4, PIKfyve, and PAK2. miR429 mimics and inhibitors were then used to assess the role of miR429 in regulating gene expression and migration. After confirming mimic and inhibitor specificity and efficiency in U937 monocytes, we found miR429 had an effect on PAK2 expression. The mimic reduced the expression of PAK2 by a fold change of 0.57, and the inhibitor increased the expression of PAK2 by a fold change of 28.6. Chemotaxis was performed to test the effect of miR429 on monocyte migration in response to monocyte chemoattractant protein (MCP‐1). The miR429 mimic treated U937 cells (n=11 replicates) had similar migration to MCP‐1 when compared to control cells treated with only transfection reagent (n=10). However, migration was decreased by 47% in U937 cells treated with the miR429 inhibitor (n=10 replicates) compared to the control.

Conclusion

Our results suggest miR429 is upregulated in RA monocytes, and that miR429 plays a role in monocyte migration by regulating genes such as the cytoskeletal regulator, PAK2.

Support or Funding Information

Hewlett‐Mellon Fund for Faculty Development at Albion College
Comparative analysis of methods to profile mRNA expression of G‐protein coupled receptors
Authors: Sriram Krishna , Zhou Shu , Lowy Andrew M. , Insel Paul A.
Published: November 2023

G‐Protein coupled receptors (GPCRs) comprise the largest family of cell surface receptors and proteins used as targets of approved drugs. Due to their low abundance, paucity of specific and sensitive antibodies and the challenge of identifying receptors in individual cell types, efforts to profile GPCR expression typically involve mRNA‐based methods and the relative quantification of gene expression. In the current study we compared in parallel the use of Affymetrix microarrays, RNAseq and qPCR‐based arrays for the measurement of mRNA expression of non‐chemosensory endogenously expressed (endo)GPCRs. We analyzed samples of patient‐derived pancreatic cancer‐associated fibroblasts (CAFs). RNAseq was done with a read depth of ~25 million reads/sample with single‐end 75bp reads. Affymetrix samples were run on the GeneChip Human Genome U133 Plus 2.0 Array chip and for qPCR, we used Taqman GPCR arrays from Life Technologies.

Our results demonstrate a high degree of correlation between results obtained from the GPCR arrays and RNAseq. In general, the number of GPCRs detected (~100 per sample), their identity and magnitude of expression were closely correlated with those 2 methods. The validity of the results was verified by independent qPCR. By comparison, the Affymetrix arrays failed to identify a large number of GPCRs (~30) detected by both RNAseq and Taqman arrays. In addition, different probe sets for the same GPCR on Affymetrix arrays sometimes resulted in drastically divergent mRNA intensities, even though both values were reported as being “statistically significant” (P<0.05). We conclude that RNAseq and qPCR‐based arrays both provide appropriate methods to assess GPCR expression (although each method has advantages and disadvantages) and are preferable to Affymetrix arrays for the detection of GPCR mRNAs, especially low abundance GPCRs. Importantly, our mRNA analyses of GPCR expression by both RNA seq and GPCR arrays indicate that CAFs and other individual cell types express >100 different GPCRS, including a large number of orphan (without known physiologic agonists) receptors, certain of which we find can regulate cell function and may be novel drug targets.

Support or Funding Information

Supported by NIH R21CA189477 and T32CA121938
The Effect of Phenylalanine Exposure on Cell Migration
Authors: Vaughan Melville B , Alkadhem Niyaf J , Seagraves Nikki J
Published: November 2023

Phenylketonuria (PKU) is an inborn error of metabolism that results when the amino acid phenylalanine (Phe) cannot be catabolized to tyrosine due to a mutation in phenylalanine hydroxylase enzyme. The inability to regulate Phe levels in individuals with PKU leads to mental retardation, seizures and other symptoms that can be avoided with early detection and a Phe‐restricted diet. When PKU mothers do not adhere to a Phe‐restricted diet during pregnancy, Phe can become teratogenic to the fetus and result in a condition known as Maternal PKU. Maternal PKU is characterized by multiple congenital defects, including cardiovascular malformations (CVM) such as coarctation of the aorta, tetralogy of Fallot, ventricular septal defects and hypoplastic left heart syndrome. However, the mechanisms responsible for Phe‐induced CVMs are poorly understood. Previous studies have shown that cardiac neural crest cells are important in formation of the outflow tract (OFT) and aortic arch arteries (AAA). Cell migration of the neural crest cells is a central process in the development of the heart.

Study Objective

Since congenital CVM of the OFT and AAA are often observed in maternal PKU, in this study we aimed to determine if exposure to high Phe levels perturbs cell migration and proliferation.

Methods

We conducted migration assays on human fibroblasts cells and chick neural crest cells tubes to determine if migration was affected by Phe. Migration was assessed by classic scratch wound assay and by Silicon elastomeric masks migration study. Additionally, in the human fibroblast cells we stained for markers of proliferation to determine if it was affected by the Phe exposure. The chick neural tubes were dissected and plated on a 3‐D collagen matrix. Migration of neural crest cells was observed over 12–24 hours and the area of migration was quantified.

Results

Phe doses tested were 0.1mM, 1mM, and 10mM. We observed migration in human fibroblast was decreased in the higher doses (1mM and 10mM) but was increased in the 0.1mM dose in both methods observed. Proliferation was decreased in the cells exposed to 0.1mM Phe but was not changed in the higher doses (1mM and 10mM). Chick neural tubes were exposed to 1.5mM Phe and we observed increase in the area of migration.

Conclusion

Phenylalanine has a dose‐dependent effect on cell migration in vitro. Additionally, it has an effect on proliferation rates. While these results are preliminary they provide a foundation for more mechanistic studies to understand the effects of MPKU on development.

Support or Funding Information

2015 Fulbright Visiting Scholar Program for Iraq, UCO CMS CURE STEM program, and UCO office of Research and Grants
Role of Skeletal Muscle in Inner Ear Development
Authors: Rot Irena , Kablar Boris
Published: November 2023

We are interested in developmental morphodynamics and the ability of muscle to provide mechanical cues for organogenesis during development. Our study of compound mouse knockout (Myf5−/−:MyoD−/) embryos that entirely lack skeletal musculature has revealed that these embryos develop abnormal phenotypes in many organs, such as skeleton (palate, mandible, clavicle), lungs, retina and inner ear. In this study we analyzed the altered development of the mouse inner ear and its sensory fields. Amyogenic fetuses lack skeletal muscles that move the chain of three middle ear ossicles responsible for the transfer of sound vibrations. They are also unable to tilt their head which prevents the perception of angular acceleration. The inner ear areas that were most affected in amyogenic embryos were vestibular sensory fields, i.e., cristae ampullaris, sensitive to angular acceleration. Our finding that a specific vestibular hair cell type 1 was absent in mutants was further used to identify a profile of genes specific for the lacking cell type. We performed a DNA microarray analysis to compare gene expression between inner ear sensory fields of amyogenic and wild type mouse embryos. This analysis generated a list of 10 up‐ and 87 down‐regulated genes in mutant inner ear tissues. We subsequently performed a detailed consultation of web‐accessible mouse databases regarding the genes with altered expression in mutants. This allowed us to identify 6 candidate genes with a possible role in the development of the inner ear sensory organs: Actc1, Pgam2, Ldb3, Eno3, Hspb7 and Smpx. They were established as candidate genes based on their altered expression in our mutant embryos compared to the wild type, as well as their previously documented expression in the sensory organs of the mouse inner ear. Further analysis of inner ear tissues of knockout embryos for these specific molecules is necessary to confirm, or rule out, the role of candidate genes in the inner ear development. Additionally, we searched for human homologues of the candidate genes since a number of syndromes in humans have inner ear abnormalities associated with them. Mutations in one of our candidate genes, Smpx, have been reported as the cause of X‐linked deafness in humans. Our current study suggests an epigenetic role that skeletal muscle might play in organogenesis, and offers an approach to finding novel genes responsible for altered morphology in the inner ear.

Support or Funding Information

Natural Sciences and Engineering Research Council of Canada

Canada Foundation for Innovation
Thermal Imaging Reveals Temperature Retention in Hindlimbs of Mice up to 4 Hours after Targeted Intermittent Limb Heating
Authors: Racine Holly L , Ion Gabriela , Kerby Jenna C , Gray Miles X , Meadows Chad A , Serrat Maria A
Published: November 2023

Our lab has previously demonstrated that daily unilateral limb heating permanently increases bone length in growing mice. We hypothesize that limb temperature remains elevated on the treated side due to a heat‐induced increase in vascular supply that accelerates bone lengthening. In order to test the temperature retention hypothesis, we conducted an extensive pilot study to determine the period of time in which limb temperature remains elevated on the heat‐treated side after each daily regimen. By collecting hourly and daily temperatures at scheduled time points throughout the trial, our goal was to determine optimal conditions for recording temperature data in our subsequent studies, as well as to identify potential technical problems that could interfere with outcomes of the heat‐treatment. Weanling female mice (N=6) were treated daily at 7am for 2‐weeks with 40C unilateral heat for 40 minutes per day following our published methods. Infrared thermal images were captured at 320 X 240 resolution using a FLIR E8 infrared camera (sensitivity <0.06C in temperature range of −20 to 250C). Surface temperatures of the heat‐treated (right) and non‐treated (left) hindlimbs were obtained from calibrated images using FLIR tools software. Statistical comparisons were made using ANOVA and paired t‐tests in SPSS. Data from multiple days were pooled at each time point since there were no significant differences among days analyzed. Common trends in temperature differences between heat‐treated and non‐treated limbs were seen throughout the day. At 6am baseline prior to treatment, the heat‐treated side was over 1% warmer than the non‐treated side (t=1.84, p=0.06). After the treatment at 8am, limb temperatures were nearly equivalent (t=1.13, ns), suggesting a systemic post‐anesthesia thermoregulatory response. By 11am, 4 hours after the treatment start, the heat‐treated side was over 2% warmer than the non‐treated side (t=8.06, p<0.001), indicating sustained heat retention. By 3pm, limb temperatures were again similar (t=0.13, ns), reflecting increased activity as mice approached the dark cycle. Excessive imaging (up to once per hour) negatively impacted daily gain in body mass, but this effect was mitigated by increasing intervals between imaging. These pilot results support our hypothesis that limb temperature remains elevated on the heated side up to 4 hours after treatment. This study is an important step toward understanding the mechanisms by which heat enhances limb elongation.

Support or Funding Information

Supported by ASBMR Grants in Aid Program and the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health (1R15AR067451‐01).
Identification and Characterization of a Novel Actin‐associated Protein Recruited to Structures at the Plasma Membrane
Authors: Chua Michael Dominic , Guttman Julian Andrew
Published: November 2023

Listeria monocytogenes is a bacterial pathogen that manipulates the host actin cytoskeleton during various parts of its infectious cycle. This bacterium invades host cells using actin‐dependent internalization mechanisms at the cell membrane and once in the cytosol, it generates an actin‐rich “comet tail” that propels the microbe within the host cytoplasm. To spread from cell‐to‐cell, L. monocytogenes uses very similar comet tail‐like structures to form listeriopods which protrude the host cell membrane during the cell‐to‐cell spreading event. Using a mass spectrometry screen designed to identify actin‐associated proteins that interact at the plasma membrane, we identified a ubiquitin‐conjugating protein (Ubc). To determine the functional role of this protein in cell membrane‐associated actin regulation, we hypothesized that Ubc would play a crucial role in Listeria monocytogenes infections as well as mammalian cell motility in general as actin‐membrane associations are also crucial for whole cell motility. To test this hypothesis, we initially immunolocalized Ubc in Listeria‐infected Caco2 cells. Ubc localized to the bacterium‐actin interface exclusively when the bacteria were along the cell membrane. To characterize the function of Ubc at these structures, we utilized both a Ubc functional inhibitor on Caco2 cells as well as mouse embryonic fibroblasts that had decreased expression of the Ubc protein. Using both strategies, we observed a decrease in 1) bacterial internalization and 2) the formation of Listeria‐induced actin structures, compared to infections with wildtype host cells. To determine if Ubc may play a role in whole cell motility, we conducted a wound healing assay on Ubc‐inhibited PtK2 cells and found that wound closure was impeded in Ubc‐inhibited cells after 24 hours as compared to controls. Our data indicates that Ubc is a novel regulator for general cell membrane‐associated actin dynamics and is a crucial factor for efficient bacterial infections.

Support or Funding Information

This study was funded through NSERC.
Phenformin Inhibits Myofibroblast Differentiation in Dupuytren's Contracture Cells Treated with TGF‐β1
Authors: McNamar Rachel Anae , Vaughan Melville B
Published: November 2023

Metformin and phenformin have been used to help treat type‐2 diabetes. Recently, these two compounds have been found to have antifibrotic effects in tissues that are proliferative and myofibroblast‐rich. Metformin and phenformin could be utilized to help treat Dupuytren's contracture by inhibiting the differentiation of myofibroblasts. Myofibroblasts are characterized by an abundance of alpha smooth muscle actin (αSMA) within stress fibers in the cytoplasm of the cell. Transforming growth factor beta 1 (TGF‐β1) has been used to upregulate the formation of αSMA and used to promote myofibroblast differentiation. Myofibroblast differentiation is thought to be inhibited by metformin and phenformin because they activate the AMP‐activated kinase (AMPK) pathway. This pathway has been found to inhibit TGF‐β1 induced myofibroblast differentiation.

This study examined the inhibitory effects of myofibroblast differentiation and proliferation that metformin and phenformin have on Dupuytren's contracture (DC) cells treated with TGF‐β1. To investigate these inhibitory effects, four treatment groups of DC cells were cultured for 48 hours and analyzed. One group acted as a control, while the other three were treated with TGF‐β1. Two of those three groups were also treated with either metformin or phenformin. Proliferation was determined using a click‐EdU culturing and staining procedure; differentiation was determined by visualizing alpha‐smooth muscle actin (αSMA) using immunocytochemistry. Through qualitative analysis of the data collected through fluorescence microscopy, the group treated with phenformin demonstrated suppression of myofibroblast differentiation and had no effect on cell proliferation. The metformin‐treated group showed no obvious effect on the suppression of myofibroblast differentiation and showed a slight increase in proliferating cells. Our future goal is to determine whether a higher dose of metformin will demonstrate similar results to phenformin and other AMP‐K activators. These results will be supplemented using other myofibroblast‐appropriate functional bioassays.

Support or Funding Information

Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number 8P20GM103447.
Bcl‐xL‐c‐FLIPL Anti‐apoptotic Interaction in Microvascular Endothelial Cells
Authors: Sawant Devendra A
Published: November 2023

Apoptosis or cell death is carried out by a series of caspase activity which can be activated either by intrinsic / mitochondrial or by extrinsic / receptor mediated pathways. The apoptotic pathway is negatively regulated at the receptor level by c‐FLIP, which blocks caspase‐8 activation, and at the mitochondrial level by anti‐apoptotic Bcl‐xL. Researchers have shown earlier that c‐FLIPL protein overexpression upregulates Bcl‐xL protein expression in the endothelial cells undergoing apoptosis during ischemia‐reperfusion injury. However, no study has shown that Bcl‐xL overexpression can regulate c‐FLIPL activity in endothelial cells. Hence, the current research study tries to elucidate a possible novel interaction between Bcl‐xL and c‐FLIPL. The objective of this study is twofold; first, to demonstrate that Bcl‐xL overexpression causes translocation of c‐FLIPL from the nucleus into the cytoplasm and second, to determine whether this translocation actually helps c‐FLIPL to demonstrate its anti‐apoptotic activity. The results from immunofluorescence study and immunoblot analysis of sub‐cellular fractionation have demonstrated that c‐FLIPL translocate in cytoplasm from nucleus in microvascular endothelial cells transfected with recombinant Bcl‐xL protein. Caspase‐8 assay and apoptotic /cell death assay using flow cytometry will further demonstrate that Bcl‐xL can regulate c‐FLIPL anti‐apoptotic activity in microvascular endothelial cells. Thus, illustrating Bcl‐xL‐c‐FLIPL anti‐apoptotic interaction could explain key pathophysiological mechanisms involved in various disease processes.

Support or Funding Information

Alderson Broaddus University College of Medical Science, Philippi, WV.
Functional Characterization of SM22 during Listeria monocytogenes Infections
Authors: Hipolito Kevin Jay , Chua Michael Dominic , Solway Julian , Guttman Julian Andrew
Published: November 2023

To cause disease, Listeria monocytogenes invades the human intestinal epithelia then spreads to other tissues. Once the bacterium invades host cells, it hijacks the host actin machinery to generate an actin‐rich structure called a comet tail that propels the bacterium within the host cells. When these motile bacteria reach the plasma membrane, these comet tails protrude the membrane forming listeriopods for cell‐to‐cell spreading of the microbes. In our previous work, we identified the 22 kilodalton smooth muscle protein (SM22) as an actin‐associated component of enteropathogenic Escherichia coli pedestals. To further characterize the role of SM22 in bacterial infections, we hypothesized that SM22 would also play a crucial role in the formation and elongation of Listeria‐induced comet tails. To test this hypothesis, we immunolocalized Listeria‐infected HeLa cells and found that SM22 localized along the full length of the comet tail. Live imaging of infected cells transfected with fluorescently‐tagged SM22 showed identical results. To study the functional role of SM22 during the infections, we used small interfering RNA (siRNA) to knock‐down SM22 protein levels. In cells with reduced levels of SM22, we observed that the actin cytoskeleton in the host cell remained unchanged, however less comet tails were evident and those that were present appeared shorter in length. Thus, depleting SM22 impairs the formation and elongation of Listeria‐induced comet tails. Taken together with our previous findings, SM22 is emerging as a general protein hijacked by a variety of gastrointestinal bacteria for their disease processes.

Support or Funding Information

This study was funded through NSERC.
Cell contractility‐driven multicellular nodule formation
Authors: Isai Dona Greta , Zach Julia , Mehes Elod , Paku Sandor , Hegedus Balazs , Dome Balazs , Czirok Andras
Published: November 2023

We demonstrate that characteristic nodules emerge in cultures of various malignant pleural mesothelioma (MPM) cell lines. Instead of excessive local cell proliferation, these nodules arise by Myosin II‐driven cell contractility. Acto‐myosin rich stress cables, lined up across cell membranes and thus mechanically linking several cells, are prevalent in sections of surgical MPM samples as well as in nodules formed by cultured MPM cells. A cell‐resolved elasto‐plastic model can explain both the formation dynamics and the observed morphologies of the nodules. The model also predicts the speed of formation as determined by the degree of mechanical tension between neighboring cells, and the stability of cell‐substrate adhesion: For small contractile forces, nodules are slow to develop and localized at the boundary of cell free areas. In contrast, high intra‐layer tension quickly transforms all cell‐coated areas into dense clusters interconnected by multicellular strands. Model simulations also indicate that a decreased stability of cell‐substrate adhesions favors the formation of fewer, but larger clusters. Accordingly, several cell types cultured on a melleable substrate, such as matrigel, exhibits this cell contractility‐driven patterning process. Used as a bioassay for intercellular contractility, analysis of the spatio‐temporal dynamics of nodule formation helps to identify functional effects of the myosin inhibitors.

Support or Funding Information

R01‐GM102801
Immunoelectron Microscopy of Endocytosis and Junction Proteins at Tubulobulbar Complexes in the Mammalian Testis
Authors: Adams Arlo , Vogl A. Wayne
Published: November 2023

The seminiferous epithelium is the site of spermatogenesis, the cellular basis for male fertility. Large junction complexes separate the epithelium into basal and adluminal compartments, as well as attach morphologically differentiating spermatids to apical regions of Sertoli cells. During spermatogenesis, turnover of these large junction complexes enables both the next generation of spermatogenic cells to move from the basal into adluminal regions of the epithelium, and mature spermatozoa to be released into the duct system of the reproductive tract. We have proposed that unique structures termed tubulobulbar complexes (TBCs) are subcellular clathrin‐based endocytosis machines that internalize intact intercellular junctions during spermatid translocation and sperm release. If this is true, then proteins associated with endocytosis in general should be associated with TBCs. Previous immunofluorescence results have indicated that Rab5 and EEA1, together with junction proteins, are present in areas occupied by TBCs. However, association with specific compartments of TBCs at the ultrastructural level has not been done. Conventional methods of immunoelectron microscopy do not provide adequate preservation of membranes to clearly localize these proteins to TBCs. We have applied a recently published pre‐embedding protocol (Melo et al. 2014, Nature Protocols 9:2382‐2394) to the rat testis and show that Rab5 associates with the bulb region of TBCs. EEA1 is associated mainly with regions after the bulbs have been internalized. Nectin‐2 associates with junctions and with TBCs. This data supports the hypothesis that TBCs are involved with endocytosis of junction proteins.

Support or Funding Information

NSERC Discovery Grant to A. Wayne Vogl
Therapeutic Potential of Mesenchymal Stem Cells on Chemically‐induced Arthritis in Rats: Role of Pro‐inflammatory and Pain‐mediating Cytokines
Authors: Atta Hazem , Noorwali Abdulwahab , Bashah Taghreed , Ganem Ahmed
Published: November 2023

Background

Osteoarthritis results from loss of joint cartilage. In Saudi Arabia, almost 2 million people are affected by osteoarthritis. The knee joint is the most commonly affected joint in cases of arthritis. Osteoarthritis of the knee is the most common cause of pain and stiffness during walking. Mesenchmal stem cells (MSCs) have been reported to be successful in repairing traumatic focal cartilage defects in human clinical trials. Their use to treat osteoarthritis needs to be studied carefully, due to the diffuse area of cartilage damage compared to focal cartilage defects. Their influence on pro and anti‐inflammatory cytokines needs to be studied.

Objective

This work aimed to study the cartilage healing effect of intra‐articular injection of autologous MSCs. The influence of MSCs on pro‐inflammatory and pain‐mediating cytokines was also examined. This may help to document and explain the reported healing capacity of MSCs. This may also help in finding a cure for the patients and save them the morbidity and the complications of drug therapy

Methods

Intra‐articular injection of monosodium iodoacetate (MIA) in the knee joint of rats followed by MSCs injection. Knee joints were surgically removed and histologiac sections from the joints were examined before and after therapy. Serum levels of interleukin‐6 (IL‐6), its soluble receptor, nerve growth factor (NGF), and substance P were measured by enzyme‐linked immunosorbent assay(ELISA).

Results

Histological examination showed healing of the affected joints by a hyaline‐like cartilage. Serum level of IL6 decreased significantly upon MSCs injection. However, the serum level of IL6‐ receptor increased by MSCs therapy. Serum level of NGF and substance P increased after MSCs therapy

Conclusions

Mesenchymal stem cells can repair damaged articular cartilage in rat models of knee osteoarthritis. Part of their action appears to be through decreasing the production of pro‐inflammatory cytokines as Interleukin‐6. They play a role in healing and pain control by influencing pain‐mediating cytokines as substance P and NGF. Their mechanism of action needs to be further studied in large animal models and in clinical trials
Mustard injured skin: are matricellular proteins involved in the delay of wound repair?
Authors: Chang Yoke‐Chen , Hahn Rita , Chang Hui‐Ying , Zhou Peihong , Svoboda Kathy K.H. , Gordon Marion , Gerecke Donald
Published: November 2023

Matricellular proteins (MPs) are involved in tissue remodeling. They create a provisional matrix prior to the formation of the mature matrix and may also mediate inflammation during wound repair. Vesicants, including sulfur mustard (SM) and nitrogen mustard (NM), induce skin blisters by cross linking basement membrane molecules; this may lead to cell adhesion alteration, enhanced inflammation and prolonged wound healing. The timing of MPs deposition to provisional matrix may play a role in modulating the delayed repair of vesication wounds. MPs [osteonectin (SPARC), SPARC‐like 1 (Hevin), and tenascin‐C (TNC)] as well as the basement membrane zone (BMZ) heterotrimer, laminin 332 (LM332) were examined in NM skin injury and wound repair. Five μmoles of NM was applied to the dorsal skin of hairless (SKH1) mice. Post wounding skin biopsies at 1, 3, 7, and 10 days were collected for further analyses. H & E histology showed epidermis‐dermis junction separation at 1 day, epidermal hyperplasia and infiltration of inflammatory cells to the dermis at 3 days. Furthermore, a severe inflammatory response at 7 days, and persistent skin wounds at 10 days post NM exposure was evident. Immunofluorescent (IF) studies showed continuous staining for LM332 in the BMZ and minimal staining of SPARC, Hevin, and TNC in naïve control skin. At 1 day post NM exposure, an interrupted staining pattern of LM332 indicated a compromised BMZ. We observed strong expression of SPARC throughout the BMZ, light staining of Hevin and TNC near the BMZ. By 3 day post exposure the LM332 was multi‐layered in appearance while Hevin had increased expression in the hyperplastic epidermis. Also there was strong expression of TNC extending deep into the dermis. Furthermore, dual‐labeling IF indicated co‐expression of LM332/Hevin in some basal epidermal keratinocytes and LM332/TNC in the dermis near the BMZ. By day 7, Hevin and TNC continued to express strongly. By day 10, Hevin persistently stained throughout the hyperplastic epidermis with a thickening expression pattern of LM332 in the BMZ; TNC expressed strongly near the BMZ, extending deep into the dermis behind the leading edge. This increased expression of SPARC and Hevin in NM injured skin was confirmed by WB data. TNC expression was also consistent with QPCR and WB studies. These data confirm the distribution of the MPs in NM injured and give us new insight into the molecules involved in delayed wound repair.

Support or Funding Information

Supported by ES005022, EY09056, and NIAMS U54AR055073
An in‐Silico Model of Xenobiotic Distribution and Metabolism in a Simulated Mouse Hepatic Lobule
Authors: Fu Xiao , Sluka James P , Clendenon Sherry , Glazier James A. , Wang Zemin , Klaunig James , Ryan Jennifer , Dunn Kenneth
Published: November 2023

Liver damage due to xenobiotics and their metabolites is often highly localized. Exposure levels vary within the liver because of the liver's complex flow pattern and because transport and metabolism vary within individual sinusoids. To analyze these coupled effects, we constructed a structurally realistic in silico model of transport and metabolism in the mouse hepatic lobule and simulated the resulting post‐exposure distribution of xenobiotics and their metabolites. Our in silico hepatic lobule architecture is based on our measurements of the ex vivo structure of mouse liver lobules. A hemodynamic model calculates blood flow rates in this simulated liver architecture, validated against intravital microscopy measurements of blood flow in vivo. Submodels simulate transport, uptake and secretion of xenobiotics and metabolites within the in silico hepatic lobule. Differential expression of metabolic enzymes along each sinusoid's portal to central axis, together with differential hepatocyte exposure to xenobiotics, induces non‐uniform production of metabolites. Using the integrated model, we varied both compound‐specific (diffusion, active transport and metabolism) and compound‐independent (temporal alteration of blood flow distribution) parameters to investigate the influence of spatial zonation of enzymes and transporters and temporally varying blood flow on the resulting local exposure to xenobiotics and their metabolites. Variations in transport mechanisms (passive diffusion and transporter‐mediated active transport) and blood flow greatly alter the regional distribution of xenobiotics and metabolites in a hepatic lobule. We conclude that heterogeneity in the biochemical and biophysical properties of xenobiotics combines with the complexity of blood flow in a lobule to create unique exposure patterns for hepatocytes in different lobular regions.

Support or Funding Information

This work was sponsored by National Institutes of Health/National Institute of General Medical Sciences grants R01 GM077138 and U01 GM111243.
Quantification of the 3D Orientation of Vascular Canals in Cortical Bone
Authors: Pratt Isaac Vorster , Cooper David Michael Lane
Published: November 2023

The orientation of vascular canals in cortical bone is thought to reflect the loading experienced by a bone during the time when those canals were formed. This orientation is also thought to be related to the growth rate of cortical bone, creating different patterns such as longitudinal, laminar, or radial. The presence of these different patterns would then reflect faster or slower growth in that region of the cortex. Most of our understanding of vascular canal orientation comes from studies using histology: a method which relies on 2D sections, and which can only calculate the orientation of canals relative to the plane of sectioning. In this poster we present a new method, using micro computed tomography and custom developed software, to quantify the orientation of vascular canals in three dimensions. Using this method, two independent angles are measured: the orientation of the vascular canal relative to the long axis of the bone (phi), and relative to a plane of section perpendicular to the long axis (theta). These two angles together can be used to classify each vascular canal in the cortex into categories such as longitudinal, circumferential, or radial, or their oblique variant. This method is the first that can measure both theta and phi. Measuring these angles provides information on the processes that shape cortical bone microarchitecture, and provides the potential to extract biological information on the growth rate and loading of the bone. Disentangling the connection between growth rate, loading, and the organization of cortical porosity is a complex problem, and this method has the potential to help investigate and aid our understanding of these relationships through both natural and controlled experiments.

Support or Funding Information

Support for this research was provided by NSERC via a Discovery grant to DMLC and a post‐graduate scholarship to IVP.
Comparative Examination of Forelimb Skeletons of Deer in South Korea
Authors: Ahn Dongchoon , Sim Jeoungha , Park Byung‐Yong , Kim In‐Shik
Published: November 2023

Two species of deer, Korean water deer (Hydropotes inermis argyropus) and Siberian roe deer (Capreolus pygargus), are the most popular wild deer in South Korea. Recently, it was reported that the Siberian roe deer living in Jeju island (Jeju roe deer) differed with the roe deer living in Korean peninsula (Inland roe deer) in morphology of body and mitochondrial gene. This study examined the morphological differences of forelimb skeletons among the 3 species of deer in South Korea. The skeletons were obtained from 23 carcasses of adult Korean water deer, 14 of Inland roe deer, and 13 of Jeju roe deer during last 10 years. The bone shapes were very similar each other, but the cranial border line scapula of Jeju roe deer was curved like ‘S’ while straight line in the other species. The lengths of skeleton of Inland roe deer were biggest among those of 3 species. Sometimes the first carpal bone was not observed in the both of Siberian roe deer, while it was always appeared in the Korean water deer. Very tiny sesamoid bone in the second and the fifth metacarpophalangeal joints were observed in two subspecies of Siberian roe deer. The numbers of proximal sesamoid bone in the joints were 1 or 2 in Inland roe deer while 0 or 1 in Jeju deer. These results suggest that the morphology of skeletons of forelimb was different in among 3 species deer, and, in addition, supported the argument that the Jeju roe deer is different subspecies with Inland deer.

Support or Funding Information

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (NRF‐2015018819).
Sternum Morphology and Locomotor Adaptation in Anthropoid Primates
Authors: Alwell Morgan Taylor , Middleton Emily R , Ward Carol V
Published: November 2023

Trunk shape varies among anthropoid primates and is thought to reflect locomotor adaptation. Hominoids, who are orthograde, below‐branch primates that rely on suspensory behaviors, have broader, deeper rib cages than do pronograde quadrupedal anthropoids. Apes have more cranially‐oriented glenohumeral joints than do humans, thought to orient the joint for use in suspensory postures during locomotion. The sternum represents a key part of the thoracic skeleton and contributes to overall rib cage morphology, yet variability in sternal shape across primates is undocumented.

In particular, hominoids should be expected to have a relatively broader manubrium and sternum than do monkeys. Further, apes should have more cranially‐oriented sterno‐clavicular joints than do humans, reflecting the higher position of the shoulder joint indicated by scapular morphology. In this study, we examined proportions of the manubrium and sternum in 199 anthropoid primates by taking linear measurements of the sternum from a series of calibrated digital photographs using the ImageJ software package. We also measured orientation of the sterno‐clavicular joint across hominoids.

Results indicate that, as expected, manubrium and sternum breadth are absolutely and relatively greater in hominoids than in both Old and New World monkeys, correlated with the transversely broader ribcages of hominoids. The semisuspensory New World monkeys Lagothrix and Ateles show similarities to hominoids in manubrial proportions, supporting a hypothesized link between thoracic breadth and suspensory locomotion. In terms of sterno‐clavicular orientation, humans have more laterally‐facing articular surfaces than most apes. Contrary to expectations, Symphalangus resembled humans in average angle of the sterno‐clavicular joint, suggesting a different configuration of the pectoral girdle in these apes that warrants further investigation. Overall, manubrial and sternal proportions appear related to locomotor adaptation in anthropoid primates, but relations between thoracic and pectoral morphology may be more varied than previously appreciated.
Havers‐Halberg Rhythms in Canis familiaris
Authors: Hogg Russell , Bromage Timothy , Hu Bin
Published: November 2023

Our objective in this study is to assess variation in Havers‐Halberg oscillation (HHO) periodicities among domestic dogs (Canis familiaris). The HHO is hypothesized to be a biological rhythm controlled by the hypothalamus, acting as a central coordinator of multiple life history variables including body size, metabolic rate, and reproductive output. The periodicity of this clock varies among and within species, but not within individuals. It has been studied in detail among primates, though very little among other mammals. In monkeys and apes, the periodicity is strongly correlated with body mass, but lemurs show a restricted range of variation in HHO rhythm despite having a comparable range of body sizes. This raises the question of how HHO rhythms and body size evolution can be decoupled. Domestic dogs represent a potential means to shed light on this area, due to their wide range of body sizes accompanied by low variation in life history variables such as lifespan. Based on this, we hypothesize that dogs will resemble lemurs in HHO variation.

To test this, we sectioned teeth of 11 previously deceased Canis familiaris from a university collection, representing a variety of breeds. Teeth preserve a histological marker of the HHO rhythm in the form of growth increments known as striae of Retzius, and the HHO rhythm can be quantified by counting the number of 24‐hour growth lines between them. Teeth were cleaned, embedded in acrylic resin, thin sectioned, and mounted to microscope slides following standard dental histology protocols. They were then imaged using circularly polarized light microscopy so that their growth increments could be measured to assess HHO periodicity. Next, HHO periodicity was regressed against body mass estimate data acquired via estimation from mandibular length measurements.

As predicted, domestic dogs have a low range of HHO periodicity variation for their body size range, falling between 4 and 6 days (with a modal value of 5). This suggests that dog body size variation is attributed to mechanisms beyond the HHO; conversely, the narrow range of variation in HHO can potentially explain the lack of variation in life history factors such as lifespan among dogs. Since body mass variation among domestic dogs is postulated to result from a mutation in IGF‐1, this provides a possible explanation for the high body mass / low HHO variation seen in lemurs as well.

Support or Funding Information

Project funded by the 2010 Max Planck Research Award, endowed by the German Federal Ministry of Education and Research to the Max Planck Society and the Alexander von Humboldt Foundation in respect of the Hard Tissue Research Program in Human Paleobiomics.
Prevalence, size, and shape of the accessory optic canal
Authors: Lambert H. Wayne , Cusick Abbey M , Zdilla Matthew J.
Published: November 2023

Anterior clinoidectomy is a neurosurgical procedure that is utilized to expose the cavernous sinus and access the clinoid segment of the internal carotid artery for management of paraclinoid aneurysms and tumors. Removal of the optic strut is oftentimes performed as part of anterior clinoidectomy in order to facilitate access to the region. However, the optic strut occasionally transmits the ophthalmic artery (OphA ) through an accessory optic canal (AOC), which predisposes the OphA to damage during surgery. Moreover, the AOC may be elusive in advanced imaging, masquerading as pneumatization of the optic strut or anterior clinoid process. Therefore, investigation of the prevalence, size, and shape of the AOC in dry bone specimens is warranted. This study analyzed optic canals and accessory optic canals (AOC) from 111 dry cranial specimens; however, 31 sides were either damaged or, in the case of hemisected skulls, absent. Therefore, a total 191 intact sides (97 right‐sided and 94 left‐sided, respectively) were analyzed. The AOC was present in 5 of the 97 (5.2%) right sides and 3 of the 94 (3.2) left sides for a total prevalence of 8 in 191 sides (4.2%). In addition to prevalence of AOC, parameters analyzed included area, perimeter, circularity, roundness, and solidity, aspect ratio, length of the major and minor axes of a best fit ellipse. An inability to recognize the presence of an AOC may lead to damage to the OphA during anterior clinoidectomy and subsequent blindness. Therefore, the information presented in this report may aid in identification of the AOC and prevent serious adverse surgical events.