bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationSupported 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 InformationThis 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 InformationInstitutional 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 InformationAlderson 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 InformationThis 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 InformationR01‐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 InformationNSERC 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

BackgroundOsteoarthritis 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.ObjectiveThis 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 therapyMethodsIntra‐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).ResultsHistological 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 therapyConclusionsMesenchymal 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 InformationSupported 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 InformationThis work was sponsored by National Institutes of Health/National Institute of General Medical Sciences grants R01 GM077138 and U01 GM111243.