bluuebunny/crossref_metadata_2025 huggingface dataset:
Therapeutically inhibiting macrophage necroptosis reduces inflammation‐driven atherosclerosis and promotes plaque stability
Authors: Karunakaran Denuja , Geoffrion Michele , Lee Richard , Wei Lihui , Gan Wei , Perisic Ljubica , Maegdefessel Lars , Hedin Ulf , Sad Subash , Guo Liang , Kolodgie Frank , Ruddy Terrence , Virmani Renu , Rayner Katey
Published: November 2023

Atherosclerosis is a maladaptive disease driven primarily by macrophages. As the atherosclerotic plaque progresses, inflammatory and macrophage cell death pathways drive the formation of complex, unstable and rupture‐prone lesions with large necrotic core(s). Necroptosis is an emerging cell death pathway involving RIP1, RIP3 and MLKL kinases that, in contrast to apoptosis, induces a pro‐inflammatory state. We hypothesize that inhibiting RIP1 and RIP3 reduces macrophage inflammation and necroptosis to reduce atherosclerosis. Oxidized LDL (oxLDL) induces necroptotic cell death in macrophages, which can be blocked by Nec‐1, a RIP1‐RIP3 inhibitor. Additionally, RIP3−/− macrophages are more resistant to oxLDL‐induced cell death. Treatment with oxLDL increased macrophage expression of necroptotic genes RIP1, RIP3 and MLKL through ROS‐dependent activation of the promoter region. ApoE−/− mice fed a western diet (WD) and simultaneously injected with 100mg/kg RIP1 anti‐sense oligonucleotides (ASO) for 8 weeks had a marked reduction in aortic lesion area compared to both control ASO‐ and PBS‐treated controls (58.8% reduction, p<0.001). In addition, serum inflammatory cytokine profiling suggested a marked reduction in circulating inflammatory cytokines (e.g. IL‐1α, IL‐3 and IL‐17A etc) in RIP1 ASO treated mice compared to control ASO. These results are consistent in vitro, where peritoneal macrophages treated with RIP1 ASO had a reduction in the gene expression of inflammatory genes (e.g. IL‐1α, TNFα) relative to control ASO. Next, in the same experimental model of established atherosclerosis (i.e. Apoe−/− mice fed a western diet for 6 weeks), therapeutic intervention with Nec‐1 these mice reduced lesion size (p<0.05) and increased markers of plaque instability, including reduced necrotic core formation (62.6% reduction, p<0.01). In addition, we developed a novel 123I‐Nec‐1 radiotracer that targets this pathway to specifically localize to and tightly correlate with atherosclerotic lesions. Lastly, in humans with unstable carotid atherosclerosis, expression of the necroptotic genes RIP1, RIP3 and MLKL is elevated and MLKL phosphorylation is detected in advanced atheromas, indicating that the necroptotic pathway is highly activated in vulnerable plaques. In conclusion, our findings offer molecular insight into the inflammatory and cell death mechanisms by which atherogenic ligands drives the formation of the vulnerable plaque and identify innovative therapeutic tools to treat atherosclerosis.
Mesenchymal Stem Cell Exosome Treatment Restores Lung Architecture and Ameliorates Pulmonary Hypertension Associated with Bronchopulmonary Dysplasia
Authors: Willis Gareth R , Fernandez‐Gonzalez Angeles , Vitali Sally H , Liu Xianlan , Mitsialis S Alex , Kourembanas Stella
Published: November 2023

Background

With no effective treatment to prevent or reverse bronchopulmonary dysplasia (BPD), a multifactorial chronic lung disease of preterm infants, the need for new therapies is urgent. Mesenchymal stem cell (MSC)‐based therapies have shown promise in numerous preclinical models of lung pathologies relevant to neonatology. We have shown that the therapeutic capacity of MSCs is comprised in their secretome, and our recent studies have demonstrated that the therapeutic vector is represented by the exosomes (extracellular vesicles; EVs) they release. Here, we aimed to purify and comprehensively characterize MSC‐EVs from both human bone marrow (BMSC) and umbilical cord Wharton's jelly (WJ) and investigate the efficacy of MSC‐EV treatments in BPD.

Methods

EVs in conditioned media were isolated by density on iodixanol gradient and characterized by electron microscopy, nanoparticle tracking analysis and western blot. Proteomic profiling was obtained by LC‐MS. Immunomodulatory capacity of MSC‐EVs was assessed by an in vitro macrophage polarization assay. Newborn FVB mice were exposed to hyperoxia (75% O2) for 7 days (postnatal day (PN) 1–7). At PN 4 mice were treated with a bolus intravenous dose of purified MSC‐EVs or human dermal fibroblast‐EVs (HDF‐EVs). After hyperoxic exposure the mice were placed in room air for one to five weeks. Histological analysis of lungs harvested at PN 14 and PN 42 was performed to assess alveolar injury, blood vessel number and vascular remodeling. At PN 42, right ventricular systolic pressure (RVSP) and right ventricular hypertrophy were assessed and pulmonary function tests were conducted.

Results

EVs immunoblots were positive for CD9, TSG101 and Alix. Proteomic profiling of WJMSC‐EVs and HDF‐EVs detected species unique or differentially enriched in MSC‐EVs. In vitro, addition of MSC‐EVs to alveolar macrophages (MH‐S cell line) significantly reduced the induction of M1 markers such as TNFα and CCL5. Hyperoxia‐exposed mice exhibited a significantly higher mean linear intercept compared to normoxic controls or to hyperoxia‐exposed mice treated with either BMSC‐EVs or WJMSC‐EVs, whereas HDF‐EV treatment had no beneficial effect on lung architecture. Hyperoxia‐exposed mice exhibited elevated RVSP compared to normoxic control mice, which was effectively reduced by treatment with BMSC‐EVs or WJMSC‐EVs.

Conclusion

We show that a bolus dose of purified human MSC‐EVs, from two different MSC sources effectively alleviates core features of hyperoxia‐induced BPD, drastically improving lung development and associated pulmonary hypertension, providing a novel platform for a new therapeutic intervention in newborn cardio‐respiratory diseases.

Support or Funding Information

This work was supported by NIH grant #RO1 HL085446 and United Therapeutics Sponsored Research
Maternal obesity programs offspring gut microbiome and liver disease in a sexually dimorphic manner
Authors: Wankhade Umesh D , Zhong Ying , Kang Ping , Chintapalli Sree V , Andres Aline , Thakali Keshari M , Shankar Kartik
Published: November 2023

Maternal obesity programs offspring weight gain and predisposition to metabolic syndrome. However, sexual differences in developmental programming are poorly understood. Here we employed a mouse model where female dams were fed either control (17% fat) or high‐fat diets (HFD, 45% fat calories) 12 wk prior to conception and then through weaning. Male and female offspring were provided ad libitum access to either control or HFD for 14 wk beginning at 4 wk of age, during which body weights were monitored weekly. At 18 wk of age hepatic histology was assessed and cecal contents were snap frozen for microbiome analysis. Male offspring gained more body weight than either control or HFD females (p<0.001). Two‐way ANOVA showed a significant effect of diet (p<0.01–0.001) and sex (p<0.001) when offspring were fed HFD. Hepatic histology also showed more severe fatty liver in male offspring relative to females. Pro‐inflammatory cytokines in serum such as IL1b, IL‐2, and IL‐6 were higher in male offspring fed HFD (p<0.05), whereas anti‐inflammatory IL‐5 was lower (p<0.001). Microbial community profiling via 16S rRNA amplicon sequencing showed that overall a‐diversity was lower in females (p<0.05) and in offspring from HFD dams (p<0.05). Two‐way ANOVA showed a significant effect of diet (p<0.001) and sex (p<0.001) on Bacteroidetes and Firmicutes when offspring were fed HFD. Enrichment of Firmicutes in males and Lactococcus, Anaerotruncus and Ruminococcus genera in female offspring were found using LefSe. When offspring were fed HFD, Bacteroidetes were lower and Firmicutes were higher in males (p<0.01). Multivariate association with linear models showed correlation between overall weight gain and abundance of families Lachnospiraceae and Clostridiaceae. Further, functional prediction using PiCRUST showed that bile acid synthesis, immune and bacterial response, protein digestion and cell cycle regulation pathways were enriched when offspring were fed HFD. Importantly, sex differences in functional categories were more pronounced by post‐weaning HFD. In conclusion, our findings suggest that prenatal HFD feeding alters hepatic steatosis and microbiome in offspring, and shows sexually dimorphic differences in response to post‐weaning HFD. Supported by USDA CRIS Project 6206‐51000‐010‐05S.

Support or Funding Information

USDA CRIS 6206‐51000‐010‐05S
High fat diet‐induced biliary lipoapoptosis, senescence, hepatic steatosis and fibrosis are reduced in secretin receptor knockout (SR−/−) mice
Authors: Kennedy Lindsey Leann , Wu Nan , Venter Julie , Meng Fanyin , Francis Heather , Zhou Tianhao , Glaser Shannon , Alpini Gianfranco
Published: November 2023

Background

Nonalcoholic fatty liver disease (NAFLD) is derived from simple steatosis, and can develop to nonalcoholic steatohepatitis (NASH) if left untreated. Recently, a cholestatic variant of NAFLD in patients has been identified as having portal inflammation and ductular reaction. Free fatty acids (FFAs) are increased in the serum of NAFLD patients and can induce lipoapoptosis. We have shown that the secretin/SR axis stimulates liver fibrosis, and it has been indicated that secretin can influence FFA uptake in adipocytes. Based on these data we aimed to evaluate the effect of SR−/− on biliary lipoapoptosis, senescence, hepatic steatosis and fibrosis following high fat diet (HFD) feeding.

Methods

WT and SR−/− mice were fed beginning at 4 weeks of age either control diet (CD) or HFD with a high fructose corn syrup equivalent for 20 weeks. Liver and serum were obtained, and cholangiocytes were isolated by laser capture microdissection. Hepatic steatosis and inflammation were evaluated by H&E and Oil Red O staining. Lipid metabolism was evaluated in total liver and cholangiocytes by qPCR for peroxisome proliferator activated receptor‐α (PPAR‐α), and the PPAR‐α‐dependent enzymatic genes carnitine palmitoyl‐transferase 1α (CPT1α) and long chain Acyl CoA synthetase (ACSL1). Lipoapoptosis was detected by TUNEL staining and qPCR for Bax in cholangiocytes. Senescence was examined by SA‐β‐gal staining in liver sections and qPCR for p18 and p21 in total liver and cholangiocytes. Liver fibrosis was measured by Sirius red staining and qPCR for TGF‐β1 and collagen‐1a in total liver. In vitro, control or shRNA‐SR transfected murine cholangiocytes were treated with 800 μM of FFAs (palmitate, stearate or oleate) added to FFA‐free media for 24 hr before evaluating lipoapoptosis and senescence.

Results

WT HFD mice had increased hepatic steatosis and inflammation, but this was significantly reduced in HFD SR−/− mice. Total liver and cholangiocytes from HFD WT mice had increased expression of PPAR‐α, CPT1α and ACSL1, but these lipogenesis markers were decreased in HFD SR−/− mice. Biliary lipoapoptosis, senescence and liver fibrosis were increased in HFD WT mice; however, these parameters were decreased in HFD SR−/− mice. In vitro, FFAs induced biliary lipoapoptosis and senescence gene expression in murine cholangiocytes, but this FFA‐induced injury was reduced in murine cholangiocytes transfected with shRNA‐SR.

Conclusion

Knockout of SR decreases PPAR‐α signaling in cholangiocytes, thereby decreasing lipogenesis. Furthermore, knockout of SR decreases HFD‐induced biliary lipoapoptosis, senescence, hepatic steatosis and fibrosis. Modulation of secretin/SR signaling may be a therapeutic option for patients suffering from NASH.
A novel hepatocyte SHP/CCL2 axis controls liver inflammation in NAFL to NASH transition
Authors: Magee Nancy S. , Zou An , Delker Don , Zhang Yuxia
Published: November 2023

Objective

Nonalcoholic fatty liver disease (NAFLD) is a burgeoning health problem worldwide, ranging from simple steatosis or non‐alcoholic fatty liver (NAFL) to a more aggressive non‐alcoholic steatohepatitis (NASH) with an increased mortality. Understanding what drives the transition from NAFL to NASH is essential to develop effective prophylactic and therapeutic strategies for NASH. Small heterodimer partner (SHP, Nr0b2) is a unique nuclear receptor important for bile acid, lipid, and glucose metabolism. The aim for this study is to explore a novel mechanism by which hepatocyte SHP regulates liver inflammation during NAFL transition to NASH.

Methods

Human liver samples from patients with simple steatosis or NASH were used to examine SHP expression. To produce a mouse model of NASH, a diet high in fat, cholesterol, and fructose (HFCF) was given to 2 month‐old male mice. Hepatocyte‐specific overexpression of SHP was achieved by tail vein injection with adeno‐associated virus 8 (AAV8) expressing SHP, driven by the alpha‐1‐antitrypsin (AAT) promoter (AAV8‐AAT‐SHP). On the other hand, hepatocyte‐specific SHP‐deficiency was induced by the administration of AAV8 expressing Cre recombinase driven by the thyroxine‐binding globulin (Tbg) promoter (AAV8‐Tbg‐Cre) on the 2 month‐old male Shpflox/flox mice. The extent of steatosis, inflammation, and fibrosis were evaluated in liver tissues. Mouse primary hepatocytes and macrophage‐like RAW 264.7 cells were included to study mechanisms.

Results

SHP was decreased in human patients with NASH and in the dietary‐induced mouse model of NASH, suggesting a biological relevance of SHP function in NASH pathogenesis. Increasing hepatic SHP level via AAV8 mediated gene delivery ameliorated diet‐induced NASH progression. In contrast, hepatocyte‐specific deletion of Shp in adult mice (Shp‐HKO) resulted in infiltration of macrophages and CD4+ T cells into the liver, which was further supported by the activation of inflammatory signaling pathways in Shp‐HKO as shown by RNA sequencing. As a result, hepatocyte‐specific Shp‐deficiency sensitized the liver to diet‐induced inflammation and fibrosis with an attenuation of steatosis. As a proof of concept, in vitro deletion of SHP in mouse primary hepatocytes increased the secretion of chemokine (C‐C motif) ligand 2 (CCL2). Most importantly, the conditioned medium from Shp‐deficient hepatocytes induced a pro‐inflammatory M1 polarization in RAW264.7 cells and promoted RAW264.7 cell migration. On a molecular level, SHP inhibited CCL2 expression through a direct inhibition of NFκB p65‐induced CCL2 transcription.

Conclusions

Our study uncovers a novel mechanism by which nuclear receptor SHP regulates hepatocyte and macrophage communication in the NAFL transition to NASH, which is mediated through inhibiting NFκB/CCL2 axis‐initiated macrophage activation.

Support or Funding Information

Grant support: NCI K22CA184146 and NIH P20 GM103549 to Yuxia Zhang.
Pro‐Inflammatory Monocyte Derived Macrophages Accumulate in Uninjured Aged Murine Livers
Authors: Stahl Elizabeth C , Brown Bryan N
Published: November 2023

In the United States, 30 million individuals suffer from chronic liver diseases, which present with increased severity and poor prognosis in the elderly population, in part due to dysfunction of the aged immune system. Immunosenescence describes the dysregulation of immune cell function with aging, which preferentially effects tissue‐resident macrophages compared to bone marrow derived macrophages. Recent findings suggest that liver macrophages can be divided into two F4/80+ subsets: CD68+ embryonically‐derived Kupffer cells and CD11b+ bone marrow derived cells, which exhibit specific cell functions. It is currently unclear how the macrophage populations residing in the liver are phenotypically and functionally impacted by aging, which may have implications for the onset and progression of inflammatory disease and liver pathology in elderly populations.

The aim of this study is to characterize the liver macrophage compartment from young (8–10 week) and aged (18–20 month) C57BL/6 wild‐type mice using immunohistochemistry and flow cytometry. First, we identified a significant increase in the total number of macrophages residing in aged livers. Preliminary in vitro studies showed that aged macrophages were primed towards a higher phagocytic capacity, regardless of M1 (LPS + IFN‐gamma) or M2 (IL‐4) stimuli, suggesting an inherent level of pro‐inflammatory activation with aging. In addition, the aged macrophage population contained a greater proportion of CD11b+ cells, suggesting an increase in bone marrow‐derived macrophages residing in the liver. To confirm this, we examined the liver macrophage profile of aged CCR2 knock‐out mice, which lack the receptor for monocyte chemoattractant protein‐1 (MCP‐1), an important signaling molecule for monocyte recruitment following injury. Interestingly, we found that mice deficient in CCR2 had a reversal in liver macrophage profile, from CD11b+ predominant to CD68+ predominant cell subsets, demonstrating that monocyte derived macrophages accumulate in the uninjured aged liver at least in part due to MCP‐1. Future studies will further characterize the functions of the CD11b and CD68 liver‐macrophages with aging, which will aid in the understanding of liver pathology onset and progression in elderly individuals.

Support or Funding Information

1. This work was partially supported by an NIH‐NIBIB training grant (T32‐EB001026) entitled “Cellular Approaches to Tissue Engineering and Regeneration” and a Pilot & Feasibility grant from the Pittsburgh Liver Research Center.
A New Role of Ronin (Thap11) in the Neural Crest and Craniofacial Development in the Mouse
Authors: Achilleos Annita , Tong Xuefei , Poché Ross A
Published: November 2023

The neural crest (NC) is a transient, multipotent, migratory cell population that is a hallmark of vertebrate evolution and gives rise to a diverse array of tissue types including sensory neurons and craniofacial skeleton. Development of the NC is a multistep process involving specification, epithelial to mesenchymal transition, migration and differentiation. Although a gene regulatory network that includes numerous transcription factors governing various stages of NC differentiation has been revealed over the last twenty years, a great deal is yet to be revealed regarding the factors essential for the lineage's multi‐potential nature in mammals. Ronin (Thap11) is a transcriptional regulator previously shown to be essential for maintaining the pluripotency of embryonic stem cells independently of the known pluripotency factors Sox2, Oct4, and Nanog. We therefore sought to investigate whether Ronin may play a role in maintaining the multipotency of the NC in a similar fashion. Here we report for the first time a conditional knock‐out of Ronin in the NC using the Cre recombinase driven by the Wnt1 promoter (termed Wnt1‐Cre+/tg;Roninflx/flx) in the mouse. Remarkably, Wnt1‐Cre+/tg;Roninflx/flx mouse embryos exhibit very severe craniofacial defects with almost all of the NC‐derived craniofacial skeleton missing. Interestingly, knock‐out of Ronin in a subset of the NC using the Pdgfrα‐Cre (Pdgfrα‐Cre+/tg;Roninflx/flx) shows a different and very unique phenotype. Pdgfrα‐Cre+/tg;Roninflx/flx mice are born and maintain a grossly normal phenotype at birth, but fail to ossify a large portion of their calvaria. To our surprise these mice, despite the extensive calvarial phenotype, do well in terms of early development indicating grossly normal brain function. Interestingly, this phenotype is reminiscent of a rare congenital disorder called acalvaria, regarding which the causative gene(s) and pathophysiology are still unknown. Moreover, Ronin has been implicated in a rare cobalamin‐deficiency disorder (termed cblX). Consistent with our findings, these patients present with craniofacial abnormalities. This is the first study to reveal a role of Ronin in the NC and craniofacial development and continued investigation using these recently generated Ronin conditional knock‐out models will be crucial in revealing the pathophysiology of the described rare birth defects as well as furthering our understanding of the mechanisms underlying the extensive multipotent developmental plasticity of the neural crest.
An Anatomical Basis for Cortical Crossmodal Plasticity
Authors: Meredith M. Alex , Lomber Stephen G.
Published: November 2023

When a major sensory system is lost, the remaining sensorymodalities functionally reorganize the vacated neural territories through a process termed “crossmodal plasticity.” The mechanisms underlying this effect have been assumed to involve the in growth of projections from novel sources, and/or the unmasking of existing but ‘silent’ connections. However, our recent experiments indicate that the anatomical basis for crossmodal plasticity after early‐deafness actually is a more complex process involving a pervasive maintenance of connections, laminar‐ and neuron‐dependent synaptic changes related toaxonal pruning/sprouting and cartographic changes that occur in a region‐specific basis.

Support or Funding Information

Supported by the National Institutes of Health Grant (NS‐39460; MAM), the Virginia Commonwealth University Presidential Research Initiatives Program (MAM) and the Canadian Institutes of Health Research (SGL).
Constitutive Cardiomyocyte Proliferation in the Postnatal Leopard Gecko (Eublepharis macularius)
Authors: Jacyniak Kathy , Vickaryous Matthew Kenneth
Published: November 2023

Although historically considered to be mitotically inactive, mammalian cardiac muscle cells (cardiomyocytes) are now widely viewed as being capable of at least low levels of homeostatic renewal throughout adulthood. Emerging evidence indicates that the extent to which cardiomyocytes can spontaneously re‐enter the cell cycle correlates with the capacity for cardiac self‐repair and regeneration. Therefore, proliferation rates may offer a predictive tool for investigating the regenerative potential of a species. Here we explore homeostatic cardiomyocyte proliferation in a representative reptile, the leopard gecko (Eublepharis macularius). The gecko heart is characterized by a trabeculated, spongy‐like ventricular lumen. This architecture is also seen in species capable of cardiac regeneration (such as zebrafish), as it provides an increased surface area for diffusion with a decreased reliance on coronary circulation. To assess cardiac cell proliferation, we employed a short duration bromodeoxyurdine (BrdU) pulse‐chase experiment (2 day pulse, 7 day chase), and immunostained for the S phase marker proliferating cell nuclear antigen (PCNA) and the mitotic marker phosphorylated histone H3 (pHH3). We determined that cardiac cells of the gecko heart continually proliferate, even into adulthood. Using double immunofluorescence, we then co‐localized the cardiomyocyte marker myosin heavy chain (MHC) with each of PCNA and pHH3. We found that ~10% of cardiomyocytes have entered the synthesis phase (MHC+/PCNA+), while ~0.5% are mitotically active (MHC+/pHH3+). Next, we performed a long duration BrdU pulse‐chase experiment (7 day pulse, 140 day chase). Unexpectedly, we observed long‐term label‐retaining (= slow cycling) cells throughout the ventricular myocardium. These data suggest that the gecko heart contains populations of both constitutively active and comparatively quiescent cells. Our results combined with a trabeculated ventricular architecture point towards the gecko as an excellent candidate to study cardiac self‐repair and regeneration.

Support or Funding Information

Natural Sciences and Engineering Research Council (NSERC) Discovery Grant 400358
Does Motivation or Learning‐Strategies Develop in a Flipped‐Classroom?
Authors: Day Leslie
Published: November 2023

Research previously conducted indicated lower achieving students prior to entering Gross Anatomy did significantly better in a flipped classroom than a control group in a traditional classroom. The question arose as to what affect the flipped‐classroom had on those students that resulted in the change of grade. The questions investigated in this study explored if motivational orientation and/or learning strategies of lower achieving students changed over the semester and if student's motivational orientation and/or learning strategies prior the course could predict student's success in a flipped‐classroom. This study analyzed pre‐ and post‐course motivational orientation and learning strategies of 98 students entering an upper‐level gross anatomy course. The data was compared against previous pre‐requisite course grades, overall GPA, Gross Anatomy course grades and a predicted model of success in the course. Results showed a significant change in many motivational subcategories (test anxiety, self‐efficacy and control belief) as well as learning strategies (peer‐learning, critical thinking) between successful and non‐successful students when compared to their predicted norm. The pedagogy of a flipped‐classroom allowed for students to gain confidence evident by motivational changes, improve study skills and critical thinking and overall performance in knowledge acquisition.
Free‐body analysis of the masticatory muscles in a caviomorph rodent, Cavia porcellus
Authors: Druzinsky Robert E. , Ulm Ashley , Vinyard Christopher J. , Iriarte‐Diaz Jose
Published: November 2023

Guinea pigs (Cavia porcellus) and other caviomorph rodents are hystricognaths. In hystricognaths, there is a groove for the pars reflexa of the superficial masseter in the ventral border of the mandible (Druzinsky, 2007). In the guinea pig (Cavia) the tendon of the pars reflexa has a dense connective tissue sesamoid that permits the tendon to glide through the groove. These South American hystricognaths are also hystricomorphs. The zygomatico‐mandibularis muscle originates on the snout and converges on a tendon that runs posteriorly through the infraorbital foramen. The tendon runs over the bony root of the zygomatic arch and turns inferiorly, to insert at the anterior end of the masseteric groove. A second sesamoid is found in this tendon, which allows the tendon to glide over the root of the zygomatic arch. Each of these elaborations of the masticatory muscles is a pinnate muscle that curves around a bony structure, acting as a pulley, and permitting the muscle to be longer than similar muscles in other mammals.

To analyze the mechanics of the caviomorph masticatory apparatus we are creating a 3D model of the skull, jaw, and masticatory muscles through contrast enhanced micro‐CT. The head of an adult guinea pig was soaked in a solution of called iodine potassium iodide (I2KI) for several weeks and then scanned at Northeast Ohio College of Medicine using the Scanco vivaCT micro‐CT. The Materialize Mimics® Innovation Suite will be used to segment the bones, teeth, and muscles to capture the detailed morphology.

Preliminary results of a free body analysis (see Reed, Iriarte‐Diaz, and Diekwisch, 2016 for methods) suggests that the pulley mechanisms of the masseter complex permit these muscles to be long, which allows them to have long excursions and produce significant forces at large gapes.
Spinal Cord Regeneration in the Leopard Gecko: Activation and Heterogeniety of Ependymal Layer Cells
Authors: Gilbert Emily Anne Beatrice , Vickaryous Matthew K
Published: November 2023

Among species capable of spinal cord regeneration, mounting evidence points towards a crucial role for a population of cells lining the central canal, herein referred to as ependymal layer cells (ELCs). In addition to their role in circulating cerebrospinal fluid, it is now understood that ELCs include a population of resident neural stem/progenitor cells (NSPCs) within the spinal cord. In taxa such as urodeles and zebrafish, ELCs are recruited in response to injury to replace lost or damaged neurons and support cells. Among other species capable of spontaneous spinal cord regeneration less is known. Here we characterized ELCs in the tail‐regenerating lizard Eublepharis macularius, the leopard gecko. As for many lizards, geckos are able to self‐detach their tail to avoid predation and then regenerate a replacement. The replacement tail includes a regenerated spinal cord with a relatively simple morphology: a monolayer of ELCs surrounded by nerve tracts. We hypothesized that ELCs of the original spinal cord include populations of NSPCs that are activated following tail loss. We conducted a detailed spatiotemporal characterization of ELCs before, during, and after tail regeneration. Prior to injury, ELCs express the hallmark NSPC marker SOX2. Following tail loss, ELCs in the original stump of the tail become highly proliferative and express an expanded panel of NSPC and lineage‐restricted progenitor cell markers, including Vimentin, MSI‐1, SOX9 and TUJ1. We also found that cells within the ependymal layer represent at least two distinct populations. Most ELCs are SOX2+, GFAP+, HuC/D−. We interpret these cells as ependymo‐radial glia. A second subset of ELCs are SOX2+, GFAP−, HuC/D+, which we identify as a neuronal‐like population of cerebrospinal fluid‐contacting cells. This complex assemblage of cells, in combination with the activation of an endogenous NSPC population may explain the remarkable regenerative abilities of the spinal cord in this species. We propose that spinal cord regeneration proceeds along a trajectory similar to development and that the completeness of the program varies across species. Ultimately, the degree with which a species is capable of progressing along the trajectory determines the degree of structural and functional fidelity is re‐established following injury.
Sonic hedgehog (Shh) in parotid saliva is a cell signaling moiety that acts as stem cells in taste buds to maintain normal taste function
Authors: Henkin Robert I , Abdelmeguid Mona
Published: November 2023

Taste bud growth and development in animals has been inhibited by the absence of Shh with destruction of taste bud architecture. Replacement of Shh reverses this effect and restores normal taste bud architecture and taste function. Vismodegib, a drug used to treat basal cell carcinoma, caused diminution of Shh secretion and caused taste loss in these patients. Drugs which inhibited Shh secretion in animals also caused taste loss. Because of our interest in taste function these results led us to hypothesize that Shh, a known cell signaling moiety, would be present in parotid saliva, the biological fluid which supports taste bud growth and development, and that its decreased secretion would be associated with taste loss (hypogeusia). To test this hypothesis we evaluated if Shh were present in parotid saliva in 27 normal subjects and in 81 patients with hypogeusia related to several etiologies. Results demonstrated that Shh was present in parotid saliva of normal subjects and that Shh levels in patients with hypogeusia of all etiologies were significantly lower than in normal subjects. These results are consistent with Shh acting as a taste bud growth factor consistent with our previous results in which specific salivary moieties act on stem cells in taste buds to develop growth and perpetuation of taste bud receptors. Without salivary Shh, taste bud receptor development in humans is inhibited, as it is in animals. Without salivary Shh, taste buds exhibit apoptosis which destroys taste bud architecture similar to results in animals. This is the first demonstration of Shh in human saliva and is consistent with this signaling moiety acting as a taste bud growth factor which maintains normal taste function by stem cell receptor activation with subsequent growth and development of taste bud receptors.

Support or Funding Information

There is no source of funding to declare.
Why mammalian cells respire?
Authors: Chandel Navdeep S.
Published: November 2023

For decades, the two major functions linked to the mitochondrial respiratory chain has been to replenish NAD or FAD thus allowing the TCA (Krebs) cycle to generate metabolites for the production of macromolecules and to generate a proton motive force for ATP production. We have elucidated that the mitochondrial respiratory chain has a third distinct role whereby it participates in cellular signaling pathways through the release of reactive oxygen species (ROS). Our work has implicated the necessity of mitochondrial ROS for multiple biological processes including hypoxic activation of HIFs, cellular differentiation, and T cell activation. Previously, the dogma in the field had been that mitochondrial ROS are only produced in pathological settings to cause both cellular and DNA damage. However, we have demonstrated that mitochondrial ROS are utilized as messengers to maintain normal biological and physiological functions. More recently, our work has shown how mitochondrial metabolites influence the epigenome. I will present our ongoing work on the mitochondria beyond their role in energy production to function as signaling organelles.

Support or Funding Information

This work is supported by AG04966, HL071643, and CA197532.
Dynamic regulation of DNA methylation
Authors: Zhu Bing
Published: November 2023

TET family enzymes successively oxidize 5‐methylcytosine (5mC) to 5‐hydroxymethylcytosine (5hmC), 5‐formylcytosine (5fC) and 5‐carboxylcytosine (5caC), which may lead to eventual demethylation. 5hmC and TET proteins occupy distinct chromatin regions, suggesting unknown mechanisms controlling the fate of 5hmC at different chromatin regions. We report the identification of 5hmC binding proteins displaying preferential association with 5hmC in vitro. In mouse ESCs, the majority of these 5hmC binding proteins localize at distal regulatory regions and their chromatin association is largely TET1 dependent. Interestingly, their binding sites are generally depleted for 5hmC but enriched for 5caC. Knockout of the 5hmC binding protein results in an ectopic accumulation of 5hmC at the original binding sites, which suggests its stimulatory role in facilitating further oxidation of 5hmC at distal regulatory regions. Moreover, at these sites, the main enzyme responsible for further oxidation of 5hmC appears to be TET2, suggesting a stepwise regulation process for 5mC oxidation.
Mitochondrial pathway of apoptosis
Authors: Wang Xiaodong
Published: November 2023

Our studies of programmed cell death in the last twenty years have taken into three interwoven directions. The first is to identify the molecular components and biochemical reactions of cell death programs; Secondly, to understand the physiological and pathological significance of programmed cell death; and finally, to derive therapeutic strategies based on the knowledge of programmed cell death to treat human diseases like cancer, autoimmune diseases, and degenerative diseases.

One of the most surprising findings of these studies is the role of mitochondria, the classical viewed powerhouse and metabolic center of eukaryotic cells, in programmed cell death in mammalian cells. Our laboratory discovered that cytochrome c, once released from the mitochondria, is able to serve as a protein ligand to trigger the activation of an apical apoptotic caspase, caspase‐9, through the formation of apoptosome. We also discovered a second mitochondrial protein Smac that neutralizes caspase inhibition imposed by the inhibitor of apoptotic proteins, IAPs, when comes to the cytosol during apoptosis. We collaborated with Yigong Shi's group to identify the N‐terminal four amino acid residues as the functional motif of Smac; and with Patrick Harran and Jef De Brabander to make a small molecule mimetic of Smac protein. The Smac mimetics not only are used as valuable research tools but different variants of the molecule are in human clinical trials for cancer.

The Smac mimetic led us to the finding that in some cell types, a caspase‐independent form of programmed cell death occurs in the presence of Smac mimetic plus caspase inhibitors in response to tumor necrosis factor family of cytokines or toll‐like receptor ligands. We subsequently worked out the core biochemical program for this form of cell death, also called necroptosis. The key signaling molecule of necroptosis is receptor interacting kinase 3, RIP3, which is activated upon induced interaction with a related kinase RIP1 or an adaptor protein TRIF, upon activation of TNF family of receptors or TLR3/4, respectively. RIP3 then phosphorylates and activates its substrate, a peudokinase MLKL, resulting in its oligomerization and translocation from the cytosol to intracellular and plasma membranes. Once on the membrane, MLKL, binds to negatively charged phospholipids and cause membrane disruption, leading to necrotic cell death. Using mouse model of RIP3 knockout and antibodies specifically recognizing the phosphorylated MLKL, necroptosis has been shown to be important for defending microbial infection and participating in tissue damage response. More recently, the data from our laboratory as well as others have shown that necroptosis is part of etiology of in several forms of degenerative disease models and aging.

In this up coming meeting, I will discuss the up‐to‐date progress from my laboratory on the molecular pathways of different forms of programmed cell death, small molecules modulating them, and potential therapeutic usages of these compounds.
Systems chemical biology: A novel approach to antibiotic discovery
Authors: Hung Deborah T
Published: November 2023

In this era of increasing antibiotic resistance, the antibiotic pipeline has failed to keep up with the need for new agents. While many factors contribute to this gap, the scientific challenges of discovering new antibiotics play prominently. Further, at one time, genomics promised to revolutionize the antibiotic discovery process; however, this promise has yet to come to fruition. We will present a novel strategy that marries chemical biology and genomics in the service of antibiotic discovery and demonstrate the power of such a strategy to identify new antibiotic candidates.
Deciphering the ZIP Codes of a Cell
Authors: Dahms Nancy M.
Published: November 2023

Lysosomes are acidified organelles capable of degrading a heterogeneous population of proteins, lipids, and glycans that are obtained from autophagic, endocytic, and phagocytic pathways. The activity of lysosomes depends upon a collection of over 60 soluble acid hydrolases that must be continually delivered to lysosomes from their site of synthesis in the endoplasmic reticulum. The acid hydrolases' N‐glycans undergo unique posttranslational modifications by two enzymes in the Golgi that results in the acquisition of phosphorylated mannose, either as a phosphomonoester (mannose 6‐phosphate, M6P) or a phosphodiester (M6P‐GlcNAc), and marks them for delivery to lysosomes by specific receptors, M6P receptors (MPRs). Failure to tag acid hydrolases with M6P results in developmental defects and the lysosomal storage diseases, mucolipidosis II and III. The phosphorylated N‐glycans are diverse in structure due to differences in their size, number of phosphorylated mannose residues (one or two), presence of M6P and/or M6P‐GlcNAc, and the location of the phosphomannosyl residues on the different branches of the N‐glycan. A subset of this diverse array of phosphorylated N‐glycans are recognized by two distinct MPRs, the ~46kDa cation‐dependent MPR (CD‐MPR) and the ~300kDa cation‐independent MPR (CI‐MPR), that carry acid hydrolases from the TGN to endosomal compartments where the acidic environment causes the MPRs to release their cargo. Additionally, cell surface internalization of exogenously supplied ligands, as occurs in enzyme replacement therapy for the treatment of lysosomal storage diseases, is dependent upon receptor‐mediated endocytosis carried out by the CI‐MPR. To determine how the MPRs carry their diverse cargo, glycan microarrays, solid phase binding assays using acid hydrolases, mutagenesis studies and structural biology of the MPRs are used to identify structural elements essential for the trafficking of acid hydrolases to lysosomes.

Support or Funding Information

NIH R01DK042267
Glycan‐Binding Proteins as Microbial Detectors
Authors: Kiessling Laura L.
Published: November 2023

Humans and other mammals live with microbial residents. A host must therefore distinguish between microorganisms that are benign (commensal), beneficial (mutualist/symbiont), or pathogenic. The mechanisms involved in surveillance of bacteria, fungi, viruses, and parasites have not been fully delineated, but lectins are emerging as important components. Indeed, the glycans displayed on mammalian cell surfaces can differ markedly from those on microbes. Microbial cell‐surface glycans therefore serve as a rich identification code that can be read by host lectins to make decisions about which microbes stay and which must go. To this end, we have examined the recognition properties of the intelectins, which are X‐type lectins that reside at mucosal barriers. This seminar will describe our recent results on intelectin recognition and on the properties of lectins that allow them to function as microbial detectors. We envision that an understanding of mucosal lectin recognition can lead to new strategies to combat pathogens and new approaches to regulate microbiome composition to promote human health.

Support or Funding Information

We thank the NIH (R01GM55984 and R01AI063596) for support.
Antibody glycosylation: an emerging biomarker of disease activity/protection
Authors: Alter Galit
Published: November 2023

Beyond their role in neutralization, antibodies mediate a broad array of functions including phagocytosis, cytotoxicity and maintaining immune homeostasis. This range of activities is controlled by two modifications to the constant domain of the antibody; genomic selection of isotype/subclass and post‐translational glycosylation. Mounting evidence suggests that antibody glycosylation changes rapidly and widely in the setting of infectious diseases, acting as a critical biomarker of disease activity. For example, agalactosylation is a marker of active human immunodeficiency virus (HIV), Mycobacterium tuberculosis (MTb) and Ebola virus infections. However, other sugars are controlled more tightly, such as fucose levels, which control antibody dependent cellular cytotoxicity. In fact, fucose levels are selectively reduced in the setting of “controlled” HIV and MTb infections, strongly arguing for different mechanisms of control of particular antibody glycan profiles. Moreover, vaccination studies demonstrate that antibody glycosylation can be actively harnessed and directed by distinct adjuvants, likely offering the humoral immune system a means to tune a broad range of antibody bio‐activities. Thus the emerging studies aimed at dissecting and defining the specific antibody glycovariants that emerge in the setting of controlled infectious diseases offers a unique opportunity to not only design more effective monoclonal therapeutics but also to define the mechanism(s) underlying pathogen control and a path to the development of next generation vaccines that may harness the post‐translational tuning of antibody effector function.
Regulation of manganese homeostasis and detoxification by the efflux transporter SLC30A10
Authors: Mukhopadhyay Somshuvra
Published: November 2023

Homozygous loss‐of‐function mutations in SLC30A10 induce familial manganese‐induced parkinsonism. We previously demonstrated that the wild‐type SLC30A10 protein functions as a cell surface‐localized manganese efflux transporter that transports manganese from the cytosol to the cell exterior, reduces intracellular manganese levels and protects against manganese‐induced neurotoxicity. In contrast, disease‐causing mutations block the intracellular trafficking and manganese efflux function of SLC30A10. I will summarize the above published findings and then present three short stories from projects that we are currently pursuing. The first project is a structure‐function study that aims to determine the mechanism of manganese transport via SLC30A10; current data suggest that the mechanism of ion coordination in SLC30A10 may be fundamentally different from related transporters in its superfamily. The second project is designed to understand the mechanism by which manganese regulates SLC30A10 activity; our studies show that increased intracellular manganese enhances SLC30A10 transcription in a metal response element‐independent manner. Finally, the third project is a mouse study that focuses on elucidating the mechanisms by which loss of SLC30A10 function at the cellular level leads to the onset of parkinsonism; for this project, I will present phenotypic characterization of full‐body and organ‐specific SLC30A10 knock‐out mice that we generated.

Support or Funding Information

Supported by NIH/NIEHS grants R00‐ES020844 and R01‐ES024812 (S.M.)
Metabolomics and Lipidomics Analysis of the NIA CALERIE Trial
Authors: Kristal Bruce S , Gathungu Rose M , Bird Susan S , Marur Vasant R , Sniatynski Matthew J , Sheldon Diane P ,   CALERIE Clinical Trial Consortium
Published: November 2023

Calorie Restriction (CR) is the most potent, robust, and reproducible known means of extending longevity and decreasing morbidity in a vast range of animal species short of primates. Despite 80 years of research, the relevance of this observation for humans remains unknown. Relevance is supported by the established link between obesity and morbidity in humans, and the health status of individuals voluntarily participating in a caloric restricted lifestyle. Other potential linkages are being directly studied in the NIA/NIH‐sponsored CALERIE (Comprehensive Assessment of Long‐term Effects of Reducing Intake of Energy) clinical trial. CALERIE enrolled 238 individuals and collected blood samples at 6 time points over 24 months. We previously examined metabolomics (via HPLC separation with coulometric electrode array‐based detection) and lipidomics (via high resolution LC‐MS‐based lipidomics) effects of the CALERIE intervention and compared these data to those previously obtained in studies of ad libitum fed and calorically restricted FBFN1 rats (males/females, different ages and extents and durations of CR). The results were, surprisingly, nearly completely negative. Retrospective Post‐Hoc analyses identified three potential reasons for these negative findings. First the original trial and our analyses intended to strictly follow trial design (i.e., intention to treat analysis), were powered for a 25% drop in caloric intake – a larger and more consistent goal than was obtained. Second, many individuals on the low calorie diet intervention lost weight in year one but gained in year two – a shift in caloric balance for which our analyses initially were, again in accordance with an intent‐to‐treat design, unprepared to compensate. Third, and most important, we determined that exposure of the forearm to high temperatures prior to some phlebotomy (termed “hot‐box”) time points — which was essential to assess one listed secondary clinical outcome of the trial — destroyed self‐similarity for our measures, rendering the baseline sample unusable for our standard normalization and control methods. Correcting for this revealed some of the expected biological/biochemical differences and provided a potentially interesting signal related to altered stress response in relationship to some aspect of caloric intake, obesity, and/or energy expenditure, consistent with the cognate literature in animal models of normal and delayed aging. We will discuss the results, the implication for metabolomics and lipidomics studies, and initial, promising re‐analysis of these CALERIE data.

Support or Funding Information

NIA/NIH