bluuebunny/crossref_metadata_2025 huggingface dataset:
Endothelial Follistatin‐like 1 Regulates The Maturation of The Pulmonary Vasculature by Modulating BMP/Smad Signaling
Authors: Tania Navessa Padma , Maarsingh Harm , Bos Sophie T , Mattiotti Andrea , Prakash Stuti , Timens Wim , Schmidt Martina , van den Hoff Maurice , Gosens Reinoud
Published: November 2023

Pulmonary arterial hypertension (PAH) is a progressive disease that is characterized by vascular remodeling and sustained vasoconstriction which consequently lead to high blood pressure in the pulmonary vasculature and right ventricle remodeling. Altered bone morphogenetic protein (BMP) signaling has been implicated in the pathogenesis of PAH as well as in pulmonary vascular maturation. The endogenous BMP antagonist follistatin‐like 1 (Fstl1) is highly expressed in pulmonary vascular endothelium of the developing lung in mice, suggesting a role in pulmonary vessel formation. In this study, the role of Fstl1 in the maturation of the pulmonary vasculature was investigated using Tie2‐cre mediated endothelial‐specific Fstl1 knockout mice. To this end, Fstl1fl/fl mice were bred with Fstl1WT/KO Tie2‐cre mice to generate Fstl1KO/fl Tie2‐cre mice and control littermates Fstl1WT/fl Tie2‐cre, Fstl1KO/fl, and Fstl1WT/fl. Heart and lung tissue was collected from pups at 1 and 3 weeks after birth. The percentage of actin‐positive small vessels per total number of vessels was determined. The expression level of Fstl1 and BMP/Smad‐regulated genes was determined in total RNA isolated from lungs using qPCR. Activation of BMP signaling was investigated using western blot. In wildtype mice, the mRNA expression of Fstl1 was higher in lung tissue at 1 week than 3 weeks (p<0.005). In line with the idea that Fstl1 is a BMP inhibitor, BMP signaling was more active at 3 weeks than 1 week, reflected by the higher level of pSmad1/5/8 at 3 weeks than 1 week. Moreover, the mRNA levels of the BMP/Smad‐regulated genes JAG1 (p<0.05) and endothelin‐1 (p<0.005) were increased at 3 weeks compared to 1 week after birth. Endothelial‐specific deletion of Fstl1 was postnatally lethal, approx. 70% of Fstl KO/fl Tie2‐cre mice died at 3 weeks after birth. Though most were found dead, some were found in respiratory distress prior to dying. In these mice, Fstl1 mRNA expression was reduced (p<0.01) compared to wildtype mice at 1 week. Endothelial deletion of Fstl1 resulted in activation of Smad1/5/8 signaling and increased BMP/Smad‐regulated gene expression of JAG1 (p<0.05), endoglin (p<0.05), and endothelin‐1 (p<0.05) at 1 week. At 3 weeks, JAG1 was reduced (p<0.01) whereas endoglin and endothelin‐1 were unchanged. Furthermore, the percentage of actin‐positive small pulmonary vessels was increased at 3 weeks old of Fstl1KO/fl Tie2‐cre mice compare to wildtype (p<0.005) whereas it was unaltered at 1 week. Loss of endothelial Fstl1 induces activation of BMP‐mediated gene expression and microvasculature remodeling, which presumably due to alteration in endothelin‐1 expression.

Support or Funding Information

This study was supported by a grant from the Netherlands Lung Foundation (grant number: 3.2.12.083)
Nucleoside reverse transcriptase inhibitors attenuate angiogenesis and lymphangiogenesis by impairing receptor tyrosine kinases signaling in endothelial cells.
Authors: Song Lin , Zhu Xiaolong , Qiu Cong , Qin Lingfeng , Chow Samson A , Yu Luyang
Published: November 2023

The successful roll‐out of antiretroviral therapy (ART) makes clinical challenges now confronting HIV‐positive patients shift significantly from acquired immunodeficiency syndromes (AIDS) to chronic noncommunicable diseases, especially cardiovascular defects. Previous reports indicated that antiretroviral therapy exacerbated arteriosclerosis by inducing dysfunction of aortic endothelial cells, while HIV‐protease inhibitor administration was associated with regression of Kaposi sarcoma by impairing tumor angiogenesis. However, the role of ART in regular blood vessel growth is unknown. In the present study, we used an integral component of ART, nucleoside reverse transcriptase inhibitors (NRTIs), to investigate their effects on major microvascular function including angiogenesis and lymphangiogenesis. Here we demonstrated that pharmaceutical dose of NRTIs (AZT, TDF and 3TC) can inhibit angiogenesis and lymphangiogenesis both in vivo and in vitro via affecting the migration but not proliferation of endothelial cells. Correspondingly, NRTIs treatment attenuated VEGFR2 and FGFR1 signaling, classical angiogenic pathways, in vascular endothelial cells and VEGFR3 signaling, classical lymphangiogenic pathway, in lymphatic endothelial cells, both in the intensity and sensitivity. However, NRTIs had little effect on endothelial Notch1 pathway, the other kind of membrane receptor signaling. Taken together, it implied a special role of NRTIs in regulating receptor tyrosine kinase (RTK) in endothelial cells. In regard to the inhibitory mechanisms targeting the RTK pathways, a special capability of AZT was observed for blocking glycosylation of these RTKs. On the other hand, both TDF and 3TC significantly restrained RTKs endocytosis into early endosome while do not affect their internalization. Furthermore, NRTI induced excessive mitoROS resulted from endothelial mitochondrial dysfunction. Reciprocally, MnSOD, mitoROS scavenger, rescued both defects of RTK signaling and angiogenic or lymphangiogenic function in microvascular endothelial cells treated with NRTIs. Therefore, we conclude that NRTIs negatively regulate angiogenesis and lymphangiogenesis by inducing vicious mitoROS generation, and subsequently impairing RTK signaling in endothelial cells.

Support or Funding Information

National Natural Science Foundation of China (Grant No. 81270357, 81422005 and 31470057), Zhejiang Provincial Natural Science Foundation of China (Grant No. LR14H020002)
Central Role for Intestinal Epithelial IL‐10R1 Signaling in Barrier Restitution
Authors: Lanis Jordi M. , Alexeev Erica E. , Kao Daniel J. , Kitzenberg David A. , Schwisow Kayla D. , Kominsky Douglas J. , Colgan Sean P.
Published: November 2023

IL‐10 is a potent anti‐inflammatory cytokine that inhibits the production of numerous pro‐inflammatory mediators in a number of cell types. Studies in animal models and in humans have demonstrated a critical role of IL‐10 signaling in a broad range of chronic inflammatory conditions, including inflammatory bowel disease. Our recent studies identified a prominent role for intestinal epithelial cell (IEC) IL‐10 signaling in inflammatory resolution during colitis. At present, little is known about how IL‐10 signaling interfaces with epithelial function in health and during inflammation. Based on our previous studies implicating enhanced restitution of epithelial monolayers in response to IL‐10, we hypothesized that IL‐10 signaling in IECs influences epithelial barrier maintenance and healing. Initial studies in IEC cell lines revealed that lentiviral‐mediated knockdown of IL‐10R1 resulted in significant defects in the formation of epithelial barrier function, measured by transepithelial resistance and paracellular flux assays (p<0.01). Studies in vivo using conditional IEC IL‐10R1 knockout mice identified a baseline defect in barrier function as evidenced by increased FITC‐dextran permeability with concomitant increases associated with ongoing inflammation (e.g. DSS colitis). Ongoing studies to identify regulatory mechanisms of IEC IL‐10R1 revealed a prominent role of the aryl hydrocarbon receptor (AHR) in induction of IL‐10R1 during inflammation. Parallel studies in AHR knockdown cell lines revealed barrier defects similar to the loss of IL‐10R1. We next utilized a scratch wound assay to define epithelial restitution and the role of this AHR‐IL‐10R1 axis. Induction of IL‐10R1 via the AHR ligand FICZ significantly increased IL‐10‐dependent wound healing. This response was abolished in cells lacking either IL‐10R1 or AHR. Together, these results provide compelling evidence on the importance of IL‐10 signaling in intestinal epithelial barrier function and restitution following injury.

Support or Funding Information

This work was supported by NIH grants DK099452, DK095491, DK50189, and by the Crohn's and Colitis Foundation of America.
Intestinal Epithelial Cell Expression of CD47 Facilitates Proliferation, Neutrophil Transmigration, and Wound Healing in vivo
Authors: Reed Michelle , Luissint Anny‐Claude , Nusrat Asma , Parkos Charles A
Published: November 2023

The cell membrane glycoprotein CD47 is a ubiquitously expressed ‘self’ antigen that is known to facilitate a wide variety of interactions involving hematopoietic cells depending on the cell type and context. Specifically, CD47 has been reported to regulate the induction of apoptosis, leukocyte transmigration, and non‐classical recognition/phagocytosis of cells by macrophages. Despite mounting evidence on CD47 function in leukocytes, comparatively little is known about the function of CD47 expression by non‐hematopoietic cells. Previous studies suggest that epithelial expression of CD47 is important for rapid neutrophil transepithelial migration in response to chemotactic agent sin vitro, yet the function of epithelial expressed CD47 has not been explored in vivo.

In this study, we utilized both epithelial‐targeted (Villin‐CreCD47f/f) and tamoxifen‐inducible epithelial‐targeted (Villin‐Cre‐ERT2CD47f/f) CD47‐deficient transgenic mice to assess the function of CD47 in the intestinal epithelium in vivo, under steady‐state conditions and during intestinal inflammation, respectively. While gross intestinal mucosal architecture and barrier integrity of Villin‐Cre CD47f/fmice was not altered under steady state conditions in vivo, CD47‐deficient crypt epithelial cells displayed decreased proliferation, as evidenced by reduced EdU uptake. Similarly, acute loss of intestinal epithelial CD47 expression in Villin‐Cre‐ERT2 CD47f/fmice produced no increase in intestinal permeability or symptoms of colitis at one month post‐tamoxifen treatment. Consistent with our previous in vitro findings of delayed neutrophil transmigration upon antibody‐mediated blockade of epithelial CD47, induced loss of intestinal epithelial CD47 expression in Villin‐Cre‐ERT2 CD47f/fmice impaired neutrophil recruitment into the intestinal lumen in response to the chemokine KC in vivo. Finally, in keeping with diminished epithelial proliferation and neutrophil infiltration observed in epithelial‐targeted CD47 deficient mice, both Villin‐Cre CD47f/fmice and tamoxifen‐treated Villin‐Cre‐ERT2 CD47f/f mice displayed increased disease activity and mucosal damage in an injury induced (DSS) model of chronic colitis.

These findings provide novel insights into the function ofCD47 expression by non‐hematopoietic cells that involves maintenance of normal epithelial cell proliferation and turnover, while enabling rapid recruitment of neutrophils under inflammatory conditions. Furthermore, given the current appreciation of CD47 as a non‐classical regulator of “don't eat me” signals such that cells deficient in CD47 are cleared by splenic macrophages, our observations that mice harboring epithelial‐targeted CD47 deficiency are healthy indicate that additional regulatory mechanisms mediate CD47 function.

Support or Funding Information

This work was funded by the National Institutes of Health grant R01DK079392.
Cortactin is Required to Maintain Intestinal Epithelial Barrier Homeostasis
Authors: Citalán‐Madrid Alí Francisco , García‐Ponce Alexander , Vargas‐Robles Hilda , Betanzos Abigail , Nava Porfirio , Rottner Klemens , Menningen Rudolf , Schnoor Michael
Published: November 2023

A stable intestinal epithelium is the basis for healthy intestines. Epithelial dysfunction causes increased intestinal permeability and bacterial translocation that may eventually cause an aberrant immune response as seen in chronic inflammatory diseases of the intestines such as ulcerative colitis (UC) and Crohn disease (CD). The actin cytoskeleton regulates epithelial barrier integrity through tight junction and adherens junction molecules, but the role of actin regulators such as cortactin (CTTN) is not well understood. Analyzing CTTN‐KO mice, we found that these animals have increased basal intestinal permeability similar to what has been demonstrated in the vasculature. Furthermore, CTTN‐KO mice were more susceptible to DSS‐induced experimental colitis with animals dying already after 5 days of DSS treatment. Histopathological analysis of CTTN‐KO colons revealed differences in the intestinal epithelial structure such as hypoplasia, edema, hemorrhage and infiltration already under basal conditions which it worsened after experimental colitis. Immunofluorescence staining of colon tissues of mice demonstrated a reduced expression and localization at junctions of ZO‐1 and E‐cadherin and intestinal epithelial crypt disassembly. Using a stable CTTN‐depleted Caco‐2 cell line, we confirmed epithelial dysfunction including reduced transepithelial electrical resistance, increased paracellular flux of 4 kDa FITC‐dextran and weaker cortical actin rings. This was accompanied by a decreased expression and internalization of ZO‐1, occludin, claudin‐1 and E‐cadherin; while claudin‐4 and b‐catenin remained unchanged. Interestingly, expression of the pore‐forming claudin‐2 was increased and neutrophils transmigrated more rapidly. While CTTN depletion did not cause increased apoptosis, we observed increased proliferation and stress fiber formation that may be responsible for the observed barrier dysfunction. Importantly, CTTN localized at cell contacts in colon biopsies of healthy individuals, whereas in IBD patients we observed strong translocation of CTTN into the cytosol and loss of colocalization with actin and ZO‐1. Our data imply that CTTN controls intestinal barrier integrity by controlling actin cytoskeleton and intercellular junction functionality causing epithelial dysfunction as seen in IBD.

Support or Funding Information

This work was supported by grants and a Ph.D stipend from the Mexican Council for Science and Technology (333396 to AFCM & 179895 to MS and a special fund PNPC.2013‐2014)
Nebulized delivery of the MAPKAP kinase 2 peptide inhibitor MMI‐0100 protects against ischemia‐induced systolic dysfunction
Authors: Brown David , Cooley Brian , Quintana Megan , Landers Cynthia , Willis Monte S
Published: November 2023

Acute myocardial infarction (AMI) results in systolic dysfunction, myocarditis and fibrotic remodeling, which causes irreversible pathological remodeling of the heart. Associated cell death and inflammation cause cytokine release, which activates the p38 MAPK signaling pathway to propagate damaging signals via MAPKAP kinase 2 (MK2). Previously we showed that intraperitoneal injection of a cell permeable peptide inhibitor of MK2, MMI‐0100, protects against fibrosis, apoptosis and systolic dysfunction in a mouse model of AMI, left‐anterior descending coronary artery (LAD) ligation. Here we tested a new route of administration of MMI‐0100: inhalation of nebulized peptide. When given within 30 minutes of AMI and daily for 2 weeks thereafter, both inhaled and injected MMI‐0100 improved cardiac function as measured by conscious echocardiography. Limited fibrosis was observed after two weeks by Massons trichrome staining, suggesting that MMI‐0100 protects the heart prior to the formation of significant fibrosis. These results support a nebulized route of administration of MMI‐0100 can protect the myocardium from ischemic damage.

Support or Funding Information

This work was supported by the National Institutes of Health (R01HL104129 to M.W.), a Fellowship from the Jefferson‐Pilot Corporation (to M.W.), and the Leducq Foundation (to M.W.).
The Role of the Duffy Antigen Receptor for Chemokines in Metabolic Disease
Authors: Benson Tyler Wendorf , Chatterjee Tapan , Joseph Joel , Popoola Orishebawo , Crowe Matthew , Yiew Nicole , Unruh Dusten , Weintraub Daniel , Pillai Ajay , Williams Jo , Mintz James , Stansfield Brian , Stepp David , Brittain Julia , Bogdanov Vladimir , Weintraub Neal
Published: November 2023

Inflammation in adipose tissues contributes to obesity‐related insulin resistance and metabolic disease, a problem with greater prevalence in African, Asian, and Hispanic populations. The Duffy Antigen Receptor for Chemokines (DARC) is a promiscuous non‐signaling receptor expressed primarily on erythrocytes and classically thought to modulate inflammation by binding chemokines such as MCP‐1 to buffer tissue inflammation and act as a chemokine reservoir. DARC allele variants protect against malaria and are prevalent in African, Asian, and Hispanic populations. We hypothesized that genetic deletion of DARC would augment adipose tissue inflammation and glucose intolerance in the setting of diet‐induced obesity.

Eight week‐old male Wild Type (WT) and DARC−/− mice (C57BL/6 background) were maintained either on chow diet or switched to high fat diet (HFD, 60% calories from fat) for up to 24 weeks. Thereafter, metabolic phenotyping was performed, mice were subsequently euthanized, and tissues and blood were collected for analysis. Bone marrow (BM) transplants were performed by irradiating eight week old WT mice and injecting BM harvested from either DARC−/− (to eliminate BM‐derived DARC) or WT mice (control).

Food intake, energy expenditure and locomotion were similar between WT and DARC−/− mice, leading to equivalent body‐weight gain and adipose tissue mass. Compared to WT mice, HFD‐fed DARC−/− mice exhibited lower plasma levels of the DARC‐bound cytokines MCP‐1, CCL11 and CXCL5, but higher adipose tissue MCP‐1 mRNA expression, more adipose tissue macrophages, and significantly larger adipocytes in the visceral adipose depots, implying enhanced adipose tissue inflammation. In addition, glucose tolerance and insulin sensitivity were significantly impaired in DARC−/− mice compared to WT mice on HFD. CD DARC−/− mice did not display increased adipose inflammation; however, they were less sensitive to insulin, exhibited larger subcutaneous adipocytes, and had higher levels of leptin mRNA and AKT phosphorylation (SER473) upon insulin stimulation when compared to WT mice. Mice reconstituted with DARC −/− BM and fed a HFD did not exhibit glucose intolerance or insulin resistance compared to mice with WT BM. Notably, we detected high levels of DARC mRNA in subcutaneous and visceral (but not brown) fat depots, and DARC protein immunoreactivity was detected in subcutaneous fat from WT but not DARC −/− mice.

We conclude that loss of DARC enhances HFD‐induced adipose tissue inflammation and metabolic disease. However, the mechanism appears to be independent of its expression on erythrocytes and its classic role as a chemokine buffer‐sink. We report for the first time that DARC is expressed on adipocytes, where it may regulate insulin signaling.
Height, Weight and Body Mass Index in low‐ and middle‐high‐income urban preschoolers in the Western Highlands of Guatemala
Authors: García‐Meza María Rosario , Doak Colleen M. , Beintema Joni Jade Serena , Orozco Mónica N. , Gwaltney Rebecca , Rolker Heike B. , Solomons Noel W
Published: November 2023

Background

Overweight and obesity prevalences are emerging rapidly among children in low‐ and middle‐income countries, especially in urban environments. In this population of urban Guatemala, chronic undernutrition is prevalent in low Socio‐Economic Status pre‐schoolers (46%) but only 6% in the higher SES children. Little is known about overweight and obesity in urban pre‐schoolers from high and low SES settings.

Objective

To compare height, weight and BMI by socio‐geographical location in two urban settings in the Western Highlands of Guatemala.

Methods

A cross‐sectional study collected data from private and public urban preschool centres of Quetzaltenango and Sololá, respectively. Informed consent was attained from the parents, and a total of 249 children, 4–7 years old, were measured. The contrast in socioeconomic status (SES) was captured by selecting public pre‐schools from a low socio‐economic area of Sololá and private pre‐schools from the relatively affluent Quetzaltenango. WHO growth references (≥5y) and standards (<5y) were used to calculate BMI‐for‐Age z‐scores. Age appropriate definitions were used, with overweight and obesity for children < 5y defined as ≥ 1 SD WHO BMI‐for‐Age Z score and for > 5y defined as ≥ 2 SD WHO BMI‐for‐Age Z score).

Results

Children from the high SES public private were on average 6.7 cm taller and 3.2 kg heavier than children from the low SES public preschools. In the youngest age group, the higher SES children had high prevalent overweight (9.6%) whereas the lower SES children had virtually no overweight (0.9%) in the same age group. The same pattern was seen in the >5y children, with 20.9% overweight in the high SES children and less than half that prevalence (9.6%) low SES children of the same age.

Conclusion

In this population of urban Guatemala, preschool children from a high SES environment are taller and heavier in comparison to their low‐SES counterparts. These results show a growing public health concern of overweight and obesity in higher SES preschool children.
Parenting Behaviors and Weight Status of Junior High School Students in China
Authors: Tian Zhengqi , Wen Ming , Wang Weidong , Xue Hong , Min Jungwon , Wang Youfa
Published: November 2023

Background

The influence of parenting behaviors on children's weight status is evident based on research in western world. However, in developing countries under dramatic economic and social changes, such as China, the parenting behaviors and related effects on Children's BMI and weight status remain understudied.

Aims

This study aimed to assess the relations between parenting and junior high school students’ weight status in contemporary China.

Methods

We used nationally representative baseline wave data from the China Education Panel Survey: the Junior High Cohorts, which was collected during 2013–2014, using stratified, multistage probability proportional to size sampling among all county level units (N=2,870) in China by the National Survey Research Center at Renmin University in China. Data on parenting and adolescent development was collected from students (N=19487) in 112 schools and from their parents (N=19007). Parenting was measured by parents’ answers to questions about their attitude and specific parenting behaviors interacting with their children; parenting was classified according to Baumrind's parenting typology as authoritative, authoritarian, permissive and neglecting; behaviors regarding family rules were categorized into strict and non‐strict. Child BMI was calculated using self‐reported weight and height. Association between parenting behaviors and child BMI and risk of overweight and obesity (defined based on the IOTF BMI reference) were studied using mixed models adjusting for age, sex, region, parental education level, birth weight and if the child being the only child in the family. This analysis included 18769 subjects (student–parent pairs).

Results

The prevalence of overweight and obesity among the students was 12.5% with large sex difference, 16.7% in boys and 8.0% in girls. Compared to children of authoritarian parents (5.6%), children of authoritative parents (85.54%) had a lower BMI (18.91±3.36 vs. 18.96±3.65, p=0.7) and lower risk of being overweight (OR: 0.86, 95% CI: 0.69,1.07). Child BMI differs significantly by family rules on Internet use (strict vs. non‐strict rule: 18.83±0.06 vs. 19.15±0.09, p<0.001). According to the estimates from mixed linear and logistic models after adjusting for age, parental education and household income level, birth weight and if the subject is the only child, parenting styles did not show significant association with child BMI.

Conclusions

Among parents of junior high students in China, authoritative parenting is the predominant style. Parenting behaviors such as imposing rules on child Internet use reflecting strictness of parenting is significantly and negatively associated with child BMI.

Support or Funding Information

Funding: NIH U54 center grant, HD070725.
Early Interventions: Promoting Success to Prevent Remediation
Authors: Schutte Audra , Kalb Elizabeth
Published: November 2023

Remediation in medical education is a widely discussed topic with limited research to support the best approaches to successfully remediate medical students in basic science courses. In the present study, two faculty‐led sessions were held with first year medical students in the first half of the fall semester in an attempt to aid students with their learning strategies. The first session was held during the second week of the semester, and attendance was voluntary. It discussed study strategies that current evidence suggests are beneficial. The second session was held during the week following the first set of examinations. Attendance was required for any student who failed an exam; students who did not fail an exam were still invited to attend the session. A few days prior to the second session students voluntarily completed a short post‐exam survey, consisting of five open‐ended questions, which was utilized to shape the second study strategies session.

In this study a thematic analysis of survey results was conducted to generate a codebook of student concerns and misconceptions following their first round of exams. The codebook allowed for quantification of these trends in student comments. Additionally, aggregate remediation trends in fall first year basic science courses from the past several years were analyzed. These remediation trends were compared to the current academic year to determine the effectiveness of these intervention sessions. As the school of medicine prepares to implement a new integrated curriculum in the 2016–2017 academic year, continued data collection and analysis will be utilized to shape future interventions.
The Methyl Donor Betaine Prevents Congenital Defects Induced by Prenatal Alcohol Exposure
Authors: Watanabe Michiko , Karunamuni Ganga , Doughman Yong Qiu , Sheehan Megan M , Ma Pei , Peterson Lindsy M , Linask Kersti K , Jenkins Michael W , Rollins Andrew M
Published: November 2023

Over 500,000 women per year in the USA drink during pregnancy, and 1 in 5 of them binge drink. 20–50% of live‐born children with prenatal alcohol exposure (PAE) present with congenital heart defects including outflow and valvuloseptal anomalies that can be life‐threatening. We established a model of PAE (modeling a single binge drinking episode during the first trimester) in the avian embryo and used optical coherence tomography (OCT) imaging to assay early‐stage cardiac function/structure and late‐stage cardiac defects. We previously reported that at early stages, alcohol/ethanol‐exposed embryos had smaller cardiac cushions and increased retrograde flow. At late stages, they presented with gross morphological defects in the head and chest wall, and smaller or abnormal atrio‐ventricular (AV) valves, thinner interventricular septae (IVS), and smaller vessel diameters for the aortic trunk branches. In other models, the methyl donor betaine (found naturally in foods such as wheat bran, quinoa, beets and spinach) ameliorates neurobehavioral deficits associated with PAE but the effects on heart structure are unknown. In the current study using this model of PAE, betaine supplementation (23.5 ng per egg) led to a reduction in gross structural defects and certain types of cardiac defects such as ventricular septal defects and abnormal AV valvular morphology. Furthermore, great vessel diameters, IVS thicknesses and mural AV leaflet volumes were normalized while the septal AV leaflet volume was increased. In summary, betaine supplementation in our PAE model can improve survival rates and reduce gross morphological defects including cardiac defects, indicating its potential as a safe periconceptional supplement for prevention of alcohol‐induced congenital defects. Future studies will focus on whether DNA methylation levels in our PAE model are being altered by alcohol and subsequently normalized with betaine. Our findings could have significant implications for public health, especially for pregnant women who are not responsive to folic acid (already present in prenatal vitamins to prevent birth defects) and require an alternative.

Support or Funding Information

Research was supported by the National Institutes of Health (NIH) award numbers R01HL083048; R01HL126747; R21HL115373. Ganga Karunamuni was supported by14POST19960016 from the American Heart Association (AHA). The content is solely the responsibility of the authors and does not necessarily represent the views of the NIH or the AHA.
Novel Findings in Rodent Pelvic Limb Anatomy
Authors: Richbourg Heather Ashley , Martin Matthew J , McNulty Margaret A
Published: November 2023

Rodent models are used for a variety of orthopedic research applications; however, rodent anatomy references are mostly artistic drawings of observed anatomy, which are subject to artistic interpretation and variation. While these drawings are useful, new imaging techniques, such as micro‐computed tomography (μCT), can provide more accurate representations of subtle anatomical characteristics. The purpose of this study was to evaluate differences in the hindlimb musculoskeletal anatomy of mice and rats, and to compare the findings to other rodent species. We hypothesized there would be no difference between mouse and rat anatomy, and the boney anatomy would be similar to other domestic species. The tarsi and femora from two adult C57BL/6 mice and two adult Sprague‐Dawley rats were scanned with μCT at 55kV, 145 μA using 6 μm or 16 μm isotropic voxels for mice and rats, respectively. In addition, an intact mouse hindlimb was stained with Lugol's iodine solution and scanned at the aforementioned parameters for visualization of musculature. Three‐dimensional reconstructions were created and boney anatomy was evaluated. General bone anatomy was similar between mice and rats; however, the mouse was identified to have a fused central and second tarsal bone. This fusion is not represented in conventional anatomy manuals of the mouse and was not observed in the rats. Both species have a medial tarsal bone, which to our knowledge is only present in rodents. Preliminary evaluation of the musculature within the mouse shows similarities to structures found in the domestic species, however further characterization of the tendinous structures crossing and attaching within the tarsal region will be completed based on the aforementioned boney findings. Current knowledge suggests that there is no intra‐species variation of tarsal bone anatomy within the mouse, so it is unclear whether the fusion observed is individual or strain variation, or an unidentified anatomical characteristic. The salamander Plethodon cinereus has been reported to have 5 tarsal bone configurations; therefore, variation within the rodent species is possible; however, no tarsal bone variations are identified within the domestic species. Further research is needed to determine if other inbred mouse strains exhibit this tarsal fusion, as well as to compare results to other rodent species, including the New Zealand white rabbit (Oryctolagus cuniculus), guinea pig, (Cavia porcellus) and nutria (Myocastor coypus). Tarsal bone configurations will influence overall joint kinematics, and fusion within the joint may be explained by evolutionary locomotion behaviors for each species. Differences in joint kinematics may impact applicability of various orthopedic research outcomes for translational studies.
The Embryology Educator Experience: A Comprehensive Survey of Faculty and the Generation of a Phenomenology of Embryology Education
Authors: Cassidy Keely
Published: November 2023

There has been a noted decrease in the time and resources devoted to embryology in medical education in the past fifty years. With more changes assured, it is necessary to investigate current patterns and establish what is truly needed for the education of future physicians. To address these concerns, the researcher distributed an online mixed methods survey to current anatomy and embryology faculty and conducted follow‐up interviews with volunteers from that cohort. Thirteen months of data collection yielded 62 participants. Of these individuals, 69% instruct students at allopathic medical schools, 13% at osteopathic medical schools, and the remaining 18% of participants teach at dentistry, other clinical or professional schools, or undergraduate four‐year institutions. The 55 represented schools are distributed widely across North America. In the United States, 20 schools were from the South, 16 from the Midwest, 9 from the Northeast, and 7 from the West. Three schools were from Canada. The survey included questions about curriculum format, teaching experience, resources, and perceptions of embryological content. While 62% of participants reported an integrated curriculum, 32% had separate courses, and 6% had another alternative curricular format. Most participants primarily taught gross anatomy and embryology, and 40% of these individuals had more than 20 years of teaching experience. The researcher used a grounded theory methodology and NVivo qualitative data analysis software to analyze the open‐ended questions regarding what faculty find most interesting or confusing about embryology, and solicitation of suggestions for the improvement of embryology education. Major themes for all three categories included visualization and resources, curricular placement, clinical relevance, and the continual addition of new embryological content via molecular research. A combined grounded theory and phenomenological approach was used to analyze the 13 follow‐up interviews. This generated a “lived experience” narrative of the phenomenon of teaching embryological content to medical students in the modern world, which led to a better understanding of the needs and challenges that face this subject matter and those who teach it. In this fluid time of medical education reform and integration, the experiences and perceptions of faculty in anatomy and embryology are invaluable to assessing the curriculum and formulating evidence‐based curriculum guidelines for the future.
Comparison of temporal variations in ultrasound training in four novice groups
Authors: Elzie Carrie , Trace Anthony Paul , Knapp Barry , Toreno Felicia , Conran Richard , Goodmurphy Craig
Published: November 2023

The use of bedside ultrasound in healthcare has greatly expanded across medical specialties over the past few decades. As a result, more medical and health professional schools are beginning to incorporate ultrasound training into their curricula. Currently, the implementation of ultrasound training varies significantly from institution to institution with key differences in the temporal placement of training within the curricula. This research project aimed to determine if the placement and timing of ultrasound training impacted skill acquisition comparing four ultrasound novice groups: 1st year medical students in gross anatomy, 4th year medical students, 2nd year physician assistant students and standardized patients. While the curricular content was identical for each novice group, the length of training varied from 16 weeks for the first year medical students, 4 weeks for the fourth year medical students, 2 weeks for the physician assistant students, and 2 days for standardized patients. Each group was given a pre‐test at the beginning of the course, a pair‐matched post‐test upon completion, and a course evaluation. Initially data was compared between the medical students. While some variation occurred in the scores on the pre‐test, similar results were achieved on the post‐test. This preliminary data suggests that the same level of proficiency can be achieved regardless if the curriculum is spread out or concentrated and is independent of year of training within medical school. These results will be compared to the courses for physician assistant students and standardized patients. Details of the curriculum will be presented along with recommendations for implementation of ultrasound training based on the pending results.
CEREBELLAR NEUROTOXICITY OF ARTEMETHER‐LUMEFANTRINE IN PROPHYLACTIC, SUPPRESSIVE AND CURATIVE EXPERIMENTAL MALARIA MODELS
Authors: Edagha Innocent Asuquo , Ekandem Gabriel J , Ekanemesang Udoudo M
Published: November 2023

Artemether‐lumefantrine (AL) is drug of choice for the treatment of uncomplicated malaria in most African and Asian countries, though cases of Plasmodium resistance have been reported. Diagnosis of cerebellar ataxia has been associated in some patients taking artemether‐lumefantrine. Here we report the potential neurotoxicity of this drug on the cerebellar histomorphology and glial pathology of Plasmodium infected mice in prophylactic, suppressive and curative experimental malaria models. All procedures adhered to the guiding principles of care and use of laboratory animals, and institutional ethics committee. 30 Swiss male mice were randomized into 6 groups, 2 groups each for the 3 models representing; the control and test. Prophylactic groups received 0.9% normal saline, and AL 5 mg/kg respectively for 3 days before inoculation with parasite; suppressive groups were inoculated with parasite, and treated immediately; while curative groups were inoculated, and treated 5 days later. Result of routine H&E for prophylactic control showed atrophy and loss of purkinje neurons, but test showed clumping of granule cells and lesions in molecular layer; suppressive control showed widespread lesions in molecular layer, atrophy and loss of purkinje cells, while test showed moderate purkinje neuronal shrinkage with mild lesions, and curative control showed severe distortion of cerebellar cell layers with severe clumping of granule cells, atrophy of purkinje cells and degeneration of neuropil in molecular layer; and test showed less severe distortions. Glial pathology of prophylactic model reveals mildly expressed glial fibrillary acidic protein; suppressive model had moderately expressed GFAP; while reactive astrogliosis was severely expressed in the curative model. In conclusion, severe cerebellar distortion and expression of GFAP in malaria models of artemether‐lumefantrine treated Plasmodium infected mice is dependent mainly on degree of parasite exposure than due to drug toxicity; still caution should be applied in the use of artemether‐lumefantrine.

Support or Funding Information

TETFUND, Nigeria
Extracellular Vesicles Derived From Mesenchymal Stem Cells for Treatment of Ocular Surface Injuries
Authors: Ritter Thomas , Lal Cerine , Treacy Oliver , O'Malley Grace , Naughton Maura , Naicker Serika , Lohan Paul , Subhash Hrebesh Molly , Ryan Aideen , Fahy Gerry , Leahy Martin , Griffin Matthew Dallas , Rani Sweta
Published: November 2023

Mesenchymal stem (stromal) cells (MSCs) are adult multipotent cells with the ability to differentiate into several cell types such as chondrocytes, adipocytes or osteocytes. However, much of the current interest in therapeutic applications of MSCs to various disease settings can be attributed to their immunomodulatory and anti‐inflammatory properties. One of the key mechanisms in MSC immunomodulation is the secretion of soluble factors with paracrine actions. Recently it has emerged that the paracrine functions of MSCs could, at least in part, be mediated by extracellular vesicles (EVs). EVs are predominantly released from the endosomal compartment and contain a cargo that includes miRNA, mRNA and proteins from their cells of origin. Compared to their parent cells, EVs may have a superior safety profile and can be safely stored without losing function thus indicating their potential as cell free therapy.

EVs were isolated from human mesenchymal stem cells derived from healthy volunteers by ultracentrifugation. After extensive quality control EVs were tested for their potential to stimulate the migration and wound healing in human corneal endothelial (HCEC) and epithelial cell (HEC) lines in vitro and corneal wound closure in a rat alkali burn injury model. We could show that EVs significantly enhanced the migration of HCEC and HCE cells leading to accelerated wound closure in vitro. Moreover, treatment of rats after alkali burn of the ocular surface led to enhanced re‐epithelisation and also maintained physiological integrity of the eye via modulating innate immune cell migration in the diseased corneas and in the draining lymph nodes.

In summary we show that EVs derived from MSCs stimulate wound healing both in vitro and in vivo and may have significant potential as a novel alternative to whole cell therapies.

Support or Funding Information

The authors are supported by grants from the Health Research Board of Ireland [grant numbers HRA_POR/2013/341 (SR, MDG and TR) and HRA_HSR/2010/63 (MDG)]; the Irish Cancer Society [grant number CRF12RYA (AER)]; Science Foundation Ireland [grant numbers 09/SRC/B1794 (MDG, TR) and 12/IA/1624 (TR)]; the European Union Framework 7 programme [Health Collaborative Project VISICORT, grant number 602470 (MDG,TR)] and by the European Regional Development Fund.
Understanding The Regulation Of Ferroptosis
Authors: Dixon Scott
Published: November 2023

Cancer cells accumulate high levels of reactive oxygen species (ROS) and as a consequence appear especially sensitive to perturbation of intracellular antioxidant networks compared to non‐transformed cells. The tripeptide glutathione (γ‐L‐glutamyl‐L‐cysteinylglycine), present at millimolar concentrations in the cytosol of most eukaryotic cells, is a crucial intracellular antioxidant required to prevent oxidative damage and cell death. We find that blockade of the cell surface cystine/glutamate antiporter system xc− results in the rapid depletion of intracellular glutathione and the onset of a non‐apoptotic, oxidative, iron‐dependent form of cell death termed ferroptosis. Interestingly, unlike apoptosis and other forms of cell death, ferroptosis does not involve activation of caspase or cathepsin proteases but rather the destruction of plasma membrane lipids by a poorly understood oxidative mechanism. We are currently exploring the biochemical regulation of this pathway and how sensitivity to ferroptosis may be regulated by the deletion of specific metabolic and tumor suppressor genes.

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This work is supported by an R00 award (R00CA166517) from the National Cancer Institute.
Integrative Methods for Elucidating the Structure & Function of Cellular Machines
Authors: Chait Brian T
Published: November 2023

The myriad events that occur in living cells (replication, organellar assembly, transport, genome organization, transcription etc.) are to a large extent carried out through dynamic associations and assemblies of macromolecules. I will describe our efforts to develop and integrate sets of tools that are designed to throw light on the evolution, structure and function of these macromolecular machines.

At the very earliest stage of analysis, we define the precise ensembles of macromolecular players that make up the assemblies of interest. Mass spectrometry has become an increasingly enabling tool for this task, owing to its sensitivity and its ability to characterize the primary structures of endogenous proteins along with any modifications that may be present. At the next stage of analysis, we apply combined proteomic‐biochemical approaches to gain insights into the architectures of protein complexes, especially those that are too large or too poorly behaved for traditional X‐ray crystallographic diffraction or NMR spectroscopic analyses. Finally, we integrate data from all these sources to generate models that can provide insights into the evolution, structure and function of these remarkable macromolecular machines. I will to discuss current capabilities and limits of this approach as well as prospects for the future.

Support or Funding Information

This work was supported in part by NIH grants GM103314 and GM109824.
Kinases as Molecular Switches: Catalytic and Non‐catalytic Functions of BRAF Reveal Novel Insights into Oncogenic Activation
Authors: Malek Shiva , Foster Scott , Whalen Daniel , Ozen Aysegul , Haling Jacob , Sudhamsu Jawahar , Yen Ivana , Yin JianPing , Hatzivassiliou Georgia , Shaw Andrey , Yu Christine , Eigenbrot Charlie , Manning Gerard , Skelton Nick , Hymowitz Sarah
Published: November 2023

Numerous oncogenic mutations occur within the BRAF kinase domain. While the majority of these mutations are activating mutations, kinase inactivating mutations in BRAF have also been reported to drive MAPK pathway signaling, suggesting that BRAF has both kinase activity dependent and independent functions. Here, we define a regulatory mechanism for MAPK pathway activation that is dependent on the conformation of the BRAF kinase domain and can be bypassed by oncogenic mutations in BRAF. Using biochemical, structural and cellular studies, we report that BRAF forms a high affinity complex with MEK1 that is independent of BRAF dimerization but sensitive to MEK1 phosphorylation. BRAF:MEK1 complexes are enriched in the cytosol of BRAFWT and KRASMT tumor cells and form a transient heterotetrameric complex with CRAF upon pathway activation. In contrast, oncogenic mutations in BRAF including point mutations and short, in‐frame deletions within the kinase domain, can activate the pathway independent of RAF dimerization. The crystal structure of BRAF harboring a short in‐frame deletion reveals that the αC‐helix is predisposed to an active conformation. Further, deletions or mutations in the β3‐αC loop render kinases resistant to αC‐helix shifting drugs such as Vemurafenib. Taken together this work underscores the allosteric and catalytic functions of BRAF as well as the importance of dimerization in pathway activation and therapeutic resistance. This has profound implications for both our fundamental understanding of MAPK biology and therapeutic design.
Interactions of branched‐chain amino acids and lipids in metabolic disease
Authors: Newgard Christopher
Published: November 2023

We seek to apply metabolomics and other “omics” tools for understanding of mechanisms contributing to pandemic metabolic diseases such as type 2 diabetes, obesity, and cardiovascular disease. We have used these tools to define mechanisms underlying development of peripheral insulin resistance and glucose intolerance in animals and humans. For example, we have identified perturbations of branched chain amino acid (BCAA) catabolism in multiple cohorts of insulin resistant humans compared to control subjects. Our studies and those of others have demonstrated the prognostic power of this signature to predict incident diabetes and intervention outcomes. These metabolites are also uniquely sensitive to the most efficacious interventions for obesity and diabetes. We have translated these findings to rodent models to demonstrate a contribution of BCAA to abnormalities in mitochondrial metabolism that contribute to the insulin resistant state, as well as to behavioral abnormalities associated with obesity. In hyperphagic Zucker obese rats, feeding of a standard chow diet partially restricted in BCAA content results in improved insulin sensitivity, with attendant changes in tissue amino acid and lipidomic profiles that suggest a relief of mitochondrial fuel overload as a contributing mechanism. Moreover, stimulation of BCAA catabolism by activation of the branched‐chain ketoacid dehydrogenase complex by small molecule or molecular interventions improves glucose homeostasis. Finally, our studies provide evidence that the gut microbiome contributes to dysregulated BCAA and lipid homeostasis in obese humans. These studies suggest that syndromes of lipid overstorage such as NAFLD and NASH are influenced by availability and metabolism of BCAA.

Support or Funding Information

Supported by NIH grants DK58398 and DK078669