bluuebunny/crossref_metadata_2025 huggingface dataset:
Removal of Glutathione Peroxidase 4 in Mouse Liver Leads to Early Lethality and Vitamin E Can Compensate for Its Loss
Authors: Carlson Bradley A. , Tobe Ryuta , Yefremova Elena , Tsuji Petra A. , Hoffmann Victoria J. , Schweizer Ulrich , Gladyshev Vadim N. , Hatfield Dolph L. , Conrad Marcus
Published: November 2023

Glutathione peroxidase 4 (GPx4) is one of several glutathione peroxidases found in mammals and its targeted deletion is early embryonic lethal. The primary functions of GPx4 are to protect cells against membrane lipid peroxidation and to govern a novel form of non‐apoptotic cell death, called ferroptosis, which appears to be relevant in a select set of organs. To study the role of GPx4 in liver tissue, a conditional knockout of Gpx4 in hepatocytes was used, along with thioredoxin reductase 1 (TrxR1) and selenocysteine (Sec) tRNA (Trsp) hepatocyte knockout mice as controls. In contrast to TrxR1‐ and Trsp‐deficient mice, Gpx4 knockout mice died within 48 hours after birth and presented extensive hepatocyte degeneration. We further found that Gpx4 knockout pups born from mothers fed a vitamin E‐enriched diet did not experience early death, demonstrating that vitamin E rescued Gpx4 knockout‐induced cell death. When vitamin E was subsequently removed from the diet, Gpx4‐deficient mice had an average survival time of approximately four weeks. We also crossed Trsp‐deficient and Gpx4‐deficient mice and were unable to obtain combined Trsp/Gpx4‐deficient mice at birth, indicating that the combined deficiency resulted in embryonic death. Combined Trsp/TrxR1‐deficient mice were born but had significantly shorter lifespans than either single knockout, suggesting that TrxR1 plays an important role in longevity of mice lacking Trsp in hepatocytes. This study demonstrates that GPx4 is critical for hepatocyte survival and proper liver function, and that vitamin E can compensate for its loss by protecting cells against lipid peroxidation.

Support or Funding Information

This work was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research.
A comprehensive nutrition program improved nutritional status among children aged 6 to <24 months in rural Malawi
Authors: Kang Yunhee , Hurley Kristen , Monclus Assumpta Bou , Ruel‐Bergeron Julie , Oemcke Rachel , Wu Lee Shu Fune , Mitra Maithilee , Phuka John , Christian Parul
Published: November 2023

To evaluate the one‐year impact of a comprehensive nutrition program—provision of daily Lipid‐based Nutrient Supplement (LNS) and a comprehensive infant and young child feeding (IYCF) and water and sanitation, and hygiene (WASH) behavior change communications (BCC) package—on the nutritional status of children in Malawi. The study design was quasi‐experimental with one program district and one comparison district. The program impact was evaluated using cross‐sectional baseline (January–March, 2014; n=2,404) and midline (January–March, 2015; n=2,378) survey data for children aged 6 to <24 months, who were measured for their length, weight, mid‐upper arm circumference (MUAC), and head circumference (HC) at each survey. Estimates of impact over one year were generated using Kernel Propensity Score Matching Difference‐in‐Differences (PSM‐DID) analyses using both baseline and midline data in the program and comparison districts. The burden of undernutrition was high at both baseline and midline with approximately half of children stunted (length‐for‐age z score, LAZ<‐2) in both districts. There was no significant difference in LAZ or in the prevalence of stunting between two districts using the PSM‐DID analysis. However, the changes in nutritional status between midline and baseline in child weight, MUAC, weight‐for‐age z score (WAZ), and weight‐for‐length z score (WLZ) favored the program district over the comparison district by 0.22kg, 0.29cm, 0.23 z‐score and 0.30 z‐score, respectively (all P<0.05). In the same way, the prevalence of children wasted, underweight, severely wasted, or experiencing moderate acute malnutrition (MAM, ‐3<WLZ<‐2) reduced by 3.9%, 6.4%, 1.1%, and 2.8%, respectively (all P<0.05). However, prevalence of overweight/obese was not increased significantly (1.3%; P=0.39). No significant differences were seen in HC. The impact evaluation showed that the LNS and IYCFWASH‐ BCC program was successful in reducing wasting, MAM, and underweight after only a year of implementation. Subsequent analysis will be key to evaluate the impact of sustained exposure (for 3.5 years) to the program on linear growth and the prevalence of stunting in Malawi.

Support or Funding Information

This study was funded by Children's Investment Fund Foundation, UK
Nutrition Messages in Picture Book Fiction for 4 to 8 Year Old Children
Authors: Matvienko Oksana A.
Published: November 2023

This study conducted a content analysis to examine the types of food and nutrition messages communicated in fictional stories aimed at children 4 to 8 years of age. The foundation of healthy eating is established at a young age. Parents play an instrumental role in the process of home‐based nutrition education. Picture books are uniquely positioned to inform young children about food, nutrition and related behaviors because they can be read multiple times and at parents’ convenience. Some reports indicate that 80 to 87 percent of parents read to their young children three or more times a week. Evidence suggests that an adult‐guided exposure to a single book makes a significant short‐term impact on child's food intake. Thus, it can be hypothesized that an assortment of age‐appropriate, engaging books may have a profound, long‐lasting influence on a child's eating habits. While some studies have examined types and frequencies of foods depicted in children's books, a comprehensive overview of a variety of food and nutrition messages in children's literature is lacking. This study attempted to fill this gap in knowledge. The Children's Core Collection and other databases were searched independently by three research assistants for fictional picture books in which food and nutrition were the main focus of the storyline. The final sample included 270 books published between 2000 and 2015. Stories were systematically coded using holistic, provisional, data‐driven, and evaluation coding methods, as discussed in The Coding Manual for Qualitative Researchers (Saldana, 2013). The mixed‐method analysis revealed four major messaging themes: food (history, life cycle, production, purchase, preparation) that represented 40% of the sample; food and nutrition behaviors (food preferences, table/dining manners, lifestyle choices, weight management), 29%; values (social skills, individuality, diversity, family, religion, food‐related traditions), 22%; sensations and emotions (hunger, food fears, cravings, enjoyment, comfort), 8%. Additionally, the examined books varied substantially in terms of their educational merit. Messages ranged from straightforward and positive to subject to interpretation/misinterpretation to questionable and potentially counterproductive, as the latter featured poor behaviors and vocabulary that some adults may find not suitable for their children. In conclusion, while there is a great variety of fictional picture books to facilitate the development of healthy eating habits in young children, the quality of their messaging about food and nutrition varies widely. Parents and educators are advised to carefully select and preview children's books to ensure that the language and information communicated in those books are consistent with one's personal values and child's maturity level.

Support or Funding Information

None
Perceived Barriers to Physical Activity during Pregnancy among Participants of the Special Supplemental Nutrition Program for Women, Infants and Children (WIC) in Southern California
Authors: Koleilat Maria , Vargas Nancy , Kodjebacheva Gergana
Published: November 2023

Regular exercise during pregnancy is known to be associated with many benefits for the mother and her baby. Pregnant women are recommended to engage in at least 30 minutes of moderate intensity physical activity on most days of the week. Nonetheless, a large number of pregnant women do not engage in the recommended amounts. Not receiving the recommended amounts of physical activity may lead to excessive gestational weight gain (GWG) and maternal obesity. Barriers to physical activity among low‐income pregnant women are not yet well understood. The objective of this study was to identify barriers to physical activity among pregnant WIC participants in Southern California using a qualitative approach and the socioecological framework. We conducted four focus groups (FGs) with pregnant participants aged 18 years or older in either their 2nd or 3rd trimester at the WIC Fullerton center. To take acculturation and BMI differences into account, the four FGs were held according to preferred language (English vs. Spanish‐speaking) and BMI category (self‐reported pre‐pregnancy body weight: Normal BMI < 25 kg/m2 and high BMI ≥ 25 kg/m2). A total of 28 participants (5–10 participants per focus group) met our study criteria and were recruited to participate. We used Atlas. Ti to analyze the focus group transcripts. The majority of the participants were married, lived with the baby's father, were working, and had another child. The Spanish‐speaking groups had at most a high school diploma while the English‐speaking participants had at most a bachelor's degree. Eighty‐six percent of participants mentioned intrapersonal factors as barriers to physical activity. The most mentioned factors were pain and other health factors, lack of motivation and energy, and concerns about exercise safety. Eight‐three percent of participants mentioned interpersonal factors as barriers to physical activity. The most mentioned factors were lack of support from their spouse and family and lack of advice from their doctor. Seven percent of participants reported the weather as an environmental barrier. Physical activity during pregnancy helps pregnant women achieve appropriate GWG and reduce the amount of postpartum weight retention. Interventions to help low‐income pregnant women overcome physical activity barriers should be guided by social ecology & family systems theories. Interventions may focus on strategies to increase motivation and social support for pregnant women. Primary care providers should provide information to pregnant women on the type and frequency of safe exercise.

Support or Funding Information

This research is supported by a CSUF intramural incentive grant.
Matrix Metalloproteinase (MMP)‐28 Activates Signal Transducer and Activator of Transcription 1 to Induce Macrophage M1 Polarization
Authors: Lindsey Merry L. , Cannon Presley L. , Flynn Elizabeth R. , Jung Mira , Iyer Rugmani Padmanabhan , DeLeon‐Pennell Kristine Y. , Ma Yonggang
Published: November 2023

Pro‐inflammatory M1 and anti‐inflammatory M2 macrophages are involved in cardiac repair after myocardial infarction (MI). Mice with matrix metalloproteinase (MMP)‐28 deletion display decreased inflammation at day 7 post‐MI. Accordingly, we hypothesized that MMP‐28 polarizes macrophages to a pro‐inflammatory M1 phenotype. In this study, we stimulated peritoneal macrophages isolated from 3 to 6 month old male C57/BL6J mice with MMP‐28 recombinant protein and evaluated cell phenotype. By quantitative RT‐PCR, the MMP‐28 catalytic domain only protein dose‐dependently upregulated pro‐inflammatory markers (Ccl3, Ccl5, Il1β, Il6, Il12a, and Tnfα, all p<0.05) 100–10,000 fold and downregulated anti‐inflammatory M2 markers (Arg1 and Tgfβ1, all p<0.05) 4–7 fold. This indicated that MMP‐28 is a potent activator of macrophage M1 polarization. Interestingly, the pro‐MMP‐28 full length recombinant protein demonstrated similar M1 polarization responses, demonstrating that MMP‐28 activity was not required. To evaluate if MMP‐28 can function as a signaling molecule to induce M1 polarization independent of its enzymatic activity, we measured the transcription factors nuclear factor (NF)‐κB and signal transducer and activator of transcription (STAT) 1, which are known M1 polarization signaling pathways. MMP‐28 catalytic domain‐stimulated macrophages demonstrated a significant activation of STAT1, evidenced by increased phospho‐STAT1 (400 fold, p<0.05). In contrast, NF‐κB activation was not significantly affected by MMP‐28 treatment. This indicates that the MMP‐28 effect is mediated through the STAT1 pathway. This study, for the first time, demonstrates that MMP‐28 functions as a signaling molecule and potently induces macrophage M1 polarization through activating the STAT1 pathway.

Support or Funding Information

This work was supported by the National Institutes of Health [HHSN268201000036C (N01‐HV‐00244), HL075360, and GM114833 to M.L.L., and P01HL051971 and P20GM104357]; the Biomedical Laboratory Research and Development Service of the Veterans Affairs Office of Research and Development Award [5I01BX000505 to M.L.L.]; and American Heart Association [14POST18770012 to R.P.I.] and [15SDG22930009 to Y.M.].
Leukocytes exhibit discrete rhythms in their recruitment patterns to arteries and veins
Authors: de Juan Alba , Druzd David , Zuchtriegel Gabriele , Kraus Kerstin , Reichel Christoph , Scheiermann Christoph
Published: November 2023

Leukocyte recruitment exhibits circadian, ~24h oscillations, which is dependent on local input by the sympathetic nervous system (SNS). Sympathetic nerves reach tissues alongside arterioles. Since smaller veins are devoid of sympathetic innervation but are the main site of leukocyte recruitment, it is unclear how these adrenergic stimuli reach veins to regulate leukocyte infiltration to tissues. Here, we demonstrate rhythmic differences in the ability of leukocytes to adhere to arteries and veins in an acute inflammatory scenario. Using quantitative in vivo imaging analyses after acute TNFα stimulation at different time points of the day, we observed a ~3‐fold circadian variation in leukocyte recruitment in carotid artery and jugular vein vessels, but with opposed phases. While peak recruitment in the artery was detected in the morning (Zeitgeber time (ZT) 1, i.e. 1h after lights on), peak recruitment in the vein occurred at night (ZT17, 5h after lights off). The same held true for the microcirculation of the cremasteric muscle, demonstrating that these inverted rhythms also occur within the same tissue. After systemic SNS denervation via 6‐OHDA treatment, oscillations were ablated in arteries and veins indicating the importance of sympathetic control. Interestingly, similar observations were found in Ng2Cre x Bmal1flox/flox mice, in which the core clock gene Bmal1 is selectively deleted in arteriolar pericytes. Thus, although arteriolar pericytes were the focus of the circadian disruption, leukocyte adhesion rhythms ceased to exist in both arteries and veins. However, when targeting myeloid cell rhythms genetically, using a Lyz2Cre x Bmal1flox/flox line, oscillations in recruitment to the carotid artery were disrupted but not in the jugular vein. In addition, expression rhythms of leukocyte adhesion molecules (CD11a and CD11b) on adherent leukocytes was in phase in both vessels types, indicating distinct contributions of the microenvironment and the leukocyte for rhythmicity in the adhesion process. Together, our data point to an important role of arteries in regulating venular leukocyte recruitment.

Support or Funding Information

Funded by the German Research Foundation (Emmy Noether SCHE 1645/2‐1 and SFB914 project B09).
Sepsis‐Associated Proteinase 3 Induces Endothelial Permeability
Authors: Patterson Eric K , Cepinskas Gediminas , Inoue Ken , Fraser Douglas D
Published: November 2023

Sepsis is a systemic inflammatory response to known or suspected infection, and associated with microvascular dysfunction and capillary leak. The cause of capillary leak may be due to substances released by activated polymorphonuclear neutrophils (PMNs) that adhere to microvascular endothelial cells. Following binding, PMN often degranulate releasing azurophilic enzymes (e.g. elastase and proteinase 3 (PR3)) at the endothelial surface and into plasma. We hypothesized that the actions of these enzymes, particularly PR3 contribute to the loss of endothelial integrity.

Patients with severe sepsis (sepsis with evidence of at least one organ dysfunctioning), were recruited on admission to the intensive care unit. Patient samples were paired with age and sex‐matched controls. The mean age was 50 ± 14 years, 3/10 males for both sepsis and controls. Septic patient pathogens were determined to be 4 gram positive, 4 gram negative and 2 culture negative; survival outcomes were 9/10 patients surviving. We employed ELISAs to determine plasma PR3 concentrations and in vitro permeability assays using Texas Red‐Dextran flow to determine the effect of PR3 on human umbilical vein endothelial cell (HUVEC) monolayer integrity.

The sepsis plasma PR3 concentration was significantly higher (p < 0.001) compared to controls (55.4 ±8.8 vs 7.7 ±2 ng/mL). For in vitro permeability assays, we employed concentrations of PR3 higher than the plasma measurements in order to more closely mimic conditions at the site of degranulation. 1 hour of PR3 treatment (5 μg/mL) significantly increased HUVEC permeability to Texas Red‐Dextran compared to controls.

The loss of endothelial integrity secondary to PR3 release by adhered PMNs could result in microvascular dysfunction and capillary leak. Endothelial injury could also exacerbate further PMN recruitment. Since other enzymes are contained in the same granules as PR3, they may also have significant actions on endothelial cells, these will be studied in the future.

We conclude that neutrophil degranulation, as evidenced by increased PR3 plasma concentrations, can lead to increased endothelial permeability through a mechanism which in part involves the actions of PR3.

Support or Funding Information

Funded by the Lawson Health Research Institute Internal Research Fund.
STAT3 regulates endothelial permeability downstream of the pro‐inflammatory cytokines IL6 and TNF‐α
Authors: Alsaffar Hiba , Martino Nina , Vincent Peter , Adam Alejandro , Lowery Anthony
Published: November 2023

The barrier function of endothelial cells (ECs) is regulated by the action of pro‐inflammatory cytokines. In human dermal microvascular endothelial cells (HDMECs), TNF‐α can induce loss of barrier function in a dose‐dependent manner. Similarly, IL6 can increase endothelial permeability in HDMECs in the presence of its soluble receptor, sIL6Rα. Activation of Src family kinases (SFKs) rendered ECs susceptible to permeability changes in response to a very low dose of TNF‐α. Correspondingly, SFK activation promoted IL6‐induced loss of trans‐endothelial electrical resistance (TEER) even in the absence of its soluble receptor. This loss of permeability in HDMEC correlated with sustained phosphorylation/activation of the signal transducer and activator of transcription (STAT3), as well as an increase in the mRNA expression of its downstream target SOCS3. siRNA‐mediated STAT3 gene knockdown prevented the increase in endothelial permeability in response to treatment of TNF‐α or IL6 with SFK activation. Consistently, pretreatment of HDMECs with ruxolitinib, a JAK kinases inhibitor, blocked STAT3 activation and the increase in permeability in response to TNF‐α or IL6 in the presence of active SFKs. Based on our findings, we conclude that STAT3 activity mediates the change in endothelial monolayer permeability in response to the pro‐inflammatory cytokines TNF‐α and IL6.

Support or Funding Information

Supported by an AHA SDG grant 13SDG17100110 to APA
Cardiac Mechanics, Hemodynamics, and Embryonic Heart Development
Authors: Rugonyi Sandra
Published: November 2023

Heart development is orchestrated by genetic programs, which are modulated by biochemical and hemodynamic conditions. Because the embryonic heart starts beating and pumping blood soon after formation of the primitive tubular heart, important cardiogenesis processes, such as formation of heart valves and chambers, occur under blood flow. Normal hemodynamic conditions are essential for proper cardiac development, and altered blood flow at early embryonic stages leads to congenital heart defects. Under normal conditions, the developing heart is constantly adapting to hemodynamic changes that accompany embryonic growth: volume flow rates continuously increase to support the development of the embryo, while ventricular blood pressure also increases to support pumping of a larger volume of blood. Abnormal blood flow conditions alter this balance and lead the heart into detrimental pathways that result in cardiac malformations. Cardiac tissue adaptations (growth and remodeling) in response to changes in blood flow during early embryonic stages are only recently starting to emerge. This is in great part thanks to advances in imaging and computational technologies that are enabling a better characterization of the mechanical and biological changes that occur in the tiny embryonic heart under normal conditions and when blood flow is altered.

To elucidate how the embryonic heart grows and remodels under normal conditions and in response to changes in hemodynamics, we use chicken embryos as animal models of cardiovascular development. We employ a combination of in vivo imaging, physiological measurements, immunohistochemistry, and computational techniques in an integrative approach to determine cardiac mechanics and how heart tissues are affected by hemodynamics. In chick embryos, cardiac beating starts at Hamburger‐Hamilton (HH) stage 10 (33–38 hrs of incubation), and septation occurs after HH24 (day 4). We focus on the looping stages of heart development, HH13 to HH24, when the heart is still tubular. During looping stages the heart is very sensitive to hemodynamic conditions, and thus this period has important repercussions for valve and chamber formation. The heart outflow tract (OFT), in particular, is a critical cardiac segment, as it gives rise to the intraventricular septum, pulmonary and aortic outlets, and their valves, and it is the site of origin for a large proportion of cardiac defects observed in human babies. We have developed techniques to reconstruct the 4D motion of the heart OFT, simulate the hemodynamics in the beating OFT, and map OFT hemodynamics and remodeling. Our results, and those of others, are starting to elucidate how hemodynamics affect early embryonic cardiac development. These new data is leading to a better understanding of embryonic cardiovascular adaptations to blood flow, and revealing how cardiac adaptations could also lead to congenital heart defects.

Support or Funding Information

NIH R01 HL094570
Divide and Conquer: On Demand Anatomy in the Medical School Curriculum
Authors: Rosenberg Martina , Hartley Rebecca
Published: November 2023

Anatomy is fundamental to clinical practice, a rite of passage in becoming a physician and key to professional identity formation. Curricular gaps in content and process combined with LCME requirements for self‐directed learning, clinical reasoning and problem‐solving prompted anatomy integration into the organ system blocks at the UNM School of Medicine. Previous reports document more positive attitudes and perceptions to teaching anatomy in context, as compared to an independent course. Yet, the effects of teaching anatomy in and out of context on anatomical knowledge has not been assessed. Although systems‐based teaching may prevent a coherent view of whole body anatomy, cognitive psychology calls for integration of new into existing knowledge (scaffolding) following the constructivist framework. We compared two medical student cohorts to determine if anatomy integration changes 1) mastery of and confidence with anatomical concepts, and 2) metacognitive awareness and professional identity. In a pre‐, post and follow up design, methods included content assessments, confidence probes, the Metacognitive Awareness Inventory, and attitude surveys informed by focus groups. Cohorts are of similar size, mean MCAT scores and study skills self report items. Results thus far show the cohorts performed comparably in pre‐instruction assessment. Data from the first cohort indicate differential gain in mastery of specific anatomical concepts. Anatomical knowledge is gained and retained. Confidence levels with anatomical concepts increased, but were not sustained at the same level. Results from surveys and focus groups highlight the impact of anatomy on learning, reflective practices and awareness of professional identity. Ongoing studies with the second cohort will reveal the impact of curricular reform on this baseline data.

Support or Funding Information

UNM Scholarship in Education Allocation Committee Grant
Deciphering How Cells Count: Molecular Control of Centrosome Copy Number
Authors: Holland Andrew , Moyer Tyler , Lambrus Bram , Levine Michelle , Clutario Kevin , Scott Phillip , Daggubati Vikas
Published: November 2023

The centrosome is a non‐membranous cellular organelle that is critical for accurate chromosome segregation and cell signaling. Defects in centrosome biogenesis are responsible for developmental disorders, chromosome segregation errors, and have been strongly linked with tumorigenesis. Aside from chromosomes, centrosomes are the only other cellular structure whose number is precisely controlled. This faithful control of centrosome number is deregulated in a wide array of tumor types where it disrupts mitosis by promoting chromosome segregation errors. These errors drive the genomic instability and rapid evolution characteristic of cancer. Our research focuses on deducing the engineering principles that allow cells to “count” the number of centrosomes produced in each cell cycle. The insights gained have implications for our understanding of cellular homeostasis and how the centrosome function may be manipulated for human benefit.
Experimental Evidence of a Hypertensive Neuropathy: Is it a new disease?
Authors: Fazan Valeria Sassoli
Published: November 2023

BACKGROUND

The sural nerve has been widely investigated in experimental models of neuropathies but information about its involvement in hypertension was not yet explored. The aim of the present study was to compare the morphological and morphometric aspects of different segments of the sural nerve in male and female spontaneously hypertensive (SHR) and normotensive Wistar‐Kyoto (WKY) rats. Rats aged 20 weeks (N = 6 in each group) were investigated. After arterial pressure and heart rate recordings in anesthetized animals, right and left sural nerves were removed and prepared for epoxy resin embedding and light microscopy. Morphometric analysis was performed with the aid of computer software, and took into consideration the fascicle area and diameter, as well as myelinated fiber number, density, area and diameter.

RESULTS

Significant differences were observed for the myelinated fiber number and density, comparing different genders of WKY and SHR. Also, significant differences for the morphological (thickening of the endoneural blood vessel walls and lumen reduction) and morphometric (myelinated fibers diameter and G ratio) parameters of myelinated fibers were identified. Morphological exam of the myelinated fibers suggested the presence of a neuropathy due to hypertension in both SHR genders.

CONCLUSIONS

These results indicate that hypertension altered important morphometric parameters related to nerve conduction of sural nerve in hypertensive animals. Moreover the comparison between males and females of WKY and SHR allows the conclusion that the morphological and morphometric parameters of sural nerve are not gender related. The morphometric approach confirmed the presence of neuropathy, mainly associated to the small myelinated fibers. In conclusion, the present study collected evidences that the high blood pressure in SHR is affecting the sural nerve myelinated fibers.

Suplementary data

A history of the beginning of the investigation of the “hypertensive neuropathy” will be presented with first results from the aortic depressor nerve and the evolution of the knowledge about how nerves are affected by hypertension, including results on the vagus nerve and phrenic nerve, and the association between hypertension and aging and hypertension and diabetes.

Support or Funding Information

FAPESP, CNPq and FAEPA
Newer Concepts in Coronary Artery Anomalies, Based on Accurate In‐vivo Imaging (IVUS)
Authors: Angelini Paolo Enzo , Uribe Carlo e
Published: November 2023

Objectives

An update on novel concepts in the understanding of coronary anomalies anatomy and their pathophysiology.

Methods

A review of our Center's multiple investigations in these fields (80 publications in 26 years).

Results

Anatomic rare variants are frequent (up to 5.6% of a general population), and need to be properly recognized. At the end, Anomalous Coronary Artery Origin off an improper sinus of Valsalva (ACAOS) results to be the main culprit of sudden cardiac death in the young athletes and/or, in a few carriers, cause of important symptoms at any age. These anomalies were observed in a recent study of ours, based on an MRI prospective study, in 1.1% of a general schoolchildren population (that translates in about 2 million people in the US). This finding calls for the need to identify any predictive markers of the risk of sudden cardiac death (SCD). Stenosis of a coronary artery is the only known means of its dysfunction. Symptoms, participation in strenuous exercises, stress testing and intravascular ultrasounds imaging (when indicated to clarify its severity and to guide indications for interventions) seem to be the main indicators of risk. Still, SCD may be the most frequent first (and, unfortunately, the last) manifestation of ischemia, pointing to the necessity of prospective and effective screening, best done by a screening‐MRI protocol, especially in young athletes. The 2 main causes of ischemic spells in ACAOS are 1. Baseline stenosis, featuring phasic and exercise‐related changes in lateral compression, inside the aortic tunica media, or intermittent spontaneous spasm of distal coronary segments.

Conclusions

CAAs are recently more frequently discovered by modern imaging, a fact that demands a prospective work up protocol to select the cases for preventive interventions. Integration of similar observation with developmental studies suggests the need to especially focus on ACAOS anomalies to better understand morphogenesis of the abnormal and the nature of functional impairment.

Support or Funding Information

Kinder Foundation and Texas Heart Institute
Intravital Microscopy Identifies Macrophages as Effector Cells for Monoclonal Antibody Therapy of Cancer
Authors: van Egmond Marjolein
Published: November 2023

Monoclonal antibodies (mAbs) have become an important addition to chemo‐ and/or radiotherapy for the treatment of cancer. They can exert multiple effector functions that can lead to eradication of tumor in vitro, such as induction of apoptosis, growth inhibition, and initiation of complement‐dependent lysis. Furthermore, mAbs can recruit immune effector cells. To investigate how mAbs mediate tumor cell elimination in vivo, and to establish which effector cells are involved we performed live cell imaging as well as intravital microscopy. We show that antibody‐dependent phagocytosis (ADCP) by macrophages is a prominent mechanism to remove tumor cells from the circulation after mAb therapy. Tumor cells were rapidly recognized and arrested by liver macrophages (Kupffer cells) even in the absence of mAbs. Furthermore, tumor cells were sampled by Kupffer cells, which was however not sufficient for elimination. By contrast, anti‐tumor mAb treatment resulted in fast ADCP of tumor cells by Kupffer cells, which was dependent on FcgRI and FcgRVI. Uptake was independent of reactive oxygen (ROS) or nitrogen (RNS) species production, and followed by degradation, which prevented the development of liver metastases. Tumor cell capture and therapeutic efficacy was lost after depletion of Kupffer cells. As such, macrophages likely play an essential role in mAb‐mediated elimination of tumor cells. Therefore, by aiming to enhance macrophage recruitment and activity mAb therapeutical strategies of cancer patients may be optimized. Currently these strategies are investigated. Additionally, the role of macrophages in eliminating tumor cells in other tissues are studied.

Support or Funding Information

This project was financially supported by the Dutch Cancer Foundation (KWF) project VU2011–4931, and the Worldwide Cancer Research project 15‐1240.
Tracking the in vivo dynamics of antigenic variation In Trypanosoma brucei
Authors: Mugnier Monica , Cross George A.M. , Papavasiliou F. Nina
Published: November 2023

Trypanosoma brucei changes its dense variant surface glycoprotein (VSG) surface coat to avoid detection by the immune system of its mammalian host. The dynamics of antigenic variation during T. brucei infection, a process that takes advantage of a genomic repertoire of over 2000 different VSG‐encoding genes, are poorly understood. How many variants appear over the course of an infection? Is there a pattern to VSG expression over time? Although some of these questions have been broached using Sanger sequencing of VSG cDNA, technical limitations have prevented a high‐resolution, quantitative study of VSG expression during T. brucei infection. We have developed a method, termed VSG‐seq, for quantitatively examining the diversity of expressed VSGs in any population of trypanosomes. Based on de novo assembly, this approach can be used for the high‐resolution study of VSG expression in any strain of T. brucei, whether in the lab or in the field. Using VSG‐seq, we have studied the dynamics of VSG expression during chronic T. brucei infections in mice, and our experiments reveal more dynamic and nuanced host‐parasite interactions than previously expected. Nearly a quarter of the full‐length VSG repertoire is sampled within only four peaks of infection, and this level of diversity persists throughout infection. We can also detect the appearance and disappearance of mosaic VSGs using this method. Mosaic VSGs are novel variants which arise through recombination events within the parasite genome during infection, and our data hint at the mechanisms by which these mosaic VSGs form. We are now applying VSG‐seq to field isolates of T. brucei as well as to populations of parasites inhabiting extravascular spaces. Altogether, these results raise many new questions about the T. brucei host‐pathogen interaction; VSG‐seq will allow these questions to be addressed in their proper context, the natural host.

Support or Funding Information

This work has been supported in part by the NIH/National Institute of Allergy and Infectious Diseases (AI085973) to F.N.P., and by an NSF Graduate Research Fellowship (DGE‐1325261) to M.R.M.
A clinical candidate targeting dihydroorotate dehydrogenase for the treatment of malaria with blood and liver stage activity
Authors: Phillips Margaret A. , Charman Susan A. , Rathod Pradipsinh K , Burrows Jeremy , Rueckle Thomas
Published: November 2023

Malaria is a deadly tropical disease that kills up to 0.6 million people yearly, mostly young children in Africa. Current drug therapies are threatened by drug resistance and the development of novel compounds that are active against drug resistant strains is essential to malarial control and eradication programs. Dihydroorotate dehydrogenase (DHODH) is an essential enzyme for the biosynthesis of pyrimidines required for DNA and RNA synthesis in the parasite. We used a combination of high throughput screening and structure guided medicinal chemistry to optimize a triazolopyrimidine‐based series of DHODH inhibitors resulting in the selection of a clinical candidate for the treatment of malaria. We have shown that DHODH inhibitors have both blood and liver stage anti‐malarial activity, that they have potent activity in both in vitro and in vivo models of malaria and that they are active against a range of clinically drug resistant parasites. A compound called DSM265 was selected from the triazolopyrimidine series for clinical development. DSM265 was shown to have excellent safety during preclinical development. DSM265 has been shown to be a long acting compound with a pharmacokinetic profile that is expected to support single dose treatment or once weekly chemoprevention.

Support or Funding Information

This work was supported by funds from the United States National Institutes of Health grants, U01AI075594 (to MAP, PKR and SAC) and R01AI103947 (to MAP and PKR) and from Medicines for Malaria Venture (MMV).
Separated at Birth, the Evolution of Kinase Dynamics Over a Few Billion Years
Authors: Kern Dorothee
Published: November 2023

I will present our recent exploration of the evolution of the fascinating rate acceleration of enzymes and the evolutionary origin of allostery. The secret of enzymes lies in their ability to partition energetic contributions among many atoms in a well‐coordinated style. To unravel these secrets, the evolution of protein dynamics in the family of protein kinases is spied on to provide a comprehensive description of enzyme catalysis in the form of an energy landscape. Ancestor resurrection combined with modern biophysical characterization combining NMR, fast kinetics and x‐ray crystallography shed light into evolution of protein function to create more sophisticated biomolecules. The evolution of energy landscapes offer a larger variety of signaling mechanisms and sophisticated enzyme catalysis compared to evolution of structure alone. Interestingly, our time‐travel delivered an additional unusual twist, the discovery of the molecular origin of the modern cancer drug Gleevec for the human tyrosine kinase Abl:

Protein kinases are attractive drug targets against cancer due to their central role in cellular regulation, but the similarity of their active site has hampered the development of specific drugs. One of the major success stories has been Gleevec, a highly selective inhibitor of Abl kinase. The molecular mechanism underlying Gleevec's selectivity for Abl has not been solved since the discovery of the drug 20 years ago despite large efforts. Gleevec's selectivity is puzzling because the drug‐binding pocket with Gleevec bound is nearly identical between Abl and Src and other homologous kinases that have very weak affinity for the drug. I will present how the characterization of the evolution of kinase dynamics over 1 billion years reveals the mechanism responsible Gleevec's selectivity. The results simultaneously shed light on the longstanding paradox of the mechanism of Gleevec specificity while offering insights into how energy landscapes evolve for catalysis. The resurrection of the tyrosine kinase ancestors explores a key event in the history of life, the development of multicellularity coinciding with the explosion of the kinome. Interestingly, gleevec takes advantage of evolutionary differences among the kinases “by accident” thereby leading to one of the most potent and specific cancer drugs to date.
Rational Optimization of Natural‐Born, Mechanism‐Based Inhibitors of the 20S Proteasomes in Humans and Mycobacterium tuberculosis
Authors: Sello Jason
Published: November 2023

It is widely appreciated that in‐depth studies of the structures and mechanisms of enzymes, receptors, and other macromolecules have enabled the rational design of drugs. Drug development has also been inspired by mechanistic studies of bioactive natural products. Research in the latter category has been particularly impactful in the development of drugs whose mode of action is inhibition of the 20S proteasome. Recently, my group and others have found that members of the syringolin family of natural products are compelling starting points for the rational design of drugs that act on this very compelling target. These inhibitory molecules irreversibly react with the catalytic threonine residue in the 20S proteasome's active sites via substrate mimicry. Using the published model for how the syringolins mimic peptides and knowledge about the substrate preferences of the proteolytic subunits of the 20S proteasomes in both humans and Mycobacterium tuberculosis, we have designed, synthesized, and evaluated syringolin analogs that may prove useful in the treatment of either cancer or tuberculosis.
The Phase of Fat: Mechanisms and Physiology of Lipid Synthesis and Storage
Authors: Farese Robert V. , Walther Tobias
Published: November 2023

Lipids play a crucial and tightly regulated role in cells as the major component of membranes. Neutral lipids, such as triglycerides, are also crucial for both cellular and physiological energy homeostasis. Membrane precursor lipids and energy storage lipids are stored as neutral lipids in cytosolic lipid droplet organelles. We are studying the molecular processes that govern the synthesis of energy storage lipids as well as their storage in and mobilization from lipid droplets. Studies on genetic factors controlling lipid droplet growth and utilization will be presented, as will relevant studies of the physiology of lipid storage.
Transformation In Biomedical Science Education For Future Physicians
Authors: Lieberman Steve
Published: November 2023

Physician education in the US is undergoing a period of rapid change unmatched in the past 100 years. Advances in the science of learning and educational technology, rapid evolution of the American health care system, projected physician shortages, and rising student debt have spurred a new wave of curriculum reform at many medical schools as well as national initiatives by major organizations including the AAMC and the AMA. Structural changes include expansion of time for clinical education and shortening of the traditional 8‐year course of study. Pedagogical changes include use of active learning methods during faculty‐student interactions combined with independent (often online) acquisition of content, emphasis on the application of science knowledge to clinical problem‐solving rather than the accumulation of decontextualized knowledge, acquisition and demonstration of competence in patient care, and education in the delivery of health care and the health care system. Many of these changes are reinforced by national exams (MCAT, USMLE).

Competency based education is diffusing from residency training to medical school and premedical programs, taking the form of entrustable professional activities (EPAs) at many medical schools. EPAs are units of clinical work (eg, developing a differential diagnosis) that learners can be entrusted to perform under progressively less supervision as they demonstrate increasing competence. Ubiquitous subsets of knowledge, skill, and attitudes (eg, biomedical and clinical science knowledge, communication) implicitly pervade the 13 Core EPAs for Entering Residency identified by an AAMC panel.

In the University of Texas System, Transformation in Medical Education (TIME) is a student‐centered, clinically focused initiative to increase the effectiveness of medical education and shorten its duration. TIME consists of 4 scalable and generalizable pilot programs in which 11 UT premedical and medical programs are partnering to create 6–7 year programs built on the principles of competency based education (CBE), intentional professional identity development, and early clinical skills training to expand the time available for authentic clinical learning. The approach to CBE includes a set of collaboratively developed competencies and shared assessments of critical transition milestones (eg, biomedical science knowledge, clinical skills). The UTMB TIME curriculum features a novel 3‐year structure that fully integrates basic and clinical science education in the classroom and on modified clerkships that are organized by life cycle stages. An essential aspect is the Student Continuity of Practice Experience (SCOPE) in which students will provide care to their own panel of patients under supervision throughout medical school.

Support or Funding Information

The TIME initiative is supported by funding from the University of Texas Board of Regents.
Molecular Visualization of the Eukaryotic Transcription Initiation Process using Cryo‐EM
Authors: Nogales Eva , Louder Robert , He Yuan
Published: November 2023

The critical step of transcription initiation requires the assembly of the general transcription factors into a pre‐initiation complex (PIC) that ensures the accurate loading of Pol II at the transcription start site, carries out the opening of the DNA duplex, and facilitates clearance of the promoter by Pol II. We are using single particle cryo‐EM to investigate the assembly, architecture and structural rearrangements of the human PIC during the process of transcription initiation. Our efforts have concentrated on studying, on one hand, the general transcription factor TFIID and its recognition of core promoter DNA, and on the other a simplified PIC using TATA‐binding protein (TBP) as a surrogate for TFIID.

Using an in vitro reconstitution system we visualized the step by step assembly of the TBP‐based human PIC, ultimately containing TBP, TFIIA, TFIIB, Pol II, TFIIF, TFIIE and TFIIH on promoter DNA. Our cryo‐EM structures provide pseudo‐atomic models that illuminate critical protein‐protein and protein‐DNA interactions with the PIC. Comparison of the closed PIC with the open complex has allowed us to shed light onto the process of promoter melting. The apparent movement of downstream DNA, together with the positioning of XPB, support the previously proposed translocase model for TFIIH. We propose that XPB would push and rotate the DNA with respect to a contact point of Pol II and the DNA, just upstream of the active site of XPB. These actions of XPB against the stably bound upstream DNA around the TATA box would in turn induce negative supercoiling near the transcription start site.

TFIID plays a central role in the initiation of RNA polymerase II‐dependent transcription by nucleating PIC assembly at the core promoter. TFIID comprises TBP and 13 TBP‐associated factors (TAF1‐13), which specifically interact with a variety of core promoter DNA sequences. Using single‐particle cryo‐electron microscopy, we determined that TFIID exist in multiple conformational states. Two major conformers, which we refer to as the canonical and rearranged states, coexist in the apo complex. Only the rearranged state is capable of binding core promoter DNA. Recently we have determined the structure of human TFIID in complex with TFIIA and core promoter DNA at sub‐nanometer resolution. Our structure shows how all the super core promoter elements (TATA, INR, MTE and DPE) are contacted by subunits of TFIID. Together with the cryo‐EM reconstruction of a fully‐assembled human TAF‐less PIC, our structures provide novel insights into the general role of TFIID in promoter recognition, PIC assembly, and transcription initiation.

Support or Funding Information

This work was funded by NIGMS and HHMI.