bluuebunny/crossref_metadata_2025 huggingface dataset:
Hair‐Bearing Human Skin Organoids from Pluripotent Stem Cells: A Novel Source of Advanced Skin Substitutes for Wound Repair
Authors: Sheets Anthony R. , Deakin CiCi , Lee Jiyoon , Koehler Karl R.
Published: May 2022

Cutaneous injuries represent a major clinical challenge domestically and globally. Indeed, chronic wounds are estimated to affect the quality of life of nearly 2.5% of the United States population, and diabetic foot ulcers have a lethality nearly equivalent to that of all cancers combined. While skin grafts remain an important component in the care of both acute and non‐healing wounds alike, there are numerous drawbacks to such procedures, including lack of acceptable donor sites, morbidity associated with harvest, potential scarring at recipient sites, and graft loss. To these ends, advanced skin substitutes have been developed in efforts to overcome some of these obstacles, however existing modalities combining dermal and epidermal cell populations fail to develop skin appendages like eccrine glands and hair follicles critical for normal skin function. Thus, there is an unmet need for advanced cellular skin substitutes that more faithfully recapitulate native human integument.

Our recent work reveals that co‐induction of epithelial and mesenchymal cell differentiation from human pluripotent stem cells gives rise to hair‐bearing skin organoids (SKOs) in vitro. These SKOs develop as spherical structures composed of mesenchymal cells surrounding stratified keratinocytes lining a central cystic cavity, producing hair follicles with bulbs oriented radially outward and hairs that grow into the cyst. Importantly, hairy human SKOs successfully engraft when applied to small incisions surgically created on the backs of immunocompromised nude mice, transitioning to a planar orientation with vertical hair outgrowth within 4‐5 weeks of transplant. Therefore, we hypothesized SKOs could similarly be applied to excisional wounds in a model system more akin to human injuries requiring skin grafts.

Here, we demonstrate that hairy human SKOs applied to murine dorsal excisional wounds at 140 days post‐differentiation successfully take and undergo cystic‐to‐planar transition (CPT) within 5 weeks of surgery. We further show that SKOs xenografted at 80 days post‐differentiation, when hair‐germ‐like buds are visible in vitro but have not yet sprouted hairs, not only readily engraft and go through CPT, but also continue to develop hairs in vivothat show vertical outgrowth within the same time frame. Moreover, human SKOs xenografted at an earlier stage of development appear less macroscopically distinct from the surrounding host mouse skin than those at the later, hair‐bearing stage, suggesting transplant at earlier time points may yield improved cosmetic outcomes. Overall, our findings reveal human pluripotent stem‐cell derived SKOs may form the basis of a novel advanced skin substitute for clinical use, with future studies focused on exploring their utility in additional models of both acute and chronic wounds, as well as understanding the molecular and cellular mechanisms underlying CPT.
Implementing Ungrading in Undergraduate Exercise Physiology
Authors: Stenson Mary
Published: May 2022

Natural science faculty are increasingly experimenting with alternate modes to assess student learning. Ungrading, popularized by Susan Blum’s edited anthology of the same name, gives instructors freedom to move beyond traditional grades for a more complete understanding of student learning. Ungrading is a student‐centered approach to assessment of learning which focuses on student self‐reflection and metacognition, and limiting instructor bias. Ungrading can be useful in physiology education by incentivizing intrinsic motivation and attention to understanding instead of memorization. Reducing grade anxiety allows students in exercise physiology to focus on understanding and applying course content.

In spring 2020, ungrading was implemented in an upper division undergraduate exercise physiology course taught over 4 weeks (N=19). In previous sections, students often commented that their grade did not reflect how much they learned. The purpose of ungrading was to more closely align grades with student learning. The lecture portion of the course occurred as a flipped classroom with small group meetings. In class, students asked and answered questions, discussed primary research, and interpreted physiological data to better understand and apply course material. Students were given details about ungrading at the beginning of the course, read selected essays about ungrading, and discussed their impressions of the new system with the instructor. One student had previous experience with ungrading.

Students completed two guided self‐evaluations to describe their learning and reflect on their challenges and successes. Twice during the course students met individually with the instructor to receive feedback about their self‐evaluations and overall performance. During the mid‐term meeting, students set specific goals to work towards for the remainder of the course.

Student self‐reflections (n=19) and student opinion surveys (n=15) were used to evaluate the ungrading practice. 89.4% (n=17) of the students indicated they agreed or strongly agreed that their learning was enhanced by ungrading. 100% of students agreed or strongly agreed that they valued their learning during the course. 87% (n=13 of 15) indicated the amount they learned in the course was excellent or good. 93% (n=14 of 15) rated the evaluative techniques and grading as excellent or good. Qualitative analysis of open‐ended reflection questions revealed students felt they were more easily able to apply knowledge with less grade based anxiety. Students reported increased curiosity and a deeper understanding of content when revising mistakes or misconceptions in their work. The instructor found ungrading very effective in improving student engagement with and understanding of course content. Overall, ungrading was effective for encouraging a deeper understanding of course content and aligning course grades with student learning.
The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Plays an Important Role in Fetal Human Colon Cell Migration and Proliferation
Authors: Mogan Cara A. , Grant Garett J. , LeBaron Kaylee , Liou Theodore G. , Helms My N.
Published: May 2022

Background‐

Cystic Fibrosis (CF) is an inherited genetic disorder resulting from mutations in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR). For reasons that are not completely clear, CF patients over the age of 40 are 4‐8 times more likely to develop colon cancer than the general population. Moreover, positive history of distal intestinal obstruction syndrome or organ transplant further increase risk of developing colon cancer by 11‐fold.

Objective‐

The objective of this research is to test the hypothesis that inherited CFTR mutations in CF patients promote colon cancer development by increasing cell migration and proliferation via altered epidermal growth factor (EGFR) signaling.

Methods‐

Scratch assays were performed on confluent fetal human colon epithelial cells (FHC) and the rate of cell migration was measured 1‐5 days after treatment with either CFTR activating (IBMX and forskolin) or inhibiting compounds (CFTRinh‐172 and CFTR siRNA). Ki67 nuclear antigen and EGFR labeling were conducted using standard immuno‐histochemical labeling and detection techniques.

Results‐

We found that both pharmacological and siRNA inhibition of CFTR activity increased the rate of wound closure and cell migration. Conversely, IBMX and forskolin stimulation of CFTR decreased the rate of FHC migration (vs untreated control rates). Inhibition of CFTR also associated with increased levels of Ki67 (a classic cell proliferation marker) and increased expression of EGFR (which has known roles in cell growth and differentiation).

Conclusion‐

Our results indicate that CFTR plays an important role in FHC colon cell migration and EGFR signaling, which could lead to a better understanding as to why CF patients have increased risk for colon cancer.
Molecular basis of sequence‐dependent differential prenylation inhibition and statin sensitivity of G proteins
Authors: Tennakoon Mithila I. , Karunarathne Ajith
Published: May 2022

Prenylation is a universal and irreversible post‐translational modification occurring on proteins involved in various cellular processes, including survival, proliferation, and migration. The prenylation process consists of enzymatically catalyzed covalent attachment of an isoprenyl lipid, either a 15‐C farnesyl or 20‐C geranylgeranyl moiety, to the Cys residue located in the four residues from the CT in the target protein via a thioether linkage. Proteins undergoing prenylation share a C‐terminally conserved consensus recognition CaaX motif, of which X determines the type of prenylation on the protein. The mevalonate (HMG‐CoA reductase) pathway synthesizes lipids for protein prenylation. The rate‐limiting enzyme; HMG‐CoA reductase of the mevalonate pathway is the primary target for cholesterol‐lowering medications and anticancer drug development. Our previous study shows that clinically used mevalonate pathway inhibitors, statins not only significantly disrupt PM localization of Gβγ but also perturb GPCR‐G protein signaling and associated cell behaviors in a Gγ subtype‐dependent manner. However, in the present work, we identified crucial residues on Gγ that regulate the efficacy of its prenylation and during post‐prenylations processing. Therefore, our findings demonstrate the molecular underpinnings of differential prenylation sensitivities of G proteins under cholesterol synthesis inhibition and hypolipidemic conditions.
The Forgotten PKAs: New Insights Revealed by the First Crystal Structure of a PKA C Beta Splice Variant, Cβ4ab
Authors: Bruystens Jessica H. , Wu Jian , Wallbott Maximilian , Bertinetti Daniela , Herberg Friedrich W. , Taylor Susan S.
Published: May 2022

A major regulatory mechanism of enzyme function in cells is protein phosphorylation that is carried out by the large protein kinase family. While the catalytic (C) subunit of cAMP‐dependent protein kinase A (PKA) has served as a prototype for this family, our enormous knowledge base is skewed towards Cα1, only one of the many tissue specific C subunit isoforms and splice variants expressed in cells. Accordingly, all published crystal structures are of Cα1. The other PKA splice variants, especially the Cβ splice variant gene products of PRKACB, have been obscure. In an effort to re‐balance our PKA perspective we have solved a 2.6 A crystal structure of the PKA splice variant Cβ4ab, which not only crystallized in a new condition but also grew in a thus far for PKA not seen space group P21 with four molecules per asymmetric unit. Compared to Cα1, the isoform with the most deposited structures in the protein data bank, the protein sequence of Cβ4ab differs by 10% or 35 out of 350 amino acids. A majority of these amino acid differences cluster in the N‐terminal and C‐terminal tails. The rest are located mostly in the N‐lobe. Some hot spots of amino acid variation nucleate two sets of C subunit dimers that interact in a novel way to create polymers within the crystal. In our structure, Cβ4ab is in a closed conformation with the inhibitor peptide IP20 bound but surprisingly no electron density is observed for either of the two Mg ions usually present with IP20 and nucleotide. SPR data for the protein kinase inhibitor provides further insights into the differences between Cβ4ab and Cα. Our structure and binding studies suggest that in addition to C beta being expressed in a tissue specific manner, they are also functionally non‐redundant. The newly revealed inter‐molecular interaction sites within the dimers highlight regions of the C subunit that have been less studied so far and are likely to have important regulatory functions.
The influence of body size and phylogeny on trabecular microstructure in the proximal femur and humerus of rodents and primates
Authors: Peters Kara D. , Kuo Sharon , Ryan Timothy M.
Published: May 2022

Life history theory states that organisms maximize their fitness via energetic trade‐offs between various biological functions. It has been well‐established that life history strategy is related to intraspecific variation in body size and that body size predicts some bone microstructural properties, but the relationship between life history strategy and skeletal variation has not been sufficiently examined across multiple mammalian clades. Generally, small‐bodied mammals have “fast” life histories and faster growth rates than large‐bodied mammals, meaning small mammals deposit bone tissue at a faster rate, which may affect skeletal morphology. Therefore, the goal of this study was to conduct a preliminary analysis of trabecular bone structure in the rodent and primate humerus and femur to evaluate the influence of life history traits, particularly body size, on bone properties in a phylogenetically diverse sample. Using a sample of 67 size‐matched adult rodent and primate individuals from 8 extant rodent genera (N=26) and 10 extant primate genera (N=41) ranging in body size from 0.04kg to 70kg, we analyzed trabecular bone structure in the proximal femoral and humeral head. Regression analyses were used to assess associations between trabecular bone properties and body size and one‐way ANOVAs were used to compare bone properties between rodents and primates across three size‐matched classes.

Regression results are consistent with previous primate and mammal studies. Bone volume fraction (BV/TV) scales with weak positive allometry in both groups and both elements except for the rodent humerus, which scales isometrically. Degree of anisotropy (DA) varies across all groups and shows no consistent relationship with body size. Trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp) increase with body size, while connectivity density (Conn.D) and bone surface density (BS/BV) decrease with body size across both clades and elements. In both groups and both bones, Tb.Th, Tb.Sp, and Conn.D scale with strong negative allometry while BS/BV scales with strong positive allometry. ANOVAs reveal that Tb.Th and Tb.Sp are higher in the primate femur than in the rodent femur for medium and large size classes, while Conn. D is higher in the rodent femur for medium and large size classes. Other bone properties do not show consistent patterns of differences between primates and rodents. Overall, while overlapping confidence intervals for all slopes suggest body size scales similarly with these bone properties across clades, ANOVA results suggest that medium and large bodied rodents have thinner, more numerous trabeculae in the femur compared to similarly‐sized primates. Further investigation is necessary to elucidate the biological significance of this result and the underlying mechanisms driving the observed phenomenon.
LPS‐induced endotoxemia promotes blood‐brain barrier permeability via TSP1‐dependent TGFβ1 activation
Authors: Bhattarai Shadikchhya M. , Frampton Gabriel , Jhawer Ashwin , Troyanovskaya Eleonora , McMillin Matthew
Published: May 2022

Introduction

During disease states, the blood‐brain barrier (BBB) can become permeable due to endothelial injury or dysregulation of tight junction proteins. Endotoxemia, which induces systemic inflammation, causes BBB dysfunction and neuroinflammation. We have shown that transforming growth factor beta 1 (TGFβ1) is upregulated during systemic inflammation and promotes BBB permeability by disrupting claudin‐5 and occludin. Therefore, we hypothesize that TGFβ1 is upregulated during endotoxemia and induces BBB permeability by disrupting tight junction proteins.

Methods

C57Bl/6 mice were administered 3 mg/kg of LPS and monitored for neurological decline. In a second set of mice 6 hours after LPS administration, an antagonist to TGFβ1 activation, LSKL, or control, SLLK, were injected (1 mg/kg/ip). Mice were euthanized 24 hours after LPS injection and tissue was collected. In a subset of animals, 3 hours prior to euthanasia, Evans Blue was injected (8%/ip) to assess BBB function. TGFβ1, claudin‐5, occludin, IL‐1β, IL6 and TNFα expression was assessed by RTPCR, immunoblotting and immunofluorescence. In vitro studies using mouse brain endothelial cells (bEnd.3 cells) were treated with LPS (10 μg/ml) or rTSP1 (1 μg/ml) to activate TGFβ1. TGFβ1, TSP1, claudin‐5 and occludin expression was assessed by immunofluorescence or RTPCR. Barrier function was determined in bEnd.3 cell monolayers using TEER or 10‐kDa FITC‐dextran diffusion. Data were analyzed using GraphPad Prism with results presented as mean +/‐ SEM. Differences between two groups were compared using Student’s t‐test and differences between three groups were determined using ANOVA.

Results

TGFβ1 expression was increased in cortex and cerebellum of LPS‐treated mice. This was associated with decreased claudin‐5 and occludin mRNA expression and immunofluorescence staining. Treatment of bEnd.3 cells with LPS increased TGFβ1 and TSP1 expression, decreased TEER, and increased FITC‐dextran diffusion. Supplementation of bEnd.3 cells with rTSP1 led to decreased claudin‐5 and occludin mRNA expression, decreased TEER, and increased FITC‐dextran diffusion across the transwell. LSKL treatment in LPS‐treated mice reduced sickness behavior, reduced Evan’s blue penetrance into the brain, restored tight junction expression and alleviated neuroinflammation when compared to SLLK‐treated mice.

Conclusions

Strategies to target TGFβ1 signaling may prove effective at alleviating endotoxin‐induced BBB dysfunction.
Evaluation of Hypothalamic Neural Stem Cell Niche – Implications on Obesity‐Induced Sympathoexcitation
Authors: Plakkot Bhuvana , Subramanian Madhan
Published: May 2022

The newly discovered hypothalamic neural stem cell niche is emerging as a key player in the regulation of systemic homeostasis. Previous studies have shown that inflammation‐associated degenerative processes in the hypothalamic neural stem cell niche impairs central regulation of metabolism and contributes to the development of metabolic syndrome in obesity. In addition to energy homeostasis, hypothalamic nuclei specifically paraventricular nucleus, is a well‐known player in the regulation of sympathetic nerve activity and in turn cardiovascular function. How obesity‐induced degeneration of neural stem cell niche affects sympathetic regulation of blood pressure is still not understood. In the present study, we aim to characterize the neural stem cell population from the hypothalamic niche. In this pilot study, 3‐month‐old C57BL/6J mice fed with standard diet were sacrificed and brains were collected for further processing. Hypothalami were freshly isolated and minced. After series of enzymatic dissociation, washing and filtration, NSCs were cultured. On 14th day of culturing, neurospheres were observed without any changes in their structural integrity. Neurosphere counts were recorded, and cells were collected for RNA extraction to perform RT‐PCR analysis for characterization. Our PCR analysis confirmed the expression of NSC markers like nestin, SOX1 and SOX2 suggesting successful isolation and culturing of NSCs in our lab. Earlier studies in our lab, suggest that HFD‐induced obesity causes cellular senescence in the PVN, a region adjacent to the neurogenic niche of hypothalamus. Senescent cells are known to impair the regenerative potential of stem cells by impairment of tissue microenvironment through their secretome. Having established these techniques, future studies will investigate the link between hypothalamic neurogenesis, senescence (in the PVN) and sympathoexcitation in obesity.
Cutaneous Microvascular Responses to Local Heating on Ventral and Dorsal Aspects of the Forearm
Authors: Patik Jordan C. , Shoemaker Leena N. , Wenner Megan M. , Edwards David G.
Published: May 2022

The cutaneous microvasculature is considered a representative vascular bed and is regularly used to interrogate the mechanisms of microvascular function in healthy and diseased humans. For instance, cutaneous dilation in response to local heating can provide an index of nitric oxide (NO) bioavailability. While the skin of the forearm is typically used for local heating studies, some studies have used the ventral forearm while others have used the dorsal forearm skin for this assessment. Since the dorsal aspect of the forearm typically has more hair and receives greater sun exposure than the ventral side, there is speculation that cutaneous dilation may differ from one side of the forearm to the other, in magnitude and/or mechanism. As such, studies utilizing different sides of the forearm may not be comparable. Therefore, we aimed to test the hypothesis that cutaneous local heating responses differ between the ventral and dorsal aspects of the forearm in response to the two most common temperature stimuli, 39°C and 42°C.

Methods

Ten young, normotensive individuals (6 men/4 women, age: 26 ± 2 years) were studied on two separate occasions separated by 2‐7 days. At each visit, 4 intradermal microdialysis fibers were placed in either the ventral or dorsal forearm, in a random order. Two fibers were perfused with Ringer’s solution to serve as control sites and the other 2 fibers were perfused with 10 mM N(G)‐Nitro‐L‐arginine methyl ester (L‐NAME) to inhibit NO synthase. Red blood cell flux (RBCF) was assessed at all sites via laser Doppler flowmetry. Each pair of sites (control and L‐NAME) was heated to either 39°C or 42°C for ~40‐45 min until RBCF plateaued. All sites were then infused with 28 mM sodium nitroprusside and heated to 43°C to establish maximal RBCF. Cutaneous vascular conductance (CVC) was calculated as RBCF / mean arterial pressure measured during the plateau and maximal phases. Plateau results are reported as a percentage of maximal CVC at each respective site. The relative NO contribution is reported as: [(control plateau – L‐NAME plateau)/control plateau] x 100.

Results

No differences were observed at the control site between ventral and dorsal aspects of the forearm when heated to 39°C (62 ± 13 vs. 62 ± 16 % CVCmax, P=0.99). Likewise, the heating response to 42°C was similar across the forearm (ventral: 90 ± 9 vs. dorsal: 86 ± 12% CVCmax, P=0.21). The relative contribution of NO to the plateau was comparable across the forearm at 39°C (ventral: 70 ± 20 vs. dorsal: 66 ± 21%, P=0.71) and 42°C (ventral: 63 ± 16 vs. dorsal: 69 ± 11%, P=0.11).

Discussion

These preliminary findings suggest that cutaneous vasodilation elicited by local heating is similar on both sides of the forearm at both 39°C and 42 °C. Additionally, it appears that the relative contribution of NO to local heating‐induced microvascular vasodilation is not different across the forearm.
Countermeasure Analysis for Galactic Cosmic Radiation Induced Damage
Authors: Adams Jessica , Haran Harry , Nikisher Bridgette , Edwards John
Published: May 2022

Galactic cosmic radiation (GCR) exposure is increased to space flight crews as travel moves beyond low Earth orbit. GCR includes high energy protons and heavy ionizing ions such as 56Fe, 28Si, and 16O and there are distinct differences in the biological response compared to low‐energy transfer events (x‐rays). Although the exposure rate is relatively low, the cumulative radiation dose is likely to have deleterious effects for long duration flight crews. The underlying mechanisms for these elevated risks are thought to include direct DNA damage as well as chronically elevated oxidant stress, and inflammation.

Given the relatively low fluence rate of GCR exposure during flight operations it might be possible to manage these deleterious effects using small molecules currently available. To that end we have utilized a FDA approved drug library to perform a high throughput screen of cultured cells following exposure to GCR. H9c2 cells were exposed to a single 75 cGy dose using the 5‐ion GCRsim protocol developed by NSRL. Following GCRsim exposure, cells were treated using 10 µM for each drug. At harvest, cells were tested for protein content, oxidant stress, cellular senescence, and mitochondrial function.

Of 725 drugs, more than 500 were ineffective in managing GCR exposure. Within the top 160 drugs, 33 were primarily designated as anti‐inflammatory. Significant variations within drug classes were observed. Only 10 of 46 drugs that interacted with histamine receptors were found useful and most were 5‐HT3 specific. The COX inhibitors deemed useful were all COX2 specific. Surprisingly, some anti‐diabetic medications observed to be useful including metformin.

The bias of mitochondrial degradation as a central readout comes from several “omics” analyses that identified mitochondrial dysfunction as a consequence of GCR exposure. Mitochondrial dysfunction is also an antecedent event in the transformation leading to carcinogenesis. And the potential for carcinogenesis is a significant risk for long duration space missions. Separate from examining mitochondrial and cellular dysfunction as readouts, we have developed a Afp‐tdTomato expressing cell line that is responsive to different carcinogenic reagents including GCR. We have observed that some of the lead candidates will at least partially rescue the cells from GCR induced Afp driven expression. Additionally adapting cell doubling time protocols we observed that GCR exposure significantly increased cell doubling time; a finding indicative of mitotic dysfunction. We also observed that this increase was partially ameliorated by some of the lead candidates. That no one class of drug dominated the response to radiation exposure speaks to the complexity of managing GCR exposure.
Biochemical Characterization of Glucan Phosphatases from Cereal Crops
Authors: Frenett Marissa , Weis Kenyon , Raththagala Madushi
Published: May 2022

Starch degradation occurs via a cyclic process of phosphorylation, hydrolysis by amylases, and dephosphorylation by glucan phosphatases. Glucan phosphatases belong to the dual‐specificity phosphatase (DSP) family of proteins within the larger protein tyrosine phosphatases (PTPs) superfamily. Plants contain two glucan phosphatases: Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2). However, little is known about the role of glucan phosphatases in agriculturally relevant cereal crops. In this study, we overexpressed and purified Oryza sativa (rice) and Zea mays (corn) SEX4 enzymes in E. Coli cells, and determined the kinetics using both generic and substrate‐specific glucan phosphatase assays. Rice and corn SEX4 display sigmoidal kinetics against soluble amylopectin substrate, a signature feature of kinetic cooperativity. We explored the mechanism for this positive kinetic cooperativity and how these enzymes can be utilized to enhance starch degradation. Our findings provide insights into the essential role of SEX4 in reversible starch phosphorylation and advance our understanding of the glucan phosphatase family of enzymes.
The DNA Binding Diversity of the SIX Homeodomain Transcription Factor Family
Authors: Rivera Barreto Anthony R. , Papa Riccardo , Hoffmann Federico , Rodríguez Martínez Jose A.
Published: May 2022

Transcription factor (TF) DNA‐binding specificity is determined by how their DNA‐binding domain (DBD) interacts with DNA. TFs are identified by the sequence homology shared with described DBDs, which allows them to be classified into families. It is highly widely accepted that similar DBDs recognize similar DNA motifs. However, changes in a TF can lead to changes in its DNA recognition. TFs are critical for development and have been found since the beginning of animals, how their DNA binding changes through time has not been fully studied. TFs members of the sine oculis homeobox (SIX) homeodomain family are found from sponges to humans and are considered atypical members of the homeodomain family. They regulate numerous developmental processes, with phenotypic features spanning from eye development in flies, red color patterning in Heliconius butterfly wings to brain development in humans. How evolutionary related TFs diversify their function has not been fully understood, especially changes to their DNA binding specificity. Using full length SIX proteins from Drosophila melanogaster, Heliconius erato, and Homo sapiens, we performed in vitro Systematic Evolution of Ligands by Exponential Enrichment (SELEX‐seq) to identify the DNA binding specificity. Our preliminary data shows the majority bind to their canonical binding motif (TGATAC), except for six4 members which prefer TGACAC. Interestingly, the way they bind to these motifs differs. Both sine oculis and six4 homologs require a 5’‐GA flanking the core motif. While optix related members prefer a shorter flaking region with less dependence on 5’‐GA. This is interesting since optix is more evolutionarily related to six4 than to sine oculis. In addition, we found that Heliconius erato optix can bind both as a homodimer with a preferred spacing of 2‐bp between binding sites or as a monomer. Our results provide new insights in the binding diversity found in the SIX family of TFs and predict genomic targets of these understudied TF family.
Convergent evolution of bacterial ceramide synthesis
Authors: Klein Eric , Stankeviciute Gabriele , Tang Peijun , Ashley Ben , Chamberlain Joshua , Hansen Matthew , Coleman Aimiyah , D'Emilia Rachel , Fu Larina , Mohan Eric , Nguyen Hung , Guan Ziqiang , Campopiano Dominic
Published: May 2022

Bacteria synthesize numerous types of sphingolipids with various physiological functions. Despite their roles in mediating host inflammation, cellular differentiation, and protection from environmental stress, their biosynthetic pathway remains undefined since several essential eukaryotic ceramide synthesis enzymes have no bacterial homologue. Using genetic and biochemical approaches, we identified the complete pathway for bacterial ceramide synthesis. Bioinformatic and phylogenetic analyses revealed the presence of these genes in a broad range of bacterial taxa and led to our discovery of the first Gram‐positive species to produce ceramides. Biochemical experiments with purified proteins support a model in which the bacterial pathway operates in a different order than in eukaryotes. Furthermore, phylogenetic analyses are consistent with the independent evolution of the bacterial and eukaryotic ceramide pathways. Current work is being done to elucidate the specific subcellular localization of the synthetic enzymes and identify additional proteins required for the transport of sphingolipids to the outer membrane of Gram‐negative bacteria.
Andrographolide‐Mediated Protection of Barrier Integrity and Permeability is Mediated Through NLRP3 Inflammasome Inhibition in Blood‐Brain Barrier Endothelial Cells
Authors: Waithe O'lisa Y. , Muthusamy Saravanakumar , Hunter Felicia A. , Childs Ed W. , Tharakan Binu
Published: May 2022

The blood‐brain barrier (BBB) that consists of the inter‐endothelial tight junctions, acts as the major blood‐brain interface and regulator of brain microvascular hyperpermeability. In the brain, the loss of BBB integrity and hyperpermeability leads to cerebral edema formation and intracranial pressure elevation in traumatic brain injury and ischemic strokes. Recent studies demonstrated that reactive oxygen species are potential signals for the NLR family pyrin domain containing 3 (NLRP3) inflammasome activation that leads to interleukin‐1β (IL‐1β) secretion. IL‐1β plays a major role in promoting BBB hyperpermeability in various brain pathologies, including TBI. Furthermore, recent studies from our lab have demonstrated a tri‐phasic role for hydrogen peroxide (H2O2) in BBB endothelial cells where it promotes, angiogenesis, hyperpermeability and apoptosis in a tri‐phasic and concentration dependent manner. Hydrogen peroxide plays a major role physiologically as the second messenger and pathologically as an inducer of oxidative stress and associated cellular signaling. We hypothesized that in BBB endothelial cells, H2O2‐induced barrier dysfunction and hyperpermeability are NLRP3 inflammasome‐dependent. The main objective of our study was to evaluate the role of NLRP3 inflammasome signaling in H2O2‐mediated barrier dysfunction and hyperpermeability in human brain microvascular endothelial cells and to test whether andrographolide, a labdane diterpenoid possessing potent anti‐inflammatory properties will attenuate such effects. Th effect of H2O2 treatment on tight junction integrity/permeability was studied using zonula occludens‐1 (ZO‐1) immunofluorescence localization, ZO‐1 immunoblot analysis, Transwell monolayer permeability assay using FITC‐dextran (10‐kDa) as a fluorescent marker and cell viability/apoptosis assay. The effect of NLRP3 inhibitor (MCC950) and andrographolide on H2O2‐induced hyperpermeability was studied using Transwell permeability assay. Our results show that, CRISPR/Cas‐9‐mediated knockdown of ZO‐1 resulted in monolayer hyperpermeability demonstrating the significance of ZO‐1 in regulating barrier functions. CRISPR‐based activation of NLRP3 induced monolayer hyperpermeability whereas CRISPR/Cas‐9‐based NLRP3 knockdown had no significant effect on permeability. H2O2 (10µM) induced monolayer hyperpermeability and the effect was decreased by MCC950 and andrographolide treatment. Hydrogen peroxide induced cathepsin B (an activator of the NLRP3 pathway) activity significantly, and the effect was decreased by andrographolide treatment. H2O2‐mediated barrier dysfunction and hyperpermeability were not due to changes in cell viability/apoptosis. These results suggest that NLRP3 is a mediator of H2O2‐induced barrier dysfunction and hyperpermeability in human BBB endothelial cells and andrographolide is an effective inhibitor of this pathway.
Synergistic Effects of Anti‐Cancer Drug Administration on Acute Myeloid Leukemia
Authors: Canavan Margaret A.
Published: May 2022

Acute Myeloid Leukemia (AML) is a malignant disease where there is a high rate of chemoresistance, or drug resistance, in patients which is a leading cause of treatment failure. Current clinical treatments of AML include Cabazitaxel and Gemcidibine, which affect DNA replication in two different ways. A potential strategy to overcome chemoresistance is to treat resistant‐AML cells with combinations of drugs that have different therapeutic targets. Since BCL‐2 proteins, which control apoptosis, are often upregulated in AML, we hypothesize that BCL‐2 inhibitors such as Venetoclax are candidate drugs to be considered for use in treating Cabazitaxel‐resistant and/or Gemcitdibine‐resistant AML. Moreover, cancer cells are also known to have dependence on glucose metabolism and therefore glucose inhibitors, such as WZB117, are also candidate drugs to overcome chemoresistance in AML. Therefore, the overarching goal of this project is to investigate the potential synergetic effects of a WZB117 and Venetoclax on chemo‐resistant AML cells Using the AML cell line HL60, baseline sensitivity to Cabazitaxel and Gemcidibine have been determined using GI50 assays. The GI50 values obtained were 1.35uM for Cabazitaxel and 0.94uM for Gemcidibine. Currently, resistant cells are being developed by treating HL60 cells with the GI10 values for each drug until their normal growth rate returns, at which time new GI50 values will be obtained and the resistant cells dosed again to perpetuate resistance. Baseline sensitivity of HL60 cells to WZB117 and Venetoclax is also ongoing. Once cells reach 5‐fold resistance for each drug, the effectiveness of WZB117 alone, Venetoclax alone, and both drugs in combination will be assessed to determine if these drugs are a viable option for inducing apoptosis in Cabazitaxel‐resistant and/or Gemcitabine‐resistant AML cells.
Vertically‐Integrated Course‐Based Undergraduate Research Experiences (CUREs) Structure a Biomedical Research Certificate Program that Promotes Inclusivity
Authors: Quick Christopher M. , Cisneros Marissa R.
Published: May 2022

Underrepresented mentors are critical for impacting the trajectory of underrepresented students toward research careers. Not only do they share lived experiences but can understand the unique challenges of navigating a predominantly white, ableist university. Undergraduates often view these mentors as role models for successful science careers and these mentors can create supportive microenvironments for underrepresented students. Although first‐ and second‐year classes offer broad access and are often the site of first contact with underrepresented faculty, teaching centered instructors are less likely to have an active research program. Minority‐serving undergraduate research programs can generate social stigma due to competitive norms of science culture and embedded bootstrapping rhetoric. Course‐based Undergraduate Research Experiences (CUREs) are often cited as a particularly promising approach to preparing and motivating undergraduates to pursue graduate study that avoids stigma. However, authentic research projects rarely can be completed in a single semester, and CUREs are not typically designed to prepare students to enter particular research labs. Furthermore, CUREs typically are only available in third and fourth years, have extensive prerequisites, and are restricted to honors students (only 19% of underrepresented students in the five biomedical majors have sufficient GPAs to be eligible for honors at TAMU). One‐semester CUREs are insufficient to prepare underrepresented students for graduate study. The Biomedical Research Certificate (BRC) Program provides vertically integrated CUREs to prepare undergraduates for the next stage of their careers. A research certificate was created to address these considerations. The BRC is a multi‐college program that allows undergraduates to perform research in collaborative teams from the students’ freshman to senior years. Open to any major, it attracts diverse students across the university interested in participating in multi‐semester health‐related research. Because there are no entry requirements (e.g., prerequisites or minimum GPA) and courses count toward degree plans, it is the only program at TAMU in which there are no barriers to participating in research starting in their freshman year. This access has radically expanded research participation of lower‐division undergraduates. Freshmen explore multiple research disciplines and join a particular research track in their sophomore year. Built to expand to meet the ever‐increasing needs of both students and research labs, the BRC has grown to include research tracks in health disparities, computational biology, in vitro experimentation, human subject research, bioinformatics, and neuroscience. These tracks not only engage undergraduates in authentic research projects, but also provide the critical skills needed to take full advantage of opportunities across TAMU in junior and senior years.
Mechanistic Elucidation of an Anti‐Epileptic for the Treatment of Glioblastoma
Authors: Yadav Anjali , Alnakhli Ali , Vemana Hari Priya , Bhutkar Shraddha , Barot Shrikant , Muth Aaron , Dukhande Vikas
Published: May 2022

Glioblastoma multiforme (GBM) is a highly proliferative grade IV malignant astrocytoma with a 5‐year survival rate of ~5%. The current treatment strategies for GBM including surgery, radiation, and chemotherapy face multiple challenges such as resistance to standard chemotherapeutic temozolomide (TMZ), sensitive location, and presence of blood‐brain barrier that hinders permeation of various chemotherapeutics. The dire need and urgency to develop a new therapeutic for the treatment of GBM led us to explore metabolic vulnerabilities of cancer. Stiripentol (STP) is an FDA approved drug used for the treatment of Dravet’s syndrome (a rare type of epilepsy) and a putative LDH inhibitor that we screened out of many metabolic inhibitors for GBM. STP showed efficacy in cytotoxicity and proliferation assay on U87 and U138 GBM cells. STP also showed efficacy in clonogenic assay and wound healing assay in GBM cells. To further explore and discover the mechanistic target of STP for the treatment of GBM, we performed apoptosis assay, cell cycle assay, and studied the expression of proteins involved in apoptosis, metastasis, and cell signaling. In addition, 3D spheroid study was performed to mimic the tumor microenvironment and the effect of STP was assessed on the same. Next, synergy studies of STP with standard of care TMZ was performed on U87 cells. Cellular toxicity studies were performed to evaluate the safety profile of STP and the combination treatment with TMZ on normal human cells. We will elucidate mechanistic basis of STP anticancer activity in GBM cells and test preclinical potential of STP in tumor xenograft model. Therefore, STP is an effective compound that can augment TMZ for GBM treatment and merits further investigation.
Toward a Validated Metabolic Systems Thinking Framework
Authors: Barton Tyler
Published: May 2022

Several recently reported research projects describe or measure student reasoning about biochemical pathway dynamics and regulation (BPDR). In this growing literature, a variety of approaches are taken to assess conceptual understanding in this area. While some studies have qualitatively investigated student misconceptions that arise in the context of research interviews, others have used test instruments constructed by biochemistry instructors to evaluate student learning quantitatively in the context of biochemistry course exams. In all cases, the focus has been on explicit biochemistry content with less emphasis on underlying general conceptual challenges. For both instructional and research purposes, a BPDR assessment framework based on underlying latent variables particularly informed by research in systems thinking is desirable.

This project builds on the insights offered by the work cited above but takes a different approach to assess BPDR reasoning by considering the problem from the perspective of domain‐general conceptual challenges in systems thinking. Drawing on systems thinking research in the learning sciences and a variety of science education research fields, a preliminary Metabolic Systems Thinking Framework (MSTF) is proposed. This framework includes five factors – organizational levels, direct relationships, indirect relationships, parallel processes, and autoregulation. These factors are proposed to represent independent conceptual factors that influence reasoning in the context of BPDR problems.

The sufficiency of the MSTF as a tool to assess student reasoning for BPDR is being evaluated through think‐aloud interviews with problems related to reasoning about glycolysis. Data from interviews with undergraduate biochemistry students has yielded evidence for the necessity of each of these factors, but whether they are jointly sufficient in this regard is currently being explored further. Additionally, a survey instrument is currently in development to establish the construct validity of the MSTF. This instrument includes multi‐item subscales for each of the factors of the MSTF. The instrument will be administered to undergraduate students enrolled in a biochemistry course covering metabolic pathways in spring 2022. Psychometric properties of the instrument will be determined to establish instrument reliability followed by confirmatory factor analysis to assess the construct validity of each factor.
Structural and Biophysical Characterization of Plasmodium falciparum Bromodomain Protein 1
Authors: Singh Ajit K. , Alkrimi Saleh , Glass Karen C.
Published: May 2022

Epigenetic regulatory mechanisms are important for the growth and survival of eukaryotic organisms. Post‐translational modifications found on histone proteins in the nucleosome function as a dynamic cell signaling mechanism that controls many essential functions such as cell growth, DNA replication, and gene expression. Bromodomains are conserved protein interaction modules that specifically recognize acetylated lysine residues on histones. Among five Plasmodium species, P. falciparum is the major cause of malaria cases worldwide. According to a WHO report in 2019 there were 409,000 deaths reported worldwide and the most affected age group was of children under 5 years of age. Interestingly, the P. falciparum genome encodes eight bromodomain‐containing proteins, and bromodomain protein 1 (PfBDP1) has been shown to play an important role in malaria pathogenesis. The domain architecture of PfBDP1 includes a single C‐terminal bromodomain and several ankyrin repeats. PfBDP1 is thought to act by tethering a transcriptional activator complex to acetylated histone H3 to control genes required for parasite invasion. Additionally, PfBDP1 forms a complex with the bromodomain protein 2 (PfBDP2) to carry out its cellular functions. We hypothesized that the bromodomain is crucial for the function of PfBDP1 via recognition of acetylated lysine modifications. We carried out several histone binding assays to identify novel histone interactions of PfBDP1 bromodomain. We also utilized a combination of structural biology and biophysical approaches to identify amino acid residues critical for ligand coordination. The rise of drug‐resistance towards P. falciparum has created an urgent need to outline the molecular mechanisms driving pathogenesis in hopes of uncovering new potential drug targets. Importantly, the results from this study may improve the development of new therapeutics to treat malaria.