bluuebunny/crossref_metadata_2025 huggingface dataset:
Revealing human carboxylesterase 1 (CES1) sequence‐dependent activity variations using fluorescent chemical tools
Authors: Beck Michael W.
Published: May 2022

Carboxylesterases (CESs) play a key role in the metabolism of many small molecule therapeutics including Remdesivir, the first drug authorized by the FDA to treat COVID‐19. The overall activity of CESs, however, can be drastically different person to person. This interindividual variability has been shown to influence the metabolism and, in some cases, clinical outcomes of patients treated with drugs that are substrates for carboxylesterase 1 (CES1). The factors that contribute to this interindividual variability have not been fully uncovered, despite the established importance of CES1 in drug metabolism. We believe that this lack of knowledge is due to the scarcity and limitations of currently available approaches to study CES1 activity in live samples. To address these challenges, we have developed new fluorescent chemical tools that can specifically report on CES1 activity in live cells. Subsequently, we deployed our CES1 fluorescent chemical tools in a new chemical biology approach to monitor CES1 sequence‐dependent activity variations in live cells. Overall, the tools and approaches we develop can help uncover factors that result in interindividual variability of CES1 activity.
Metagenomics Analysis of Viral Families Carried by Aedes Mosquitos in Houston
Authors: Silguero Martin , Pham Franklin , Hernandez Ana , Strobel Sarah , Le Angelina , Witt Lauren , Najarro Michelle , Ngo Ava , Gonzalez Paola , Larios‐Sanz Maia , Rosell Rosemarie
Published: May 2022

Mosquitos are known vectors of many serious viral diseases, but little is known about the complete collection of viral strains that they might carry. Historically, we have relied on culturing techniques to study microbial diversity, but genomic analyses have demonstrated that there is much more out there than initially suspected. In this study, we used a metagenomic approach to characterize the viral populations carried by different species of mosquitos found in Houston. Specifically, we sequenced the entire genetic material in pooled mosquito samples, followed by analyses with computational tools to identify each sequence obtained. We collected mosquitos from various locations in Houston and separated them by both species and sex using morphological characteristics and DNA barcoding. Total DNA and RNA were used in order to characterize both DNA and RNA viruses. RNA samples were converted to cDNA to facilitate sequencing and the combined DNA/cDNA libraries were sequenced using 454 technology. We used a modified BLAST workflow developed in‐house to analyze the resulting sequences. Our first analysis involved samples of male and female Aedes aegypti.Our results showed that we are able to detect both DNA and RNA viral sequences in our samples, as well as thousands of unknown or novel sequences. A total of 51 different species of viruses were identified in the female sample, and 40 different species in the male sample. Those of interest include West Nile and Dengue, as well as lesser known viruses, like Nam Dinh. Interestingly, the female sample carried more RNA viruses than the male sample, although further work will reveal if this finding is significant or an experimental artifact. Currently, we are in the process of building libraries from new samples of mosquitos, including other species typically found in our area, such as Culexand Anopheles. We plan on further analyzing the sequence libraries obtained, with particular focus on the unknown sequences, many of which may be viral in origin. These studies will help us better understand the viral collections carried by different mosquitos in the Greater Houston area.
Triple Negative Breast Cancer (TNBC) Cells Growth is Inhibited by High Dose Ascorbic Acid
Authors: Bennett Lunawati L.
Published: May 2022

Objective

According to the World Health Organization report, the incidence of breast cancer mortality is very high worldwide with 2.1 million women are being affected each year and death are estimated at 627,000 women. There are several subtypes of breast cancer. One of these subtypes is known as triple‐negative breast cancer (TNBC). TNBC doesn’t have the receptors for the hormone estrogen, nor progesterone, nor express HER2/neu; therefore, TNBC doesn’t respond to hormonal nor targeted therapies which makes TNBC difficult to treat.

The desired outcome of the cancer therapies is to decrease the proliferative activity of the cancer cells, to minimize drug resistance, and to decrease the toxicity of the anticancer drugs. Ascorbic acid has gained popularity as preventive agent for cancer due to its minimal cytotoxic to normal cellular tissue; however, the use of ascorbic acid for the treatment of cancer has controversial history. Taking oral ascorbic acid had no or little effect on cancer treatment, but recent discovery that intravenous ascorbic acid can provide 100‐fold higher plasma levels than oral intake has renewed interest on the role of ascorbic acid in the cancer treatment.

Hypothesis

At high dose (pharmacological concentrations), ascorbic acid may act as a prodrug to deliver extracellular H2O2 that is selectively toxic to cancer cells, but non‐toxic and well tolerated to normal cells. It is postulated that TNBC cells growth will be inhibited by high dose of ascorbic acid.

Methods

Ascorbic acid was buffered to pH 7 with sodium hydroxide and prepared immediately before use. MDA‐MB‐231 cells was used as TNBC model. MTT assay, Hoechst33342, H2DCFDA, and Rhodamine123 staining using fluorescent microscope were conducted to determine cell viability, chromatin inducing apoptosis, intracellular generation of ROS, and mitochondrial membrane potential, respectively. Western Blot to detect different signaling protein expressions such as Poly (ADP‐Ribose) polymerase (PARP), Casp‐3, Casp‐9, P‐53, Bax, Bcl2, Apaf‐1, MMP2, MMP 9, BRCA1, BRCA2, Bim, Map kinases were conducted to test the hypothesis that ascorbic acid was effective to kill TNBC cells.

Conclusion

This finding suggests that high dose ascorbic acid has a potential candidate as effective anti‐cancer for TNBC. At 1.6 mM, ascorbic acid killed MDA‐MB‐231, a TNBC cells by causing increased in caspase 3‐9 activities and by decreasing cell cycle regulatory proteins, Mitogen activated kinase 1‐4, and reactive oxygen species.
Structure of a pathologic amyloid nucleus determined by rational genetic deconstruction of an intracellular nucleation barrier
Authors: Halfmann Randal , Kandola Tej , Venkatesan Shriram , Zhang Jaihui , Lerbakken Brooklyn , Blanck Jillian F. , Wu Jianzheng , Unruh Jay , Berry Paula , Lange Jeffrey J. , Von Schulze Alex , Box Andrew , Cook Malcolm , Sagui Celeste
Published: May 2022

A long‐standing goal of the study of amyloids has been to characterize the structural basis of the rate‐determining nucleating event. However, the ephemeral nature of that event has made it inaccessible to classical biochemistry, structural biology, and computational approaches. Here, we addressed that limitation by using Distributed Amphifluoric FRET to measure the dependence of amyloid formation on concentration and conformational templates in living cells, whose volumes are sufficiently small to resolve the outcomes of independent nucleation events. We characterized numerous rationally designed sequence variants of polyglutamine (polyQ), a polypeptide that precipitates Huntington’s and other amyloid‐associated neurodegenerative diseases when its length exceeds a characteristic threshold. This effort uncovered a pattern of approximately twelve Qs, only for polypeptides exceeding the clinical length threshold, that allow for amyloid nucleation to occur spontaneously within single polypeptides. Nucleation was inhibited by intermolecular phase separation. Using atomistic molecular dynamics simulations, we found that the pattern encodes a minimal steric zipper of interdigitated side chains. Lateral growth of the steric zipper competed with axial growth to produce “pre‐amyloid” oligomers. By illuminating the structural mechanism of polyQ amyloid formation in cells, our findings reveal a potential molecular etiology for polyQ diseases, and may provide a roadmap for the design of new therapies.
Let's Play Games: A comparison of case‐based learning approach with gamification technique
Authors: Zafar Umema , Khattak Madiha , Zafar Hamna , Rehman Huma
Published: May 2022

Case based learning technique (CBL) has been in practice throughout the world for a few decades now. Our institute adopted it some four years back, when it shifted towards modular system of teaching. It is the main technique being used for conducting small group discussions. We decided to introduce a new technique called the gamification technique of conducting small group discussion. Need was there to know the effectiveness of the new technique as well as to assess the factors for its preference so it could be modified to be more productive. The aim of this research was to assess the effectiveness of gamification teaching technique in comparison to the traditional CBL technique both quantitatively and qualitatively.

This is a mixed‐method randomized controlled trial, conducted in Khyber Medical College on First Year Medical Students from June to October 2021. Group based teaching involving both CBL and gamification approach was used in this study in a cross over manner. Addressing the ethical concerns, and after informed consent pre‐ and post‐testing was done to quantify the performance and an open‐ended survey was disseminated after the sessions to check the perception of the students.

The study recorded (quantitatively) that post testing mean score of gamification teaching technique was 3.41±0.982, while for CBL the mean was 3.55±1.055, recording an insignificant difference with p‐value of 0.608. In qualitative analysis, about 12 (80%) students preferred the gamification technique, their perception being that it instills competitiveness, increases the involvement of students in class and their motivation level. This research further revealed that the CBL approach had the advantage of quick learning via the facilitator presentation, and due to the handouts, it was easy to follow. Some of the negative points of CBL reported were that; the participants found it a boring and monotonous way of learning. The drawback of gamification reported was, that the students were unsure about the accuracy of the information they prepared initially as it wasn’t being directed by the facilitator. The study concluded an insignificant difference among the two techniques quantitatively; however, qualitatively the students preferred gamification as a better technique for learning.
Human Cadaveric Head Disarticulation, Bisection, and Pharynx & Larynx Dissection; A Student‐to‐Student Perspective
Authors: Seely Kevin D. , Berry Adam , Adamson Colby , Jones Jordan , Troy Amanda
Published: May 2022

Cadaveric dissection has long been a central component of medical school curricula in both allopathic and osteopathic training. However, presumptive and lengthy dissection instructions in commonly used laboratory guides are among the limiting factors in foundational dissection experiences, causing a steep learning curve. This educational study aims to produce a more precise and practical dissection guide for the historically difficult dissection of the human head; including disarticulation, bisection, and the dissection of the pharynx & larynx. Hypothesized to provide memory and learning anchors due to ease of association, osteopathic correlations and anatomical considerations are provided throughout the guide, particularly in the disarticulation process. In addition, the guide aims to allow a student to succeed on their first attempt at dissecting by providing students’ perspectives on the procedures involved; including photos, pitfalls, and tips. Clinical correlations are also provided throughout. Medical students are encouraged to begin reasoning from their understanding of anatomy, and this dissection manual is intended to guide them in doing so.
Genomic Study of Dopamine Receptor Ligand Binding Sites of the Bivalve Crassostrea virginica
Authors: Small Shatema , Catapane Edward J. , Hinkley Craig , Carroll Margaret A.
Published: May 2022

Gill lateral cells of Crassostrea virginica are innervated by dopamine (DA) and serotonin nerves. DA slows down lateral cell cilia beating rates and serotonin accelerates them. DA receptors are classified as D1R and D2R. Physiology and cell biology work of our lab found the DA receptors involved in gill lateral cell cilia inhibition are D2R‐like in the gill cells and D1R‐like in the cerebral and visceral ganglia. Our HPLC studies found DA in various tissues, including gill, cerebral and visceral ganglia of Crassostrea virginica. Using immunofluorescence techniques, we showed the presence of DA neurons in cerebral and visceral ganglia as well as D2R‐like postsynaptic receptors in gill lateral cells and D1R‐like postsynaptic receptors in cerebral and visceral ganglia. Recently the genomes of C. virginica and other bivalves have begun to be mapped. By conducting searches of the NCBI (National Center for Biotechnology Information) database using DNA and protein sequences of C. virginica and other invertebrate and mammalian species we found matches for D1R genes on chromosomes 4 and 5, and D2R genes on chromosomes 3 and 5 of C. virginica. BLASTS of the receptors found matches with very low Expect Values (E values) and high Percent Identity of the D1R and D2R receptors to those in other bivalves, gastropods, insects, mice, rats and humans. Various invertebrates had Percent Identity above 60%, while humans and mice had Percent Identity of 30 ‐ 40%. We hypothesize that the ligand binding sites (LBS) for D1R and D2R receptors in C. virginica are evolutionarily conserved and will closely match those of other animals. To study this, we searched the NCBI database for D1R and D2R LBS of C. virginica and compared them to other animals. We found D2R LBS contained 17 amino acids (W, D, V, S, F, T, L, S, S, S, W, F, F, N, F, T, Y) with very highly conserved (70 ‐ 100%) to LBS of other bivalves, gastropods, insects, mice, rats and humans. D1R LBS have not yet been identified in C. virginica, nor in the other animals we searched for, except for humans where it contained 17 amino acids (W, D, I, S, T, S, A, S, S, S, W, F, F, N, F, V, W). The study complements our physiology and cell biology studies demonstrating the presence and function for DA in C. virginica, and shows the genome of C. virginica contains genes to produce DA receptor LBS that are similar to those of other animals. This new information is valuable as it shows that the simple nervous system of C. virginica can be used to expand studies on DA neurotransmission.
The Microsomal Triglyceride Transfer Protein Inhibitor, Lomitapide, Improves Vascular Function in Obesity
Authors: Munkhsaikhan Undral , Kwon Young‐In , Sahyoun Amal M. , Ait‐Aissa Karima , Kassan Modar , Kassan Adam
Published: May 2022

Objectives

In this study, we investigate the effect of lomitapide, a microsomal triglyceride transfer protein inhibitor, on cardiovascular function in obesity.

Hypothesis

We hypothesize that lipid level downregulation by lomitapide is a fundamental mechanism that improves cardiovascular function.

Methods

Eight‐week‐old C57/b6 mice were fed with a high fat diet (HFD) for 12 weeks in the presence and absence of lomitapide. Lomitapide was administered by gavage (1mg/Kg/Day) for the last 2 weeks of HFD feeding. Bodyweight, blood glucose, body composition, and lipid profile were determined. Vascular function and endothelial function markers were studied in aorta and mesenteric resistance arteries (MRA).

Results

Lomitapide treatment reduced the body weight in obese mice. Blood glucose, % of fat mass, total cholesterol, and LDL levels were significantly reduced and the % of lean mass was significantly increased after lomitapide treatment. Aortic and mesenteric arteries vascular response to sodium nitroprusside (SNP) was similar among groups. However, the vascular response to acetylcholine (Ach) was improved in the treated group. This was associated with a decreased level of vascular endoplasmic (ER) reticulum stress, inflammation, and oxidative stress.

Conclusions

Treatment with lomitapide attenuated the increase in body weight in obese mice and restored the lipid profile and vascular function. These effects were accompanied by a decrease in inflammation and oxidative stress.
Therapeutic Potential of Justicia secundaon Oxidative Stress Indices in N‐Acetyl‐P‐Benzoquinone Imine Induced‐ Hepatotoxic Rats
Authors: Nosiri Chidi , Oguike Victor , Nwaogwugwu Caleb , Obiwuru Ikenna , Nosiri Prinz , Friday Aaron
Published: May 2022

The therapeutic potential of Hot Water Leaf Extract of Justicia secunda(HWEJS) on oxidative stress indices in paracetamol (N‐Acetyl‐p‐Benzoquinone Imine) induced hepatotoxic rats was investigated. Justicia secunda is a blood boosting plant used traditionally with so many medicinal activities. Five groups of 10 rats each were used for the study, GpD was the control given n‐saline and the test grps A,B and C were administered with 2000mg/kg N‐Acetyl‐p‐Benzoquinone Imine (NAPQI) and after 24hrs, groupsB and C were administered with 200 and 400mg/kg of HWEJS respectively for 7 days. The body weights were also measured. Results of the study showed a relative weight loss of all the groups except the control in the first 2 days thereafter a relative increase was observed throughout the duration of the experiment. This could be as a result of the ameliorative effect of the extract against NAPQI overdose. Oxidative stress indices in the animals treated with NAPQI (as shown in the figure) increased the levels of the oxidative enzymes while the 200 and 400mg/kg HWEJS reduced them except in Malondealdehyde which showed a reduced level with NAPQI with increased levels on the treated groups. Gpx showed a significant (p<0.05) decrease at 200mg/kg and 400mg/kg. CAT and SOD decreased significantly (p<0.05) at 400mg/kg and non‐significant (p>0.05) at 200mg/kg respectively relative to control. MDA, an indicator for lipid peroxidation recorded non‐significant (p.>0.05) levels for all the test groups when compared to the control. These results suggest that the oral administration (p.o) of hot water extract of the leaves of J.S have antioxidant activities at the doses of 200 and 400mg/kg in the hepatotoxic rats. The combination of SOD, CAT and GPx enzymes constitute a first line antioxidant defense system that plays a crucial role in total defense mechanisms during metabolic processes. In the extract treated animals, SOD showed that it may have removed superoxide anions from oxidative insult by helping in breaking down potentially cellular harmful oxygen molecules while the CAT and GPX protected the cells from damage due to free radicals like the peroxides. This result could suggest that the hot water extract of Justicia secunda can be used in conditions where harmful oxygen molecules are believed to play a role in a disease or in oxidative stress‐related diseases as a therapeutic agent.
The beneficial effect of SCFAs on the cardiovascular system during hypertension
Authors: Kwon Youngin , Munkhasaikhan Undral , Sahyoun Amal M. , Kassan Modar
Published: May 2022

Hypertension, which is associated with gut dysbiosis, is an independent and preventable risk factor for the development of cardiovascular diseases. The gut microbiota and its metabolites such as short‐chain fatty acids (SCFAs) play an important role in host physiology. SCFAs can regulate blood pressure in both rodent models and humans. However, the exact mechanism by which SCFA regulates hypertension is unknown. Our data showed that SCFAs can regulate miR‐204, a non‐coding RNA that is expressed in vessels. We recently showed that miR‐204, is reduced in vessels during hypertension. Additionally, miR‐204 targets inositol 1, 4, 5‐triphosphate receptor type1 (IP3R1) and therefore disrupt the calcium release into the cytosol leading to exacerbated vascular constriction. Thus, we hypothesize that SCFAs can regulate blood pressure and vascular constriction through miRNA‐204/IP3R1/Ca2+ axis. To test our hypothesis, we treated hypertensive mice with SCFAs cocktail (67.5 mM acetate, 40 mM butyrate, 25.9 mM propionate) in drinking water for 21 days. Increased blood pressure from hypertensive mice was reduced after SCFAs intake. Aortas and mesenteric arteries from hypertensive mice display increased vasoconstriction in response to phenylephrine, U46619, and angiotensin II. Treatment with SCFAs reduced vasoconstriction in response to all agonists. During hypertension, aortas display low levels of miR‐204 expression which was recovered after SCFAs intake. Additionally, IP3R1 was significantly up‐regulated in hypertensive mice and decreased after SCFAs supplementation. Furthermore, our in vitro data showed that SCFAs can directly affect the vascular miRNA‐204/IP3R1/Ca2+ axis. Our results suggest that SCFAs can regulate Ca2+ homeostasis and vascular contraction through the miR‐204/IP3R1 axis. The outcome of this study will establish novel treatment strategies conferring the benefits of SCFA control while circumventing the potential risks of delivering live biologics.
Developing Assessments to Gauge Biomolecular Visual Literacy: A 5 step approach to ensuring accuracy and reliability
Authors: Procko Kristen , Engelman Shelly , Beckham Josh , Dries Dan R. , Franzen Margaret A. , Jakubowski Henry , Novak Walter R. , Roberts Rebecca , Roca Alberto I. , Shor Audrey C.
Published: May 2022

BioMolViz is a community of educators, assessment experts, and biomolecular visualization enthusiasts dedicated to improving biomolecular visual literacy. A central goal of the project is to create a repository of validated assessments that instructors can use in their classrooms to gauge students’ visual literacy. Through three‐day workshops, we convene faculty to craft and peer review biomolecular visualization assessments in teams. These workshops are often the first step in our assessment development process. In this presentation, we will describe a five‐step model for designing and validating assessments that consists of: 1. Item creation 2. Item revision 3. Expert panel review 4. Field testing and 5. Dissemination through the online BioMolViz repository. Results from the first three phases of the process will be reported, and our plan for field testing and disseminating assessments will be discussed. In 2020, our workshops and assessment development model moved online, which opened up new pathways for sustained engagement with workshop participants. In particular, we will share how a new working group emerged when we established our remote workflow. A subset of the group, The BioMolViz Fellows, continued working with our community for several semesters and has become integral to the iterative assessment revision process. Alongside our validation process, we will highlight insights for building online communities, establishing remote workflows, and sustaining long‐term participant engagement.
Structural insight into key mevalonate pathway enzymes
Authors: Kung Yan , Ragwan Edwin
Published: May 2022

The mevalonate pathway produces the universal biosynthetic precursors of steroids and isoprenoids, the largest and most structurally diverse class of natural products. Two key enzymes control metabolic flux through the pathway: HMG‐CoA reductase (HMGR), which catalyzes the committed and rate‐determining step, and mevalonate kinase (MK), a target of feedback regulation in many organisms. Although HMGR inhibition by cholesterol‐lowering statin drugs is well studied, critical mechanistic and structural features are still unknown. With MK, large variations in the structures of MK homologs remain uncharacterized, even as greater structural insight may shed light on varying strategies of MK feedback inhibition. Using biochemical and X‐ray crystallographic methods, we have revealed large conformational movements in HMGR that control substrate and cofactor binding and release during the reaction, in addition to uncovering the structural basis for NAD(P)H cofactor specificity. We have also solved crystal structures of new MK homologs, suggesting novel structural strategies for MK inhibition. Overall, this work significantly advances our structural and mechanistic understanding of the two key enzymes of an important metabolic pathway.
Nonstructural Protein 1 of SARS Coronavirus Interacts with Stress Granule Protein G3BP1 and Accumulates in the Stress Granule
Authors: Nag Anita , Gerassimovich Yevgeniy A. , Bridges Kaitlin M.
Published: May 2022

The nonstructural protein 1 (nsp1) of severe acute respiratory syndrome coronavirus (SARS‐CoV and SARS‐CoV‐2) orchestrates a multi‐pronged mechanism to suppress host gene expression, also known as the host shutoff. The C‐terminus of nsp1 competes for the mRNA binding site in the 40S ribosome, preventing host translation from host mRNAs. Additionally, nsp1 triggers selective cleavage of host mRNAs while keeping the viral RNA intact. Nsp1 of SARS‐CoV and SARS‐CoV‐2 share 84% sequence identity, prompting us to believe they share a similar mechanism of host shutoff. So far identification of nsp1‐interacting partners has isolated stable complexes of nsp1 with the 40S and 60S ribosomal proteins. However, these complexes do not reveal any transient interaction nsp1 might have with other host proteins to suppress cellular function. Using proximity‐dependent biotinylation to capture the transient interactions of nsp1 with other cellular proteins, we found multiple subunits of the nuclear pre‐mRNA processing complex and cellular stress granule‐associated proteins. Here, we show that nsp1 interacts with the majority of these pre‐mRNA processing and stress granule‐associated proteins through its C‐terminal domain, the region responsible for binding the 40S ribosome. In addition, nsp1 co‐immunoprecipitates with stress granule‐associated protein G3BP1 and eIF4G. Upon induction of stress, these proteins accumulate in nonmembranous organelles called stress granules that are known to store stalled translation complexes. Using both immunofluorescence and immunoblot against stress granule‐associated proteins we show that nsp1 disrupts the maturation of stress granules.
Effect of miRNA: MRE Complementarity for SBP‐1 Gene Silencing in a Caenorhabditis Elegans Apoptosis Model
Authors: Cocquyt Alexander J. , Wee Abigale H. , De Lucas
Published: May 2022

Small Interfering RNAs (siRNA) are a type of post‐transcriptional gene regulator with perfect complementarity to its target gene/mRNA. microRNAs (miRNA) are a type of post‐transcriptional gene regulator that silences a number of genes based on sequence complementarity to the miRNA Response Element (MREs) on the target mRNA. siRNAs of varying complementarity to their target sequence will be used as a proxy to [investigate/disprove] the existence of a miRNA complementarity level that maximizes gene silencing of SBP‐1 which regulates lipid homeostasis in C. elegans.

The lipid homeostasis and fatty acid synthesis pathway in C. elegans is homologous to the human SREBP pathway. Red Nile staining and body size measurements provide proxy measurements for C. elegans lipid homeostasis and fatty acid synthesis in response to glucose feeding and siRNA introduction via soaking. SBP‐1, fat‐2, fat‐5, fat‐6, fat‐7, and elo‐2 mRNA expression in C. elegans is measured via RT‐PCR.

In this study, we determine whether higher siRNA:mRNA complementarity increases siRNA silencing of the Bcl‐2 and Sbp‐1 pathways in C. elegans following the introduction of high and low complementarity siRNAs as a proxy for investigating the gene silencing effects of miRNA:MRE complementarity.
Development and characterization of an aging rat model of sustained Gulf War Illness
Authors: Ramirez‐Sanchez Israel , Navarrete‐Yanez Viridiana , Villarreal Francisco
Published: May 2022

Gulf War Illness (GWI) afflicts ~30% of the US military personnel who served in the 1990‐91 Persian Gulf War. Prominent symptoms include cognitive deficits such as anxiety, memory impairment and fatigue. Currently, no treatments are available for GWI and the afflicted Veteran population has aged by an additional 30 years to an average age of mid‐50s. As per the expected effects of GWI plus aging, one would hypothesize that cognitive and muscle related symptoms would be further aggravated. With this goal in mind, we implemented an aging rodent model of GWI whereby young male rats. Animals were exposed to GWI chemicals/stress for 4 weeks or vehicles/no stress (controls) and allowed to age under normal conditions and feeding for an additional 15 months so as to replicate the current aging profile of Gulf War Veterans. Cohorts of 3 month old male Wistar control and GWI rats were implemented with an average initial body weight of ~260 g. Over a period of about 70 weeks, animals approximately doubled their body weight with no significant differences between both groups. Figure 1 reports on an average change in front limb strength of control rats over a period of 58 weeks. Limb strength is recorded in Newtons (N) and is normalized to animal weight (g). As can be observed, a clear trend for decreases in normalized front limb strength occurs over time. Figure 2, plots front limb strength recorded over a period of 58 weeks in control and GWI rats. As can be observed, a sustained difference is noted following a 4 week exposure to GWI chemicals (week 0‐4) that starts at week 8 and is sustained in a significant manner over a period of ~50 weeks. Thus, exposure to GWI chemicals/stress triggers a sustained long‐term phenotype that can be detected by measures of muscle strength. Studies were also implemented to assess changes in neurocognitive behavior as recorded at week 58. GWI animals demonstrate a greater level of anxiety as per significant differences noted in time spent in central vs. peripheral (wall) areas. Measures of short and long‐term memory were obtained using the object preference test. GWI rats exhibit a significant decrease in short‐term memory which was not evident in long‐term measurements. In summary, limited exposure of healthy young rats to GWI chemicals/stress triggers as in GWI patients, a sustained disease phenotype in muscle and neurocognitive measures that may be amenable to treatment with agents that stimulate mitochondrial bioenergetics and/or reverse muscle atrophy. This premise is currently being tested in this cohort of aging control and GWI rats using the cacao flavanol epicatechin.
Malate Dehydrogenase‐Citrate Synthase Multienzyme Complex Dynamics Is Affected By TCA Cycle Flux In Living Yeast Cells
Authors: Omini Joy J. , Krassovskaya Inga , Obata Toshihiro
Published: May 2022

A multienzyme complex is formed by enzymes that catalyze sequential reactions of a pathway, such as malate dehydrogenase (MDH) and citrate synthase (CS) of the Krebs tricarboxylic acid (TCA) cycle. The MDH‐CS multienzyme complex is assumed to be dynamic, but the relationship between its assembly and cellular metabolic flux is not yet established. We hypothesize that the dynamic MDH‐CS interaction is related to the TCA cycle flux. A time‐resolved in vivo protein‐protein interaction study was performed by split‐luciferase assay using nanoBIT system (Promega) in Saccharomyces cerevisiae (BY4741) cells. Cells were grown on different carbon sources, and the interaction of MDH‐CS multienzyme complex was measured as a function of luminescence. To induce change in metabolic flux, various carbon sources, electron transport chain inhibitors, and fermentation inhibitors were applied. We found that cells grown on glucose showed a significantly lower association of the complex compared to raffinose or acetate grown cells. Glucose, which represses the TCA cycle and oxidative phosphorylation via the Crabtree effect, triggered dissociation of the complex when it was added to raffinose grown cells, and the addition of a fermentation inhibitor restored this interaction. On the other hand, acetate which stimulates TCA cycle flux, enhanced the association of the complex. Glycerol/lactate also caused an abrupt increase in the MDH‐CS complex association. Interestingly, the electron transport chain inhibitors altered the MDH‐CS complex dynamics in varying ways. Taken together, these results show that the dynamics of the MDH‐CS complex is related to TCA cycle flux, and the MDH‐CS complex may play a role in regulating metabolic flux through the TCA cycle.
Cytotoxic Effect of Synthetic Peptides on Normal and Cancerous Cells
Authors: Nyman Karl , Kerns Hailey , Torres Daniel , Sheehan Hugo , Martin Rachael , Borysov Sergiy I.
Published: May 2022

This research explores anti‐cancer activities of synthetic peptides that inhibit the CMG helicase, the major DNA replicative helicase. Targeting CMG helicases for cancer treatment provides a unique opportunity to develop a cancer treatment that effectively eradicates cancer cells without affecting normal healthy tissues. This approach exploits a natural difference between activation of CMG helicases in normal and cancer cells. Collective evidence shows that normal cells – compare to cancer cells – possess higher levels of CMG helicase reserves available for activation throughout the S phase. Cancer cells, on the other hand, activate the majority of their helicases at once, thereby exhausting their helicase reserves. Based on this, we hypothesized that our peptides – by targeting the CMG helicase – would lead to the death of cancer cells, while spare corresponding normal cells.

In this study, we utilized two types of cancerous and normal cell lines: pancreatic cancerous Hs766T and normal hTERT‐HPNE cell lines, as well as glioma M059J and normal glial SVGp12 cell lines. Cells were plated in 96 well plates at a density of 3,000 cells/well and treated with different concentrations of studied peptides. Viabilities of cells were scored by a colorimetric CCK‐8 assay. According to our IC50 calculations, both cancerous cell lines were much more sensitive to the peptide treatments compare to their normal counterparts. Indeed, we observed that normal cell lines required almost 25 times higher concentrations of peptides to reach the same cytotoxic effect as the cancerous cells. Interestingly, we contrast these results with our studies with Gemcitabine, a therapeutic agent that is currently used for cancer treatment. Unlike our studied peptides, treatments with Gemcitabine led to an equal demise of both cancerous and normal cells.
Quantitative Mass Spectrometry Reveals a Proteome‐wide Role for Cyclin A and Cks1 in Multisite, Non‐Proline Directed Phosphorylation by CDK1
Authors: Ly Tony , Al‐Rawi Aymen , Endicott Jane , Korolchuk Svitlana
Published: May 2022

Cdk1 is a master kinase of mitotic cell division, phosphorylating thousands of substrates in a highly temporally regulated manner. How Cdk1 phosphorylates the right substrate at the right time remains an open question.

Cdk1 activity requires complex formation with a cyclin subunit: cyclin A or cyclin B. Additionally, active Cdk1 is found in complex with a small adaptor protein called Cks1. Cyclin subunits activate Cdk1 and tune Cdk1 substrate choice through interactions with short linear motifs on substrates. While Cks1 is essential in all organisms tested to date, a general function for Cks1 in Cdk1 substrate choice has not been demonstrated.

In this study, we investigated the roles of the non‐catalytic subunits of Cdk1 in substrate phosphorylation in human cells. We designed an in vitro assay in which we induced protein phosphorylation in fixed and permeabilized human lymphoblasts using active recombinant Cdk1 either in complex with cyclin A, cyclin B, or cyclin B+Cks1. Global phosphorylation levels were then measured using quantitative mass spectrometry.

Our data demonstrate that, contrary to the textbook model of Cdk1 as a proline‐directed kinase, both cyclin A and Cks1 facilitate widespread Cdk1 phosphorylation of sites lacking a +1 proline motif. Our results also suggest a role for Cks1 in multisite phosphorylation. We propose a model whereby cyclin A and Cks1 have indispensable functions in promoting non‐proline directed, multisite phosphorylation of Cdk1 substrates to mediate dynamic, switch‐like changes in protein function required for mitotic entry, proper chromosome segregation and cell division.
CB2 Receptor Potentiation Mediates Antipsychotic‐like Efficacy in Mice
Authors: Ferranti Anthony S. , Ishmam Tazeen , Swann Isaiah C. , Du Jennifer , Mariadoss Janet , Craig Lindsley W. , Rook Jerri M. , Conn Peter J. , Foster Daniel J.
Published: May 2022

Although the cannabinoid type‐2 receptor (CB2) is highly expressed in the immune system, emerging evidence points to CB2 playing a key role in regulating neuronal function in the central nervous system (CNS). Recent preclinical literature indicates that CB2 is expressed on numerous neuronal subtypes including midbrain dopamine neurons projecting to the striatum. The ability of CB2 to regulate mesolimbic dopamine signaling could make it an intriguing target with which to modulate mood regulation, substance abuse, motivation, and psychomotor behaviors in rodents. These findings are interesting in light of clinical studies showing that lower CB2 expression is linked to an increased risk of schizophrenia in Japanese populations and that Caucasian suicide victims exhibit altered CB2 expression. Here, we demonstrate that a CB2 positive allosteric modulator (PAM) has antipsychotic‐like efficacy in reversing amphetamine‐disrupted prepulse inhibition (PPI) in C57BL/6N mice, and this effect was blocked by administering the CB2 antagonist AM630 prior to the CB2 PAM. Furthermore, administration of the CB2 PAM reversed amphetamine and MK‐801 induced hyperlocomotion, indicating that CB2 potentiation can reverse deficits induced by pharmacological manipulation of dopaminergic and glutamatergic systems relevant to schizophrenia. The CB2 PAM did not show efficacy in reducing immobility time during Forced Swim Test (TST) and Tail Suspension Test (TST). These findings suggest that CB2 PAMs may represent a novel therapeutic strategy for treating patients with schizophrenia, and warrant further investigating of CB2 as a target for schizophrenia‐relevant behaviors in preclinical models.
Determining the role of the Ras family GTPase Rap1 in regulating mTORC2 activity and function in cell migration
Authors: Cahigas Genesis M. , Charest Pascale
Published: May 2022

Directed cell migration is an important biological process that is necessary for the embryonic development, maintenance of multicellular organisms and immune response to external stimuli. Dysregulation of cell migration is implicated in the onset and progression of diseases including cardiovascular diseases and cancer metastasis. Many studies have identified genes and proteins that are important for directed cell migration, but the process remains incompletely understood. The mechanistic Target of Rapamycin Complex 2 (mTORC2) is a complex of proteins that have been identified as important players in cell migration. mTORC2 is known to have a role in regulating F‐actin organization and actomyosin (MyoII). Moreover, there is increasing evidence that shows a correlation between mTORC2 and cancer cell migration and invasion. Despite this knowledge, the role and regulation of mTORC2 is not fully understood. Recently, we found that Rap1, a member of the Ras superfamily of small GTPases with conserved roles in cytoskeleton remodeling and cell adhesion, is a conserved binding partner of mTORC2 component RIP3/SIN1. We previously showed that Rap1 positively regulates mTORC2 activation in response to the chemoattractant cAMP in the model organism Dictyostelium discoideum. Moreover, we recently found that expression of a constitutively active Rap1 mutant (Rap1CA) potentiates the insulin‐induced activation of mTORC2 in human HEK293 cells. We then undertook a study to investigate if Rap1 regulation of mTORC2 is part of a mechanism involved in regulating human cell migration. We observed that basal mTORC2 activity, as well as that induced by several potential promigratory signals, including insulin, epidermal growth factor (EGF), insulin‐like growth factor‐1 (IGF‐1), and lysophosphatidic acid (LPA), was increased by the overexpression of wild‐type Rap1 or Rap1CA. Therefore, these observations suggest that Rap1 plays a role in positively regulating mTORC2 activation in HEK293 cells in response to stimulation of promigratory signals, similar to our previous observations made in Dictyostelium. Ongoing studies now aim at determining how Rap1 regulates the function of mTORC2 in cell migration.
miRNA Regulation of α‐2,6‐ Sialylation: Comprehensive Analysis of ST6GAL1 & 2
Authors: Jame Chenarboo Faezeh , Mahal Lara K.
Published: May 2022

MicroRNAs (miRs) are endogenous non‐coding RNAs that modulate gene expression at either the transcriptional or translational level through interaction with untranslated regions of mRNA. Works from the Mahal Lab and others have shown miRs as major regulators of glycosylation. ST6GAL1 and ST6GAL2 decorate glycoproteins with α‐2,6‐sialic acid, an epitope with important roles in immunology and cancer biology.

Herein, we analyze miRs that regulate ST6Gal1 and ST6Gal2 gene expression through interaction with 3′UTR regions of target mRNAs. Using miRFluR, a high throughput fluorescent sensor‐based method recently introduced by the Mahal Lab, we analyzed miR regulation of ST6Gal1 & 2 with miR mimics representing the currently known human miRome (~2700 miRs). Our analysis identified 89 miR regulators of ST6GAL1 and 102 regulators of ST6GAL2. Selected hits were validated by Western blot analysis and RT‐PCR. Finally, we took advantage of SNA staining assay using fluorescence microscopy to validate the sialylation outcome by ST6GAL1 after treatment with specific miR mimics. Our data reveals miRNA regulation of α‐2,6‐sialylation that is associated with pancreatic and other cancers.
Imaging mRNAs with corrected RNA stability
Authors: Li Weihan , Maekiniemi Anna , Singer Robert H.
Published: May 2022

MS2 system is widely used to image mRNAs in living cells, where the spatial distribution of mRNAs regulates various aspects of cell biology, including translation and RNA decay. To image an mRNA of interest, the mRNA’s 3’ untranslated region (UTR) is genetically fused with an array of MS2 stem loops, which recruit fluorescently labeled MS2 coat proteins. And each mRNA molecule appears as a diffraction‐limited spot under fluorescent microscope. Here, we report that the MS2 system renders some of the tagged mRNAs less stable and being degraded by the nonsense‐mediated mRNA decay (NMD) pathway. To resolve this issue, we improved the MS2 system by fusing the MS2 coat protein with the translation termination factor eRF3, which protects the mRNA from being degraded through the NMD pathway. Our improved MS2 system faithfully reports the mRNAs’ stability and localization, and will see wide applications in future studies of RNA biology.
Structure of cysteate acyltransferase, the condensation enzyme that initiates sulfonolipid biosynthesis by inhabitants of the gut microbiome
Authors: Radka Christopher , Rock Charles
Published: May 2022

Sulfonolipids (SL) are unusual lipids found in the outer membranes of Gram‐negative bacteria in the phylum Bacteroidetes. SL and its deacylated derivative, capnine, are sulfur analogs of ceramide‐1‐phosphate and sphingosine‐1‐phosphate, respectively. The structural similarity to key mammalian signaling molecules led us to investigate the biochemistry and distribution of SL production in inhabitants of the gut microbiome. Alistipes finegoldii, an anaerobic gut commensal contains two genes related to the bacterial serine palmitoyl transferase (Spt) identified in Sphingobacteriia. The two proteins were purified and the Afl_1224 gene product catalyzed the condensation of [14C]acyl‐acyl carrier protein and cysteate (3‐sulfo‐alanine). The product of cysteate acyltransferase (AfCat) was identified as 3‐ketocapnine by mass spectrometry. AfCat has a bound pyridoxal phosphate cofactor (PLP) that undergoes a spectral blue shift when exposed to cysteate consistent with the transition of a lysine‐aldimine complex to a substrate‐aldimine complex. We solved the crystal structure of dimeric AfCat using SAD phasing. AfCat has the prototypical fold enveloping the PLP cofactor bound to Lys‐251 found in Spt and has an electrostatic surface patch for ACP binding. AfCat is distinguished from Spt by its carboxy terminal domain that extends to interact with the amino terminus, an enlarged substrate binding site to accommodate and stabilize the negatively charged sulfono head group of cysteate, and the HAS motif in Spt is HAC in AfCat. These unique structural elements were used to identify SL producing organisms in metagenomic sequencing data from the gut microbiome that are responsible for SL and capnine in mammalian stool samples. Supported by GM034496.
UBE3A Hyperactivity as a Driver of Neurodevelopmental Disease
Authors: Weston Kellan , Gao Xiaoyi , Zhao Jinghan , Kim Kwang‐Soo , Harrison Joseph S. , Wu Kuen‐Phon , Yi Jason
Published: May 2022

UBE3A is a HECT (homologous to E6AP C‐terminus) domain E3 ubiquitin ligase that targets substrate proteins for degradation through the ubiquitin‐proteasome pathway. The UBE3Agene is of unique interest for its gene dosage‐dependent effect in the developing brain: Precise deletion or null mutation of the maternal copy of UBE3A causes a severe intellectual disability known as Angelman syndrome; meanwhile, duplication or triplication of the gene region in which UBE3A resides is linked to a prevalent syndromic form of autism known as Dup15q syndrome. However, little is known about the effects of missense variants which cause a single amino acid change in the enzyme, and prediction of disease outcomes for a given variant remains a challenge. Here, we pose that investigating variants’ effects on UBE3A functional activity levels is critical for predicting disease. In order to identify if precise mutations in UBE3A are sufficient to drive disease, we devised a high‐throughput assay to screen the functional consequence of UBE3A missense variants. We screened over 150 variants and identified distinct functional classes of UBE3A mutants based on their effect on enzymatic activity. Importantly, we identified over a dozen novel gain‐of‐functionvariants that aberrantly hyperactivate UBE3A enzyme activity. Through collaborations with clinical centers, we confirm that individuals possessing hyperactivating UBE3A variants exhibited phenotypes that were distinguishable from Angelman. Mice carrying a specific hyperactivating mutation on the maternal allele exhibited aberrant motor and early communication defects, as well as microcephaly. Finally, we mapped the results of our screen to the UBE3A protein structure to reveal a previously‐undefined allosteric regulatory exosite within the catalytic domain that we show to act as a charge‐dependent regulator of enzymatic activity. We found additional HECT domain enzymes to possess disease‐associated variants within their exosites, suggesting that exosite dysfunction is a common mechanism underlying a set of neurodevelopmental disorders. Together, our study indicates that excessive UBE3A activity increases the risk for neurodevelopmental pathology and suggests that deep structure‐functional analysis of protein variants can uncover disease‐relevant regulatory mechanisms.