bluuebunny/crossref_metadata_2025 huggingface dataset:
Tgfb1‐mediated Regulation of Microglia Maturation and Activation
Authors: Spittau Björn
Published: April 2020

The pleiotropic cytokine transforming growth factor‐beta 1 (TGFβ1) has been shown to play pivotal roles in a variety of different cell types including immune cells such as T cells, monocytes/macrophages as well as microglia. As the resident immune cell population of the central nervous system (CNS), microglia are essential effector cells during physiological and pathological events. Microglia originate from mesodermal progenitors that arise from the yolk sac and colonize the embryonic CNS where they adopt a mature microglia‐specific phenotypic pattern including expression of P2ry12, Tmem119, Olfml3, Siglech, Gpr34, Hexb, and Fcrls. The differentiation and maturation of postnatal microglia, as well as the regulation of the complex functional repertoire of mature microglia, needs to be carefully orchestrated. Here, we demonstrate that the deletion of Tgfbr2 in early postnatal microglia results in a loss of microglia maturation whereas silencing of TGFβ signaling in adult microglia does neither result in impairment of the microglia‐specific gene expression signatures, nor is microglial survival and maintenance affected. Tgfbr2‐deficient microglia were characterized by distinct morphological changes and transcriptome analysis using RNAseq revealed that loss of TGFβ signaling results in the upregulation of microglia activation and priming markers. Taken together, recent studies employing sophisticated transgenic approaches to silence microglial TGFβ signaling have revealed that TGFβ1 is indispensable for microglia maturation and the establishment of the microglia‐specific gene expression pattern as well as the homeostasis of adult microglia and the control of excessive microglia activation.
Illuminating the Biochemical Activity Architecture of the Cell
Authors: Zhang Jin
Published: April 2020

The complexity and specificity of many forms of signal transduction are widely suspected to require spatial microcompartmentation and dynamic modulation of the activities of signaling molecules, such as protein kinases, phosphatases and second messengers. We have developed a series of fluorescent biosensors to probe the compartmentalized signaling activities in living cells. In this talk, I will focus on cAMP/PKA and PI3K/Akt/mTORC1 signaling pathways and present studies where we combined genetically encoded fluorescent biosensors, superresolution imaging, targeted biochemical perturbations and mathematical modeling to probe the biochemical activity architecture of the cell.

Support or Funding Information

The work is supported by NIH
Sex as a Biological Variable in Bronchopulmonary Dysplasia
Authors: Lingappan Krithika
Published: April 2020

The incidence of bronchopulmonary dysplasia (BPD) is higher among premature male neonates even after adjusting for other confounders. Despite the well‐established sex‐specific differences in the incidence of BPD and impaired lung function in males, the molecular mechanism(s) behind this are poorly understood. Mammalian cells have intrinsic differences based on sex and respond differently to stressors irrespective of the past or current concentrations of sex hormones. Our research investigates the effect of sex on neonatal hyperoxic lung injury and the mechanisms underlying these differences. Alveolarization and angiogenesis is preserved in female neonatal mice exposed to hyperoxia compared to males. Differences in the lung transcriptome and the epigenome may contribute to these sex‐specific differences. We have shown that differential expression of miRNA (miR‐30a) and their downstream targets have also been shown to modulate this process. Sex‐specific differences at the cellular level in the pulmonary endothelial cells and fibroblasts will also be discussed. To distinguish sex differences caused by gonadal hormones versus sex chromosome complement (XX versus XY), we used the four Core Genotype mice (FCG). The FCG model can discriminate between sex differences determined by gonadal type (versus those determined by the effects of sex chromosomes). Sex‐specific transcriptomic changes were observed in numerous genes for each genotype at in room air and after hyperoxia exposure. Sex‐hormones and sex chromosomes may cause sex‐specific differences in the pathogenesis of BPD in premature neonates. The presentation will address knowledge gaps in the molecular mechanisms behind the sexually divergent incidence of BPD and will discuss novel molecular pathways for future therapeutic interventional strategies to combat BPD.

Support or Funding Information

This work was supported by grants from NIH: K08‐HL127103; R03‐HL141572 and R01‐HL14775 to KL.
Current Progress of hiPSCs‐based Therapy in non‐human Primates and Human Clinical Trials
Authors: He Jia-Qiang
Published: April 2020

Since the first successful creation of mouse induced pluripotent stem cells (miPSCs) in 2006 by Dr. Shinya Yamanaka and the subsequent generation of human iPSC (hiPSCs) in 2007, independently, by Dr. Yamanaka and Dr. James Thomas, iPSCs have been drawing exceptional attention compared to human embryonic stem cells (hESCs), mainly because hiPSCs can be easily generated from virtually any embryonic or adult somatic cells of the body without demolishing human embryos, thus offering an immense promise for personalized regenerative medicine; In contrast to the fetal or adult stem cells (FSCs, ASCs), hiPSCs demonstrate unlimited abilities of self‐renewal and differentiation potency to become almost all somatic cells in the body. Over the past 14 years, about 17,364 papers regarding iPSCs have been published (PubMed search between 2006/01/01 and 2019/11/12), which cover a wide spectrum ranging from in vitro basic researches, computer modeling, in vivo animal and non‐human primate studies to human clinical trials. Up to the present, systemic protocols involving iPSC reprogramming, purification, large‐scale culture expansion, and lineage‐specific differentiation have been successfully developed or optimized to increase the efficiencies of production. Meanwhile, iPSCs and its derivatives have also been widely utilized for mechanistic studies of cellular and molecular development, disease modeling, new drug discovery and toxicity testing. Along with it, a vast type of small‐, large‐animals, and non‐human primate‐models have been exploited to test the therapeutic potentials of these cells in treatments of numerous human diseases. More excitingly, 53 clinical trials of iPSC‐based therapy have been registered in the https://clinicaltrials.gov as of today. Although most of them are still in the “Recruiting” stage, the substantial increase of the registered trials signifies a critical movement towards future clinical practice in all aspects, such as ethical and regulatory compliance under surveillance of U.S. Food and Drug Administration (FDA), quality control of cell products, current good manufacturing practice (cGMP)‐guided cell storage, culture, purification, and lineage‐specific differentiation as well as in vivo assessments of efficacy and safety in a short and long‐term follow‐up. In this talk, we will present an overview of the current progresses, challenges, and concerns of hiPSC‐based treatments for various major diseases (e.g. neural disorders, cardiovascular diseases, and diabetes) using animal models; especially, we will focus on the recent progress acquired from the studies of non‐human primates and human subjects.

Support or Funding Information

This work was supported by the National Institute of Health (NIH 1R15HL140528), the Virginia Tech Commonwealth Health Research Board (CHRB, HE208‐09‐16), and the IRC seed grant from the College of Veterinary Medicine at Virginia Tech.
Role of anti‐receptor autoantibodies in tissue remodeling.
Authors: Smith Terry
Published: April 2020

The ocular manifestation of Graves’ disease (GD), known as thyroid‐associated ophthalmopathy (TAO), is an autoimmune process where the tissues of the upper face and orbits become inflamed and undergo remodeling. TAO remains a disfiguring and potentially blinding disease for which no medical treatments has yet to be approved by the FDA. Initiating TAO are thought to be immune responses to autoantigen(s) shared by the thyroid and orbit. The two leading candidate autoantigens in this disease are the thyrotropin receptor (TSHR) and insulin‐like growth factor receptor (IGF‐IR). Autoantibodies against TSHR drive thyroid over‐production of thyroid hormones in GD while anti‐IGF‐IR antibodies can be detected in patients with GD and TAO. Anti‐IGF‐IR antibodies have yet to be characterized and their role in GD remains controversial. Here I will discuss the current evidence supporting autoantibodies as driving the development of TAO and the downstream consequences of cell‐surface signaling they initiate. Further, identification of these antibody‐dependent processes has led to the development of promising therapeutic candidates for TAO, a vexing and poorly treated disease.
Regulatory Dynamics of Midfacial Growth in Evolution and Disease
Authors: Selleri Licia , Welsh Ian , Hansen Karissa , Osterwalder Marco , Losa-Llabata Marta , Wells Julie , Bult Carol , Mohun Timothy , Hu Diane , Marcucio Ralph , Visel Axel , Swigut Tomek
Published: April 2020

Craniofacial morphogenesis demands coordinated outgrowth of spatially‐separated facial prominences. Underscoring this complex choreography, 1/3 of all human birth defects affect craniofacial development. Both orofacial clefting and malocclusions are common birth abnormalities that affect the midface. During midfacial development, regulatory genes control the dynamics of transcriptional activity, tissue patterning, and intercellular signaling to modulate the timing of morphogenetic and growth processes. Variation in these dynamics underlies both evolutionary divergence in midfacial outgrowth and the etiology of craniofacial birth defects. In non‐avians, the main variable driving species‐specific midfacial length is the maxilla of the upper jaw, while in avians it is the premaxilla. Also, avians have cleft palate in physiological conditions. Despite the availability of datasets of regulatory elements from craniofacial tissues and cranial neural crest cells, the majority of regulatory networks that control embryonic midfacial outgrowth are unknown with respect to their in vivo spatiotemporal context‐dependence. We are establishing regulatory landscapes that control different morphogenetic and outgrowth characteristics of midfacial elements at the organismal level in chick, mouse, and pig embryos, species characterized by remarkably different midfacial lengths. To this end, we are combining 3D gene expression profiles obtained via High Resolution Episcopic Microscopy (HREM) of embryos processed by whole mount in situ hybridization as well as genome‐wide transcriptional regulatory activity and epigenetic regulation assessed by RNA Seq, ATAC‐Seq, and chromatin mark ChIP‐Seq of embryonic midfacial anlagen. By this approach, we have identified candidate genes that regulate maxillary‐specific morphogenetic programs in avians versus mammals. One of these genes, which encodes a transcription factor (TF) bearing zinc finger motifs and a homeobox motif, is expressed meristically along the AP palatal shelf of mouse embryos at E11.5–14.5, but is not expressed in the palatal shelves of avian embryos at the corresponding developmental stages. Mice we generated with CRISPR/Cas‐mediated loss‐of‐function of this TF show cleft palate and stunted snout. Strikingly, children with a deletion of this TF exhibit high‐arched palate and cleft palate. These findings establish mechanisms underlying tolerance to morphological variation that separate normal development and evolution of craniofacial features from malformations associated with birth defects.

Support or Funding Information

UCSF Recruitment Funds
Incorporating Retrieval Into Classroom Activities: Setting Good Habits
Authors: Anderson Lisa Carney
Published: April 2020

Retrieval is a learning strategy in which the student attempts to recall concepts from memory without consulting course notes, a textbook or the internet. Classical studies of retrieval support retrieval as an effective study technique. However, when students have had previous success with storage techniques such as listening to videos, reading textbook, or re‐reading course notes, it is difficult to convince students to use evidence‐based techniques.

Three instances of introducing retrieval to students in clinical physiology are presented. In all three cases, students heard about the classical experiments of Karpicke and Roediger. In addition, the instructor shared her own retrieval success. In the first instance, students were exposed to retrieval practice questions throughout class time activities, mainly power point lectures interspersed with active learning exercises. Students completed an anonymous survey about their attitudes about retrieval as part of their final exam. In the second instance, students in a systems physiology course were given retrieval exercises at the beginning of each of a series of 5 lectures on endocrine physiology. After student completed the exam on this material, students were invited to complete an online survey of their use of and attitudes toward retrieval. In the third instance, student in a clinical physiology course worked to answer a series of short answer questions without the benefit of any course resources. After 30 minutes of work, the instructor led a discussion of the activity in terms of retrieval and confidence.

These three classroom experiences provide evidence that there is an association between positive attitudes towards and use of retrieval and exam performance. For example, in a sample of 200 physiology students, agreement with the statement I used retrieval methods as I studied for this exam, is significantly correlated with exam performance (r= 0.33, p < 0.00001). However, there was no significant correlation between agreement with the statement, I think retrieval is an effective study technique supported by evidence and exam performance. In addition, student comments indicate that students may require multiple opportunities to learn about and practice retrieval before they are ready to incorporate retrieval into their regular study habits.
Mesenchymal stem cells in tissue homeostasis and regeneration
Authors: Chai Yang
Published: April 2020

The human skull is composed of twenty‐two bones connected by sutures, which are fibrous joints that contain skeletal progenitor cells. The calvarial sutures allow compression of the skull during childbirth and dramatic postnatal growth. Craniosynostosis is a common congenital defect characterized by premature suture fusion, which can cause severe outcomes including abnormal growth of the skull, increased intracranial pressure, retarded brain development and impaired neurocongnitive function. Currently, the only treatment option for infants with craniosynostosis is surgery that involves cutting the calvaria into pieces, manually reshaping them, and fixing them in place mechanically, necessitating blood transfusion and complex post‐surgical care. In many cases, the calvarial bones fuse again, which requires repeated operations to relieve the constriction on the brain during the sensitive period of development in order to improve physiological function. Clearly, there is an immense need for a better approach to the treatment of craniosynostosis and prevention of re‐occurrence.

Recent studies have shown that Gli1+ cells are an indispensable mesenchymal stem cell (MSC) source within the cranial sutures. Ablation of Gli1+ cells leads to fusion of all craniofacial sutures in adult mice; we have also demonstrated there is a premature loss of Gli1+ cells prior to coronal suture fusion in Twist1+/− mice, which provide an important, clinically relevant craniosynostosis model of Saethre‐Chotzen syndrome. Here we took advantage of the Twist1+/− mouse model, developed an innovative scaffold combined with Gli1+ MSCs and were able to regenerate a coronal suture and improved the morphology and function of Twist1+/− mice. This discovery provides a potential new approach for treating patients with craniosynostosis.

Support or Funding Information

Supported by the NIDCR, NIH R01 DE026339; U01 DE 026914
Spatial Ability and Learning: Implications for Anatomy Education
Authors: Uttal David H.
Published: April 2020

It is now well known that spatial ability is correlated with many aspects of scientific learning and practice, including the learning of surgical anatomy. However, because this data is only a correlation, we do not know whether it represents a causal relation. For example, is having high spatial abilities an intellectual pre‐requisite for succeeding in learning anatomy? I will explore the role of spatial ability in learning in spatially rich field, such as anatomy, and will consider when and why spatial thinking may affect learning. Even when there is a high correlation between spatial ability and performance, this alone does not mean that spatial ability is always involved in learning new information. I will also consider ways in which students with lower levels of spatial ability may do well.
Effective Near‐peers in Anatomy
Authors: Quiroga-Garza Alejandro , Elizondo-Omaña Rodrigo Enrique , Salinas-Alvarez Yolanda Enrique , Guzman-Lopez Santos Enrique
Published: April 2020

With the breaking down of 700–1,000 student semesters into discussion groups of 6–8 students, effective near‐peers are needed. Gross Anatomy is a 2nd‐semester course of a 12‐semester Medical School program. 3er‐semester students may apply as near‐peers and undergo a selection process. After this, they begin their training in accordance with seniority. Between 40 and 60 near‐peers aid professors in class and practice hours. Weekly sessions are scheduled to discuss the clinical relevance of the anatomy being taught that week. The theory is reviewed by anatomists, and clinical cases by specialists from different fields (Surgery, Gynecology, Orthopedics, Sports Medicine, Radiology, Otorhinolaryngology, Ophthalmology, Neurosurgery, Neurology, Psychiatry, ect.), according to the topic. Cadaveric dissections are guided amongst peers with the most seniority and surveillance by professors. Near‐peers, are exposed to clinical scenarios and practice, to better understand the topics, and transmit the knowledge to 2nd‐semester students during clinical sessions.

With a high volume of students and a low ratio of professors, near‐peers have been an effective solution. 70.7% perceived sessions with near‐peers to have no difference from that imparted by a professor. Only 12% reported some aspects of the sessions to be confusing when given by near‐peers. Satisfaction and acceptance were high. With a high volume of near‐peers, scholarships or economic remuneration is only available to the 20 most senior and participative near‐peers. A “payment with knowledge” method and integration activities have been beneficial in keeping near‐peers satisfied and committed to the Department. Specialized courses are offered. All near‐peers from their first semester to their last may take the advanced dissection, advanced suturing, and basic fundamentals of ultrasonography courses offered by experts. Those in their 3er semester are offered laparoscopic anatomy and ultrasound‐guided vascular access courses. In their 7th semester, virtual arthroscopy, and minimally invasive surgical skill courses are added. If the near‐peers complete all 7 courses offered during their time in the Department, a Clinical Anatomy and Surgical Training Diploma are emitted.

Social activities between the Department staff and near‐peers are also promoted. Social gatherings are scheduled every semester. Breakfasts are sponsored by the department after every test (3 exams and 1 final per semester). Student sports teams are encouraged, with jerseys made for all near‐peers in solidarity. Department chief and professors integrate with near‐peers. Near‐peer satisfaction and loyalty are high. Most visualize Gross Anatomy as the best depart to be a near‐peer of and are inspired to become anatomists.

Support or Funding Information

None
Breaking Down Large Anatomy Groups
Authors: Elizondo-Omaña Rodrigo Enrique , Quiroga-Garza Alejandro , Salinas-Alvarez Yolanda Enrique , Guzman-Lopez Santos
Published: April 2020

Our School of Medicine has over 10,000 applicants annually, accepting between 1,000 and 1,200 per semester. Every 6 months, the Human Anatomy Department (a second‐semester course) receives between 700 to 1,000 students for a staff of 16 professors. The students are broken down into groups of 30–45 students for dynamic teaching classes in which students actively participate using intellectual abilities to promote clinical reasoning. Professors facilitate information and guide the classes, while students discuss regional anatomy with group participation. With the aid of qualified near‐peers, students are split into small 6 to 8 student groups and distributed between clinical cases, imaging study cases, ultrasound anatomy, and prosection/dissection modules, promoting clinical reasoning for solving anatomical oriented cases. With a high student/low cadaver ratio, the IFA (identify, function, application) method has proven effective in prosection time.

Questionnaires have demonstrated students’ satisfaction with the course distribution of time, and exposure to clinical cases and imaging to be high with a 94% approval.

Support or Funding Information

None
Addressing Craniofacial Anomalies through International Collaborations and Capacity Building
Authors: Butali Azeez
Published: April 2020

Craniofacial anomalies especially orofacial clefts (OFC) are one of the most common congenital anomalies in Humans. The global prevalence of OFC is 1/700 live births but could be as high as 1/500 in Asians or as low as 0.5/1000 in Africans. These differences in prevalence suggest that there may be different genetic and environmental contributions which requires collective experts across the world for the identification and understanding of genetics, genomics, and environmental factors as well as the interplay between these factors. International collaborations literally mean multi‐ethnic research to identify the causes, risk factors and opportunities for prevention of OFC by collecting data and samples from individuals around the world. The clinical ‐basic‐translational research required for OFC means requisite experts around the world, which then means training and capacity building. In this talk, the audience will have the opportunity to listen to how international collaborations around OFC is driving new discoveries and capacity building in Africa. Additional information on how multidisciplinary expertise in clinical medicine/dentistry, human genetics, developmental biology, anthropology, epidemiology, bioinformatics and implementation science is setting the stage for prevention of OFC will also be shared.

Support or Funding Information

DE022378 and Robert Wood Johnson Foundation Grant number 72429
Is Being Stressed Really that Bad? Mechanistic Pathways and Interventions for the Stress‐health Relationship
Authors: Crosswell Alexandra D.
Published: April 2020

There is now considerable evidence from epidemiological and experimental studies demonstrating the profound impact that extensive periods of psychological stress have on physical and mental health. The impact is so profound that analyses using the Midlife in the United States (MIDUS) nationally representative longitudinal study have demonstrated that those in the highest quartile of a cumulative index of stress exposure across their lifetime compared to those in the lowest quartile have a 20% increased risk of dying over the next 12 years. More immediately, chronic stress exposure decreases engagement in core health behaviors like exercise and sleep, increases negative mood and conflicts with others, limits one’s ability to pay attention, and increases the risk of developing the common cold. All of these in turn negatively impact productivity and performance in school and work. The biological mechanisms linking psychological stress to these negative mental and physical health effects are now well documented and include the immune system, cellular aging, neuroendocrine system, and autonomic nervous system. In recent years, there has been important headway made on understanding how to combat the negative effects of stress – both through psychological interventions that increase psychological resilience to stress, and through targeting biological processes that are negatively impacted by stress. In particular, contemplative practices such as meditation and yoga have been shown to reduce stress and boost stress‐related biomarkers of disease, likely through the activation of the parasympathetic nervous system. When the body feels safe, such as in relaxed states of focused attention and when one feels a sense of belonging in a group setting (both of which are often the case in contemplative practice), their parasympathetic system is activated, allowing the body to enter a state of relaxation. Activation of the parasympathetic nervous system also puts the breaks on the sympathetic nervous system which is responsible for putting the body in the ‘fight or flight’ highly activated response. It is in this space of relaxed, parasympathetic activation, that the body can heal itself from the damage done by chronic stress, thus pointing to a key aspect to target with stress‐reduction interventions. This talk will focus on the biological underpinnings of the stress‐health relationship and the cutting edge science showing what we can do to reduce the negative effects of chronic psychological stress.
Salvador‐Warts‐Hippo Genes Methylation status in Osteosarcoma Cell Lines
Authors: ALOAKIL MAJED S. , Al-HAZMI HAZIM S. , ALENAD AMAL M. , ALNAAMI ABDULLAH S. , Al-DAGHRI NASSER M.
Published: April 2020

Osteosarcoma is an aggressive malignant bone tumour greatly prevalent in children and adolescent. The Salvador‐Warts‐Hippo signalling pathway is a highly conserved and is important in regulating organ size, cell proliferation and cell death. Tumorigenesis has been extensively studied along with the genetic alterations of the promoters of Salvador‐Warts‐Hippo genes which promote poor prognosis. Although, the epigenetical modifications, which present a fundamental controlling points for genetic regulations, remain inadequately clear. Such as the methylation status of CpG islands which are associated to key components genes of the hippo signalling pathway which includes MST1, MST2, LATS1, LATS2, WW45 and SAV1. We applied two pairs of primers were needed with one pair specific for methylated DNA (M) and the other for unmethylated DNA in several bone cancer cell lines including SaOS2, HT1080 and Ewing’s sarcoma A673. Combined Bisulfite Restriction Analysis (CoBRA) carried out on these cell lines and the methylation patterns were varied between osteosarcoma and Ewing’s sarcoma cells. This approach of combination techniques provides a clear note of the methylation status profile of CpG islands that governs the expression of these Salvador‐Warts‐Hippo genes in osteosarcoma cell lines. In addition, the level of expression of Salvador‐Warts‐Hippo pathway genes during demethylating agents treatment play a corresponding evidence of the biological relevance that the methylation status has on the transcriptome. The outcomes of the present study offer distinguishable means that allow the ability to differentiate between several epigenetic variations in different osteosarcoma cell lines. We conclude that Salvador‐Warts‐Hippo needs further investigation in osteosarcoma progression.

Support or Funding Information

Deanship of graduate studies, King Saud University, Riyadh, Saudi Arabia
Best Practices for Inclusion of Deaf and Hard‐of‐Hearing Scientists at Conferences
Authors: Craig Paul A. , Samar Vincent , Womack Kathryn , Listman Jason
Published: April 2020

Deaf and Hard‐of‐Hearing (Deaf/HoH) scientists use a very broad range of communication modes: some use American Sign Language while others use Signed English, and still others use spoken English. A series of interviews were conducted with Deaf/HoH scientists about their experiences in attending national scientific conferences. These scientists are highly visual in their communication and they provided some great insights on adjustments at conferences that would dramatically improve the experiences of all scientists at conferences. These suggestions included specific approaches to conference planning and preparation, making arrangements for interpreters with discipline‐specific training, seating arrangements, captioning and interpreting services for plenary sessions, and the availability of floating interpreters that would enable Deaf/HoH scientists to more spontaneously and fully participate in poster sessions and conversations with vendors. It should be noted that these adjustments for Deaf/HoH attendees usually benefit other attendees as well ‐ for example live captioning benefits people who aren’t native English speakers and people whose hearing is declining as they age. In addition to best practices for Deaf/HoH participation at conferences, some specific comments from the scientists will be shared to add depth and perspective to these practices.

Support or Funding Information

The RIT RISE Scholars program is supported by the NIH Research Training Initiative for Student Enhancement #1R25GM122672.
Characterization of Proteins from Zebra Mussel Shells
Authors: Freiermuth Margaret
Published: April 2020

Wisconsin and Minnesota lakes are under attack from invasive Zebra Mussels, Dreissena polymorpha, a freshwater mussel species native to Eurasia. The Zebra Mussels were introduced to the Great Lakes around 1985–1986 and have continued to aggressively invade the surrounding areas of Minnesota and Wisconsin. Outcompeting native species, including fish, for food sources, Zebra Mussels are thriving. Oddly, this confluence of attributes, i.e., fast growth but undesired presence, offers a unique opportunity to capitalize on the economic potential of this invasive species’ presence in Wisconsin. Previously we have extracted and optimized the protocol for extracting proteins from Zebra Mussel shells. The extracted proteins will be analyzed to find their identity using MALDI‐TOF MS characterization. This will be the next step in determining a possible commercial use in a large pool representing an underutilized potential resource that is also a pest.
L‐lactate dehydrogenase A selectively interacts with phosphatidic acid species containing stearic acid at the sn‐1 position
Authors: HOSHINO FUMI , SAKANE FUMIO
Published: April 2020

Introduction

Phosphatidic acid (PA) acts as a lipid second messenger and is involved in a wide variety of cellular events. PA consists of various molecular species that have different acyl chains at the sn‐1 and sn‐2 positions, and consequently, mammalian cells contain at least 50 structurally distinct PA species. However, the different roles of each PA species are poorly understood.

Results

In the present study, we attempted to identify 16:0/16:0‐PA (X:Y = the number of carbon atoms:the number of double bonds)‐binding proteins from the mouse skeletal muscle via a screening method using 16:0/16:0‐PA‐containing liposome precipitation and MS/MS analysis. Intriguingly, we have found for the first time that
l‐lactate dehydrogenase A (LDHA) bound to 16:0/16:0‐PA. LDHA catalyzes conversion of pyruvate to lactate and is considered to be a important checkpoint of anaerobic glycolysis. Moreover, its expression level is elevated in many types of cancers and has been linked to tumor growth, maintenance, and invasion.

Further characterization revealed that LDHA specifically bound to PA and did not associate with other phospholipids, such as phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol and cardiolipin. Interestingly, in addition to 16:0/16:0‐PA, PA species containing stearic acid (18:0) at the sn‐1 position, 18:0/18:0‐PA, 18:0/20:4‐PA and 18:0/22:6‐PA, also interacted with LDHA in vitro and their binding activities were significantly stronger than 16:0/16:0‐PA. However, LDHA failed to associate with 16:0/18:1‐PA, 16:0‐lyso phosphatidic acid (LPA) and 18:0‐LPA. We are now examining whether the 18:0‐containing PAs affect the localization and activity of LDHA.

Conclusion

We demonstrated for the first time that LDHA is a PA‐binding protein. Moreover, we revealed that LDHA is a very unique PA‐binding protein that possesses exceptional preference to PA species containing 18:0 at the sn‐1 position. We have already reported that creatine kinase muscle type and α‐synuclein bound to saturated fatty acid‐ and/or monounsaturated fatty acid‐containing PAs (e.g. 16:0/16:0‐ and 16:0/18:1‐PA) and monounsaturated fatty acid‐containing PAs (e.g. 18:1/18:1‐PA), respectively. Taken together, these results indicate that there are many functionally distinct PA‐binding proteins having different selectivities to PA species. Therefore, it is likely that each PA species individually regulate diverse physiological functions through different PA‐binding proteins.
Focused Library Screening for Isoform Selective Adenylyl Cyclase 1 Inhibitors as Non‐Opioid Alternatives for Chronic and Inflammatory Pain
Authors: Hayes Michael P. , Wiernicki Todd R. , Martinez-Grau Maria Angeles , Burris Kevin D. , Watts Val J.
Published: April 2020

Due to the socioeconomic burden associated with chronic pain conditions and the fact that opioids have severe side effects, there is a need for new approaches and drug targets for the treatment of chronic pain. An increasing body of evidence suggests that inhibition of neuronal Adenylyl Cyclase 1 (AC1) may be one way to achieve this goal, as mice lacking AC1 are significantly less responsive in neuropathic and inflammatory pain models. ACs catalyze the formation of cAMP from ATP following stimulation by a variety of cellular inputs, like G protein subunits, kinases, calmodulin, and the exogenous small molecule forskolin. Here we used a recently developed cell model which lacks forskolin stimulated cAMP accumulation due to genetic ablation using CRISPR/Cas9 of AC3 and AC6, the major forskolin responsive AC isoforms in parental HEK293 cells. Following stable overexpression of recombinant human AC1, we screened over 20,000 compounds from the Eli Lilly Open Innovation Drug Discovery program against calcium‐calmodulin stimulated AC1 activity. The hit rate from the preliminary screen was less than 1%, highlighting the utility of this cell model as compared to parental HEK293 cells. Primary screen actives were retested against AC1 to confirm activity in dose‐response experiments. Counter‐screening was performed against representative AC isoforms AC2, AC5, and AC8, leading to the identification of two novel, promising scaffolds with distinct AC1 inhibition profiles. Subsequently, we evaluated the mechanism of inhibition of these lead compounds as compared to previously identified AC inhibitors in our cellular model, isolated membrane fractions, and mouse hippocampal homogenates in response to various stimuli, including GPCR‐Gαs, forskolin, and calcium‐calmodulin. Further, we have also initiated structure activity relationships for these two scaffolds, as well as begun examining their selectivity for AC1 over other unrelated neurological targets. Taken together, these scaffolds may hold promise as tool compounds for further interrogating the role of selective AC1 inhibition in pain neurobiology and for the development of AC1‐targeting therapeutics in the future.

Support or Funding Information

This work was supported by the Eli Lilly Open Innovation Drug Discovery program, Rick and Anne Borch Research Award, and Purdue University College of Pharmacy.
Analysis of Oligomerization and Protein‐Protein Interactions within the Conjugation Machinery of Bacillus subtilis
Authors: Ragucci Adelyn , Berkmen Melanie
Published: April 2020

Bacterial mating, or conjugation, is the transfer of DNA from one bacterial cell to another. Conjugation is significant to bacterial evolution and plays a large role in the spread of antibiotic resistance. The conjugation machinery of the Bacillus subtilis integrative and conjugative element ICEBs1 is a specialized DNA translocation channel known as a Type 4 Secretion System (T4SS). T4SSs are composed of multiple interacting oligomeric proteins that span the donor cell membrane. Previously, we found that the B. subtilis T4SS protein ConE (VirB4) interacts with itself and another T4SS protein, ConB (VirB8). These protein‐protein interactions were identified using a bacterial two hybrid method (BACTH) in which the proteins are fused to either the T18 or T25 domains of adenylate cyclase. Given that characterized VirB8 homologs oligomerize, we hypothesized that ConB interacts with itself, likely forming trimers or larger oligomers. To test this hypothesis, the conB gene was amplified by PCR and ligated into vectors that encode either the T18 or T25 domains. The plasmids were transformed into Escherichia coli and verified by sequencing. Subsequent BACTH assays in which the plasmids were co‐transformed into E. coli cells verified the interaction between T25‐ConB and T18‐ConB. We have performed site‐directed mutagenesis on the conB gene and intend to determine which domains of ConB are required for oligomerization and for interaction with ConE. Our work should provide greater insight into the structure and function of the T4SSs of B. subtilis and other Gram‐positive bacteria.
Differential Regulation of DJ‐1 in Glial Cells upon MHV‐A59 ‐Induced Oxidative Stress
Authors: Kundu Soumya , Anthony Gisha Rose , Kumar Rahul , Raveendran Vineeth , Pal Debnath , Sarma Jayasri Das
Published: April 2020

Astrocyte and microglial activation are two hallmarks associated with oxidative stress induction in neural cells. Generation of reactive oxygen species (ROS) is one of the molecular outcomes of neurotropic Mouse Hepatitis Virus A59 (MHV‐A59) infection in the Central Nervous System in an experimental animal model for human demyelinating disease Multiple Sclerosis. MHV‐A59 infection has been found to cause encephalitis, meningitis, and hepatitis in acute stage (Day 5–7 post‐infection) and progressive demyelination in chronic stage (Day 30 post‐infection). The virus has shown glial cell tropism leading to its activation, although the role of oxidative stress in this aspect is not well understood. Therefore, our work focuses on the regulation and function of important anti‐oxidative markers like DJ‐1, Nrf2, and HMOX‐1, upon viral infection in animal CNS tissue, isolated cells from tissues, primary culture and cell culture‐based studies. All three genes were found to be upregulated in vivo in mice brain at acute stage (Day 3 and 5) and chronic stage (Day 30) post‐infection. Similar upregulation was also observed in isolated microglia from mice brain, primary astrocytes, and microglia cultured from neonatal mice brain. In vitro studies with DBT astrocytoma cell line and N9 microglia cell line showed oxidative stress generation and upregulation in all three genes at initial time points of viral infection; however all the genes were found to be downregulated at higher time points. DBT cells exhibited decrease in cell viability and oxidative stress generation upon viral infection, and such changes had been observed to be ameliorated when DJ‐1 was stably transfected into DBT cells. Literature studies indicate that XBP1, an ER stress marker, upregulates DJ‐1 expression upon oxidative stress induction. In silico and in vitro studies showed that XBP1 was differentially activated in both DBT and N9 cells and was associated with DJ‐1 promoter upon viral infection in N9 cells. MHV‐A59 has shown tropism for glial cells resulting in their activation, and oxidative stress‐related cellular mechanisms involving these genes can contribute to the activation process of these glial cells.

Support or Funding Information

This study was supported by the Department of Biotechnology (DBT) and Indian Institute of Science Education and Research Kolkata (IISER Kolkata). Soumya Kundu was supported by University Grant Commission (UGC). Gisha Rose Anthony, Vineeth A. Raveendran, and Rahul Kumar were supported by DST‐INSPIRE fellowship under the banner of IISER Kolkata.
The Relationship between ER Stress and Protein Quality Control at the Translocon
Authors: Broshar Courtney Leigh , Buchanan Bryce , Mehrtash Adrian , Runnebohm Avery , Snow Brian , Scanameo Laura , Hochstrasser Mark , Rubenstein Eric
Published: April 2020

Multiple diseases are associated with elevated levels of endoplasmic reticulum (ER) stress, including some forms of cancer, neurodegeneration, and heart disease. ER stress arises when ER‐resident proteins misfold and accumulate. Cells respond by activating multiple mechanisms which reduce ER stress. Our objective is to understand the relationship between ER stress and protein quality control at the endoplasmic reticulum, using Saccharomyces cerevisiae as a model organism. The ubiquitin‐proteasome system (UPS) is responsible for the majority of degradation of misfolded and aberrant proteins at the ER (such as those arising during ER stress) through ER‐associated protein degradation (ERAD). We have found ER stress differentially impairs ERAD pathways, including those mediated by the same ubiquitin ligase. For example, ER stress impairs degradation of translocon‐associated proteins and of proteins with luminal degradation signals (ERAD‐T and ERAD‐L substrates, respectively). Degradation of these substrates is mediated by the Hrd1 ubiquitin ligase. In contrast, degradation of proteins with intramembrane degradation signals (ERAD‐M substrates), which also requires Hrd1, was not impaired by ER stress. We compared the effects of ER stress caused by protein misfolding and of ER stress caused by disruption of lipid homeostasis on the degradation of ERAD‐T substrates. We observed that ER stress caused by protein misfolding impaired ERAD‐T. On the contrary, ERAD‐T was unaffected by ER stress caused by disruption of lipid homeostasis. We then investigated the effects of different forms of stress on ERAD‐T. We observed that oxidative stress and heat shock did not impair ERAD‐T. We found that none of four characterized ER stress‐sensing mechanisms were required for ERAD‐T or its impairment by ER stress. Taken together, our results indicate that ER stress differentially impairs ER protein quality control mechanisms and suggest the existence of a novel ER stress‐responsive mechanism that impairs ERAD‐T. Understanding how the cell responds to ER stress may inform the treatment of diseases associated with stress.

Support or Funding Information

This work was supported by Ball State University ASPiRE research awards to BWB, CLB, and AMR; a Ball State University Department of Biology Sigma Zeta research award to BJS, an NIH grant to MH (GM046904), a Ball State University Faculty Advance research award to EMR, an Indiana Academy of Science Senior Research Grant to EMR, and an NIH grant to EMR (GM111713).
Functional cooperativity between the trigger factor chaperone and the ClpXP proteolytic complex
Authors: Houry Walid A.
Published: April 2020

A functional association was uncovered between the ribosome‐associated trigger factor (TF) chaperone and the ClpXP degradation complex in bacteria. Bioinformatic analyses demonstrated conservation of the close proximity of tig, gene coding for TF, and genes coding for ClpXP suggesting a functional association. Effect of TF on ClpXP‐dependent degradation varied based on the nature of substrate. While degradation of some substrates was slowed down or unaffected by TF, surprisingly, TF increased the degradation rate for a third class of substrates. These include λ phage replication protein λO, master regulator of stationary phase RpoS, and SsrA‐tagged proteins. Globally, TF enhances the degradation of about 2% of newly synthesized proteins. TF was found to interact through multiple sites with ClpX in a highly dynamic fashion to promote protein degradation. This chaperone‐protease cooperation constitutes a unique and likely ancestral aspect of cellular protein homeostasis in which TF acts as an adaptor for ClpXP.

Support or Funding Information

Canadian Institutes of Health Research
Association of DRD2 Gene Ser311Cys Polymorphisms, DAT 40bp VNTR and Cannabis Dependence
Authors: Olasore Holiness Adedeji , Osuntoki Akinniyi Adediran , Magbagbeola Olubunmi Abiola
Published: April 2020

Cannabis as the most widely used illicit drugs among Nigerians has been associated with psychiatric disorders. Studies have implicated genetic predisposition in drug abuse and dependence. We therefore aimed at finding out the relationship between some dopaminergic gene polymorphisms and cannabis dependence.

One hundred and four patients attending a psychiatric facility in Lagos, Nigeria who were diagnosed of cannabis dependence according to ICD‐10 were recruited for the study. Ninety six subjects who were not smoking cannabis were also recruited as a comparative group. Genotyping was carried out for Ser311Cys polymorphism of the dopamine receptor D2 (DRD2) gene and the 40bp VNTR of the dopamine transporter (DAT) gene.

The Cys allele was more frequent than the Ser allele among the smokers (0.878 and 0.122 respectively), compared to the non‐smokers (0.611 and 0.389 respectively). Most of the smokers were homozygous Cys/Cys (0.433) while most of the non‐smokers were heterozygous Ser/Cys (0.798). Individuals with DRD2 Ser311 allele were more than five times as likely to use cannabis as those with Cys311(p = 0.001, OR = 4.575, 95%CI = 2.727–7.678). The 9 and 10‐repeats (9R and 10R) of the DAT VNTR were the most common repeats. Other repeats were very few and were even found in heterozygous form in combination with either 9 or 10 repeats. The 10R allele was more frequent than the 9R among the smokers (0.560 and 0.440 respectively) and also among the non‐smokers (0.726 and 0.274). While most of the smokers were heterozygous 9R/10R (0.559), most non‐smokers were homozygous 10R/10R (0.595). Individuals with at least one 10R were almost twice as likely to use cannabis as those with the 9R (p = 0.004, OR = 1.915, 95%CI = 1.225 – 2.995).

The Cys311 allele of the DRD2 and the 10R of the DAT VNTR were significantly associated with cannabis smoking as individuals with these alleles were more likely to smoke cannabis.
Feeling Fragile: Chemosensitization of Metastatic Breast Cancer Cells Through the Use of 4EGI‐1 and Etoposide
Authors: Patel Anya , Jacobson Blake , De Luke , Ronan Jehnna , Kratze Robert
Published: April 2020

Breast cancer is the most common cancer among women worldwide. High quantities of Etoposide (ETP), a topoisomerase II inhibitor, have been shown to have considerable adverse effects, therefore making chemosensitization an appropriate area to research. Combination therapies with the drug have been largely unexplored. In the past, ETP has been used to treat metastatic breast cancer in conjunction with other drugs, specifically chemosensitizers. Combining ETP with another drug as a chemosensitizer could increase its efficacy. In past studies, non‐small cell lung cancer has been treated with a novel drug compound, 4EGI‐1, which is a translation inhibitor. Combining the drugs by using 4EGI‐1 to treat metastatic breast cancer as a chemosensitizer could decrease the amount of ETP needed as well as increasing its efficacy. Here we examine the relationship between different concentrations of ETP and 4EGI‐1 and their effect on immobilized breast cancer cell culture. Cell death was used as an assay to determine the efficacy of the drug combination. We hope to show the synergistic effects of ETP and 4EGI‐1 on breast cancer cells. The cell line used was MDA‐MB‐231, a human epithelial breast cancer cell, derived from a 51‐year‐old female diagnosed with mammary adenocarcinoma. The goal of our in vitro studies is to eventually increase patient comfort in a clinical setting by reducing the necessary amount of ETP.

Support or Funding Information

University of Minnesota, The Nueva School