bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel and convenient bacterial platform for production of encapsidated double‐stranded RNA targeting white spot syndrome virus in shrimp
Authors: Wuthisathid Kitti , Chaijarasphong Thawatchai , Chotwiwatthanakun Charoonroj , Somrit Monsicha , Sritunyalucksana Kallaya , Itsathitphaisarn Ornchuma
Published: April 2020

White spot syndrome virus (WSSV) is a highly infectious virus that can cause 100% mortality in farmed shrimp in the matter of days, leading to significant economic losses to shrimp aquaculture worldwide. To mitigate the adverse impact of WSSV, effective antiviral strategies are necessary. Currently, double‐stranded RNA (dsRNA), which can silence viral gene expression through an RNA interference (RNAi) pathway, is among the most promising therapeutic tools for WSSV, but its farm‐scale utilization is held back by high costs and the lack of delivery approach. Recently, virus‐like particles (VLPs) have been applied as nanocarriers to deliver dsRNA into shrimp tissues, resulting in prolonged half‐life of the encapsidated dsRNA and stimulation of shrimp innate immunity. However, recombinant VLPs and dsRNA are traditionally expressed in two different E. coli strains: a protease‐deficient BL21(DE3) strain and an RNase III‐deficient HT115(DE3) strain, respectively. The ability to produce both VLPs and dsRNA simultaneously in a cell will reduce the costs and steps associated with the production, but attempts at VLP‐dsRNA coexpression in each of these strains resulted in degradation of either VLP or dsRNA due to the presence of proteases or RNaseIII, respectively. Here, to streamline VLP‐dsRNA production and allow simultaneous expression of VLP and dsRNA in the same cell, P1 transduction was employed to generate a novel E. coli strain which is both protease‐ and RNase III‐deficient. The newly engineered E. coli strain is able to express both MrNV‐VLP and VP28 dsRNA efficiently. Therefore, this new expression platform will pave the way for development of effective and low‐cost production of VLP‐encapsulated dsRNA for protection against viral infection in farmed shrimp.

Support or Funding Information

The authors would like to thank the following institutions for supporting research and scholarship to graduate student: Thailand Graduate Institute of Science and Technology (TGIST), National Science and Technology Development Agency (NSTDA) and Mahidol University.
Hypoxia‐induced annexin A6 expression underlies the Metabolic Vulnerability and response of TNBC cells to EGFR‐Tyrosine kinase inhibitors
Authors: Williams Stephen De’Shawn
Published: April 2020

PURPOSE

Triple‐negative breast cancer (TNBC), and the basal‐like TNBC subtype, accounts for ~20% of all breast cancer (BC) cases, and is particularly prevalent in younger patients of African‐American and Hispanic descent. The importance of annexin A6 (AnxA6) as a tumor suppressor in various cancers has been widely investigated, but the mechanisms by which AnxA6‐induced basal‐like triple negative breast cancer progression remain largely unknown. Physiological changes such as hypoxia, metabolic acidosis, and oxidative stress in the tumor microenvironment (TME) during cancer progression endow cancer cells with malignant properties, ultimately leading to metastatic dissemination that remains the major cause of mortality in cancer patients. By linking the altered expression status of AnxA6, with changes in metabolic output of the tumor cells and the efficacy of the otherwise ineffective epidermal growth factor receptor‐tyrosine kinase inhibitors (EGFR‐TKIs), these metabolic vulnerabilities can be targeted to attenuate the growth and/or motility of this hard‐to‐treat breast cancer subtype.

DESIGN METHODS

AnxA6‐high (BT‐549) and AnxA6‐low (MDA‐468) TNBC cell lines were cultured in hypoxic conditions (94%N2; 1%O2; 5%CO2; 37°C) using a Bactrox Hypoxia Chamber (Terra Universal Inc., Fullerton, CA). TNBC cells were treated with EGFR‐TKI Lapatinib (Biovision Inc., Milpitas, CA).

RESULTS

We demonstrate that exposure of AnxA6‐high (BT‐549) and AnxA6‐low (MDA‐468) TNBC cell lines to acute hypoxia (1% O2; ≤24 hrs.) is associated with down‐regulation of AnxA6 while chronic hypoxia (1% O2; >24 hrs.) led to a consistent up‐regulation of AnxA6. AnxA6, FABP4, and HIF‐1α downstream genes GLUT1 and PGK‐1, mRNA levels were upregulated in chronic hypoxic conditions. AnxA6‐dependent metabolic studies reveal that down regulation of AnxA6 in TNBC cells increased lactate production, glucose consumption, ATP‐synthesis and lipid metabolism under hypoxic conditions. In addition, chronic hypoxia sensitized TNBC cells to EGFR‐TKIs.

CONCLUSION

These data suggest that the transition from AnxA6‐high to AnxA6‐low may be dependent on hypoxia in the TME and consequently, TNBC cell survival and resistance to EGFR‐targeted therapies.

Support or Funding Information

The project is supported by NIH 1SC1CA211030 (AS)
Uncovering Dysregulated Micro‐RNA Expression in Senescent Human Vascular Smooth Muscle Cells: Impacts on Cellular Communication and Vascular Aging
Authors: Nguyen Diem Duong Ngoc , Chilian William , Pung Yuh Fen
Published: April 2020

Vascular aging is highly associated with cardiovascular morbidity and mortality and vascular smooth muscle cell (VSMC) senescence is one of its key contributors. However, intracellular and extracellular signaling and communication of senescent VSMCs are not fully elucidated. Understanding micro‐RNA (miRNA) regulation during VSMC senescence may elucidate the molecular mechanistic link between vascular aging and early adverse vascular remodeling. This study aimed to identify dysregulated miRNA expression within senescent VSMCs as well as senescent VSMC‐derived exosomes. Healthy and senescent human VSMCs (hVSMCs) were cultured in vitro and exosomes secreted by these cells were isolated via ultracentrifugation. Subsequently, cellular and exosomal miRNAs were extracted and used for library preparation of whole‐genome small RNA next‐generation sequencing (NGS). Post‐sequencing and differential expression analyses compared the miRNA profiles between healthy and senescent hVSMCs as well as their derived exosomes. Thereafter, NGS data were validated using real‐time polymerase chain reaction (qPCR). Additionally, target prediction and pathway analysis were performed. In senescent hVSMCs, eight significant differentially expressed mature miRNAs were identified (n= 4, q <0.05). Bioinformatic analyses showed correlation between upregulated miRNAs and activated ubiquitin proteasome pathway, a senescence hallmark. Meanwhile, downregulated miRNAs might associate with cell cycle arrest at G1/S phase via the TGF‐β/SMAD signaling pathway, promoting hVSMC entry into senescence. On the other hand, three significant differentially expressed miRNAs were found in senescent hVSMC‐derived exosomes. Data suggested that the upregulated exosomal miRNA was involved in the formation of advanced glycation end‐products, known to play a role in vascular complications. Concurrently, downregulation of exosomal miRNAs promoted cellular senescence, thus advanced aging. Interestingly, there was a consistent observation of hsa‐miR‐155‐5p downregulation in both senescent hVSMCs and their secreted exosomes. Further analyses with the other dysregulated miRNAs suggested hsa‐miR‐155‐5p involvement in the TGF‐β/NF‐κB signaling pathway. The dysregulated miRNAs in senescent hVSMCs and senescent hVSMC‐derived exosomes offered valuable insights on the mechanisms involved in hVSMC intracellular and extracellular signaling during senescence, thus contributing to our understanding of vascular aging in humans.

Support or Funding Information

The study was funded by Ministry of Higher Education Malaysia grant, FRGS/1/2015/SKK08/UNIM/03/2 to YFP.
Allosteric Modulators of CDK4 for Lung Cancer Therapeutics
Authors: MORRIS May Catherine , Bihel Frederic , Diot Sebastien , Peter Clovis , Bouclier Celine , Bricard Jacques , Pellerano Morgan , Hellal Malik , Prével Camille
Published: April 2020

Lung cancer is the leading cause of cancer‐related death worldwide. CDK4/Cyclin D kinase is a key cell cycle regulator and constitutes an attractive pharmacological target for development of anticancer therapeutics, in particular in KRAS‐mutant lung cancer. Although several ATP‐competitive inhibitors targeting protein kinases have been developed as anticancer therapeutics, including several recently FDA‐approved CDK4 inhibitors, these compounds suffer from limited efficacy, selectivity and toxicity due to their conventional mechanism of action. Alternative strategies to overcome limitations associated with conventional kinase inhibitors include targeting protein/protein interactions and conformational transitions which are essential for kinase activation.

Using a CDK4‐specific conformational biosensor that discriminates against ATP‐pocket binding compounds and that we implemented to screen a small chemical compound library of original small molecule scaffolds, we identified and validated a druggable and patentable compound that tampers with CDK4 function. This compound inhibits Rb phosphorylation, blocks proliferation of several cancer cell lines, and does not bind the ATP pocket of CDK4 nor any of 456 other kinases. An ongoing hit‐optimization process led us to identify derivatives exhibiting greater anti‐proliferative activity than Abemaciclib or Palbociclib in the A549, KRAS‐mutated NSCLC cell line. Interestingly, these non‐ATP CDK4 inhibitors only have a cytostatic effect, and do not induce cytotoxicity even at high doses. Further studies aimed at characterizing the mechanism of action of this compound have highlighted an unexpected and atypical mechanism of inhibition targeting CDK4. Our compounds constitute attractive leads as next generation of CDK4‐specific inhibitors and lung cancer therapeutics.

Support or Funding Information

This work was supported by the SATT Connectus, Strasbourg France and awarded the 2019 Best Academic Project of MATWIN (https://matwin.fr/en/cdk4ppi‐awarded‐2019‐best‐academic‐project‐by‐the‐matwin‐international‐board/)
Embryonic hyperglycemia causes a reduction in photoreceptor cells and increased oxidative stress in the developing retina
Authors: Titialii Kayla , Morris Ann
Published: April 2020

Poor blood glucose regulation has been linked to various long‐term metabolic disruptions in adults, such as tissue damage in the eye. Yet, little is known about the effects of embryonic hyperglycemia on retinal development. To address this question, our lab has established a protocol for inducing hyperglycemia in zebrafish embryos by submerging zebrafish embryos in fish water containing 50mM glucose +/− 10μM dexamethasone from 10 hours post fertilization (hpf) until 120 hpf. Following treatment, larval heads were either fixed for sectioning or used for RNA extraction and the remaining body was used to quantify glucose concentration. The fixed heads were processed for sectioning and immunohistochemistry to label and quantify cell types in the retina. Treatment with 50mM glucose + 10μM dexamethasone reliably produced hyperglycemic larvae, with at least 2.5‐fold increased whole‐body glucose concentration compared to control‐treated larvae. Our results show that hyperglycemic larvae had a significant reduction in rod and cone photoreceptors with abnormal morphology, and significant increase in reactive oxygen species (ROS) production in the retina. In addition, Müller glia cells showed abnormal morphology, but an increase in number. Finally, genes related to the pentose phosphate pathway were also up‐regulated in hyperglycemic eyes. Our results suggest that embryonic hyperglycemia results in abnormal photoreceptor development via ROS production, which is accompanied by an elevation in pentose phosphate pathway activity. As the population of women with uncontrolled diabetes during pregnancy continues to grow, it is imperative to pinpoint the effects of embryonic hyperglycemia. The similarities between humans and zebrafish will allow us to study these effects, which will aid in the development of therapeutic strategies.

Support or Funding Information

Gertrude F. Ribble Mini Grant, Department of Biology, University of Kentucky Diabetes Research Center, Washington University, Grant No. 5P30 DK020579
The Anatomy, Bioacoustics, and Neural Physiology of Dolphin Biosonar
Authors: Mulsow Jason , Houser Dorian S. , Finneran James J.
Published: April 2020

Light is rapidly attenuated in sea water, and the habitats of dolphins and other toothed whales are characterized by reduced visual stimuli relative to terrestrial habitats. Sound is efficiently transmitted under water; however, and these species have undergone significant modifications to head structures that facilitate both passive and active (echolocation) acoustic sensing of marine environments.

The nasal anatomy involved in the creation of dolphin biosonar signals is intricate. Structures immediately beneath the blowhole – termed the phonic lips – create short‐duration, directional pulses that travel through a fat‐filled melon (forehead) and into the water. The exact mechanism by which these “clicks” are generated in the phonic lips is not well understood. Recent electrophysiological measurements suggest that neural potentials may precede the production of every click. Thus, dolphins may actively mediate click generation on a click‐by‐click basis. It is not known if this is possible at the highest rates observed in dolphin echolocation (hundreds of clicks per second), as this would require very rapid motor activity. Dolphins also appear to control the direction of the outgoing biosonar beam independent of large head movements. This may be facilitated by deformations of the melon or manipulation of a complicated system of air sacs surrounding the click generation structures.

One of the most obvious anatomical modifications to sound reception structures is the complete absence of external pinnae, and only a small remnant of the ear canal is apparent on the surface of the skin. Although lower frequencies (below 20 kHz in the case of bottlenose dolphins) may primarily be received in this region, higher‐frequency sound (up to approximately 140 kHz) is thought to primarily travel through the mandible. The mandible is partially hollow and filled with specialized “acoustic fats” that connect to the auditory bullae. The cochlea is similar in construction to those of terrestrial mammals; however, there are bony laminae present that result in increased basilar membrane stiffness that facilitates very high‐frequency hearing. Recent data suggest that these laminae may drastically increase the traveling wave speed along the high‐frequency basal end of the basilar membrane, and result in near simultaneous stimulation of the 40–140 kHz region that is critical to echolocation. Refinement for echolocation is further reflected in the neural anatomy of dolphins; e.g., the auditory nerve and brainstem are hypertrophied for rapid transmission of sonar signals. Ongoing research is aimed at determining how the auditory regions of the cortex process acoustic stimuli, but little is currently known about these phenomena.

Support or Funding Information

Funded by the Office of Naval Research
The Diverse Sensory Specializations of Cetacea
Authors: Reidenberg Joy S. , Laitman Jeffrey T.
Published: April 2020

Cetaceans (whales, including dolphins and porpoises) have many unique sensory adaptations that enable them to perceive and respond to stimuli underwater. These adaptations include audition, vocalization, vision, balance, chemoreception, and somatosensation.

Cetaceans generate and receive sounds at both ends of the frequency spectrum, and use these sounds for both communication and echolocation. Toothed whales (odontocetes) specialize in high frequency sounds, while baleen whales (mysticetes) focus on low frequency sounds. High frequencies have short wavelengths, and thus enable odontocetes to detect minute features in the reflected echoes related to geometry, texture, density, vector, velocity, etc. Nasal structures generate two different frequencies simultaneously (stereo echolocation). Sounds are received in jaw fats and transmitted to the ears. These features help odontocetes target and catch prey, but can also be used for short‐range navigation and communication.

The mysticete larynx generates low frequency sounds. Echoes from these large wavelengths can reveal large features at a distance, facilitating navigation through the ocean landscape during migration. As low frequencies also attenuate more slowly, they can be used for long distance communication. Low frequencies present a hearing dilemma, as wavelengths can be larger than the whale! However, using the large skull as an antenna allows mysticetes to perceive change along most of the wavelength, and relay that information through bony conduction to the ear. Males use highly patterned vocalizations (songs) that advertise sexuality. Songs can be shared with and repeated by new whale groups, thus indicating sophisticated cultural exchange.

Vision is another sense that is adapted to life underwater. Cetacean eyes allow visual acuity both above and below water. These include changes to the lens and retina that accommodate light filtering at various depths, and differences in the refraction of light in water versus air. The sclera is also thickened, and may be an adaptation that prevents the eye from deforming under high pressures during diving.

Much less is known about the senses of touch, balance, smell, or taste. Cetacean skin is very sensitive, but little is known about its specific somatosensory detection abilities (vibration, water flow, pressure, electromagnetic fields, etc.). Balance seems essential for navigating in three‐dimensional space, yet paradoxically, cetacean semicircular canals are reduced. Smell and taste appear useless to a predator that feeds underwater and swallows prey whole, yet olfactory bulbs and taste buds appear to persist at least in baleen whales. These unique features present open areas of inquiry in cetacean sensory ecology.
Craniofacial Phenotypic Plasticity in Mice Exposed to Various Temperatures
Authors: Heuzé Yann , Maréchal Laura , Devlin Maureen J.
Published: April 2020

Influences of environmental factors such as temperature on skeletal phenotype have been hypothesized and demonstrated for several decades. Animal models, mostly rodents, exposed to cold temperatures display significant reduction of long bone length (endochondral ossification), tail length, ear size, and foot size, without change in body mass. Regarding the craniofacial phenotype, subtle changes in facial shape and a relative decrease in the volume of the maxillary sinuses and nasal cavity were reported in rats but, to the best of our knowledge, never reproduced with other models or with different temperatures. Here we propose to compare craniofacial phenotypes of male C57BL/6J laboratory mice that were exposed from 3–12 weeks of age to 16°C, 22°C, and 26°C (N=4–5/group). The mice were sacrificed at the age of 12 weeks. Reconstructions of the 3D microCT images were performed and 58 anatomical landmarks were measured on the craniofacial skeleton. Landmark three‐dimensional coordinates were then analyzed using geometric morphometrics. Our results confirmed significant shape differences between the mice exposed to different temperatures. The anatomical regions displaying the most intense shape variation depending on temperature are the anterior aspect of the snout (nasal bones and premaxilla), sphenoid, and parietal bones, with mice exposed to 16°C displaying an overall less globular skull. Bones forming through endochondral ossification do not appear to be more affected than those forming through intramembranous ossification. These findings confirm that phenotypic plasticity plays a significant role in the so‐called cold adapted phenotype.
Interspecies transcriptome analyses identify genes that control the development and evolution of limb skeletal proportion
Authors: Cooper Kim , Saxena Aditya , Sharma Virag , Neufeld Stanley , Tran Mai , Gutierrez Haydee , Erberich Joel , Birmingham Amanda , Cobb John , Hiller Michael
Published: April 2020

Despite the great diversity of vertebrate limb proportion and our deep understanding of the genetic mechanisms that drive skeletal elongation, little is known about how individual bones reach different lengths in any species. Here, we directly compare the transcriptomes of homologous growth cartilages of the mouse (Mus musculus) and bipedal jerboa (Jaculus jaculus), which has extremely long metatarsals of the feet and ‘mouse‐like’ arms. When we intersected gene expression differences in metatarsals of the two species with expression differences in forearms, we found that about 10% of all orthologous genes are associated with disproportionate elongation of jerboa feet. Among these, Shox2, has gained expression in jerboa metatarsals where it is not expressed in other vertebrates that have been assessed. This transcription factor is necessary for proximal limb elongation, and we show that it is sufficient to increase mouse distal limb length. Unexpectedly, we also found evidence that jerboa foot elongation occurs in part by releasing latent growth potential that is repressed in mouse feet. In jerboa metatarsals, we observed higher expression of Crabp1, an antagonist of growth inhibitory retinoic acid, lower expression of Gdf10, an inhibitory TGFβ ligand, and lower expression of Mab21L2, a BMP signaling inhibitor that we show is sufficient to reduce limb bone elongation. By intersecting our data with prior expression analyses in other systems, we identify mechanisms that may both establish limb proportion during development and diversify proportion during evolution. The genes we identified here therefore provide a framework to understand the modular genetic control of skeletal growth and the remarkable malleability of vertebrate limb proportion.

Support or Funding Information

This work used the Extreme Science and Engineering Discovery Environment(XSEDE) at SDSC, which is supported by National Science Foundation grant number ACI1548562. This work was also supported by Natural Sciences and Engineering Research Council grant RGPIN/355731‐2013 to JC and by a Searle Scholar Award from the Kinship Foundation, a Pew Biomedical Scholar Award from the Pew Charitable Trusts, a Packard Fellowship in Science and Engineering from the David and Lucile Packard Foundation, and the National Institutes of Health under award number R01AR075415 awarded to KLC.
The Effect of Modified Curcumin Compounds on the Numbers and Morphology of Microglia
Authors: Gyengesi Erika , Ullah Faheem , Asgarov Rustam , Niedermayer Garry , Muench Gerald
Published: April 2020

Chronic neuroinflammation is a promising therapeutic target in many neurodegenerative and age‐related brain disorders. In this study, we investigated two highly bioavailable curcumin preparations, Longvida (LC) and Meriva (MC) for their anti‐inflammatory properties, using the GFAP‐IL6 mouse model. We hypothesized that the highly bioavailable curcumin, as a potent cytokine‐suppressive anti‐inflammatory drug, will decrease microglial and astroglial activation measured by cell number and morphology, and consequently improve motor and cognitive functions.

Methods

GFAP‐IL6 and wild type mice were fed with LC and MC for 1 and 5 months, respectively. LC fed mice were tested on the elevated walking beam, Rotarod, Barnes maze and Novel object recognition tests before all the mice were sacrificed. Immunohistological staining for microglia and astrocytes were carried out and unbiased stereological analysis was completed for all groups on microglia(labelled by Iba‐1 and TSPO) and astrocytes numbers (GFAP) as well as the morphology of the cells were analyzed using the MBF Biosciences system (StereoInvestigator and Neurolucida 360).

Results

Short term MC oral administration lead to a dose‐dependent reduction in neuroinflammatory markers. Moreover, MC decreased the number of activated microglia and astroglia, as well as changed the microglial morphology from activated to resting state. Long term LC treatment led to a significant decrease in the number of microglia in the hippocampus and cerebellum and in turn, led to significant improvement of both motor and cognitive function. Both short‐ and long‐term consumption of modified curcumin preparations significantly downregulated microglial activation GFAP‐IL6 mice. Spatial memory and recognition memory improvements were also found in mice fed with LC in comparison to control counterparts. Our results promise potential therapeutic applications of both Longvida and Meriva curcumin against brain inflammation and associated cognitive and motor decline.

Support or Funding Information

The present study was supported by a project grant (#P00023016) by Indena S.P.A, Italy to GM and EG. F.U has been supported by a postgraduate scholarship by Western Sydney University.
The Genetic Architecture of Alveolar Formation in the Lung in the Context of Bronchopulmonary Dysplasia
Authors: Minoo Parviz , Gao Fremont , Li Changgong , Chao Cho-Ming , Bellusci Saverio
Published: April 2020

Alveologenesis is the most important and the least understood phase of lung development. Alveolar defects underlie both neonatal and adult lung diseases. Their arrested development is found in infants with BPD (BronchoPulmonary Dysplasia), while their destruction is characteristic of emphysematous lungs in adult COPD. We have developed 3 parallel, postnatal genetically‐based models to analyze the molecular basis of alveologenesis. Conditional postnatal inactivation of genes encoding receptors for TGFb, or PDGFA or IGF1 in a unique cell type we have identified as Secondary Crest Myofibroblasts, or SCMF blocks alveologenesis & produces a phenotype similar to human BPD. We reasoned that due to nearly identical phenotypes, the three BPD phenocopy models must share genetic elements whose role in alveologenesis is indispensable. To test this hypothesis, we have isolated mutant and control SCMF and identified their transcriptomic profile by RNA sequencing (RNAseq). Bioinformatic analysis of the sequencing data identified “pathway‐specific” differentially expressed genes (DEG) for TGFb, PDGFA/PDGFRa and IGF1 mutant models. Most importantly however, as predicted, we identified a cluster of “shared” genes which we propose contains conserved core alveologenesis regulatory genes. The accumulated evidence reveals a constellation of genes that makeup the genetic architecture of alveologenesis in mice with relevance to pathogenesis of BPD in human preterm neonates.

Support or Funding Information

NIH/NHLBI & the Hastings Foundation
Hippo‐signaling in Heart Development and Regeneration
Authors: Martin Jim
Published: April 2020

The mammalian heart regenerates poorly and damage commonly leads to heart failure. The Hippo pathway is an evolutionarily conserved kinase cascade that regulates heart size during development and prevents adult mammalian cardiomyocyte regeneration by inhibiting the transcription cofactor Yap. Recent work indicates that deletion of the Hippo pathway component, Salv, in cardiomyocytes can reverse ischemic heart failure in a mouse model. Further experiments have uncovered Yap target genes and provided insight into the mechanisms by which Yap regulated cardiomyocyte gene expression.

I will discuss our latest findings that reveal new insights into heart size control and regeneration.
Retrieval Enhances Higher and Lower Order Thinking in Anatomy and Physiology Students
Authors: Dobson John
Published: April 2020

Retrieval practice is a learning strategy that was developed by cognitive scientists and is based on the act of recalling information after exposure. Over the last two decades, many dozens of studies have found that retrieval‐based strategies can enhance learning and memory more effectively than more commonly‐used strategies such as repetitive reading. Although the advantages of retrieval practice have been demonstrated in both well‐controlled laboratories and real‐world settings such as middle school and university classrooms, the evidence also indicates that most educators and students remain unaware of the benefits of the strategy. It is especially unfortunate when such an effective learning strategy is underutilized in courses that cover anatomy and physiology information because many students find those subjects to be particularly complex, overwhelming, and difficult to learn. The purpose of this presentation is to review key studies that examined the effects of retrieval practice on lower and higher orders of thinking with anatomy, physiology, and medical information. Lower orders of thinking include the abilities to identify, define, and distinguish; whereas higher orders of thinking include the abilities to transfer, critique, and evaluate. The evidence reviewed in this presentation demonstrates that retrieval practice is a relatively effective means by which to increase both higher and lower order thinking with anatomy and physiology information.

Support or Funding Information

None
Genetic and Tissue Interactions in the Embryogenesis of the Diencephalon and Circumventricular Organs
Authors: Kiecker Clemens
Published: April 2020

The posterior forebrain (diencephalon) is one of the vesicles that emerges in the anterior neural tube early in embryogenesis. It gives rise to the thalamus (the major relay station of the brain), the prethalamus and pretectum, the epithalamus including the habenular complex, the hypothalamus including the posterior lobe of the pituitary gland and the retinas of the eyes. It also generates a number of circumventricular organs, highly specialised sensory and secretory structures that lack a blood‐brain barrier and that allow for the exchange of substances between the brain and the circulatory system. I will give an overview of the genetic mechanisms that regulate the embryonic development of the diencephalon with a focus on (a) the role of secreted signals and transcription factors and (b) interactions between tissues. The pituitary gland proper develops through an interaction between neural (hypothalamus‐derived) and non‐neural (placode‐derived) tissues, and our recent work indicates that the pineal organ forms through a similar interaction between neural and placodal tissues, suggesting a common theme in the establishment of diencephalon‐derived neurosecretory and sensory structures.

Support or Funding Information

This work has been supported by King’s College London.
Interventional Strategies to Delay Aging Related Diseases & Conditions of the Musculoskeletal System
Authors: Huard Johnny
Published: April 2020

Aging leads to several geriatric syndromes including frailty, a condition characterized by loss of functional reserve and tissue regeneration repair capacity. Frail individuals exhibit significant mobility and psychological deficits resulting in significant healthcare costs. Thus, identifying strategies to delay aging, or prevent the progressive loss of tissue homeostasis and functional reserve associated with frailty, will dramatically restore function and independence in millions of elderly patients and significantly improve quality of life. We have demonstrated that bone marrow‐derived mesenchymal stem cells (MSCs) and muscle‐derived stem/progenitor cells (MDSPCs) become dysfunctional with age. A fundamental property of aging is the accumulation of senescent cells. Senescence is a cell state defined by loss of proliferative capacity, increased metabolic activity, and importantly, resistance to apoptosis. Senescent cells can release pro‐inflammatory cytokines/chemokines, proteases, and other factors known as the senescence‐associated secretory phenotype (SASP). The SASP can adversely affect not only neighboring cells, but also likely contributes to driving systemic aging. Compounds that selectively target and kill senescent cells were recently identified and characterized. These senolytic drugs target and inhibit anti‐apoptotic pathways that are upregulated in senescent cells thereby inducing apoptotic cell death and abrogating systemic SASP factors. Importantly, senolytic drugs have a favorable safety profile being either FDA approved (dasatinib) or over the counter flavonoid supplements (quercetin, fisetin), positive findings may be more readily translatable to Phase I–II clinical trials. Further, it has been reported that exposure to factors present in the serum of young mice restores the regenerative capacity of aged progenitor cells, through parabiotic pairing. We believe that pregnancy represents a unique biological model of a naturally‐shared circulatory system between young and old organisms. Our preliminary data have demonstrated improved musculoskeletal tissue healing after injury in pregnant mice and improved differentiation capacity of aged muscle progenitor cells (MPCs) after stimulation with serum from pregnant mice. We hypothesize that circulating factors from fetal microchimeric cells (FMCs) during pregnancy have beneficial effects on maternal wound healing, such as reduction in fibrosis for optimal tissue regeneration. In summary, we believe these novel interventional strategies could be used to restore the endogenous stem cell populations in age‐related disorderst to promote healthy aging & to delay age‐related disorders.
Effect of biglycan on apoptosis of neurons in acute cerebral infarction in rats
Authors: Du Yanrong , DUAN HUBIN , Qiao Xiaohong , Xue Wen , cheng junjun , Hao Chunyan , Li Youlian , Liu Wenyun , Guo Jianxun
Published: April 2020

Objective

To investigate the effect and mechanism of biglycan (BGN) on apoptosis after experimental acute cerebral infarction (CI) in rat.

Methods

A total of 110 clean grade Wistar male rats were randomly divided as follow: A total of 80 clean grade Wistar male rats were randomly divided into sham group and CI group consisted of 3h, 6 h, 12h, 24h, 48h, 72h, and 7d (n=10). The expression of BGN in neuron and its time course were detected. A total of 30 clean grade Wistar male rats were randomly divided into sham group, sham + control lentivirus group, sham + BGN lentivirus group, CI + control lentivirus group, and CI + BGN lentivirus group (n=6). The silence efficiency of BGN and the effect of BGN on neuronal apoptosis and neurological score after CI were detected. CI model was induced by reforming longa method. Lentiviral vector was administered intracerebroventricularly 7 days before CI. The expression of protein and mRNA of BGN were detected by western blot and real‐time quantitative PCR (qRT‐PCR). Immunofluorescence was conducted to explore the expression of BGN on neuron. Neurological score was detected by the modified Garcia score, beam balance tests and Longa’s Five‐Point Pacification Low. The TUNEL was used to detect neuronal apoptosis.

Results

The expression of protein and mRNA of BGN were peaked at 24h after CI (P<0.05). A majority of BGN were detected on neuron at 24 h after CI. Compared with CI + control lentivirus group, the modified Garcia score, beam balance tests and the Longa’s Five‐Point Pacification were significantly improved in CI + BGN lentivirus group (P<0.05), as well as the TUNEL‐positive neurons ratio was significantly decreased ( P<0.05).

Conclusion

Inhibition of BGN can significantly reduce early neuronal apoptosis after CI, and attenuated neurological deficits.

Support or Funding Information

This study was supported by the National Natural Science Foundation Youth Fund (30600637), China Postdoctral Foundation (2014M561207, 2019T120195), the Shanxi Province Basic Research Program (2010021034‐4, 201601D011119), the Shanxi Scholarship Council of China (2011‐096 and Key Projects No.4), Department of Human Resources and Social Security of Shanxi province, The science and technology research projects of health department of Shanxi Province (2016D011119), The Teaching Reform Program of Shanxi Higher Education, The Education Reform Project of Postgraduate Students in Shanxi Province, Lvliang people’s hospital fund, The Education Reform Project of Shanxi Medical University.
Younger Age of First Exposure to Soccer Heading is Associated with Decreased MicroRNA‐7844‐5p
Authors: Muñoz Eric R , Caccese Jaclyn B , Wilson Brittany E , Shuler Kyle T , Santos Fernando V , Yamaguchi Felipe , Jeka John J , Hudson Matthew B
Published: April 2020

Younger age of first exposure (AFE) to American football has been associated with later‐life cognitive deficits in former National Football League players experiencing cognitive and behavioral deficits, but not in other cohorts of high school, college, amateur, or professional contact/collision sports athletes. Although several studies evaluated clinical outcomes, limited information exists among these cohorts and subclinical measures, such as blood biomarkers may serve as an early indicator of future impairments. Extracellular vesicles, including exosome‐like vesicles (ELVs), are released from cells and contain a variety of molecular cargo from the originating cell, and have emerged as reliable biomarker candidates.

OBJECTIVE

The purpose of this study was to compare plasma ELV concentration, size, and microRNA (miRNA) levels between soccer players reporting AFE to soccer heading before age 10 years (AFE≤10) and soccer players reporting AFE to soccer heading after age 10 years (AFE>10).

METHODS

College‐aged soccer players self‐reported AFE to soccer heading. Participants were divided into two groups based on their responses: AFE>10 (n=9, 5 males, 12.33±0.24 years) and AFE≤10 (n=18, 11 males, 7.94±0.46 years). Plasma ELV concentration and size were assessed using nanoparticle tracking analysis. Total RNA was isolated from ELVs, followed by performing miRNA first strand cDNA synthesis and subsequently qPCR. Comparisons were made via unpaired t‐test (p<0.05).

RESULTS

Data are presented as mean ± SE. AFE≤10 was associated with decreased miR‐7844‐5p when compared to AFE>10 (~60%, p=0.03). No changes in miRNA were observed for miR‐92b‐5p, miR‐423‐5p, miR‐24‐3p, miR‐144‐5p, miR‐221‐5p, and miR‐22‐3p. Groups did not differ in concentration (AFE≤10, 8.34 ×109 ELVs/ml plasma ±0.96 ×109 vs AFE>10, 8.39 ×109 EVs/ml plasma ±0.98 ×109) or size (AFE≤10, 117.58 nm ±7.12 vs AFE>10, 106.40 nm ±4.14) of circulating ELVs.

CONCLUSION

Collectively these data demonstrate that younger AFE to soccer heading was associated with decreased circulating miR‐7844‐5p. Decreased circulating miR‐7844‐5p may be an early indicator of future pathology.

Support or Funding Information

Supported by NIH‐NINDS R01 NS102157‐01, NIH P20 GM113125, NIH P20 GM103446, NIH R03 HD094594
Structural Characterization of the Chondroitin Sulfate motif binding to VAR2CSA in Placenta and Cancer
Authors: Spliid Charlotte Bredo , Toledo Alejandro Gomez , Esko Jeffrey , Clausen Thomas Mandel
Published: April 2020

Malaria parasites have evolved to express certain proteins called VAR2CSA on the surface of infected erythrocytes. This allows them to adhere to unique chondroitin sulfate (CS) motifs, which are only found in the placenta. It was recently discovered that VAR2CSA also binds to over 95% of all types of cancer. The exact structure of the CS chains, termed oncofetal CS (ofCS), has previously been reported to be mostly non‐sulfated with low levels of 4‐O‐sulfation in the placenta. In this study, we found that ofCS was ~90% 4‐O‐sulfated with the remaining ~10% being mostly 6‐O‐sulfated and 2‐O‐sulfated. Although previously thought to be purely glucuronic acid (GlcA) we found that ~25% of the uronic acid content consisted of iduronic acid (IdoA). Additionally, we identified that the CS quantity and chain length were significantly increased in placenta and colon tumors compared to healthy organs and colon. This increased CS availability along with the unique ofCS motif is likely what causes high specificity of VAR2CSA to placenta and cancer.

Support or Funding Information

VAR2 PharmaceuticalsGangstedfondenLouis‐Hansen Fonden
Membrane Phospholipid Composition Governs Pah1 Phosphatidate Phosphatase Activity
Authors: Kwiatek Joanna , Carman George
Published: April 2020

In yeast and higher eukaryotes, all membrane phospholipids and the storage lipid triacylglycerol, are derived from phosphatidate (PA), which is also implicated as an activator of cell growth and proliferation, vesicular trafficking, secretion, and endocytosis. PA also regulates expression of lipid synthesis genes via the Henry (Opi1/Ino2‐Ino4) regulatory circuit, where the key step is the PA‐mediated translocation of the Opi1 repressor between the nuclear/ER membrane and the nucleus. Data indicate that several enzymes play important roles in the PA‐mediated regulation of lipid synthesis that occurs at the nuclear/ER membrane. Of the enzymes that metabolize PA, the PA phosphatase enzyme, which is encoded by the PAH1 gene, has emerged as key regulator of PA homeostasis. Indeed, cells lacking this enzyme exhibit several phenotypes that include elevated levels of PA, derepression of phospholipid synthesis genes, increased synthesis of membrane phospholipids, and the abnormal expansion of the nuclear/ER membrane. These mutant cells also exhibit a drastic reduction in TAG and lipid droplets. The PA phosphatase, which catalyzes the conversion of PA to diacylglycerol, must translocate from the cytoplasm to the ER membrane where its substrate PA resides to function in lipid synthesis. Thus, studies to examine mechanisms for the enzyme reaction at the membrane surface are warranted. In this work, we implemented defined unilamellar vesicles with a diameter of 100 nm composed of the major phospholipids within the ER membrane. Vesicles composed of phosphatidylcholine (PC) and 10 mol% PA supported PA phosphatase activity. The addition of phosphatidylethanolamine (PE) or phosphatidylinositol (PI) stimulated the activity, but phosphatidylserine (PS) had little effect. In more complex vesicle compositions, the maximum activity was observed with vesicles containing PC/PE/PS/PA or PC/PE/PI/PA. Yet, the activity was reduced when vesicles contained PC/PE/PI/PS/PA. The PA phosphatase activity was dependent on vesicle number as well as the surface concentration of PA within the vesicles. This vesicle system is being used to assess the roles of phosphorylation/dephosphorylation of Pah1 on membrane interaction and PA phosphatase activity.

Support or Funding Information

Supported by NIH grant GM028140
Engineered High‐Specificity Affinity Reagents for the Detection of Glycan Sialylation
Authors: Yang Loretta , Wu Sheng-Cheng , Meng Lu , Gerner-Smidt Christian , Woods Robert J.
Published: April 2020

As alternatives to established carbohydrate‐detection reagents (antibodies and lectins), Lectenz Bio is developing engineered proteins (known as Lectenz®), that target glycan sequences for with existing reagents either do not exist or are sub‐optimal. Lectenz® are engineered proteins derived from catalytically inactivated glycan‐processing enzymes that have been optimized for high affinity towards specific glycan sequences. The conversion of such enzymes into affinity reagents is facilitated by computationally‐guided directed evolution. Lectenz® are being developed for a variety of glycan detection and enrichment applications including affinity chromatography, Western blot, and immunohistochemistry. Here we demonstrate the performance and specificity of two novel sialic acid recognizing Lectenz® engineered from a sialidase enzyme.

Support or Funding Information

NIH R44GM113351, R44OD024964
Oxidative stress‐induced germ cell apoptosis: The NOX‐ER stress connection.
Authors: Al-Saleh Farah , Al-Maghrebi May Abdulsamad , Khashab Farah Ahmed , Fadel Fatemah Mohammed , Al-Kandari Nora Yaqoub
Published: April 2020

Testicular torsion and detorsion (DTT) can seriously damage the testes and cause infertility issues if left untreated. In animal models, it is represented as a testicular ischemia reperfusion injury (tIRI). During tIRI, excessive generation of reactive oxygen species (ROS) induces oxidative DNA damage (ODD) leading to germ cell apoptosis (GCA). The study objectives include investigating the role of NADPH oxidase (NOX), a source of cellular ROS, in instigation of ODD and subsequent GCA during tIRI and finding out NOX’s role in inducting endoplasmic reticulum (ER) stress. Male Sprague‐Dawley rats (n=36) were divided into three groups: sham, tIRI only and tIRI + apocynin (50 mg/kg), a NOX inhibitor. The tIRI rats underwent ischemia for 1 hour followed by 4 hours of reperfusion prior to rat sacrifice. Apocynin was administered 30 minutes post ischemia. Harvested testes were evaluated for spermatogenic damage by histological analysis, while biochemical and molecular modulations were assessed using biochemical assays, ELISA, immunofluorescence staining and real time PCR. Spermatogenic arrest was associated with increased lipid and protein peroxidation and decreased superoxide dismutase activity as a result of tIRI. The tIRI‐induced ODD was indicated by a significant increase in the levels of DNA strand breaks, 8‐OHdG formation, ph‐H2AX and ph‐ATM. The ASK1/JNK/survivin apoptosis pathway was significantly activated in response to tIRI. Finally, a significant increase in the immunoexpression of the unfolded protein response (UPR) pathway components like CHOP, GRP78, caspase 12 and ph‐eIF2‐alpha1 supported the occurrence of ER stress during tIRI. NOX inhibition by apocynin protected against ODD, GCA and ER stress. Our results indicate that NOX‐mediated ROS generation directly contributes to the tIRI‐induced ODD and GCA. In addition, NOX inhibition suggested its direct influence on inducing ER stress during tIRI.

Support or Funding Information

This study was supported by Kuwait University Grants YM 16/17 (College of Graduate Studies ) and SRU02/13 (Research Administration).