bluuebunny/crossref_metadata_2025 huggingface dataset:
Signaling mechanisms involved in protease‐activated receptor‐1 mediated nitric oxide production.
Authors: Watts Vabren L , Motley Evangeline D
Published: June 2021

Protease‐activated receptors (PARs) are G protein‐coupled receptors that are known to regulate endothelial nitric oxide synthase (eNOS) activity by phosphorylating the enzyme at various sites. Phosphorylation of eNOS‐Thr495 negatively regulates eNOS activity by causing a decrease in nitric oxide (NO) production. Currently, the mechanisms for eNOS‐Thr495 phosphorylation by PAR agonists are unknown. In this study, we used a specific synthetic PAR‐1 activating peptide, TFLLR, and thrombin to assess the role of PAR‐1 involvement in NO production in endothelial cells. Both agonists phosphorylated Thr495 in a time‐ and concentration‐dependent manner. Also, cells pretreated with a PAR‐1 inhibitor, showed a significant decrease in thrombin and TFLLR‐induced phosphorylation of eNOS Thr495. Rho and Rho‐Kinase inhibitors also caused a significant decrease in PAR‐1‐induced Thr495 phosphorylation. In addition, we observed that both PAR‐1 agonists caused a significant decrease in nitrite production, whereas inhibition of PAR‐1, Rho, and ROCK caused a significant increase in PAR‐1 agonist‐mediated nitrite production. This suggests that PAR‐1 induces phosphorylation of eNOS‐Thr495 and inhibition of NO production via a Rho/ROCK dependent pathway. These findings will contribute to understanding the signaling pathways that regulate eNOS‐induced nitric oxide production. NIH‐NCRR 2G12RR03032.
Smooth Muscle Cell Seeding on Decellularized Porcine Saphenous Vein Scaffolds –A Step Towards Functional Tissue Engineered Blood Vessels
Authors: Machingal Masood A. , Merritt Heather P.M. , Neff Luke P , Van Dyke Mark , Soker Shay , Geary Randolph , Christ George J.
Published: June 2021

The need for improved autologous tissue engineered blood vessels (TBEV) for vascular replacement is unequivocal. To this end, the over‐riding goal of our vascular research program is to develop TEBV that more closely approximate native vasculature. To this end, we have utilized an approach that combines dual‐seeding of endothelial and smooth muscle cells on decellularized arterial scaffolds (DAS) followed by bioreactor preconditioning. A major barrier to this approach is the limited migration of SMC into the medial layer of the DAS. To overcome this limitation, the current studies explored the potential of decellularized venous scaffolds (DVS). DVS were statically seeded with SMC for 2 hours and preconditioned in a bioreactor with either pulsatile flow (60 bpm /80‐120 mm Hg) or steady flow (100 mm Hg) for two weeks. The effect of cyclic strain preconditioning on attachment, proliferation and migration of SMCs was evaluated with H&E, Trichrome and DAPI. Our preliminary data suggest that seeded SMC adhere to and proliferate on DVS. We also demonstrated that cyclic preconditioning using pulsatile flow improves SMC migration and integration/ingrowth into the medial layer of the scaffold. Although further study is required, these initial data are encouraging and indicate that in vitro conditioning of DVS may produce TEBV that more closely mimic the architecture and physiological characteristics of native arteries.
When Selecting an Adeno‐associated Viral Vector Serotype, Cell Monolayer Transduction Efficiency Does Not Accurately Predict Tissue Transduction Efficiency in Equine Synovial Tissues
Authors: Mason Jeffrey B. , Vandenberghe Luk H. , Wilson James M. , Richardson Dean W.
Published: June 2021

Low transduction efficiency of synovial tissues is a key concern in osteoarthritis gene therapy approaches. To determine the most efficient gene transfer strategy for effective in‐situ treatment of articular tissues, eight adeno‐associated viral vector (AAV) serotypes were assessed in monolayer‐cultured equine synovial cells and synovial tissue explants. Of the eight serotypes tested at 1x102 genome copies (GC) per cell, AAV2/2 produced the highest transduction efficiency in monolayer‐cultured cells followed by vector AAV2/5. When GC per cell was increased 10‐fold, again vector AAV2/2 produced the highest transduction efficiency followed by vector AAV2/5. However, when the same candidate vectors were used to transduce freshly‐collected synovial tissue explants, the hierarchy in transduction efficiency had changed. At approximately 3x102 GC per cell, vector AAV2/6.2 now produced the highest transduction efficiency followed by vector AAV2/5. When the GC per cell was increased 10‐fold, again vector AAV2/6.2 produced the highest transduction efficiency followed by vector AAV2/5. We conclude that vector AAV2/6.2 appears to be a superior vector for transduction of fibrous synovial joint tissues and that cell monolayer transduction efficiency does not accurately predict tissue transduction efficiency. Research support was provided by Brushwood Stables.
In‐vitro comparison of sensors and amplifiers to measure left ventricular pressure in mice
Authors: Reddy Anilkumar K. , Taffet George E. , Hartley Craig J.
Published: June 2021

Several companies market catheters to measure left ventricular (LV) pressure in mice, and it is often assumed that all sensors are similar, but there may be important differences in fidelity, stability, and immunity to noise and artifacts. We used an electronic signal generator, a dynamic pressure generator, and a laboratory scale to measure the frequency and step responses, immersion response, stability, accuracy, linearity, and sensitivity to side forces of amplifiers and 4 solid‐state sensors each from Millar, Scisense, and RADI. All sensors were stable after immersion with drifts of <1 mmHg/hr over 5 hrs, had flat frequency responses to >1 kHz, and were accurate and linear to within ±2 mmHg from 0‐300 mmHg. The amplifiers from Millar, Scisense, and RADI had time delays of 0.2, 3.2 and 10.6 ms; rise times of 300, 50, and 250 mmHg/ms; and 6 dB‐down frequency responses of 1200, 300, and 600 Hz respectively. The Scisense (but none of the other) catheters showed a 5.3 mmHg/g response to lateral forces applied to the sensor tip. Given that all of these semi‐rigid catheters straighten the normally curved vascular path upon insertion, the dynamic cardiac forces could generate significant "pressure" signals. We conclude that there are significant differences in solid‐state mouse pressure sensors and amplifiers which could generate unrecognized offsets, time delays, and distortions of raw signals and derivatives in‐vivo.
Acute spinal cord stimulation alters cytokine gene expression in the meninges
Authors: Tilley Dana , Vallejo Ricardo , Benyamin Ramsin , Vogel Laura , Kramer Jeffery
Published: June 2021

Spinal cord stimulators represent an advanced medical device based therapy that can effectively treat chronic neuropathic pain conditions. We hypothesized that spinal cord stimulation may impact the expression of cytokines in the meninges, thereby offering one potential mechanism by which these devices produce analgesia. Rats were anesthetized and multipolar, micro‐circumferential leads connected to a clinically utilized impulse generator were epidurally placed. Leads were placed between L2 and L5. Rats were stimulated for 2 hours at 50% motor threshold (20 Hz, .2 ms pulse duration). Following either stimulation or sham stimulation (leads placed in the epidural space for two hours without current application) animals were sacrificed and the meninges underlying the leads were harvested. Utilizing RT‐PCR, we found that after two hours, both TNF‐alpha and IL‐10 transcript levels were significantly elevated, while IL‐6 and IL‐1beta levels were unchanged. These preliminary results suggest that spinal cord stimulation can provoke changes in cytokine gene expression in the meninges. Further studies are warranted to see if the meninges are releasing these into the CSF to potentially modulate spinal function.
Agenesis of the Right Internal Carotid Artery Associated with Persistence of the Primitive Olfactory Artery. A Case Report.
Authors: Veras Wilson Rafael
Published: June 2021

Unilateral agenesis of the internal carotid Artery (ICA) is an extremely rare developmental anomaly. The absence of an ICA was first described by Tode in 1787 after being discovered during a postmortem examination. Only approximately 100 cases have been reported in the literature with a left predominance (3:1). When a unilateral ICA is absent, collateral circulation is sufficient to maintain cerebral function with few or no neurologic symptoms. Most patients with ICA agenesis present with focal neurologic signs, such as convulsions, headache, or transient ischemic attack, and ICA agenesis may be associated with aneurysm, hemorrhage, cerebral Hypoplasia, hemangioma, or anomalous vascular anastomosis. During dissection carried out in the Anatomy laboratory an absence of the right internal carotid artery was found. The specimen consists of a brain from a male cadaver with absence of the right internal carotid artery. A common trunk arises from the anterior aspect of the left internal carotid artery to supply the territory corresponding to the right internal carotid artery. Just anterior to the right middle cerebral artery this common trunk gives rise to two small vessels and then splits into two terminal branches. These two small vessels probably correspond to a persistent primitive olfactory artery (PPOA, embryological vessel). Six different branching patterns of circulation in cases of absence of internal carotid artery have been described for many authors; this paper describes a branching pattern of circulation previously undescribed.
Localization of the sural nerve in relationship to calcaneal (Achilles) tendon repair
Authors: Atsas Stavros , Blackmon Joseph A , Campbell Ferrell R , Wells Michael J , Lambert H. Wayne
Published: June 2021

The sural nerve is a major cutaneous nerve of the lower limb that supplies the posterolateral leg and the lateral margin of the foot. It is at risk of iatrogenic injury even during minimally‐invasive surgical procedures to repair the calcaneal (Achilles) tendon. This cadaveric study investigated the point at which the sural nerve crossed the lateral boundary of the Achilles tendon to enable clinicians to identify the location of the sural nerve within 90% and 95% confidence intervals. In most cases, the sural nerve crossed the lateral border of the Achilles tendon 8‐10 cm along the vertical length of the tendon. Based upon the results of 107 leg dissections, surgeons can approximate the location of the sural nerve with confidence and minimize the damage to this important cutaneous nerve.

Grant Funding Source

AAA Student/Postdoctoral Travel Award
Developmental stages of Coturnix coturnix japonica, the Japanese quail
Authors: Ainsworth Sophie Jane , Stanley Rachael , Evans Darrell
Published: June 2021

Coturnix coturnix japonica (Japanese Quail) is an avian species that has been extensively used in developmental studies for many years. Unlike the domestic chicken, Gallus gallus domesticus, the development of the quail has not been accurately staged, despite being used as an important developmental tool. The chick was accurately staged over 50 years ago in a seminal paper by Hamburger and Hamilton and although there have been several papers looking at developmental stages of the quail, the process has not been comprehensive or definitive and therefore researchers have often used the chick staging criteria. One of the most obvious and dramatic differences between quail and chick is hatching times, with the quail hatching some 4‐5 days before the chick. This more rapid developmental cycle means that the use of the Hamburger and Hamilton stage series for all quail developmental studies may not be advantageous. Therefore, we have constructed a comprehensive stage series of quail embryo development, which covers the whole incubation period. It is hoped that such a series will provide researchers with an accessible manual, when using the quail model. Funded by Brighton & Sussex Medical School.
An inverse dynamic model of the plantar aponeurosis: the influence of walking speed
Authors: caravaggi paolo , Pataky Todd , Savage Russel , Crompton Robin
Published: June 2021

An inverse dynamics approach has been used to gain an insight into the mechanical behaviour of the plantar aponeurosis (PA) at different walking speeds. To our knowledge only one study has addressed the dynamic behaviour of the PA with relation to the Achilles tendon force (Erdermir, 2004). The aims of this study were to create a multibody model to quantify PA tension and to examine how PA tension changes with walking speed.

Ultrasonography was employed to determine the origin of the PA at the inferior aspect of the calcaneus and the radii of the metatarsal heads for three subjects who volunteered in the study. Commercial software for multibody dynamics analysis (MSC.Adams) was used to build the subject‐specific model geometry and to calculate the elongation trajectories for five segments of the PA based on stance phase kinematics data.

The PA was pre‐tensioned during early stance phase in all subjects. The amount of pretensioning and the maximum elongation of the slips of the PA decreased from medial to lateral. Peak PA tension occurred in late stance phase (~1.5 BW) for all walking speeds and was similar in magnitude to cadaver simulation.

Preliminary results demonstrate model validity and reveal an interesting relation between PA tension and walking speed. The increase in PA tension with walking speed during early stance may represent a pre‐loading protective mechanism.

This study was supported by the ASGBI.
Podosome‐like tubulobulbar complexes internalize intact junctions in the seminiferous epithelium
Authors: Young J'Nelle Sarah , Asis Marc De , Vogl A. Wayne
Published: June 2021

Tubulobulbar complexes are actin‐rich double‐plasma membrane protrusions with associated vesicles that form at intercellular junctions in the seminiferous epithelium of the mammalian testis. One role that has been proposed for these 'podosome‐like' structures is that they internalize 'intact' intercellular junctions during the translocation of spermatocytes through basal junction complexes between Sertoli cells, and during sperm release from Sertoli cells at the apex of the epithelium. If tubulobulbar complexes internalize junctions, then components of tubulobulbar complexes that contain junction proteins also should be associated with endosomal markers. In this study, we probed apical tubulobular complexes in fixed epithelial fragments of rat and mouse testes for adhesion molecules (alpha‐6 integrin in rat, and nectin 2&3 in mouse) and early endosomal markers (Rab 5 in rat and EEA1 in mouse). The antibody to Rab 5 labeled the distal ends of tubulobulbar complexes that also labeled strongly with the antibody to alpha‐6 integrin. EEA1 surrounded vesicles at the ends of tubulobulbar complexes that also were positive for nectin 2 and nectin 3. These results are consistent with the proposal that tubulobulbar complexes are subcellular machines that internalize intact intercellular junctions in the testes.

Grant Funding Source

NSERC
Regulation of pericyte migration by Secreted Protein Acidic and Rich in Cysteine (SPARC)
Authors: Rivera Lee Benjamin , Brekken Rolf Andrew
Published: June 2021

Pericyte recruitment is required for stabilization of blood vessels. We observed that orthotopic tumors grown in SPARC‐null mice exhibited a reduction in pericyte‐associated blood vessels compared to tumors grown in wildtype mice. These data implicate SPARC as a regulator of pericyte recruitment. To investigate how SPARC affects pericyte behavior we examined primary pericytes isolated from wildtype and SPARC‐null animals and 10T1/2 cells in vitro. Using a transwell assay, we found that reduction of SPARC activity by the use of RNAi or a neutralizing antibody reduced the migration of 10T1/2 cells towards fibronectin. Additionally, we found that pericytes isolated from SPARC‐null mice migrated significantly less than wildtype counterparts. Treatment with a TGFβ neutralizing antibody or an ALK5 inhibitor abrogated these effects. Furthermore, we found that SPARC interacts with endoglin as demonstrated by coimmunoprecipitation and solid phase binding assays. Finally, we determined that endoglin expression is required for SPARC to effect pericyte migration. These data suggest that SPARC functions through endoglin to control TGFβ‐mediated inhibition of pericyte migration. Funding was provided by RO1CA118240.
Renshaw cell loss in a transgenic mouse model of amyotrophic lateral sclerosis
Authors: Blanchard Stephane Robert , Al‐Marsoummi Sarmad , Carr Patrick Arthur
Published: June 2021

The distribution and abundance of immunohistochemically‐identified Renshaw cells was studied in the cervical and thoracic spinal cord of an amyotrophic lateral sclerosis (ALS) mouse model. Transgenic G93A‐SOD1 mice displayed ALS‐like disease onset at 70 to 90 days of age with paresis at 100 to 130 days of age. Gephyrin immunofluorescence was used to identify putative Renshaw cells in tissue from mice at 45, 70 and 112 days of age. Preliminary results suggest that in this model of ALS, functional deficits are preceded and accompanied by a profound loss of gephyrin‐labeled putative Renshaw cells.

In sections of cervical spinal cord, means counts per ventral horn were: at 45 days ALS=0.70 and control=2.60; at 70 days ALS=0.90 and control=2.12; and at 112 days ALS=1.00 and control=2.61. In sections of thoracic spinal cord, means counts per ventral horn were: at 45 days ALS=0.73 and control=1.07; at 70 days ALS=0.73 and control=0.98; and at 112 days ALS=0.2 and control=0.96.

Differences were significant (p<0.05; Mann‐Whitney Rank Sum test) for all ages and spinal cord levels. A significant decrease in the number of Renshaw cells prior to the onset of the disease suggests that, in this model, the loss of motor‐related interneurons precedes clinical progression of the disease. Supported by NIH 5R01HL075034‐05.
New parameters for analysis of changes microglial morphology using stereology and histomorphometry
Authors: Lyck Lise , Bulavina Larissa , Finsen Bente
Published: June 2021

New parameters for analysis of changes microglial morphology using stereology and histomorphometry

In histological evaluation of experimental and pathological specimens, the degree of microglial activation is often evaluated on the basis of morphological features of the cells including the size and shape of the cells, the number and length of cell processes, as well as by increased expression of markers such as CD11b, CD45 (LCA) and Iba‐1. From stereological studies it is known that microglial activation is followed by proliferation of microglial cells and change in the morphology of the cells.

To extend the list of morphological parameters that can be measured quantitatively for comparative studies, we combined digital image analysis and stereological methods for analysis of the morphology of microglia labeled by Iba‐1. In the molecular layer of fascia dentata of the hippocampus from 6 male C57Bl6‐mice the total number of microglial cells was 16 906 cells, the mean cell size was 151.3 µm3, and the average length of cell processes was 80.0 µm/cell. We found microglial cell bodies were ovoid in shape, as judged by the ratio of the shortest and longest diameter (ratio = 0.61) and that on average 5.65 cell processes were extending at the equatorial plane of the cell.
Subregions of the inferior parietal lobule are associated with progression to Alzheimer's disease in participants of the Alzheimer's Disease Neuroimaging Initiative
Authors: Greene Sarah Judith , Killiany Ronald
Published: June 2021

Several regions within the brain have been associated with progression from normal to Alzheimer's disease (AD) based on magnetic resonance imaging (MRI) studies, including the hippocampus, entorhinal cortex, and more recently, the inferior parietal lobule (IPL). In this study, the IPL was divided into three regions: the gyrus (G‐IPL), the banks of the sulcus (B‐IPL), and the fundus (F‐IPL) to determine if these regions were associated with the progression from normal to AD. Participants of the ADNI (n=12, 50% male, average age 78) underwent a structural MRI scan and neuropsychological exam (NP), and were categorized as normal controls, mild cognitively impaired (MCI), or AD. FreeSurfer was used to measure these regions. Once scans were processed, each IPL region was manually traced based on automated curvature intensities and corrected for head size. Multivariate analysis of variance determined that the B‐IPL was different among groups (p<0.05) in the right (RH) and left (LH) hemisphere, and G‐IPL was different in the LH. Correlations between these regions and NP scores were as follows (p<0.05): LH B‐IPL with Auditory Verbal Learning immediate and delayed, and both LH and RH G‐IPL with the categories test. These findings suggest that the banks and gyral portions of the IPL are impacted by AD pathology before the fundus of the IPL in individuals with MCI and AD.

Grant Funding Source

None
Spatial map of sensory inputs in the human insular cerebral cortex.
Authors: Johnson John Irwin , Morris John Andrew , Fobbs Archibald J.
Published: June 2021

To reveal the ordering of sensory projections regions in insular cortex, we reviewed fMRI studies of human brains, supplemented by electrophysiological and neuroanatomical studies in other species. The resulting map shows a distribution of sensory inputs supporting our idea of the insula as a hub of the primary sensory regions of the cerebral cortex. The Olfactory region, in the antero‐inferior‐most insula, lies next to, and has strong interconnections with, the extra‐insular primary olfactory cortex. This insular Olfactory region has agranular cortex: the 4th (inner granular) cortical layer is absent. Posterior to, and interacting with this Olfactory region, is the primary Taste cortex, dysgranular cortex: the 4th layer is intermittent. Around the Taste region are sensory inputs from Visceral organs, such as the heart, digestive tract, and urinary bladder. The posteriormost insula has granulous cortex: a well‐developed continuous 4th layer. Superiorly within this granulous region are somatotopically organized Thermosensory and Mechanosensory projections. Behind and below these, in the posteriormost insula, are Vestibular and Visual inputs. Inferior to these are Auditory projections, adjoining the auditory regions of the temporal lobe. Within the insula, there are extensive interactions between the respective sensory regions, as well as between sensory projections and efferent neurons.
Short‐term bilateral whisker removal from birth causes long‐lasting changes in the structure and function of the somatosensory system
Authors: Lee Li‐Jen , Chen Wan‐Jung , Chuang Ya‐Wen , Wang Yu‐Chun
Published: June 2021

The sensory areas in the mammalian brains are topographically arranged. The body map in the primary somatosensory cortex is organized corresponding to the peripheral receptors that contact the external word. Notably, the cortical map can be modified by the sensory experience particularly during early postnatal period. Sensory deprivation has been used to demonstrate the structural and synaptic plasticity in the developing brain, however, little is known about the behavioral changes after the recovery of early‐life sensory deprivation. Here we cut the major whiskers of male rat pups bilaterally from the date of birth (P0) to postnatal (P) day 3 and examined the behavioral performance during P28 to P32. At this age, all cut whiskers had regrown to normal length; however, the sensory deprived rats exhibited significant shorter crossable distance in the whisker‐specific gap‐crossing test implying the tactile dysfunction. The cortical areas in the layer IV somatosensory region in relation to the trimmed whiskers were enlarged and the spiny stellate neurons within had larger dendritic span. With abnormal sensory map, the neonatal whisker‐clipped rats also showed higher explorative activity and more social behaviors. Our results demonstrated the early short‐term sensory deprivation perturbed the developmental program of the brain and affected later behaviors in perceptual, executive and social aspects.
Error‐Free Repair of CAG/CTG Hairpins via Endonucleolytic Incisions in Human Nuclear Extracts
Authors: Li Guo‐Min , Hou Caixia , Chan Nelson , Tian Lei , Gu Liya
Published: June 2021

Expansion of trinucleotide repeats (TNRs) is associated with progression of several human neurological diseases including Huntington's disease. The proposed mechanism of TNR expansion involves replication‐associated slippage, leading to the formation of TNR hairpins. Thus, factors preventing or removing TNR hairpins are predicted to inhibit disease progression and promote TNR stability. To determine how human cells process TNR hairpins, circular plasmid DNA substrates containing a (CAG)25 or (CTG)25 hairpin were constructed and analyzed for repair by human nuclear extracts. We show that human cells catalyze error‐free repair of (CAG)25 and (CTG)25 hairpins in a nick‐directed PCNA‐dependent manner. The repair is initiated by incisions specifically targeting the repeat sequences in the nicked strand, followed by repair DNA synthesis using the continuous strand as a template, thereby ensuring TNR stability. Depletion of PCNA blocks repair prior to TNR incision. However, distinct incision products were observed in reactions containing TNR hairpins with different strand locations and secondary structures, indicating utilization of distinct endonucleases in different hairpin repair. Our findings on TNR hairpin repair will provide significant insight into the mechanisms of TNR expansion and the etiology of human diseases caused by TNR expansions.
Regulation of DNA damage responses by the polymerase manager proteins UmuD and UmuD′
Authors: Beuning Penny J , Sefcikova Jana , Fang Jing Susan , Engen John R
Published: June 2021

Organisms experience DNA damage from numerous sources. When bacterial cells experience DNA damage and other stresses, the SOS response is induced, leading to upregulation of at least 50 genes in E. coli. Among the upregulated genes are specialized Y family DNA polymerases, which replicate damaged DNA and play a role in tolerance to DNA damage. The activity of these potentially mutagenic proteins is tightly regulated. The manager proteins UmuD2 and the cleaved form UmuD'2 provide one regulatory mechanism. The umuD gene products interact with both Y family polymerases, the replicative DNA polymerase, and the beta processivity clamp. The goal of this study is to determine how the conformation and dynamics of the umuD gene products regulate the cellular response to DNA damage. We are characterizing the effects of UmuD and UmuD' on DNA polymerase III activity and probing their conformations using fluorescence resonance energy transfer (FRET) and hydrogen‐deuterium exchange mass spectrometry (HXMS). In HXMS experiments, backbone amide hydrogens that are solvent‐accessible exchange with deuterium over time, whereas inaccessible hydrogens do not exchange. Our HXMS results reveal that the N‐terminal arm of UmuD, which is not present in the cleaved form UmuD′, exhibits local partial unfolding. Additionally, there are substantial regions of stable conformation in both proteins.

This work is funded by a Dreyfus Foundation New Faculty Award.
The Joint Genome Institute's Microbial Genome Annotation Program for Undergraduates
Authors: Kerfeld Cheryl A.
Published: June 2021

The JGI has developed a set of tools and faculty training modules to integrate microbial genome annotation across the undergraduate life sciences curriculum, from introductory molecular biology to capstone microbiology and biochemistry courses. The annotation platform includes IMG‐EDU, a new version of IMG with special features such as six frame translation viewer. IMG‐EDU and links to other databases are embedded in a web/wiki portal that guides students through the annotation process and records their findings. The annotation platform also provides tools for instructors to assign genes and pathways and to evaluate students' annotations. This will enable colleges and universities nationwide to "adopt a genome" for manual curation and student research. Students at 12 colleges and universities nationwide are currently part of the pilot phase of the program. Expansions of this program into metagenomics and functional genomics are currently being developed.
Structural basis for the recognition of pri‐mir‐18a by hnRNP A1
Authors: Ding Lei , Ma Jin‐Biao
Published: June 2021

MicroRNAs (miRNAs) are a highly conserved class of small (18‐24 nucleotides) non‐coding RNAs that regulate a broad spectrum of biological processes. Aberrations or corruption of miRNA functions lead to mis‐regulated cell proliferation, tumorgenesis, and ultimately, cancer. Recent studies showed that miRNAs that are potential oncogenes and tumor suppressors are specifically regulated at post‐transcriptional level by RNA‐binding proteins, such as hnRNP A1. However, post‐transcriptional regulatory mechanisms of these RNA‐binding proteins in miRNA processing still remain unsolved. It is expected that the structure of the pri‐miRNA is altered by RNA‐binding proteins, which may facilitate or block the processing of a specific pri‐miRNA by Drosha microprocessor complex. We have identified a novel fragment in the terminal loop of pri‐miR‐18a that contains conserved sequence motifs recognized by two RRM domains of hnRNP A1 (UP1). Crystals of the RNA fragment and UP1 complex that diffract X‐rays to 2.4 Angstroms resolution have been obtained and the structure was determined by molecular replacement. We have defined the speficity of the recognition using mutagenesis and in vitro binding assay based on structural study. The molecular mechanism for the facilitating of pri‐miR‐18a processing by hnRNP A1 are further investigated by mutagenesis and biochemical in vitro pri‐miRNA processing assay.
In vivo analysis of ribosome structure and dynamics
Authors: McGinnis Jennifer , Weeks Kevin M.
Published: June 2021

The ribosome catalyzes protein synthesis according to an mRNA template in a highly accurate, fast, and dynamic process called translation. Ribosomal RNA (rRNA) has been implicated in playing a crucial role in the structural, catalytic, and dynamic aspects of translation.Although rRNA appears to play a large role in ribosomal dynamics, direct in vivo measurements of RNA structure during translation have not been possible. Here, we use SHAPE (selective 2′‐hydroxylacylation analyzed by primer extension) chemistry to interrogate the local structure of 16S rRNA at single nucleotide resolution in the mid‐log phase of Escherichia coli growth. SHAPE chemistry is an effective tool for examining RNA structure in vivo and yields structural information for over 1400 16S nucleotides. In many regions, SHAPE chemistry exactly recapitulates the secondary structure derived from comparative sequence analysis. However, in several key areas, the time‐averaged rRNA structure is not consistent with the accepted static structure. Local, but highly significant, rearrangements in the RNA secondary structure thus appear tocontribute to ribosome function. A full understanding of dynamic RNA structure at nucleotide resolution will be important for complete understanding of the mechanics of translationin vivo.
Ubiquitination of HEXIM1 by HDM2
Authors: Chao Sheng‐Hao , Lau Joanne , Diribarne Gaelle , Dey Anwesha , Bensaude Olivier , Lane David P
Published: June 2021

Hexamethylene bis‐acetamide inducible protein 1 (HEXIM1) is an inhibitor of positive transcription elongation factor b (P‐TEFb), which controls RNA polymerase II transcription and human immunodeficiency virus Tat transactivation. In cells, more than half of P‐TEFb is associated with HEXIM1 resulting in the inactivation of P‐TEFb. Recently, we found that nucleophosmin (NPM), a key factor involved in p53 signaling pathway, interacts with HEXIM1 and activates P‐TEFb‐dependent transcription. Here we report that HDM2, another important regulator of p53, ubiquitylates HEXIM1 specifically. We also identify the lysine residues, which are located with the basic region of HEXIM1, required for ubiquityl modification. Interestingly, the HDM2‐induced HEXIM1 ubiquitination does not lead to proteasome‐mediated protein degradation. Our results demonstrate that HDM2 functions as a specific E3 ubiquitin ligase for HEXIM1, suggesting a possible role for HEXIM1 in the regulation of p53 signaling pathway.
Small angle neutron scattering: a powerful tool to study polyglutamine aggregate formation
Authors: Perevozchikova Tatiana , Stanley Christopher , McWilliams‐Koeppen Helen P. , Berthelier Valerie
Published: June 2021

Formation and accumulation of amyloid protein aggregates in selected neurons is a hallmark of many neurodegenerative disorders, including polyglutamine (polyGln) disorders like Huntington's disease (HD). PolyGln disease proteins contain an abnormally long sequence of glutamine repeats leading to the formation of fibrillar aggregates with highly stable and organized β‐sheet structures. However, there is still an open debate as to how aggregation correlates with pathogenesis and if neuronal toxicity can be associated with early stages of protein aggregation. To investigate early aggregates and the aggregation pathway we performed small‐angle neutron scattering (SANS) on synthetic polyGln peptides. SANS is a useful technique for probing structural changes on the nanometer length‐scale in solution. Measured changes in the radius of gyration and mass per length demonstrated large‐scale structural changes occurring during aggregate growth kinetics. We also investigated the effects of protein context, glutamine length, and osmolytes on aggregation behavior. The influence of osmolytes is of particular interest since the osmotic stress created by osmolyte addition allows probing hydration and thermodynamics accompanying structural changes.
Recombinant expression and characterization of Epulopiscium sp. Type B glyceraldehyde‐3‐phosphate dehydrogenase
Authors: Troy Gina Marie , Piefer Andrew John
Published: June 2021

The objective of this project is to clone, express and kinetically characterize glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) from the bacterium, Epulopiscium sp. Type B. This huge, uncultured bacterium (~250 µm) is visible to the naked eye and grows in the gut of the herbivorous surgeonfish Naso tonganus, where it may assist in the fishes' algae digestion. Epulopiscium is significant because it is larger than most animal cells yet it does not have the cellular complexity of eukaryotes. To study this protein, and gain further insight into the physiology of the bacterium, the gene encoding GAPDH was amplified using PCR and cloned into Escherichia coli for subsequent protein production. GAPDH plays an essential role in glycolysis where it converts glyceraldehyde 3‐phosphate and NAD to 1,3‐bisphosphoglycerate and NADH. This represents the first energy producing step of glycolysis. Most characterized GAPDHs are homotetramers and contain conserved cysteine and histidine residues in the active site. GAPDH from Epulopiscium was compared to the GAPDH of Clostridium botulinum, E. coli, and Homo sapiens. The 27 amino acid spacing between the active site cysteine and histidine residues was conserved in Epulopiscium GAPDH. Furthermore, it was found that there was 92% amino acid consensus among all four GAPDHs examined. This project was funded by the Hartwick College Department of Chemistry.
The spatiotemporal dynamics of a modular metabolic network that regulates longevity in yeast
Authors: Titorenko Vladimir I , Goldberg Alexander , Gregg Christopher , Boukh‐Viner Tatiana , Kyryakov Pavlo , Bourque Simon , Beach Adam , Burstein Michelle , Richard Vincent , Rampersad Sonia , Milijevic Svetlana
Published: June 2021

The yeast Saccharomyces cerevisiae is a valuable model for unveiling the mechanisms of cellular aging in multicellular eukaryotes. Yeast aging can be slowed down by calorie restriction (CR), a low‐calorie dietary regimen that extends life span and delays age‐related disorders in a wide spectrum of organisms. To establish the mechanisms underlying the anti‐aging effect of CR, we assessed the effect of a CR diet and numerous mutations extending life span on the metabolic history and age‐dependent organelle dynamics of chronologically aging yeast. We found that yeast merge a number of cellular processes, which we call modules, into a metabolic longevity network. Our findings imply that 1) yeast establish a diet‐ and genotype‐specific configuration of the network by setting up the rates of the processes taking place within each of its modules; 2) the establishment of a network's configuration occurs before yeast enter a non‐proliferative state; and 3) different network's configurations established prior to entry into a non‐proliferative state define different rates of survival following such entry. Thus, by designing a specific configuration of the modular longevity network prior to reproductive maturation, yeast define their life span. Implementing our knowledge, we identified five groups of novel anti‐aging small molecules that greatly extend yeast longevity by remodelling two key modules of the network.
Tetraploidy impairs human aortic endothelial cell (HAEC) function and is prevented by nicotinamide phosphoribosyltransferase (Nampt)
Authors: Borradaile Nica M , Pickering J Geoffrey
Published: June 2021

Senescent EC are found in atherosclerotic arteries and contribute to impaired vasodilation and inflammation. Polyploid EC are also found in atherosclerosis, but the consequences of this change are unclear. In HAEC, we found that tetraploidy precedes replicative senescence, is worsened during growth in high glucose, and is delayed by the NAD+ regenerating enzyme, Nampt. However, the impact of tetraploidy on EC function, independent of senescence, is unknown. Likewise, the effect of Nampt on EC genomic stability during replicative aging and glucose overload is unknown. We induced senescence‐independent tetraploidy by incubating HAEC with nocodazole for 48 h. After a 24 h recovery, these cells were 70% tetraploid, with no change in senescence‐associated β‐galactosidase activity. Growth rate and eNOS protein were decreased (‐38%, ‐23%) and ICAM‐1 protein was increased (19%). The effect of Nampt on tetraploidy associated with replicative senescence and glucose overload was assessed in HAEC expressing eGFP or eGFP‐Nampt and grown in basal (5 mM) or high (30 mM) glucose to the end of their lifespan. Nampt completely prevented aging‐ and glucose overload‐induced tetraploidy. We conclude that tetraploidy can independently impair HAEC function and that the emergence of tetraploidy during replicative aging and glucose overload can be prevented by enhancing NAD+ salvage. Research support: HSFO T5675, CIHR FRN11715.
Association of Sets of Alleles of Gene Encoding Lipoprotein Lipase Enzyme With Type 2 DM and Its Macrovascular Complications
Authors: Hemimi Neveen Salah El Din , Abd El Salam Mona Mohamed , Abd Elwahab Mahmoud A
Published: June 2021

Background

Lipoprotein Lipase (LPL) increases the uptake and catabolism of triglycerides rich lipoproteins (TRLs) by hydrolyzing triglycerides in chylomicrons and VLDLs and also by serving as a bridge mediating the binding of TRLs to cell surface via heparan sulphate proteoglycans leading to their uptake by a variety of receptors.

Aim

To detertmine whether two LPL polymorphism (LPL Hind III and LPL S447X) can be considered as predictors for DM and its macrovascular complication.

Methods

176 normal individual, 42 non‐complicated type 2 diabetic patients and 78 macrovascular complicated type 2 diabetic patients were enrolled in the study. The LPL Hind III and LPL S447X genotypes were identified by RFLP‐PCR of peripheral blood DNA samples. Analysis of data was done by using SPSS program.

Results

Allele frequencies for H+ and S were 80.8% and 93.3% respectively in the diabetic group compared to 64.2% and 90.9% in the control group. The carriers of either (H+/H+) or (SS) variant of LPL were at high risk of developing type 2 DM (OR= 2.6; 95% CI= 1.6‐4.3 and OR= 1.4; 95% CI= 0.7‐2.7) respectively. Furthermore they were at much higher risk of developing macrovascular complications (OR= 3.7; 95% CI= 1.6‐8.2 and OR= 3.3; 95% CI =1.1‐10.0) for carrier of (H+/H+) and (SS) genotype respectively.

Conclusion

(H+/H+) and /or (SS) genotype were associated with Type 2 DM and its macrovasular complications.

Source of research support

Dubai Medical College for Girls
Genetic Polymorphism of Ectoenzyme Nucleotide Pyrophosphate Phosphodiesterase (ENPP1) and Susceptibility to Type 2 DM and Its Complications
Authors: Hemimi Neveen Salah El Din , Abd El Salam Mona Mahmoud , Abd Elwahab Mahmoud A
Published: June 2021

Background

A functional missense DNA polymorphism in exon 4 of ENPP1 gene causes amino acid change from lysine to glutamine at codon 121 (K121Q). The Q variant of PC1 is associated with reduction of insulin receptor autophosphorylation and therefore a stronger inhibitor of insulin signaling.

Aim

To test the hypothesis that ENPP1 K121Q polymorphism is associated with type 2 DM and its complication.

Methods

167 normal individual, 30 non‐complicated type 2 diabetic patients and 76 complicated type 2 diabetic patients were enrolled in the study. The ENPP1 K121Q genotypes were identified by RFLP‐PCR of peripheral blood DNA samples. Analysis of data was done by using SPSS program.

Results

Allele frequency for Q was 36.8% in the diabetic group compared to 26% in the control group. The carriers of XQ genotype (KQ and QQ) were at high risk of developing type 2 DM (OR= 1.8; 95% CI = 1.1‐2.9) when compared with the carrier of KK genotype. Furthermore they were at much higher risk of developing its complications (OR= 2.1; 95% CI=0.9‐4.9).

Conclusion

Q 121 allele is a genetic determinant of type 2 DM and related atherogenic phenotypes.

Source of research support

Dubai Medical College for Girls
Alteration of PEPCK‐C gene expression in liver and white adipose tissue results in insulin resistance
Authors: Croniger Colleen Marie , DeSantis David , Hsieh Chang‐Wen , Millward Carrie A
Published: June 2021

PEPCK‐C gene is expressed primarily in the liver, kidney, and adipose tissue. Using a mouse model with a deletion of the PPAR binding site in the endogenous PEPCK‐C gene promoter (PPARE‐/‐ mice), we have altered the regulation of the PEPCK‐C gene expression in the liver and ablated gene expression in the white adipose tissue (WAT). The hepatic expression of PEPCK‐C is reduced during fasting which protects the mice from developing diabetes. The mice, however , develop moderate insulin resistance as measured by hyperinsulinemic‐euglycemic glucose clamps and 2‐deoxyglucose uptake. Treatment of the mice with rosiglitazone either with normal chow or high fat feeding worsened the insulin resistance in the PPARE‐/‐ mice, however treatment with pioglitazone improved insulin resistance in the PPARE‐/‐ mice. These data suggest that the PPAR binding site in the PEPCK‐C promoter is an important regulator of the glyceroneogenesis pathway and dysregulation of PEPCK‐C contributes to development of insulin resistance.
Blunted Hepatic Glucose Production from Lactate in Leptin‐Treated Streptozotocin‐Induced Diabetic Rats
Authors: Kang Yuan , Wernette Catherine M. , Judd Robert L. , White B. Douglas
Published: June 2021

The aim of this study was to compare the hepatic glucose output of leptin‐ and vehicle‐treated diabetic rats in response to lactate via an isolated perfused liver system. Wistar rats were made diabetic with an IV injection of STZ (50 mg/kg). After confirmation of hyperglycemia, rats were administrated a daily ICV injection of either leptin (5 microgram/day) or vehicle. Once blood glucose concentrations of the leptin‐treated diabetic rats returned to normal, each rat was fasted and administered lactate (16.6 mmol/kg) via gastric gavage. Blood glucose concentrations were determined every 15 min for 2 hours. Rats were fasted as before and the livers perfused at a rate of 35 ml/min with Krebs‐Henseleit‐Bicarbonate (KHB) buffer for 30 min, followed by KHB buffer supplemented with lactate (2 mM) for one hour. Perfusates were collected in 30‐second fractions and glucose concentrations in selected fractions determined. Leptin‐treated rats had nearly a 5‐fold lower glucose response to lactate gavage than vehicle‐treated rats. Hepatic glucose production from the fasted leptin‐treated rats was also severely blunted as compared to vehicle‐treated rats. These data suggest that chronic leptin administration leads to an inhibition of gluconeogenesis, which may result in normalization of blood glucose concentration in STZ‐induced diabetic rats. (Supported by the Alabama Agricultural Experiment Station)
Myo‐inositol catabolism in Drosophila melanogaster: implications for diabetes and development
Authors: Nguyen Tien Duc , Cabrera Ilva E. , Eldon Elizabeth D. , Klig Lisa S.
Published: June 2021

Myo‐inositol is a six‐carbon sugar that can serve as a carbon/energy source, an osmolyte, and a precursor of phospholipids. It has been suggested to play a pivotal role in diabetes mellitus. High levels of the myo‐inositol catabolic gene product, myo‐inositol oxygenase (MIOX), has been seen in diabetic tissues. More than 75% of genes involved in human diseases have orthologs in the model organism Drosophila melanogaster. This study identified the D. melanogaster MIOX gene using bioinformatic and molecular genetic tools. This gene is located on chromosome 3L, and has 4 exons and 3 introns. The predicted protein is 33 kDa and is 52.2% identical (67.7% similar) to human MIOX. In silico experiments have also revealed many upstream regulatory sequences responsive to inositol, osmotic and/or oxidative stress. To determine if perturbation of this gene results in a developmental phenotype, D. melanogaster mutant strains heterozygous for a P‐element insertion into MIOX gene were crossed. Preliminary results suggest that homozygotes with disrupted MIOX genes are viable on rich media. Another component of this study was to determine where the MIOX gene product is localized throughout development. Preliminary data from western blots, confocal microscopy, and immunohistochemistry, indicates that MIOX may be located in the gut/malpighian tubule region.
Targets for covalent protein modification by 4‐hydroxynonenal/4‐hydroxyhexenal‐mediated carbonyl stress in the mitochondria
Authors: Rauniyar Navin , Prokai‐Tatrai Katalin , Prokai Laszlo
Published: June 2021

Protein carbonylation is associated with various neurodegenerative diseases. Characterization of selectivity of 4‐hydroxy‐2‐nonenal (HNE) and 4‐hydroxy‐2‐hexenal (HHE) modifications, major components of carbonyl stress, helps to understand its impact on protein function and/or activity, as well as its effects on downstream targets or other interacting proteins. Rat brain mitochondrial proteins modified by reactive carbonyl species representing end‐products of lipid peroxidation such as HNE and HHE were investigated using high performance liquid chromatography (LC) and tandem mass spectrometry (MS/MS). Shotgun‐based approach involving conventional data‐dependent acquisition on a hybrid linear ion trap‐Fourier transform ion cyclotron resonance mass spectrometer was employed after prior enrichment of HNE‐/HHE‐modified peptides by solid‐phase hydrazide strategy. Selective enrichment of substoichiometric levels of modified peptides from complex mixtures followed by LC‐MS/MS identified several peptides that were modified with HNE or HHE with amino acid resolution. Additionally, bioinformatics (Ingenuity Pathway Analysis) was used to identify biological pathways, networks, and functions significantly altered by such modifications. This research was supported by the grant AG025384 from the National Institutes of Health.