bluuebunny/crossref_metadata_2025 huggingface dataset:
Intra‐element variation in osteocyte lacunar density and morphology in healthy young male cortical bone
Authors: Carter Yasmin , Suchorab Jessica L. , Thomas C. David , Clement John G. , Cooper David M.L.
Published: June 2021

We previously reported considerable intra‐element variation in osteocyte lacunar properties, with density differences up to 30% across a cross‐section of the male (20 yrs) femoral cortex related to the biomechanical axes. The purpose of this research was to determine whether this pattern of variation occurs in a larger sample. Anterior (A), posterior (P), medial (M) and lateral (L) blocks (2×2 mm) were prepared from the proximal femoral shaft of 7 healthy males aged 20–34 and synchrotron micro‐CT imaged. Average lacunar density (± sd) from the APML regions were 22,961 ± 1991, 28,264 ± 4186, 32,082 ± 7731 and 30,525 ± 1977 per mm3 of bone, respectively. Consistent with the pattern observed in the previous study, density was significantly different between the A and the L region (p<0.05). The density of the combined A and P regions was also lower (p=0.006) than that of the combined M and L regions. The differences in density were mirrored by shape differences; with the combined AP region having more elongate (p=0.004) and flattened (p=0.001) lacunae, than those of the ML region. The consistent appearance of this pattern of variation in a larger sample suggests considerable normal variation occurs in the lacunar properties of healthy young males. The underlying functional significance of the observed differences likely relates to localized variations in loading conditions as the pattern corresponds well with mechanical axes.

Grant Funding Source: Saskatchewan Health Research Fund
Comparative tissue stainability and localization of active staining principle in fractions of Lawsonia inermis (Henna) leaves
Authors: Alawa Judith Nkiruka , Kauche Eunice , Ahmed Bilkisu , Adetiba Bamidele
Published: June 2021

The crude aqueous and ethanolic extracts of the leaves of Lawsonia inermis (Henna) have demonstrated cytoplasmic staining properties in animal tissues. However, to improve the staining efficacy on tissues and to localize the active dyestuff, the extracts were fractionated sequentially in n‐hexane, ethyl acetate, n‐butanol and distilled water using column chromatography. The soluble fractions were subjected to thin layer chromatography to identify the active compounds. Tissue stainability of the fractions was evaluated in formalin‐fixed and paraffin embedded tissues (liver, kidney, cardiac muscle, testis, lungs, brain and intestine) obtained from Wistar rats. Ethyl acetate was the most effective and showed better tissue stainability compared to n‐Butanol, characterized by the deep brown colour intensity on the tissues and remarkable contrast with haematoxylin comparable to haematoxylin and eosin stain. This corroborated the TLC analysis which demonstrated that the ethyl acetate and n‐butanol fractions showed brownish spots which were absent in the hexane and distilled water fractions. Ethyl acetate spot showed deeper colouration than the n‐Butanol spot suggesting that the active staining principle known as lawsone is localized in these fractions. The n‐hexane and distilled water fractions showed weak to no staining activity on tissues. This indicates that the active staining principle of Henna is localized in the ethyl acetate and n‐butanol fractions and these will serve as good solvents for the further purification of the active staining compound.

Grant Funding Source: A.B.U Zaria
Quantitative Immunostaining: 3D X‐ray Microscopy for Visualizing and Measuring Protein Distribution in Three Dimensions
Authors: Merkle Arno , Metscher Brian , Yun Sylvia , Lau SH
Published: June 2021

Immunostaining has become an important tool for biological and biomedical research, providing information on the distribution of gene products such as proteins and ribonucleic acids, and has been primarily developed through optical microscopy‐based techniques, including serial sectioning histology and optical projection tomography (OPT). However, due to the limited penetration depth of visible light, these techniques are often limited to either small samples ranging from tens to hundreds of microns or to lower resolutions (10 μm).

We describe a three‐dimensional (3D) imaging technique using a laboratory based 3D X‐ray microscope (XRM) for quantitative characterization of proteins in small animal embryos at high resolutions of <1 μm spatial resolution (<200 nm pixel). By using non‐destructive X‐rays that achieve large sample penetration, we demonstrate whole mount imaging capabilities of antibody probes in developing embryos. We will additionally discuss software techniques to measure X‐ray attenuation within the sample to provide quantitative mapping of protein densities. Future directions include correlative studies using compatible molecular probes for light and electron microscopy and counterstaining organs and cells with additional heavy metal stains.
Time‐dependent Development of Social Stress Caused by Repeated Exposures to Aggressors Simulating Features of Post‐traumatic Stress Disorder (PTSD)
Authors: Chakraborty Nabarun , Meyerhoff James , Gautam Aarti , Muhie Seid , Hammamieh Rasha , Jett Marti
Published: June 2021

A utility criterion of a PTSD mouse model is recruiting a stressor with systematically variable intensity. An ideal stressor should maintain a ‘dose‐response relationship’ with the subjects’ behavioral shift. A typical PTSD model includes a brief exposure to foot or tail shock.

We developed a model that involves repeated exposures (either 5‐day or 10‐day) to trained conspecific aggressors (Agg). A contextual reminder study evaluating a cluster of ethogram identified PTSD‐like (acute‐ and persistent‐) syndromes such as incubation, extinction and desensitization of fear responses. Pathophysiological consequences supported the model.

This model reflects the combat‐like situation where life‐threatening events occur repeatedly and randomly. The direct relationship of PTSD‐risk with deployment frequency further justifies the model.

Agg induced stress intensity can only be elevated by prolonging the exposure. The risk of habituation can thereby defeat the ‘dose‐response’ relationship. We addressed this concern by evaluating the time‐dependent behavioral shift of the subject mice (C57BL/6j). Subsets of subject mice were withdrawn from the Agg‐exposure (Agg‐E) schedule at regular intervals and their psycho‐patho‐physiological characters were evaluated. A regression model elucidated the temporal relationship of Agg‐E stress with the psychological alteration.
BNIP3 regulates mitophagy and apoptosis in delayed neuronal death in stroke
Authors: Shi Ruoyang , Kong Jiming
Published: June 2021

Physiological level of autophagy promotes neuronal survival, while apoptosis contributes to delayed neuronal death in ischemic stroke. In this study, we show that the proapoptotic BCL‐2 family member BNIP3 is an upstream regulator for both processes. Specifically, deletion of BNIP3 gene is neuroprotective by affecting mitophagy and apoptosis pathways. We performed IHC, western blot, Co‐ip, ELISA, and cell transfection to analyze the BNIP3's regulation on mitophagy and apoptosis in cortical neurons and ischemic brains. Both BNIP3 wild‐type and knock‐out mice were used. In primary neurons exposed to OGD/reperfusion, BNIP3 was highly expressed, with the time course and expression levels of apoptosis‐related proteins (i.e. active caspase‐3, cytochrome C, and BAX) and autophagy‐related proteins (i.e. LC3, Beclin‐1, and LAMP‐2) positively regulated. Promoting or inhibiting autophagy pharmacologically didn't affect the expression patterns of BNIP3, indicating it is an upstream regulator. We also measured the brain damage of neonatal stroke in transgenic mice. TTC staining showed the infarct volume of ischemic brains was significantly reduced in BNIP3 KO mice compared to WT mice upon 3–7 days recovery. Silence of BNIP3 gene in cortical neurons of KO mice activated a robust autophagic response and decreased apoptosis, coordinately contributing to the neuroprotection in the KO animals after stroke.

Grant Funding Source: Canadian Institute of Health Research, Canadian Stroke Network, Manitoba Health Research Council, Manitoba Institute of Child Health
Myenteric Dogiel II Neurons Express Nerve Growth Factor
Authors: Kawaja Michael D , Petrie Casey N
Published: June 2021

We have generated lines of transgenic mice that express enhanced green fluorescent protein (EGFP) under the control of the murine nerve growth factor (NGF) promoter. These mice afford us unique temporal and spatial insight into those cellular populations that display NGF promoter activity from early mammalian embryogenesis to adulthood. These mice also offer us the opportunity to assess those cells that express NGF promoter activity in response to injury and/or disease. We have made particular note of a population of EGFP‐positive cells in the colon of these transgenic mice. In adult animals, these EGFP‐positive neurons can be unequivocally identified as a population residing within the myenteric plexus. These neurons have large smooth cell bodies, with two or three axonal processes that give rise to a dense plexus of fibers within the enteric ganglia (these axonal plexuses are also visualized by EGFP). Based on these anatomical features, we have speculated that these EGFP‐positive neurons are primary afferent (or Dogiel type II) neurons. Preliminary assessments of the neurochemical features of these EGFP‐positive neurons reveal that most express the calcium‐binding protein calretinin. We will present additional data regarding the localization of other biomarkers for Dogiel type II neurons, including calcitonin gene‐related peptide. We will also show how these EGFP‐positive cells react to chemically‐induced colitis in adult mice. These data provide strong evidence that primary afferent neurons of the murine colon are a hitherto unexpected source of NGF in the normal and inflamed bowel. This work was supported by the Canadian Institutes of Health Research.
Levels of nitric oxide synthase and cholecystokinin mRNA in the upper gastrointestinal tract of rats following experimental spinal cord injury
Authors: Swartz Emily , Holmes Gregory M.
Published: June 2021

In addition to the loss of motor and sensory function, SCI causes immediate changes to gastrointestinal (GI) tract physiology and reflex function. Reflex control of the stomach is dominated by vago‐vagal circuits which remain anatomically intact following SCI. Our previous reports indicate that SCI a) reduces gastric motility, tone, and emptying; b) diminishes vagal afferent sensitivity to GI peptides such as cholecystokinin (CCK); and c) diminishes mesenteric blood flow.

The mechanisms, interrelationships, and temporal patterning of post‐SCI gastroparesis remain poorly defined. We tested the hypothesis that acute SCI induces GI inflammation and a resulting reduction of CCK mRNA expression.

We used qRT‐PCR to quantify the levels of CCK, iNOS, and nNOS in the GI tract, 1 week post‐SCI. Our data show a significant increase in CCK and iNOS in the duodenum; nNOS shows an upward non‐significant trend. Jejunal expression of CCK is reduced while both iNOS and nNOS levels show a non‐significant upregulation. Our data are similar to experimental ischemia‐reperfusion of the GI tract, in which the superior mesenteric artery (SMA) occlusion causes a dramatic inflammatory response within the GI tract. We conclude that our model of SCI represents a continuum of GI ischemia and that the increase in inflammatory mediators may play a role in decreasing the sensitivity of the gastric vagal afferents.

Grant Funding Source: NINDS 49177
Ethanol down‐regulates microglia inflammatory responses augmented by endoplasmic reticulum stress
Authors: Goral Joanna , Meyer Alice
Published: June 2021

Rationale

Endoplasmic reticulum (ER) stress is involved in pathology of neuroinflammatory disorders, multiple sclerosis among them. Ethanol is known to affect immune function, including inflammation. In this study we examined the effect of ER‐stress inducer thapsigargin on inflammatory responses of BV2 mouse microglia. We also examined whether ethanol and inflammatory agents could induce ER stress in BV2 cells.

Methods

BV2 microglia were stimulated with LPS (100 ng/ml) with or without ethanol (100 mM) for 24 h. The cells were treated with thapsigargin for 2–18 h. The inflammatory response was evaluated by measurement of nitric oxide (NO) production. XBP1 and CHOP, proteins induced by ER stress, were detected by western blot.

Results

Thapsigargin did not induce, but significantly increased (x 2.5), LPS‐induced NO production by BV2 microglia. This effect was attenuated by ethanol (by 38%). XBP1 and CHOP were detected in BV2 cells exposed to thapsigargin, LPS, and ethanol.

Conclusions

The presence of XBP1 and CHOP shows that ER stress can be induced in BV2 cells by thapsigargin, LPS and ethanol. LPS‐induced inflammatory responses can be augmented by additional ER stress. However, ethanol – ER stress‐inducing agent itself – can reduce thapsigargin‐augmented inflammatory responses.
Inhibition of Pokeweed Antiviral Protein (PAP) by Turnip Mosaic Virus Genome‐Linked Protein (VPg).
Authors: Domashevskiy Artem , Goss Dixie J
Published: June 2021

Pokeweed antiviral protein (PAP) from Phytolacca americana is a ribosome inactivating protein (RIP) and an RNA N‐glycosidase that removes specific purine residues from the sarcin/ricin (S/R) loop of large rRNA, arresting protein synthesis at the translocation step. PAP is also a cap‐binding protein, and is a potent antiviral agent against many plant, animal, and human viruses. To elucidate the mechanism of RNA depurination, and to understand how PAP recognizes and targets various RNAs, the interactions between PAP and Turnip mosaic virus genome linked protein (VPg) were investigated. VPg can function as a cap analog in cap‐independent translation, and potentially target PAP to uncapped IRES‐containing RNA. In this work, fluorescence spectroscopy and HPLC techniques were used to quantitatively describe PAP depurination activity and PAP‐VPg interactions. PAP binds to VPg with high affinity (29.5 nM); the reaction is enthalpically driven and entropically favored. Further, VPg is a potent inhibitor of PAP depurination of RNA in wheat germ lysate, and competes with structured RNA derived from tobacco etch virus (TEV) for PAP binding. VPg may confer an evolutionary advantage by suppressing one of the plant defense mechanisms, and also suggests the possible use of this protein against the cytotoxic activity of RIPs.
Modulation of Mcm2–7 activity by Cdt1
Authors: DaSilva Lance F , Kolaczyk Tomasz , Ma Xiaoli , Davey Megan J
Published: June 2021

Genome duplication occurs once and only once in each cell cycle. During G1 phase, pre‐replicative complexes (pre‐RC), composed of ORC, Cdc6, Cdt1 and Mcm2–7, are assembled at replication origins. Mcm2–7 is the replicative helicase in eukaryotic cells; however, it does not unwind DNA in the pre‐RC. It is only in S‐phase, after activation of Mcm2–7, that DNA unwinding is detected. Cdt1 co‐purifies from yeast cells in a complex with Mcm2–7. Beyond being an escort for entry of Mcm2–7 into the nucleus and into the pre‐RC, the role of Cdt1 in DNA replication is not known. Using purified components from bacterial expression systems, we assembled Mcm2–7•Cdt1 complexes and compared its activity to that of Mcm2–7 alone. We show that Mcm2–7•Cdt1 has lower ATPase activity than Mcm2–7. In addition, DNA unwinding by Mcm2–7•Cdt1 is significantly lower than by Mcm2–7 alone. Furthermore and consistent with published reports, in vitro pre‐RC reconstitution experiments showed loading of Mcm2–7 to origins was dependent on ORC, Cdc6 and Cdt1. Together, the effects of Cdt1 on Mcm2–7 suggest mechanisms by which Cdt1 may carry out its essential role in the initiation of DNA replication.

Research funded by CIHR
RNA Polymerase primed DNA synthesis in T7 Bacteriophage system
Authors: Nandakumar Divya , Patel Smita S
Published: June 2021

DNA replication initiation is divided into three steps: Recognition of the replication origin by the initiator protein, melting of the duplex DNA, and synthesis of RNA primer. Primer synthesis is required as there are no known prokaryotic DNA polymerases that can carry out DNA synthesis de novo. In the T7 bacteriophage system and in mitochondrial DNA, the RNA polymerase catalyzes all three steps to initiate DNA replication. The primary replication origin in the T7 genome has two T7 RNA polymerase promoters separated by 52 bp sequence and followed by an AT rich region. There is almost nothing known about the mechanism of RNA‐primed DNA synthesis, including the length of the RNA primer synthesized by T7 RNA polymerase, the mechanism of switching from transcription to DNA synthesis, and why these events occur only at specific promoters in the T7 genome. Whether specific interactions exist between the two polymerases is also not known. Our results suggest that on short DNA substrates, one of the two promoters at the replication origin is sufficient for initiation of DNA synthesis. The RNA polymerase synthesizes primers of 8–12 nucleotide length that are utilized by the DNA polymerase. In the T7 bacteriophage system, this is the critical length at which the transition from transcription initiation to elongation occurs. Our results also show that an actively transcribing RNA polymerase is necessary for the primers to be utilized by the DNA polymerase.

Source of research support: NIH
DNA mismatch synthesis complexes provide insights into base selectivity
Authors: Xia Shuangluo , Wang Jimin , Konigsberg William
Published: June 2021

Current hypotheses that attempt to account for the high degree of base selectivity exhibited by replicative DNA polymerases (pols) concur that ternary complexes formed with incorrect dNTPs are destabilized. Knowing what accounts for this destabilization is likely to be the key to understanding base discrimination. To address this issue, we have determined crystal structures of ternary complexes with all twelve mismatches using an engineered RB69 pol variant that enabled us to capture nascent mispaird dNTPs. These structures show that mismatches in the Nascent base‐pair Binding Pocket (NBP) of the pol differ markedly from mismatches embedded in either isolated DNA duplexes or in primer‐templates complexed with the pol. Repositioning of the templating nucleotide residue and the enlarged NBP in qm ternary complex play important roles in accommodating incorrect incoming dNTPs. From these structures, we propose additional reasons as to why incorrect dNTP are incorporated so inefficiently by wild type RB69 pol: (i) steric clashes with side chains in the NBP after Fingers closing; (ii) weak interactions or large gaps between the incoming dNTP and the templating base; (iii) burying a protonated base in the hydrophobic environment of the NBP. All of these possibilities would be expected to destabilize the closed ternary complex so that incorporation of incorrect dNTP would be a rare event.(NIH RO1‐GM063276‐09)
SAMHD1, a new actor in the regulation of DNA precursors in mammalian cells
Authors: Bianchi Vera , Franzolin Elisa , Rampazzo Chiara , Ferraro Paola , Miazzi Cristina , Pontarin Giovanna , Reichard Peter
Published: June 2021

SAMHD1 was discovered in the immune system where it restricts HIV‐1 infection. Mutations lead to mitochondrial DNA deletions in Aicardi‐Goutièrez syndrome. The protein is a deoxynucleoside triphosphate triphosphohydrolase. We study the participation of catabolic enzymes in the regulation of DNA precursors (dNTPs) in cultured mammalian cells. We found that SAMHD1 and its mRNA are present in various cell types, including skin fibroblasts. Downregulation of SAMHD1 by siRNA transfection increases all 4 dNTP pools and changes their relative proportions, leading to a block of cell growth with G1 arrest. We suggest that SAMHD1 fulfills a general regulatory function beyond restriction of lentiviral growth. Supported by Italian Telethon (GGP09019), Italian Association for Cancer Research (grant 1091) and the University of Padova (Startegic Projects 2008).
Characterization of the role of Srs2 human orthologs in triplet repeat maintenance
Authors: Nguyen Jennifer , Freudenreich Catherine
Published: June 2021

Triplet repeats can form hairpin structures that can lead to chromosomal instability, breakage, and fork stalling during replication. Helicases unwind these structures to maintain genome integrity. Srs2 is a PCNA interacting non‐replicative helicase and anti‐recombinase that can displace Rad51. We have previously shown that Srs2 is important in maintaining CAG repeat stability, preventing chromosome fragility at expanded repeats, and facilitating replication through repeats in yeast. Since Srs2 plays such an important role in repeat maintenance, we want to identify and characterize potential human orthologs. Two candidates are Fbh1 and Rtel. Both of these helicases have little sequence homology to Srs2 but are functional similar in that they both antagonize homologous recombination. We introduced the cDNA sequences of each of these potential orthologs into the endogenous Srs2 locus for complementation studies. We found Fbh1 and Rtel can rescue srs2Δ sensitivity to MMS and CPT, respectively. We found that Fbh1 cannot complement repeat fragility but can rescue repeat expansions that occur in srs2Δ. On the other hand Rtel partially complements both repeat fragility and instability. These results suggest that Rtel may be the closer human ortholog but in higher eukaryotes the different Srs2 functions may be divided among a number of different helicases. This research is funded by the Tufts Biology department
Histone Deacetylase Inhibitors Upregulate Natriuretic Peptide Receptor‐A Gene Transcription via Histone Modifications and Sp1 Acetylation
Authors: Kumar Prerna , Neerukonda Smitha , Nguyen Vickie , Pandey Kailash N
Published: June 2021

Atrial natriuretic peptide (ANP) exerts its antihypertensive effects by binding to guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA), which generates the second messenger cGMP. The present study was aimed at understanding the epigenetic signaling governing Npr1 (coding for GC‐A/NPRA) gene transcription. The mouse mesangial cells were cultured in DMEM containing 10% FBS and ITS (insulin, transferrin, and sodium selenite), transfected using Lipofectamine‐2000, and treated with histone deacetylase (HDAC) inhibitors. Luciferase assay showed that trichostatin A (TSA, pan‐inhibitor) and mocetinostat (MGCD0103, class I inhibitor) induced Npr1 promoter activity by 8‐fold and 10‐fold, Npr1 mRNA levels by 4‐ and 5.3‐fold, and protein expression by 2.5‐ and 3‐fold, respectively. TSA and MGCD0103 inhibited HDAC activity by 25% and 50%, respectively. Furthermore, TSA and MGCD0103 enhanced acetylation and binding of H3‐K9/14, H4‐K12, and Sp1 to Npr1 promoter. Taken together, the present results demonstrate that TSA and MGCD0103 enhanced Npr1 gene expression via inhibition of HDAC1 and HDAC2 and increased histone H3, H4, and Sp1 acetylation. The present findings provide a novel regulatory mechanism for Npr1 gene transcription, an important player in the control of hypertension and cardiovascular homeostasis. This work was supported by the NIH grant (HL57531).
gln3 Mutations Dissociate Responses to Nitrogen Catabolite Repression and Rapamycin Inhibition of TorC1
Authors: Rai Rajendra , Tate Jennifer J. , Cooper Terrance G.
Published: June 2021

The GATA‐family transcription activator, Gln3 responds to the cell's nitrogen requirements and environmental resources. In nitrogen excess (YNB‐Gln medium), Gln3 is sequestered in the cytoplasm and the transcription it mediates is minimal. In contrast, during nitrogen‐limitation (YNB‐Pro), Gln3 relocates to the nucleus and activates transcription of genes required to scavenge poor nitrogen sources; these responses are designated nitrogen catabolite repression (NCR). Since rapamycin (Rap) addition also elicits nuclear Gln3 localization [Rap is an inhibitor of the global regulator TorC1], TorC1 has been thought to be responsible for NCR‐sensitive Gln3 regulation. Accumulating evidence, however, suggests that GATA factor regulation occurs by two separate pathways, one TorC1‐dependent and the other NCR‐sensitive. Here, we identify Gln3 alterations that dissect the individual contributions of these two pathways to overall Gln3 regulation. Gln3 C‐terminal truncations or three Gln3 amino acid substitutions on one face of a putative 11 residue α‐helix completely abolish the response of Gln3 localization to Rap addition, but have no affect on its response to nitrogen limitation. This suggests that the two regulatory systems can be effectively separated. However, overall wild type control of intracellular Gln3 localization requires the contributions of both individual regulatory systems. NIH GM‐35642.
Dimer interface of Deinococcus radiodurans HucR communicates DNA and ligand binding
Authors: DEOCHAND DINESH KUMAR , Perera Inoka Chinthana , Grove Anne
Published: June 2021

Deinococcus radiodurans HucR (Hypothetical Uricase Regulator) is a transcriptional regulator belonging to the MarR family of proteins. HucR binds to promoters of hucr and uricase genes leading to repression of both. In presence of the ligand urate, the repression of both genes is relieved. The crystal structure of HucR reveals that it is a dimer, each monomer with the topology α1‐α2‐β1‐α4‐α5‐β2‐β3‐α6‐α7. The framework of the dimerization domain is provided by α2/α2′, in which the imidazole rings of His51 and His51′ are stacked. DNA binding affinity assessed by EMSA for HucR and its mutant H51F at pH 8.0 and 5.0 shows that the HucR‐H51F mutant is less sensitive to pH. Analysis of tryptophan intrinsic fluorescence spectra shows that HucR binds urate with negative cooperativity whereas HucR‐H51F binds with positive cooperativity. Moreover, thermal shift assays of HucR and HucR‐H51F demonstrate that HucR is completely unfolded at pH 5.0 and has a Tm ~ 52 °C at pH 8.0 while HucR‐H51F is only modestly destabilized at pH 5.0 (Tm ~ 45 °C). Notably, DNA binding stabilizes HucR at pH 5.0 whereas HucR‐H51 is destabilized on binding DNA. We suggest that histidine residues at the dimer interface act as a pH sensor and that DNA and ligand binding is communicated through the dimer interface.
Transcriptional regulation of trocarin D, a prothrombin activator from Tropidechis carinatus
Authors: Han Xia , Kwong Shiyang , Ge Ruowen , Kolatkar Prasanna , Kini R. Manjunatha
Published: June 2021

Trocarin D from the Tropidechis carinatus venom (TrTD) is a prothrombin activator sharing similar structure and function with blood coagulation factor Xa (FXa). It is probaly evolved from its plasma counterpart (TrFX) by gene duplication. The gene sequences of TrTD and TrFX are highly similar, except for the promoter and intron 1 regions. The insertion in the promoter region of TrTD (VERSE—VEnom Recruitment/Switch Element) accounts for elevated but not tissue specific expression of TrTD. The insertions/deletions intron 1 were examined for their “silencing” role in non‐venom gland cells. Using a series of deletion luciferase reporter assays, the strongest silencing cis‐elements were located in a 193 bp fragment within the insertion 2 segment (1975 bp). This segment has multiple silencing cis‐elements that turn off gene expression synergistically. Several silencer binding transcription factors (TFs) were identified using DNA affinity chromatography and mass spectrometry. The findings here highlight the importance of intron 1 for TrTD venom gland‐specific expression, and will contribute to understanding the transcriptional silencing mechanisms of TrTD in non venom gland tissues.

Source of research support: Academic Research Grants from NUS
DNA Based Folding Bio‐Sensors That Are Capable of Detecting Transcription Factors Involved In Cancer Progression By Expressing Recombinant TATA Binding Protein
Authors: Reyna Yerelsy C , Bonham Andrew J
Published: June 2021

Transcription factors bind to specific genomic sequences to control the flow of genetic information from DNA to mRNA. The preinitiation complex of these proteins includes TATA binding protein (TBP), which specifically recognizes the TATA box, a highly conserved region at the beginning of many genes. As many cancers result from the aberrant regulation of transcription factors, there is great interest, both academically and diagnostically, in a better understanding these binding events, and TBP is a core factor that can help guide the design of novel probes of transcription. Here, we report on the design and use of DNA‐based folding biosensors that are capable of quantitatively and sensitively detecting TBP using custom optical and electrochemical probes that are activated by TBP's intrinsic DNA‐binding activity. These sensors will ultimately allow us to investigate the role that protein‐protein interactions and inhibitory drugs have on gene expression.
Rab4 GTPase regulates cell surface expression of hERG channels through Nedd4–2
Authors: Cui Zhi , Zhang Shetuan
Published: June 2021

The human ether‐a‐go‐go‐related gene (hERG) encodes the pore‐forming subunits of the cardiac rapidly activating delayed rectifier K+ channel (IKr) which is important for cardiac repolarization. Dysfunction of IKr causes long QT syndrome (LQTS), a cardiac electrical disorder which can lead to cardiac arrhythmias and sudden death. Currently, trafficking mechanisms of the hERG channel are poorly understood. We investigated the effects of Rab4 GTPase on the function and expression of hERG channels using Western blot, patch clamp and immunofluorescence staining. Our data revealed that Rab4 selectively decreased the hERG expression on the plasma membrane. Mechanistically, Rab4 did not directly target the hERG protein. Instead, it increased the expression of ubiquitin ligase Nedd4–2, which mediates hERG ubiquitination and degradation. Mutations that disrupt the Nedd4–2 targeting site in hERG completely abolished the effect of Rab4. Our data further revealed that Rab4 inhibited Nedd4–2 self ubiquitination, leading to a decreased degradation rate of Nedd4–2. We conclude that Rab4 regulates the cell surface expression of hERG channels through Nedd4–2.

Supported by the Canadian Institutes of Health Research – CIHR.
Peptidylarginine deiminase modulates the physiological roles of enolase via citrullination
Authors: Choi Eun‐Kyoung , Jang Byungki , Jeon Yong‐Chul , Ishigami Akihito , Maruyama Naoki , Carp Richard I , Kim Yong‐Sun
Published: June 2021

The citrullination of enolase by PAD (peptidylarginine deiminase) has emerged as an important post‐translational modification in human disorders; however, the physiological function of citrullination remains unknown. Here, we report that citrullination diversely regulates the biological functions of ENO1 (α‐enolase) and NSE (neuron‐specific enolase). We developed three mouse IgG1 monoclonal antibodies with specificity to the following: anti‐CE1 (citrullinated enolase 1), anti‐CE1/2, and anti‐CE2. The levels of citrullinated ENO1 and NSE were elevated, and their immunoreactivities were also increased in cortical neuronal cells or around blood vessels in patients with sporadic Creutzfeldt‐Jakob disease and Alzheimer's disease compared with controls. In a time‐ and dose‐dependent manner, PAD negatively regulated enolase activity via citrullination, and enolase in diseased patients was more inactive than in controls. Interestingly, the citrullination of enolase effectively promoted its proteolytic degradation by calpain‐1. Surprisingly, the citrullination of enolase enhanced its plasminogen‐binding affinity, which was blocked by the lysine analogue ε‐aminocaproic acid. These findings suggest that PAD‐mediated citrullination regulates the diverse physiological activities of enolase and that CE may be a candidate diagnostic/prognostic factor for degenerative diseases. [This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (NRF‐2011–619‐E0001)]
Activation of muscarinic receptor increases cardiac potassium hERG channel through phosphorylation of E3 ubiquitin ligase Nedd4–2
Authors: Wang Tingzhong , Zhang Shetuan
Published: June 2021

The human ether‐a‐go‐go‐related gene (hERG) encodes the rapidly activating delayed rectifier potassium channel (IKr) which is critical for repolarization of cardiac action potential. Loss of function of hERG channels due to gene mutations or drug blockade causes long QT syndrome. Presently, little is known about the restoration or enhancement of hERG channel function. In the present study, we found that muscarinic receptor agonist, carbachol, specifically increased hERG expression and IhERG, but had no effect on IKs, IEAG and IKv1.5 channels in HEK expression systems. Carbachol treatment decreased hERG‐ubiquitin interaction and hERG degradation. It is known that hERG channels are degraded by the Nedd4–2, we found that disrupting the binding sites of Nedd4–2 by the Y1078A point mutation or C‐terminal truncation Δ1073 mutation in hERG completely eliminated the effects of carbachol on hERG channels. Our data further indicate that carbachol treatment inhibited Nedd4–2 activity by enhancing its phosphorylation level. Another muscarinic receptor agonist, oxotremorine, increased hERG function, and the muscarinic receptor antagonist 4‐DAMP abolished the effects of both carbachol and oxotremorine on hERG channels. Enhancement of hERG function by muscarinic receptor activation represents a novel pathway in hERG regulation, and this finding may have potential for the management of long QT syndrome patients.

Supported by: Canadian Institutes of Health Research (CIHR).
Landscape of the PARKIN‐dependent ubiquitin modified proteome in response to mitochondrial depolarization defined through quantitative proteomics
Authors: Sarraf Shireen A , Raman Malavika , Guarani‐Pereira Virginia , Sowa Mathew E , Huttlin Edward L , Gygi Steven P , Harper J Wade
Published: June 2021

Parkinson's Disease (PD) results from loss of dopaminergic neurons of the substantia nigra, in part due to mitochondrial malfunction. The protein kinase PINK1 and E3 ubiquitin ligase PARKIN, found mutated in hereditary and sporadic forms of PD, are thought to control mitochondrial homeostasis. Upon mitochondrial damage, activation of PINK1 on the mitochondrial outer membrane (MOM) recruits PARKIN to ubiquitylate Porin, Mitofusin, and Miro proteins on the MOM; however, the full repertoire of PARKIN substrates and how their ubiquitylation alters mitochondrial homeostasis via mitophagy is poorly understood. Large‐scale quantitative diGLY capture proteomics identified hundreds of ubiquitylation sites in dozens of proteins upon mitochondrial depolarization, with strong enrichment for mitochondrial proteins. Physical association of PARKIN with a cohort of ubiquitylation targets and the proteasome was dependent upon mitochondrial depolarization. Structural and evolutionary analysis of PARKIN‐dependent ubiquitylation events revealed conservation of target sites in vertebrate and D. melanogaster MOM proteins. These studies provide a resource for elucidating how the PINK1‐PARKIN pathway re‐sculpts the mitochondrial proteome via ubiquitylation to maintain mitochondrial homeostasis. Supported by NIH grants GM070565 to JWH, GM067945 to SPG, and a grant from the Michael J Fox Foundation to JWH.
Ubiquitin and cell cycle regulation of Golgi membrane dynamics
Authors: Wang Yanzhuang , Tang Danming , Zhang Xiaoyan
Published: June 2021

Partitioning of the Golgi membrane into daughter cells during mammalian cell division occurs through a unique disassembly and reassembly process. Several converging lines of evidence have suggested that monoubiquitination plays an essential role in the regulation of post‐mitotic Golgi membrane fusion. Monoubiquitination, as a regulatory signal, occurs during mitotic Golgi disassembly and is required for subsequent Golgi reassembly. The AAA ATPase p97 and its adapter protein p47 are involved in membrane fusion during post‐mitotic Golgi reassembly. The p97/p47 complex binds to monoubiquitin through the UBA domain of p47, and this interaction is required for p97‐mediated Golgi membrane fusion. Proteasome activity is not involved in either Golgi disassembly or reassembly. We have identified a Golgi‐localized ubiquitin E3 ligase HACE1 which is involved in this process. We have also discovered the p97/p47 binding protein VCIP135 as a deubiquitinating enzyme whose activity is required for post‐mitotic Golgi reassembly. These data suggest that cycles of addition and removal of ubiquitin to and from substrates regulate Golgi membrane dynamics during cell division.
Cross‐Presentation of peptides Chaperoned by Heat Shock Protein gp96
Authors: Phillips Shantell Sherie , Binder Robert J
Published: June 2021

Cross‐presentation is the ability of Dendritic Cells (DCs) to uptake and process extracellular antigens and present them on their major histocompatibility class I molecules (MHC). Heat shock protein (HSP)‐chaperoned peptides are cross‐presented by DCs. It has been demonstrated that CD91 acts as a cell surface receptor for heat shock protein gp96, binding gp96 directly and allowing the HSP‐peptide to be up taken by the cell into the endosome. Previous studies have shown that HSP90 is critical for the translocation of peptides from the endosome to the cytosol for proteasomal degradation. These peptides are then translocated to the endoplasmic reticulum where they are loaded onto the MHCs for cell surface display. The fate of gp96 after endocytosis is unknown and is the focus of this study.

In the current study, we investigate the fate of gp96 after internalization. We used selective cell fractionation of DCs to identify the location of gp96. Through this procedure, we were able to initially separate the intracellular membranes and plasma membranes of the DCs and showed that gp96 was primarily associated with intracellular membranes. With further fractionations, we were able to isolate the cytosol, mitochondria, and nucleus and showed that at 30mins gp96 was primarily located in the cytosol and not the nucleus or mitochondria. These study results show the route of trafficking gp96 associated with cross‐presentation.
Casein kinase 1δ/ε regulates clathrin‐ and actin‐mediated endocytosis
Authors: Peng Yutian Connie , Grassart Alexandre , Lu Rebecca , Michelot Alphée , Wong Catherine C. L. , Yate John , Barnes Georjana , Drubin David G
Published: June 2021

While much has been learned about the mechanisms by which clathrin‐coated vesicles are formed during endocytosis, much less is known about how their formation is regulated. Here, we investigate the role of protein phosphorylation in regulation of clathrin‐mediated endocytosis. We found that in both budding yeast and mammalian cells the casein kinase 1δ/ε, Hrr25, associates with and regulates the endocytic machinery. In budding yeast, Hrr25 is recruited to endocytic patches early in the pathway by the C‐terminus of the yeast Eps15‐like protein, Ede1, where it phosphorylates Ede1 and several other endocytic proteins. An analog‐sensitive allele was created and used to show that Hrr25 kinase activity is required for the kinase's recruitment to endocytic sites. Loss of the kinase activity impairs initiation of endocytosis and causes abnormal patch dynamics. Furthermore, our results in mammalian cells provide evidence for conservation of casein kinase 1δ/ε functions in more complex organisms. Ongoing studies are aimed at elucidating the detailed mechanisms and specific in vivo phosphorylation targets underlying Hrr25's regulatory functions in endocytosis.
Stabilizing rescued delF508 CFTR at the plasma membrane by potentiation of its interaction with Na+/H+ exchanger regulatory factor 1
Authors: Arora Kavisha , Moon Chang Suk , Zhang Weiqiang , Ren Aixia , Yarlagadda Sunitha , Sinha Chandrima , Naren Anjaparavanda P.
Published: June 2021

Cystic fibrosis (CF) is a lethal genetic disease and warrants effective therapeutic interventions. Preclinical studies screened CF‐correctors based on their ability to partially correct the processing defects of ΔF508 mutation of cystic fibrosis transmembrane conductance regulator (CFTR), the most common CF causing mutation. The CF‐corrector VX‐809 underwent Phase II trials in combination with VX‐770 or Ivacaftor and was shown to significantly improve lung function in CF patients. In this study, we demonstrate the significance of protein‐protein interaction in enhancing the stability of mutant channel protein at the plasma membrane. We show that VX‐809 partially stabilizes ΔF508‐CFTR interaction with scaffolding protein Na+/H+ exchanger regulatory factor 1 (NHERF1) at the plasma membrane. We demonstrate in cystic fibrosis bronchial epithelial cells that VX‐809 increases half‐life of ΔF508 CFTR at the plasma membrane by potentiating its otherwise weak interaction with NHERF1. Single particle tracking analysis further strengthens our observation as VX‐809 restricts the mobility of ΔF508‐CFTR at the plasma membrane while the effect is mitigated in ΔF508 CFTR His10 (lacks the PDZ motif and hence the ability to interact with NHERF1). Therefore, our studies demonstrate the importance of macromolecular complex in rescuing mutant CFTR to the plasma membrane and will further help device newer generation of more effective CF‐correctors.
Quantitative internalization kinetics of the green fluorescence protein‐tagged guanylyl (guanylate) cyclase/natriuretic peptide receptor‐A in Human Embryonic Kidney‐293 cells
Authors: MANI INDRA , Garg Renu , Nguyen Vickey A , Pandey Kailash N
Published: June 2021

Cardiac hormone atrial natriuretic peptide (ANP) modulates blood pressure and blood volume by activation of the guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) and subsequent generation of intracellular second messenger cGMP. Advances in the development of proteins conjugated with the enhanced green fluorescence protein (eGFP) provide fluorescence based analysis of protein trafficking in individual cells. The aim of this study was to quantify internalization kinetics of NPRA in human embryonic kidney‐293 (HEK‐293) cells through confocal microscopy. Cells were stably transfected with cDNAs encoding NPRA‐eGFP. HEK‐293 cells were treated with natural ligand ANP at different time course (0, 5, 10, 15, and 30 minutes) and internalization of receptor was analyzed through confocal immunofluorescence microscopy. In control cells without ANP treatment, all receptors were localized on plasma membrane. Internalization of receptor from the plasma membrane was analyzed as 58.4% at 5 min, 69.0% at 10 min, 70.8% at 15 min, and 75.6% at 30 min with compare to controls. Similarly 51.4% receptor population was localized in cytoplasm at 5min, 62.5% at 10 min, 65.5% at 15 min and 69.9% at 30 min. In conclusion these data demonstrate that after ligand binding, NPRA is rapidly internalized and trafficked from the cell surface to cell interior.

This work was supported by the NIH grant (HL57531).
Biochemistry and immunology of Gram‐positive bacterial heme acquisition systems
Authors: Shipelskiy Yan , Wang Zhiguo , Rosinski Justin , Buckles Tom , Mahale Dilip , Dragosjlvic Steve , Newton Salete M. , Klebba Phillip E.
Published: June 2021

Gram‐positive pathogenic bacteria scavenge heme from their hosts during infection. Six heme binding proteins exist in L. monocytogenes (Hbp1 and 2) and S. aureus, (IsdA, B, C and H) that contain NEAT‐domains which presumably interact with heme during its uptake. We cloned the structural genes, purified and characterized the corresponding proteins to understand their specificities, affinities and interactions. We used affinity purified, 6H‐tagged proteins to generate murine polyclonal and monoclonal antibodies. The resulting high‐titer antisera showed specificity for the original immunogens, as well as cross‐reactivity to other NEAT‐domain containing orthologous and paralogous proteins. For example, anti‐Hbp2 recognized Hbp1 as well as IsdH by both ELISA and western immunoblot. These sera inhibited the uptake of hemin (Hn) and hemoglobin (Hb) by L. monocytogenes and S. aureus, and retarded the pathogenesis of these strains in a mouse sepsis model system. Anti‐Hbp1 and 2 sera were cross‐reactive with IsdH and attenuated the virulence of L. monocytogenes. Cross‐reactivity between NEAT‐domain containing proteins suggests that antibodies generated against the iron acquisition system of one Gram positive organism may provide immunity to infection by other Gram positive organisms, likely because the sera inhibit heme acquisition. Lastly, we introduced cysteine at key exposed residues, and labeled the Cys sulfhydryls with extrinsic fluorophores to evaluate localization of these proteins in the bacterial cell wall. This research was supported in part by Trellis Biosciences, S. San Francisco, CA.
Manipulation of host cell signaling by bacterial FIC proteins
Authors: Pieles Kathrin , Harms Alexander , Dehio Christoph
Published: June 2021

Fic proteins are found in all kingdoms of life and are defined by their conserved FIC domain that catalyzes the transfer of an AMP moiety onto target proteins. A subset of Fic proteins has been shown to adenylylate small GTPases of the Ras superfamily resulting in their inactivation leading to cytoskeletal collapse and cell death. Pathogens belonging to the genus Bartonella translocate effector proteins into the host cell cytoplasm in order to establish chronic infections. As most of these effectors harbor a FIC domain, our lab is studying its role in bacterial pathogenesis by identifying targets in the eukaryotic host cell. Here, we present an example of highly selective adenylylation among the class of small GTPases. In contrast to other previously described Fic proteins, we found that Bep1 of Bartonella rochalimae does not adenylylate a broad spectrum of Rho family GTPases but exclusively targets Rac1 indicating a more subtle subversion of cellular functions which is consistent with the stealthy infection strategy of Bartonella. We hypothesize that Rac1 adenylylation leads to cytoskeleton rearrangements promoting bacterial entry and survival. Research was supported by SNSF grant 31003A‐132979 and Werner Siemens Foundation.
Measuring bacterial S‐acylation in Pseudomonas putida
Authors: Milam Claire E. , Johnson R. Jeremy
Published: June 2021

In eukaryotes, reversible palmitoylation of cysteine residues via a thioester linkage allows dynamic shuttling of proteins between intracellular compartments. Regulation of protein localization by palmitoylation is involved in key cellular processes including cell signaling, ion transport, and neuronal communication. These dynamic cycles of palmitoylation and depalmitoylation are controlled by two classes of cellular enzymes, protein acyl transferases and acyl‐protein thioesterases. While bacterial proteins are S‐acylated and homologues of human acyl protein thioesterase are present in many gram‐negative bacterium, dynamic cycles of S‐acylation have not been observed in prokaryotes. In this study, we utilized azide mimics of palmitic acid to measure the level of protein S‐acylation in Pseudomonas putida. S‐acylated proteins were then detected by click chemistry using either biotin or fluorophore labeled alkynes. Specific S‐acylated proteins were identified in P. putida and their identities are currently being confirmed. Using deletion strains for potential bacterial palmitoylation machinery, we plan to search for proteins that regulate levels of S‐acylation in P. putida. Funding provided by Butler University Holcomb research fund.