bluuebunny/crossref_metadata_2025 huggingface dataset:
miR‐709 regulates miR‐15a/16 biogenesis at post‐transcriptional level in nucleus: an implication of a microRNA hierarchy system
Authors: Tang Rui , Li Limin , Cao Tin , Hou Dongxia , Chen Junyuan , Zhang Chenyu , Zen Ke
Published: June 2021

MicroRNAs are endogenous non‐coding RNAs of 21–23 nt which regulate target gene expression at the posttranscriptional level typically through either translational repression or mRNA cleavage. Recent developments have revealed that there is extensive of miRNAs as well as miRNA pathways related proteins in cell nucleus, leading us to hypothesize that nucleus microRNAs might have their own functions. We observed that a specific microRNA, miR‐709, is detected in nucleus fraction preparing from various mouse cell lines as well as tissues samples. In cell nucleus miR‐709 will meet and bind its target, pri‐miR‐15a/16‐1, which is a primary transcript for another microRNA cluster: miR‐15a and miR‐16. Such binding will prevent pri‐miR‐15a/16‐1 processing in nucleus and thus regulate miR‐15a/16 maturation at the pri‐miRNA to pre‐miRNA stage. Furthermore, nucleus miR‐709 participates cell apoptosis through BCL2‐miR‐15a/16 pathway. Our data indicates that one microRNA can directly target another microRNA's primary transcript in nucleus and regulate its biogenesis via this pathway. In conclusion this association may further suggest a “microRNA hierarchy” system in biological world.
Hydrogen peroxide regulates splicing by modulating the protein levels of select splice factors
Authors: Sharina Iraida , Zhu Wen , Murad Ferid , Cote Gilbert
Published: June 2021

In our previous studies we demonstrated the existence of an alternatively spliced soluble guanylyl cyclase (sGC) isoform C‐α1 resistant to oxidative stress‐induced protein degradation. We extended these studies to address if sGC RNA splicing can be regulated by H2O2 exposure in human breast carcinoma MDA468 cells which constitutively express sGC. RT‐PCR demonstrated that treatment with 1 mM H2O2 for 24h induced the C‐α1 sGC transcript level. To identify potential sGC splicing regulators we performed an in silico analysis using ASD search engine from EMBL. Binding sites for SRp20, hnRNP 2AB1, HuR and PTB1 splice regulators were identified proximal to the regulated and canonical splice acceptors within intron 2. Thus, we investigated if the exposure to H2O2 affects the abundance of these predicted splice regulators. Using Western blotting we observed a significant decrease in levels of PTB1 and hnRNP A2/B1, but not HuR or SRp20 proteins. Next we demonstrated that the expression of PTB1 is affected on the protein, not mRNA level. We also found that oxidative stress‐induced degradation of PTB1 is not mediated by Caspase‐3. These data suggest that H2O2, a ubiquitous molecule associated with oxidative stress, modulates the expression of a select subset of splicing factors, which might play an important role in cellular stress response and adaptation.

Research is supported by the Welch and JS Dunn Foundations to F.M.
Alternative splicing of Caspase 9 is modulated by the PI3K/Akt pathway via phosphorylation of SRp30a
Authors: Shultz Jacqueline Coates , Goehe Rachel W. , Wijesinghe Dayanjan S. , Murudkar Charuta , Hawkins Amy , Shay Jerry W. , Minna John D. , Chalfant Charles E.
Published: June 2021

Increasing evidence points to the functional importance of alternative splice variations in cancer pathophysiology. Two splice variants are derived from the CASP9 gene via the inclusion (Casp9a) or exclusion (Casp9b) of a four exon cassette. Here we show that alternative splicing of Casp9 is dysregulated in non‐small cell lung cancers (NSCLC) regardless of their pathological classification. Based on these findings we hypothesized that survival pathways activated by oncogenic mutation regulated this mechanism. In contrast to K‐RasV12 expression, EGFR overexpression or mutation dramatically lowered the Casp9a/9b splice isoform ratio. Moreover, Casp9b downregulation blocked the ability of EGFR mutations to induce anchorage‐independent growth. Furthermore, Casp9b expression blocked inhibition of clonogenic colony formation by erlotinib. Interrogation of oncogenic signaling pathways showed that inhibition of PI3K or Akt dramatically increased the Casp9a/9b ratio in NSCLC cells. Finally, Akt was found to mediate exclusion of the exon 3,4,5,6 cassette of Casp9 via the phosphorylation state of the RNA splicing factor SRp30a via serines 199, 201, 227 and 234. Taken together, our findings demonstrate that oncogenic factors activating the PI3Kinase/Akt pathway can regulate alternative splicing of Casp9 via a coordinated mechanism involving the phosphorylation of SRp30a.
Optimization of fluorescence based Group I ribozyme studies
Authors: Zaborowicz Matthew A. , Aprile Kayla , Rohlman Christopher E.
Published: June 2021

Group I introns are self‐splicing RNA's. They can be made into ribozymes through modifications which creates an active site at the location of the initial splice site. This ribozyme can then act on a substrate strand of RNA with a base sequence complementary to the active site. These ribozymes are used to study the function of the intron itself. Fluorescent tags can be attached to the substrates so that we can study their kinetic properties. A machine that can be used is a genetic sequencer. The ABI 310 sequencer operates through capillary electrophoresis. The laser is very sensitive and can detect the fluorescent substrate in the nanomolar range. The ABI 310 is also fully automated and faster than typical bandshift gel assays. Multiple samples can be made and loaded into the instrument at a time. The data gives us information for quantification of reaction completion at a certain time point. The conditions of the ribozyme induced cleavage reaction can then be altered in an attempt to further understand the intron's function. We are working to adapt this system to a range of Group I model systems. This research was supported by the Albion College Foundation for Undergraduate Research, Scholarly and Creative Activity.
Microtubule dependency of stress granule dynamics
Authors: Mason Twila Annette , Kolobova Elena , Goldenring James R
Published: June 2021

Stress granule formation is an important mechanism for cell survival during stress that remains largely uncharacterized. We studied the role of microtubules in the formation, maintenance, and disassembly of stress granules. During stress, small, diffuse granules initially form throughout the cytoplasm. These granules then coalesce near the nucleus into larger granules that disassemble once the cellular stress is removed. Complete stress granule formation is dependent upon microtubules. Human cervical cancer (HeLa) cells, pre‐treated with nocodazole for microtubule depolymerization, formed only small, diffuse stress granules upon arsenite treatment. Upon removal of nocodazole and arsenite, the small granules coalesced near the nucleus, completing formation, prior to disassembly. An organic arsenical, ZIO‐101 (darinaparsin), as single agent also induced the formation of small, diffuse stress granules, having an effect similar to that of nocodazole/arsenite. ZIO‐101 inhibited the polymerization of microtubules both in vivo and in vitro. Upon removal of ZIO‐101, the small, diffuse stress granules completed formation prior to disassembly. In conclusion, stress granules must complete formation prior to disassembly, and completion of stress granule formation is dependent upon microtubules. Research support is provided by NIH grant RO1DK43405 and a gift of ZIO‐101 from Ziopharm.
Thermodynamic Examination of Leadzyme
Authors: Frost Arian Cai , Grover Neena
Published: June 2021

The leadzyme is a small, double‐stranded, artificially created ribozyme that contains an asymmetrical 4×2 bulge. This bulge region contains four unpaired bases and at least one non‐canonical base pair. The bulge cleaves in the presence of lead and can be competed out by Mg2+. The leadzyme contains three conserved bases (C6, G9, and G24) that are implicated in divalent metal ion binding and structure stabilization. NMR data suggests the formation of an A25+‐C6 non‐canonical base pair with two hydrogen bonds while the crystal structure of the leadzyme shows one hydrogen bond. The active site bulge in the leadzyme may be flexible under different ionic conditions and pH environments. Thermal denaturation experiments were used to compare the relative stability of the wild type RNA in the presence of various concentrations of Mg2+ ions in pH 5.5 and 7.5. Our data shows that the second hydrogen bond between A25‐C6 is not forming. The ion binding properties of the leadzyme are being examined in RNA constructs where the conserved sites have been modified. Results of leadzyme stability and ion binding properties will be presented.
The gene TDAG51 facilitates the phosphorylation of eIF2α in mouse embryonic fibroblasts
Authors: Carlisle Rachel E , Dickhout Jeffrey G , Austin Richard C
Published: June 2021

Unfolded protein accumulation in the endoplasmic reticulum (ER) results in ER stress. This causes the binding of GRP78 to unfolded proteins, auto‐phosphorylation of PERK and the phosphorylation of eIF2α, leading to changes in the rate and characteristics of protein synthesis, including an upregulation of CHOP/GADD153 expression. TDAG51 is an ER stress response gene and has been shown to regulate translation. We hypothesized that TDAG51 influences protein translation by mediating the phosphorylation of eIF2α. Mouse embryonic fibroblasts (MEFs) treated with ER stressors, tunicamycin (Tm; 5 μg/ml) or thapsigargin (Tg; 1 μM), or a phosphorylated eIF2α dephosphorylation inhibitor, salubrinal (30 μM), or combined treatments demonstrated an increase in eIF2α phosphorylation. TDAG51−/− MEFs showed a decrease in eIF2α phosphorylation to drug vehicle, Tm plus salubrinal, Tg and Tg plus salubrinal treatment. It was determined that TDAG51−/− MEFs have reduced CHOP/GADD153 expression compared to wild type cells. Therefore, TDAG51 knockout may regulate CHOP/GADD153 expression by reducing the phosphorylation of eIF2α, which in turn may lead to decreased expression of GADD34, essentially inhibiting the GADD34/PP1 dephosphorylation complex. This mechanism is also being investigated through the use of siRNA‐mediated knockdown. Supported by CIHR MOP‐67116.
Participation of de novo sphingolipid biosynthesis in the regulation of autophagy in Kdo2‐Lipid A stimulated RAW264.7 macrophages
Authors: Sims Kacee , Haynes Christopher , Kelly Samuel , Allegood Jeremy , Wang Elaine , Momin Amin , Leipelt Martina , Reichart Donna , Glass Christopher , Sullards M. Cameron , Merrill Alfred
Published: June 2021

Activation of RAW264.7 cells with a lipopolysaccharide specific for the TLR4 receptor, Kdo2‐Lipid A (KLA), causes a large increase in cellular sphingolipids‐‐from 1.5 to 2.6 × 109 molecules per cell in 24 h, based on the sum of subspecies analyzed by “lipidomic” mass spectrometry. Thus, this study asked: What is the cause of this increase, and is there a cell function connected with it? The sphingolipids arise primarily from de novo biosynthesis; with the exception of ceramide, which is also produced from pre‐existing sources. Nonetheless, the activated RAW264.7 cells have a higher number of sphingolipids per cell because KLA inhibits cell division, thus, the cells are larger and contain increased numbers of membrane vacuoles termed autophagosomes. Indeed, de novo biosynthesis of sphingolipids performs an essential structural and/or signaling function in autophagy because autophagosome formation was eliminated by ISP1 in KLA stimulated RAW264.7 cells; furthermore, an anti‐ceramide antibody co‐localizes with autophagosomes in activated RAW264.7 cells versus the Golgi in unstimulated or ISP1 inhibited cells. These findings establish that KLA induces profound changes in sphingolipid metabolism and content in this macrophage‐like cell line, apparently to produce sphingolipids that are necessary for formation of autophagosomes, which are thought to play important roles in the mechanisms of innate immunity.
The M‐domain controls the Hsp104 protein disaggregating activity
Authors: Sielaff Bernhard , Lee Sukyeong , Tsai Francis T. F.
Published: June 2021

Yeast Hsp104 and bacterial ClpB are ring‐forming, ATP‐dependent protein disaggregases that, together with the cognate Hsp70 system, rescue proteins from a previously aggregated state. Hsp104 possesses an N‐terminal domain, an M‐domain, and two AAA+ domains. The M‐domain is located on the Hsp104 exterior, as it is in ClpB, which was confirmed by our cryoEM structure of a functional Hsp104‐T4 lysozyme chimera. Unexpectedly, we found that our Hsp104 chimera has gained function and can solubilize heat‐aggregated proteins in the absence of the Hsp70 system. To determine the function of the M‐domain, we engineered different Hsp104 variants and chimeras, and showed that the M‐domain is essential for protein disaggregation but dispensable for the ATPase and substrate translocating activities. Remarkably, we found that replacing the yeast Hsp104 M‐domain with that of bacterial ClpB, and vice versa, switches the species‐specificity so that our chimeras now cooperate with the non‐cognate Hsp70 system. Our results demonstrate that the M‐domain controls the Hsp104 protein remodeling activities in an Hsp70‐dependent manner. This work was supported by grants from the NIH and the Welch Foundation (Q‐1530).
Appropriate interaction of Ecm29 with the proteasome is necessary for proteasome functional integrity and requires the Not4 E3 ligase
Authors: Collart Martine A
Published: June 2021

In this study we determine that Not4 is important for ubiquitin turnover and affects proteasome integrity, in part through Ecm29, a protein that contributes to the assembly of the proteasome. Deletion of Not4 leads to accumulation of polyubiquitinated proteins, reduced levels of free ubiquitin and synthetic lethality when combined with the deletion of the Doa4 or Ubp6 deubiquitinating enzymes. Consistently, purification of the proteasome in the absence of Not4 reveals functional, salt‐resistant interaction between the proteasome regulatory particle, RP, and the proteasome core particle, CP, and instability of all other forms of RP. Purification of proteasome holoenzyme via RP reveals co‐purification of Ecm29 from wild type cells, but much less from not4 cells. Indeed, in the absence of Not4, Ecm29 interacts less well with the proteasome, gets ubiquitinated and degraded Interestingly, Ecm29 was mostly identified in purified RP‐containing complexes that are inactive, and are not detectable in cells lacking Not4. On the other hand, Ecm29 co‐purifies with the Ccr4‐Not complex and co‐immunoprecipitates with Not4. Our results characterize Ecm29 as a proteasome chaperone whose appropriate interaction with the proteasome requires Not4.

This work was supported by the Swiss Natinal Science Foundation.
Benchmarking a fluorescence microplate‐based aggregation assay using subvisible protein particle standards
Authors: Shen Dee , Chan Eric , Dai Lijun , Coleman Jack , Patton Wayne F
Published: June 2021

Aggregation represents one of the most significant obstacles to protein‐based pharmaceutical development. During drug formulation, aggregation can impact product quality in terms of biological activity and immunogenicity. Improved methods are required to detect and monitor aggregation during manufacturing. We report upon a fluorescence microplate assay employing a novel fluorescent probe specifically devised to detect aggregated proteins and peptides. The probe is nonfluorescent in solution, but becomes brightly fluorescent when bound to the tertiary structure of aggregated proteins. The assay is capable of detecting less than 0.2% aggregated protein in a concentrated antibody formulation, with a linear dynamic range of two‐orders of magnitude. Sub‐visible oligomeric particles are readily detected without a need for sample separation, solvent exchange or dilution. Subvisible protein particle standards were developed to benchmark the fluorescence microplate assay, as well as orthogonal characterization methods, including laser‐based nanoparticle tracking and micro‐flow Imaging. Advantages of the microplate‐based assay include simple implementation with widely available instrumentation, results achievable in 30 minutes or less and robust assay performance. The assay provides a convenient, complementary orthogonal method for cross‐validation of instrument‐intensive techniques.

This work was supported by ENZO Life Sciences.
Computational study of bound water in a human collagen II model peptide and its variants
Authors: Miles Andrew Thomas , Anderle Gloria
Published: June 2021

The human collagen II sequence P02458 (GFO GER GSO GAQ GLQ GPR GLO GTO GTD GPK) was obtained from the UniProtKB/Swiss‐Prot database and was used to construct collagen II trimers based on the 1CGD.pdb crystal structure. This sequence is of interest because it contains two human mutations at residue 717 (the GPR triplet above) that cause congenital bone disorders. The G717S mutation causes femoral head necrosis, a degenerative disease, while G717V causes achondrogenesis; neonatally lethal. The AMBER10 molecular dynamics package was used to simulate the resulting peptides in explicit water at 310 K. Sites of bound water were identified using VMD (Visual Molecular Dynanics), a visualization package used to observe the trimeric protein and its bound waters. These bound waters are sequestered by polar amino acid residues that form cage‐like structures that contribute to the stability of the trimer via hydrogen bonding. In the second and third nanosecond of the wild type (G717G), three cages were found. Nanosecond two of G717S and G717V had two cages and one cage, respectively. These findings suggest that the G→S and G→V mutations both lead to decreased stability, but the G717V shows a higher degree of unfolding at the C‐terminus. Department of Chemistry and Pharmaceutical Science, Becton College, Fairleigh Dickinson University, Madison, NJ
Analysis of the role of PHLPPs in pancreatic β‐cell function
Authors: Larson Kara L. , Gao Tianyan , Ozcan Sabire
Published: June 2021

Pleckstrin Homology Domain Leucine Rich Repeat Protein Phosphatases (PHLPP) are a pair of recently discovered PP2C‐type serine/threonine phosphatases in the PPM phosphatase family. They dephosphorylate the hydrophobic motifs of Akt at serine 473 and protein kinase C at serine 660. PHLPP has been shown to have a role in cancer cells but has not been studied in many other cell types. Overexpression of the protein in cancer cells causes increased apoptosis as well as reduced tumor size via downregulation of Akt signaling. To determine whether PHLPPs have a role in regulation of glucose homeostasis, we have performed glucose tolerance tests using wild type and whole body PHLPP1 knockout mice and found that mice lacking PHLPP1 have improved glucose tolerance compared to wild type. This may be due to increased insulin secretion or improved insulin action in the PHLPP1 knockout mice. Preliminary data suggest that PHLPP1/2 may play a role in regulation of insulin secretion in pancreatic beta cells. Experiments are under way to dissect the molecular mechanisms by which PHLPPs regulate insulin secretion and thereby influence glucose homeostasis. Supported by NIH grant R56 DK067581‐06
ATP release induced by electrical stimulation increases glucose uptake via AKT in skeletal muscle
Authors: Osorio‐Fuentealba Cesar , Altamirano Francisco , Espinosa Alejandra , Jaimovich Enrique
Published: June 2021

Insulin activates downstream pathways in skeletal muscle including PI3K and Akt. On the other hand, ATP is released by contractile activity in skeletal muscle cells and extracellular ATP is able to act as an autocrine or paracrine factor in muscle cells.

We determined the effects of extracellular ATP in Akt signaling and glucose uptake. Adult muscle fibers and rat myotubes were exposed to electric field tetanic stimulation (400 pulses of 1 ms at 45 Hz), 100 nM insulin or 100 ìM ATP. Electrical stimulation induced ATP release and Akt phosphorylation in myotubes. We measured uptake of the fluorescent hexoses 2‐NBDG and 6‐NBDG at room temperature using confocal microscopy. The hexose dyes were excited at 488 nm and images were collected at 505–550 nm. Fluorescent images were analyzed and glucose uptake was normalized to the basal condition. Tetanic electrical stimulation, insulin and ATP were able to induce glucose uptake in both myotubes and adult fibers. ATP, before insulin addition, increased glucose uptake induced by insulin. 10 μM each Akt VIII inhibitor and LY 294002 inhibited the increase in glucose uptake in myotubes.

Results suggest that electrical stimulation potentiates insulin signaling in skeletal muscle via ATP, which may play an important role as a mediator of glucose uptake during muscle activity.

FONDAP 15010006, FONDECYT 11090301, AT‐24100067
Energy metabolic block induces accelerated senescence in human cancer cells
Authors: Krynetskiy Evgeny
Published: June 2021

Glycolytic pathway is an essential source of energy in cancer cells; therefore, it can be selectively targeted by anticancer pharmacotherapy. The pivotal enzyme in this pathway is glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) which executes both enzymatic and regulatory functions. We evaluated the role of GAPDH in the control of energy homeostasis in isogenic (p53+/+ and p53−/−) human carcinoma cells by using RNA interference, site mutagenesis, and confocal microscopy. Knockdown of GAPDH induced accelerated senescence of cancer cells via p53‐regulated pathway, in the absence of DNA damage. Depletion of GAPDH by short interfering RNA decreased protein level, GAPDH enzymatic activity, and ATP. In response to reduced ATP, AMPK pathway was activated leading to p53 stabilization, and cell proliferation arrest. GAPDH‐depleted cells acquired senescence phenotype revealed by changes in morphology and senescence‐associated markers. Rescue experiments aimed to reinstate energy metabolism (by adding the downstream metabolite pyruvate), or GAPDH level partially restored ATP level but not cell proliferation suggesting that energy crisis by GAPDH depletion may not be the only cause of accelerated senescence. These results indicate that accelerated senescence induced by GAPDH depletion may provide a strategy to control proliferation of cancer cells.

This work was supported in part by NCI grant R01 CA104729.
Modulation of lipid metabolism in interacting visceral adipocytes and ovarian cancer cells
Authors: Nieman Kristin M , Kenny Hilary A , Zillhardt Marion R , Penicka Carla , Ladanyi Andras , Buell‐Gutbrod Rebecca , Gwin Katja , Lengyel Ernst R
Published: June 2021

Ovarian cancer (OvCa) is the 5th leading cause of cancer‐related deaths in women. The primary site of OvCa metastasis is the adipocyte (ADI)‐dominated omentum. A growing body of evidence supports metabolic dysregulation as a cancer hallmark. This research was aimed at investigating metabolic changes in interacting OvCa cells and ADIs. Human omental ADIs were plated on OvCa cells for coculture experiments and protein detection. Fatty acid (FA) oxidation was assessed by radioisotopic assay. FA binding protein 4 knockout (FABP4−/−) and wild‐type (WT) mice were inoculated with OvCa cells and sacrificed 10 weeks later. Human OvCa and omental metastatic tissues were utilized for IHC. Lipid accumulation and transfer, from ADIs into OvCa cells, were evident following coculture. FA oxidation was elevated in cocultured OvCa cells in a time‐dependent manner. Increased activated hormone‐sensitive lipase abundance in cocultured ADIs indicates upregulated lipolysis. FABP4 deficiency reduced tumor growth in FABP4−/− mice compared to WT mice. FABP4 was evident in OvCa cells at the ADI interface of omental metastasis but absent in the primary tumor. These results suggest ADIs promote OvCa metastasis and alter metabolic activity. FABP4 may mediate trafficking of FAs, providing energy for OvCa survival and metastasis. These findings may identify novel therapeutic targets for OvCa treatment. Support: NIH‐NCI T32 CA 9594‐21.
Anti‐angiogenic effects of zoledronic acid on osteotropic breast cancer cells
Authors: Contreras Cynthia M. , Morshedian Shaghayegh , Hamud Walid A. , Dhakal Bishal , Khanal Bijay R. , Bush Jason A.
Published: June 2021

Emerging data suggest the bisphosphonate, zoledronic acid (ZOL), exerts both indirect and direct anti‐tumoral effects by decreasing tumor cell proliferation, increasing apoptosis, and inhibiting angiogenesis. Recent data indicate that anti‐angiogenic factors exert cellular effects via suppression of mitochondrial oxidative phosphorylation/respiration and elevation of intracellular ROS levels. This project intended to define proteins involved in response to ZOL‐treatment of an osteotropic breast cancer cell line, MDA‐231BO. Results demonstrate that MDA‐231BO cells are more sensitive to ZOL cytotoxicity and salutary effects are both media‐ and calcium‐dependent. To investigate pro‐apoptotic mechanisms of ZOL, total RNA, whole cell protein lysates, or mitochondrial‐enriched fractions were isolated from MDA‐231BO cells and using a combination of RT‐PCR, 1D/2D‐PAGE separation and MALDI‐MS/MS, we identified several angiogenesis inhibitors that were upregulated and pro‐angiogenic factors that were downregulated after ZOL treatment. Functional studies demonstrate the improved efficacy of ZOL on MDA‐231BO compared to MDA‐MB‐231 controls. Taken together, these results further support a model that ZOL tips the ‘angiogenic switch’ in favor of anti‐angiogenic factors in cancer cells with a propensity for bone metastases. Partial research support came from NIH 5P20MD002732.
Silencing of Wnt5a during colon cancer metastasis involves histone modifications of the gene
Authors: Li Qian , Chen Hong
Published: June 2021

Colon cancer is the third most common cancer in the United States. Approximately 90% of colon cancer deaths arise from the metastasis of primary tumors. Aberrant expression of Wnt5a, one of the WNT signaling factors, has been reported during colon cancer development and progression. This study tested the hypothesis that silencing of Wnt5a contributed to the colon cancer metastasis. We used the cell line models SW480 and SW620, which are poorly and highly metastatic colon cancer cell lines, respectively. SW480 was established from a primary human colon cancer, and SW620 was from a lymph node metastasis in the same patient. Wnt5a mRNA expression was more than 1,000 times lower in the metastatic SW620 than in SW480. Chromatin immunoprecipitation showed less association of RNA polymerase II with Wnt5a promoter region in SW620 compared to SW480, confirming the low transcriptional activity in SW620. There were much lower levels of acetylated histone H3 and H4 at the promoter region of Wnt5a. Although the demethylation reagent 5‐aza‐cytidine caused induction of Wnt5a expression, methylation of the promoter CpG island was not affected. Histone deacetylase inhibitors trichostatin A and sodium butyrate both significantly increased the mRNA expression of Wnt5a in SW620. In conclusion, our study suggested that the silencing of Wnt5a in highly invasive colon cancer might result from decreased histone acetylation.
Epigenetics in Maize: Using a novel PCR technique to sequence bisulfite treated DNA
Authors: Staltari Giuseppe
Published: June 2021

Epigentics is a form of gene regulation that involves a change in phenotype that is due to either a change in chromatin structure or DNA modification but does not cause a direct change in DNA sequence. One specific epigenetic phenomenon, paramutation, describes an epigenetic interaction between alleles of the same locus wherein one allele causes a change in expression of another. This research used bisulfite sequencing to determine the pattern of cytosine methylation in a promoter region in the r1 locus. The allele that undergoes silencing, R‐r:standard, is composed of two transcriptional units in opposing orientation, which flank a promoter region called Sigma. Primer bias was minimized through a novel PCR protocol that “jumps” from specific annealing temperatures. This protocol has proved to be successful, and four epigenetic profiles were sequenced and characterized.
Role of Glutathione S‐Transferase Activity in Endoplasmic Reticulum Chaperone DsbA‐L in the Assembly of Adipocyte Homrone Adiponectin
Authors: Kartchner Laurel B. , Malinowski Pamela , Tsao Tsu‐Shuen
Published: June 2021

Adiponectin is an adipocyte‐derived hormone with anti‐inflammatory and insulin‐sensitizing actions. Adiponectin circulates as trimers, hexamers, and higher molecular weight (HMW) species. Studies have shown HMW adiponectin is the most active, but the assembly of HMW adiponectin is poorly understood. DsbA‐L is a protein present in the endoplasmic reticulum (ER) that has been shown to increase production of HMW adiponectin. DsbA‐L is also known as mGST‐k with glutathione S‐transferase activity (GST). To determine the mechanism by which DsbA‐L promotes HMW adiponectin assembly, we examined whether DsbA‐L's ability to increase HMW adiponectin is dependent upon its GST activity. DsbA‐L with S16A substitution in the critical SXXS motif could not promote HMW adiponectin oligomerization but S19A substitution could. We recombinantly expressed and purified his‐tagged wild‐type (WT), S16A, and S19A DsbA‐L using nickel affinity chromatography and will test whether they have GST activity. If GST activity is found in WT and S19A DsbA‐L but not in S16A DsbA‐L, then it supports the hypothesis that GST activity is necessary for increased adiponectin oligomerization due to DsbA‐L. If S16A DsbA‐L retains normal GST activity, then it likely serves as a chaperone for adiponectin. These studies could potentially uncover the causes responsible for decreased concentrations of HMW adiponectin associated with type 2 diabetes.
Potential Role of PDE3B in Acquisition of Brown Fat Characteristic by White Adipose Tissue in Mice
Authors: Guirguis Emilia , Hockman Steven , Manganiello Vincent
Published: June 2021

Cyclic nucleotide phosphodiesterase 3B (PDE3B) regulates energy metabolism. PDE3B knockout (KO) mice in SvJ129 background have less white adipose tissue (WAT) than their wild type (WT) counterparts. Furthermore, their white fat shows characteristics of brown adipose tissue (BAT). This phenotype is less pronounced in PDE3B KO mice in C57/BL6 background, which is prone to obesity. We have studied genetic and protein profiles of both KO strains to dissect genes involved in WAT transdifferentiation. Thus, certain BAT‐genes, e.g. peroxisome proliferator‐activated receptor (PPAR) á and PRDM16 were up‐regulated in KO as compared to WT in both strains. However, increased expression of BAT‐related uncoupling protein (UCP) 1 and PPARã coactivator (PGC) 1á, as well as decreased expression of WAT‐related p107 and receptor interacting protein (RIP) 140, were seen only in SvJ129 PDE3B KO. After treatment of C57/BL6 mice with the â3‐adrenergic agonist, CL316243, expression of WAT differentiation program genes, e.g. RIP140 and p107, was reduced. In response to CL316243, some BAT‐genes were up‐regulated (CCAAT‐enhancer‐binding protein â, cyclooxygenase‐2); others, down‐regulated (PRDM16, C‐terminal binding protein). Importantly, proteins related to fatty acid oxidation and thermogenesis (PGC1á, PPARä, UCP1) were increased after treatment of C57/BL6 PDE3B KO mice with CL316243.
Ketone body utilization is regulated by leptin signaling in hypothalamic neuron
Authors: Yamasaki Masahiro , Hasegawa Shinya , Narishima Ryota , Fukui Tetsuya
Published: June 2021

Previously, we showed that the gene expression of acetoacetyl‐CoA synthetase (AACS), the ketone body‐utilizing enzyme for lipid synthesis, was suppressed by leptin deficiency‐induced obesity in white adipose tissue. In this study, to clarify the effects of leptin on ketone body utilization in the central nervous system, we examined the effects of leptin signaling on AACS expression. In situ hybridization analysis of ob/ob and db/db mice revealed that AACS mRNA level was reduced by leptin deficiency in the arcuate nucleus and ventromedial hypothalamic nucleus in hypothalamus but not in other brain regions. Moreover, Leptin increased AACS mRNA level both in primary mouse neural cells and in neural cell line (N41). In N41 cells, AACS level was decreased by AMPK activator (AICAR) but increased by its inhibitor (compound C). These results suggest that the up‐regulation of AACS expression by leptin is due to the suppression of AMPK activity via neural leptin signaling and that the deficiency of this regulation may be responsible for neurological disorders in central appetite control.
Analysis of altered protein trafficking in schizophrenia by targeted LC‐SRM/MS
Authors: Peterman Scott , Prakash Amol , MacDonald Matthew , Ciccimaro Eugene , Banerjee Anamika , Blair Ian A , Hahn Chang‐Gyu
Published: June 2021

N‐methyl‐d‐aspartate (NMDA) receptor hypofunction is currently a leading hypothesis for the etiology of schizophrenia. Recently, our lab directly observed decreased NMDA receptor activity in postmortem prefrontal cortex tissues of patients and linked this dysfunction to altered protein‐protein interactions at the receptor. The NMDA receptor complex is composed of an estimated 100 proteins and is localized to the post‐synaptic density, a thickening of the cell wall at the dendrite rich in receptors, scaffolding proteins and signaling molecules. We hypothesize that alterations to trafficking of the receptor and members of the receptor complex contribute to this dysfunction. To test this hypothesis we have developed a liquid chromatography – selected reaction monitoring/mass spectrometry (LC‐SRM/MS) method utilizing both synthetic stable isotope labeled peptide standards and a surrogate Stable Isotope Labeled Proteome (SILAP) derived from mouse brain tissue labeled with L‐Lysine (13C6). We have used this method to quantify 30 target proteins across several sub‐cellular fractionations of human postmortem pre‐frontal cortex from schizophrenic and matched control subjects. To date we have identified several scaffolding proteins with altered trafficking. We are currently investigating an additional 150 proteins.
Large‐scale phosphoproteomic profiling of native rat renal collecting duct epithelium: Tyrosine phosphorylation
Authors: Zhao B , Pisitkun T , Hoffert J , Chou CL , Knepper M
Published: June 2021

To systematically identify the phosphotyrosine (pY) sites in the renal collecting duct epithelium, we used LC‐MS/MS (LTQ‐Orbitrap) after pY peptide enrichment. Suspensions of renal inner medullary collecting ducts (IMCDs) from rat kidneys were pre‐incubated for 10 min with a physiological saline solution to which was added 0.1 mM sodium pervanadate to maximize tyrosine phosphorylation. Isolated proteins were subjected to pY immunoprecipitation followed by Ga3+‐immobilized metal affinity chromatography. Using target‐decoy analysis to limit false positives to <1%, we identified 503 unique pY peptides, corresponding to 273 phosphoproteins, which have been added to the online IMCD Phosphoproteome Database (http://goo.gl/oYKu). Site/sequence conservation analysis using custom software indicated that over 84% of the sites are highly conserved, suggesting functional roles. Analysis of the pY‐containing protein set relative to the whole IMCD transcriptome showed highly significant enrichment of proteins present in the KEGG “Adherens Junction” Pathway and highly significant enrichment of proteins with the following Gene Ontology Biological Process terms: “cell adhesion”, “actin cytoskeletal organization”, and “cell migration”. These associations point to likely roles of tyrosine phosphorylation‐dependent signaling pathways in the renal collecting duct epithelia. (BZ was a 2010 NIBIB‐BESIP summer intern from the U. of Michigan.)
Mapping‐based temporal pattern mining algorithm (MTPMA) identifies unique clusters of phosphopeptides regulated by vasopressin in collecting duct
Authors: Saeed Fahad , Hoffert Jason , Pisitkun Trairak , Knepper Mark
Published: June 2021

We developed a new algorithm, MTPMA, for clustering time‐course patterns following step‐inputs in biological systems. We tested the algorithm with data from large‐scale quantitative phosphoproteomics experiments done as follows: Inner medullary collecting duct (IMCD) samples were incubated in the presence or absence of 1nM dDAVP (vasopressin) for 0.5, 2, 5, and 15 minutes (N=3 pairs) followed by LC‐MS/MS‐based phoshoproteomic analysis. Quantification used 8‐plex iTRAQ and commercially available software. Of the 12,533 phosphopeptides identified, 3,298 were found in at least 2 out of 3 time courses and had quantifiable iTRAQ ratios. These phosphopeptides were analyzed with MTPMA in order to identify groups that changed in abundance with similar temporal responses after vasopressin addition. The algorithm maps the data from a Cartesian plane to a discrete binary plane and uses an efficient dynamic programming technique to mine similar patterns after mapping. The mapping allows clustering of similar time‐courses that are temporally closer to each other than to other time‐courses. The algorithm identified 30 clusters of phosphopeptides with distinct temporal profiles, including peptides that significantly increased or decreased in abundance in response to vasopressin. These time‐course clusters provide a starting point for modeling the signaling network involved.
The urinary biomarker database
Authors: Shao Chen , Wei Lilong , Zhu Lisi , Yang Fan , Li Menglin , Jia Lulu , Mu Yi , Wang Jiangning , Guo Zhengguang , Li Xundou , Zhang Dan , Sun Wei , Gao Youhe
Published: June 2021

Urine is an important source of biomarkers. Present urinary biomarker studies face two major problems. First, since it is not possible to compare all diseases that may cause the changes of urinary proteome, a large number of currently identified biomarker candidates do not have disease‐specificity. Second, the confidences of some biomarker candidates are not satisfied, due to the limits of current experimental techniques or the biological variations of urinary proteome among healthy people. Here we set up the Urinary Biomarker Database by comprehensively collecting and manually curating of all published literatures of urinary biomarker discovery. Researchers can analysis their data conveniently and comprehensively by comparing them with experimental results stored in our website, so that to study physiological functions of urinary system or select the most likely biomarker candidates for further validation, with reduced experimental costs and higher research efficiency. By analyzing the collected data, current achievements and directions for future studies of urinary biomarker discovery can be figured out. The URL of our database is http://www.bmicc.org/web/english/search/hupd.
Differential proteomic analysis of the brain synaptic plasma membrane proteins from DHA‐deficient and adequate mice using 16O/18O labeling
Authors: Sidhu Vishaldeep K , Huang Bill , Kim Hee‐Yong
Published: June 2021

Polyunsaturated fatty acids, especially docosahexenoic acid (DHA, 22:6n‐3) plays an important role in normal brain development in mammals. DHA promotes hippocampal neurite growth and synaptogenesis while DHA‐ deficiency causes impairments in hippocampus‐dependent learning and memory. In this study, we compared the synaptic plasma membrane (SPM) proteins obtained from DHA‐adequate and deficient mice using 16O/18O labeling to provide insights into the role of DHA in synaptic protein expression and function. Synaptosomal preparations from DHA‐deficient and adequate mice cortex were subjected to osmotic shock to obtain SPM. The SPM fraction was analysed by SDS‐PAGE coupled to nanoLC‐MS/MS. For quantification, we differentially labeled the peptide samples from DHA‐adequate and deficient mice using O16/O18 labeling technique. The heavy to light ratios (18O/16O) were calculated using Mascot Distiller. This strategy allowed us to detect more than 400 proteins from the SPM fraction including various receptors, kinases and transporter proteins. The 18O/16O ratios revealed differential expression of several pre‐ and postsynaptic proteins involved in neurotransmission. Some of these proteins were validated by western blot analysis. Altered levels of synaptic proteins important for synaptic transmission suggest a role of DHA in proper brain development and function.

National Institutes of Health, MD.
Protease networks and pathway interconnectivity in Gram‐positive bacteria
Authors: Panikkar Anupama , Knapp Merrill , Kodukula Krishna , Galande Amit , Talcott Carolyn
Published: June 2021

The emergence of Gram‐positive drug resistant bacteria such as methicillin‐resistant Staphylococcus aureus (MRSA), Streptococcus pneumoniae and Enterococcus represent a serious public health problem. Novel drug targets are needed and bacterial proteases are potential candidates as they are implicated in virtually every important biological process. To understand how proteases participate in pathology, it is important to elucidate the degradome, its function and its effect on biological networks. With this goal, we are developing a comprehensive computational model of protease networks and pathway interconnectivity of multiple Gram‐positive bacteria using Pathway Logic. Pathway Logic is a systems biology approach to biological processes as integrated systems rather than isolated parts based on formal methods and rewriting logic. As an initial step, we developed a model of interaction networks with 30 proteases from 7 different Gram‐positive bacteria. Our results demonstrate interconnectivity between proteases. The comparison feature of Pathway Logic tool identified key proteases participating in multiple pathways within a large network and across multiple networks that could be “choke points”. Our results suggest novel roles for proteases that need to be validated using experimental methods. This research is supported by funding to SRI International from the Commonwealth of Virginia.
The Functional Amino Acid Navigator
Authors: Cargill Jason K , Solimeo Renee , Gomez Sal , Palenchar Peter
Published: June 2021

The purpose of Functional Amino Acid Navigator (FAN) is to create a database that captures information about how enzymes function with respect to how they interact with small molecules, and then use that database to address questions related to how enzymes function, genome annotation and evolutionary changes in allosteric sites. FAN can give a detailed picture of enzymes interacting with small molecules at the atomic level where possible. The data in FAN is gathered from primary published literature detailing specific enzymes function. Information can also be added to FAN by outside individuals after registration. FAN will also analyze BLAST protein sequence alignments to help ensure correct protein annotation by identifying amino acids for which FAN contains a function and determining if they are conserved based on the BLAST alignment. The data collected in FAN has been used to compare and contrast changes in allosteric sites as compared to catalytic sites from multiple species to gain information about evolutionary adaptations.
Genetic effect of single‐nucleotide polymorphisms in the PPARGC1B gene on airway hyperreactivity in asthmatics
Authors: Lee Shin‐Hwa , Jang An‐Soo , Park Sung Woo , Park Jong‐Sook , Kim Yang Ki , Uh Soo‐Taek , Kim Yong Hoon , Chung Yup , Park Byung‐Lae , Shin Hyung Doo , Park Choon‐Sik
Published: June 2021

Peroxisome proliferator‐activated receptor gamma coactivator 1 beta (PPARGC1B) is a co‐activator for intracellular receptors such as the ER, PPAR, and GR, which are involved in asthma development. Association of single‐nucleotide polymorphisms (SNPs) in PPARGC1B gene with risk of asthma and airway hyperreactivity (AHR) was investigated. Direct sequencing of DNA from 24 Korean was performed to identify PPARGC1B SNPs. Genotyping was done in 264 controls and 949 asthmatics. 19 SNPs were identified, and 7 SNPs were genotyped. No significant difference existed in the distribution between the asthmatics and controls. However, allele frequency of −427C>T showed a significant association with logPC20 methacholine values in the asthmatics (p = 0.005). Real‐time PCR demonstrated higher PPARGC1B mRNA levels in asthmatics having the −427CC allele than in those having −427TT or CT alleles. Luciferase reporter assays revealed that −427C allele caused higher promoter activity than −427T allele. EMSA demonstrated that −427C allele exhibited stronger binding activity to a nuclear protein in 293T cells than did the −427T allele. The polymorphisms of −427C>T on the promoter the PPARGC1B gene may affect the development of AHR through the modulation of PPARGC1B gene products.
Improvements to the JProfileGrid software for visualizing very large multiple sequence alignments
Authors: Roca Alberto I , Abajian Aaron C
Published: June 2021

In 2008 the JProfileGrid software was released for visualizing multiple sequence alignments of protein families with hundreds of sequences (http://www.biomedcentral.com/1471‐2105/9/554). Upon analyzing virus protein families such as influenza hemagglutinins with thousands of sequences, it became apparent that the larger datasets were taxing certain design limits of the software. Here we describe new features of the JProfileGrid Java software to accomodate very large alignments. For example, the software now allows a user to sort and search the menu list of sequence names. In addition, an “overview” mode allows one to visualize the entire ProfileGrid. A new double‐pane window feature facilitates dissecting an alignment to detect structural patterns. The ProfileGrid window panes can be independently color‐coded and/or used for selecting subsets of sequences. Finally, the ProfileGrid can be saved as PNG image files for incorporation in sequence databases such as Pfam and the NCBI Influenza Virus Sequence Database. [AIR supported by the Erasmo Foundation (grant TSC13702).]
QUEEN OR WORKER? AN ESSAY ABOUT CASTES DETERMINATION IN Schwarziana quadripunctata (LEPELETIER, 1836) (HYMENOPTERA, APIDAE, MELIPONINI)
Authors: Hyodo Vivian Cristina Costa Castilho , Ribeiro Marcia Fátima , Imperatriz‐Fonseca Vera Lucia
Published: June 2021

Stingless bees are highly eusocial bees characterized by massive feeding. This food is offered to larvae in individual cells which are provided with liquid food. Their division in two groups (Melipona and Trigona) is also based in queen rearing in special larger cells, called royal cells in Trigona, and its absence in Melipona, where all bees (males, workers and queens) are reared in similar sized cells. Nevertheless, intermediate situations occur, as in Schwarziana quadripunctata, that produces queens of different sizes, reared in normal or in royal cells. We investigate three possibilities of caste determination in those bees. The brood cells volume, the quantity and the protein content of the larval food in individual cells, the size and protein content of the eggs produced by the queen (by Bradford essay). Data were analyzed concerning colony, seasons and colony condition (strong, medium and weak colonies). Results were also statiscally analyzed. We found variations in eggs' size and concentration of protein, in brood cells volume per colony. Moreover, extremely large variations were found in total protein concentration of larval food in individual cells. According to the analysis performed, there was a highly correlation on the size of queens to their produced eggs size.