bluuebunny/crossref_metadata_2025 huggingface dataset:
Replacing Arg70 by Histidine in The Cytochrome Aa3‐600 Menaquinol Oxidase from Bacillus Subtilis Changes The Nitrogen Interacting with The Semiquinone Formed at The Qh Site But Does Not Eliminate Catalytic Function
Authors: Yi Sophia , Taguchi Alexander , Dikanov Sergei , Gennis Robert
Published: June 2021

Cytochrome aa3‐600 is a terminal oxidase in the electron transport pathway that contributes to the electrochemical membrane potential by actively pumping protons. A notable feature of this enzyme complex is that it uses as its electron donor menaquinol instead of cytochrome c, when it reduces dioxygen to water. The enzyme stabilizes a menasemiquinone radical (SQ) at a high affinity site that is important for catalysis. One of the residues that interacts with the semiquinone is Arg70. We have made R70H mutant in the high affinity quinone binding site (Qh) and have characterized the menasemiquinone radical by advanced EPR in X‐ and Q‐band. The bound SQ state at Qh site has a strong isotropic hyperfine coupling (14A ~ 2.1 MHz) with a hydrogen bonded nitrogen. This nitrogen originates from a histidine side chain, based on determined quadrupolar parameters (κ = 0.36 MHz, η = 0.25) typical for protonated imidazole nitrogens. In contrast, the SQ stabilized in the wild type is hydrogen bonded with Nε from Arg70 side chain. The mutation also changes the number and strength of the hydrogen bonds in the SQ environment, based on the analysis of the 1H 2D ESEEM spectra. Despite the alterations in the immediate environment of the SQ, the R70H remains catalytically active. This contrasts to the equivalent mutation in the close homologue, the cytochrome bo3 ubiquinol oxidase from E. coli, where the R75H mutation eliminates function.
Anti‐Tumour Flavonoids Regulation on Oestrogen and Progesterone Receptor Genes in Cervical Cancer Cell Lines (HELA)
Authors: Fadaka Adewale , James Ayorinde , Magbagbeola Olubunmi , Alakija Abiola
Published: June 2021

Cervical cancer is the second most common cancer among women in the world. It is still the most common gynaecological cancer worldwide. Plants have remained an important source in the search for novel anti‐cancer compounds of which several polyphenolic flavonoids are interesting candidates. The present study was carried out to evaluate the modulatory effect of isolated flavonoids (free and bound) from Hibiscus Sabdariffa on oestrogen and progesterone receptor genes in cervical cancer cell lines (Hela). Growth inhibition concentration (IC50) assay was done using MTT cell viability assay. Hela cells were treated with 200µg/ml of both free and bound flavonoids respectively. Total RNA was extracted and mRNA was converted to cDNA; semi‐quantitative polymerase chain reaction using densitometric analysis was carried out for both oestrogen and progesterone receptor genes. The results obtained showed that oestrogen receptor gene was down regulated by both the free and bound flavonoids while there was an up regulation of the progesterone receptor gene. In conclusion, this study therefore provides the scientific evidence for the therapeutic genetic modulation of sex hormone receptor genes of cervical cancer.
ERCC1 and Thymidylate Synthase as Prognostic Biomarkers in Malignant Mesothelioma
Authors: Mezzapelle Rosanna , Miglio Umberto , Rena Ottavio , Mercalli Francesca , Veggiani Claudia , Paganotti Alessia , Buosi Roberta , Rinaldi Maurizio , Bianchi Marco , Boldorini Renzo
Published: June 2021

The standard of care for malignant pleural mesothelioma (MPM) is a combination of platinum compounds and Pemetrexed. Still, the median survival of MPM patients is low (about 12 months). Excision Repair Cross Complementing Group 1 (ERCC1) acts by removing DNA adducts formed by platinum compounds, whereas Pemetrexed inhibits Thymidylate Synthase (TS), which is involved in DNA synthesis.

Pleural biopsies were collected from 140 MPM patients (98 males and 42 females, mean age 68 years) before treatment initiation. Average disease specific survival was 13 months (range 1‐60). mRNA was extracted from formalin fixed paraffin blocks, reverse transcribed and assayed by qRT‐PCR. Immunohistochemistry with anti‐ERCC1 and anti‐TS mAbs was scored semi‐quantitatively.

Of 140 patients, 102 were treated, the remaining were not because of bad performance status or advanced age. Patients with low ERCC1 protein expression survived longer (24.3 vs 12.5 months, P=.03). Likewise, patients with low levels of TS mRNA had better survival (P=.01); there was no advantage, however, in the treated sub‐group, suggesting that Pemetrexed treatment and low TS expression had no additive value.

ERCC1 protein expression and TS gene expression both seem important prognostic markers; we suggest their evaluation in routine biopsies from MPM patients.
Association between DAPK1 Promoter Methylation and Cervical Cancer: a Systematic Review and a Meta‐analysis
Authors: Agodi Antonella , Barchitta Martina , Quattrocchi Annalisa , Maugeri Andrea , Vinciguerra Manlio
Published: June 2021

DNA methylation, a major epigenetic mechanism, is involved in various biological processes including cancer. Particularly, promoter methylation can inactivate Death‐Associated Protein Kinase1 (DAPK1), a tumor suppressor gene which is often methylated during the carcinogenesis of cervical cancer. In the present systematic review and meta‐analysis, we evaluated the association between DAPK1 promoter methylation and cervical cancer risk. A literature search, using the PubMED database, was performed to select studies published up to July 2014. Subgroups analyses were performed. A total of 27 studies were included. All studies evaluated DAPK1 promoter methylation in squamous cell carcinoma and 12 studies also in adenocarcinoma. The “gold standard” method of promoter methylation evaluation was methylation specific PCR. Twenty studies, evaluating DAPK1 promoter methylation both in tumor and in healthy controls samples, were included in the meta‐analysis (1092 from cancer patients and 837 from controls). Meta‐analysis showed a significant association between DAPK1 promoter methylation and cervical cancer (pooled OR: 21.20; 95% CI: 11.14, 40.35) with the random effects model. The association was confirmed in specific subgroups. In conclusion DAPK1 promoter methylation might play an important role in the pathogenesis of cervical cancer. The promoter methylation of DAPK1 is a frequent event in cervical carcinogenesis and may have potential clinical application as a cancer biomarker.

This work was conducted with the research support of Bench Srl, University of Catania.
TG‐interacting‐factor–induced malignant progression of hepatocellular carcinoma cells depends on superoxide production from Nox4
Authors: Huang Huei‐Sheng , Liu Zi‐Miao , Tseng Hong‐Yu , Su Fang‐Cheng
Published: June 2021

Hepatocellular carcinoma (HCC) is one of the most deadly malignancies worldwide due to its high recurrence rate, high metastatic potential, and resistance to drugs. Elucidation of the mechanisms underlying malignancy in HCC is needed to improve diagnosis, therapy, and prognosis. Previously, we showed that TG‐interacting factor (TGIF) antagonizes arsenic trioxide‐induced apoptosis of HepG2 cells and is associated with poor prognosis and progression of urothelial carcinoma in patients after radical nephroureterectomy. To determine whether TGIF plays a role in HCC tumorigenesis, we compared the expression of TGIF, its downstream targets, and ROS levels between HCC HepG2 cells and the more invasive SK‐Hep1 cells. Superoxide production, phosphorylation of c‐SrcY416 and AKTS473, and expression of TGIF and NADPH oxidase (Nox) were higher in invasive SK‐Hep1 cells than in HepG2 cells. TGIF‐overexpressing HepG2 xenograft tumors markedly promoted tumor growth and metastasis to the lungs. Overexpression of TGIF in HepG2 cells increased superoxide production from Nox4, MMP expression, invadopodia formation, and cellular migration/invasion ability. Conversely, knockdown of TGIF in SK‐Hep1cells attenuated these processes. Using gene knockdown and pharmacological inhibitors, we demonstrate that c‐Src/AKT is the upstream signaling that regulates TGIF‐induced Nox4 activation and subsequent superoxide production. Taken together, our results implicate TGIF as a potential biomarker for prognosis and target for clinical therapy in patients with advanced HCC.
Structural Basis for Akt Translocation to the PM in Breast Cancer Cells
Authors: Agamasu Constance , Ghanam Ruba , Saad Jamil
Published: June 2021

Akt is a serine/threonine kinase that plays a key role in the regulation of cell survival, apoptosis and oncogenesis. The translocation of Akt to the plasma membrane (PM) for phosphorylation and subsequent activation is a critical step in the Akt activation pathway. It has been established that membrane binding of Akt is mediated by direct interactions between it's pleckstrin homology domain (PHD) and lipids such as phosphatidylinositol‐(3,4,5)‐trisphosphate (PI(3,4,5)P3). There is now evidence that Akt activation in breast cancer cells is also modulated by the calcium‐binding protein, calmodulin (CaM). Upon epidermal growth factor (EGF) stimulation of these cells, CaM co‐localizes with Akt at the PM to promote membrane anchoring and activation. Although previous studies have shown that both PI(3,4,5)P3 and CaM may modulate Akt activation, the molecular mechanisms governing these interactions have not been well characterized. We hypothesize that PI(3,4,5)P3 and CaM act in synergy by binding to Akt(PHD) simultaneously to facilitate anchoring to the PM. In this study we employ biochemical, biophysical and nuclear magnetic resonance (NMR) techniques to characterize the interaction between CaM and PI(3,4,5)P3 to Akt(PHD). Our results demonstrate that there is a direct interaction between CaM and Akt(PHD) and suggest that Akt(PHD) undergoes significant conformational changes upon CaM binding. We also provide evidence that both PI(3,4,5)P3 and CaM bind to Akt(PHD) simultaneously. Elucidation of the structural basis for Akt‐CaM and Akt‐PI(3,4,5)P3 interaction is critical for understanding the activation mechanism, which may provide insights for new potential targets to control the pathophysiological processes of cell survival.
Biological Effects of Rosemary Extract in Human Lung Cancer Cells
Authors: Moore Jessy , Naimi Madina , Tsiani Evangelia
Published: June 2021

Lung cancer accounts for the most cancer‐related deaths. Non‐small cell lung cancer (NSCLC) represents a very aggressive form of lung cancer and is characterized by resistance to chemo‐ and radiotherapy thus, finding new ways to inhibit NSCLC cell proliferation and survival is very important. Many phytochemicals have emerged as having potential chemotherapeutic effects and rosemary extract (RE) has been shown to have anticancer properties in vitro and in vivo however, limited evidence exists regarding its effect in NSCLC cells. The objectives of the present study were to examine the effects of RE on NSCLC cell proliferation, and investigate its effects on the phosphatidylinositol 3‐kinase (PI3K)‐Akt and AMP‐activated protein kinase (AMPK) signaling pathways which regulate cell proliferation and survival. Various NSCLC cell lines (A549, H1299, H460) with different histologies and mutations were treated with varying concentrations of RE. Cell proliferation was measured by crystal violet assay, clonogenic assays were performed to examine the effects of RE on cell survival, and immunoblotting with phospho‐ specific antibodies was performed to examine signaling events. RE dose‐dependently inhibited cell proliferation in all cell lines. Significant inhibition of cell proliferation was seen at concentrations as low as 5µg/mL. Maximum inhibition (% of untreated control ± SE) was seen at 50µg/mL (21.9% ± 2.6, p<0.001) in A549, 50µg/mL (30.7% ± 3.0, p<0.001) in H460, and 25µg/mL (23.3% ± 1.3, p<0.001) in H1299 cells. RE (50µg/mL) inhibited total and phosphorylated levels of Akt, while phosphorylated levels of AMPK were enhanced. Clonogenic survival was also inhibited by RE. Our data suggest that RE may have potent anticancer properties in NSCLC and warrants further investigation.
The Role of IL33 in Glioblastoma Multiforme
Authors: Demircan Kadir , Yukselten Yunus , Bal Merve , Bunsuz Merve , Akcora Yildiz Dilara , Sunguroglu Asuman , Ugur Hasan
Published: June 2021

Gliomas show the majority of primary brain tumors in adults and are part of the most malignant tumors. Although there are surgical resection and conventional therapy,Glioblastoma multiforme (GBM) patients' survivals are approximately 12‐15 months.Cytokines secreted by either cancer cells themselves or by the surrounding cells and infiltrating microglia/macrophages play a critical role in glioma malignancy. Lately IL‐33 is associated to tumorgenesis and are cytokines which play a key role on tumor pathogenesis. The aim of study is to search protein levels of IL‐33 and ADAMTS5, which is also effective at tumorigenesis in GBM.Western blot analysis showed over‐expression both of IL‐33 and ADAMTS5 in GBM.According to these results we showed that expression of IL‐33 and ADAMTS5 in GBM increased simultaneously. We treated cytokine of IL‐33 in GBM primary cell culture to prove these finding in vitro conditions. 0‐10‐30 ng/ml IL33 cytokine cells were treated for 48 h in order to show the effect on GBM primary cells. Exposure to IL‐33 treatment for 48 h resulted in dose dependent increase of ADAMTS5 protein levels in human GBM primary cells.Cytokines secreted by either cancer cells themselves or by the surrounding cells and infiltrating microglia/macrophages play a critical role in glioma malignancy. Our present findings, the abundant expression of proliferation‐associated IL‐33 is a key characteristic of human GBM primer cells, and these are involved in enhanced cell proliferation and tumorigenicity. Thus, the present study indicated that high expression of IL‐33 in glioblastoma cells may contribute to glioma progression.
Anti‐Proliferative Effects of Selected Dietary Polyphenols on Human Colon Carcinoma Cell Line, T84
Authors: Javed Fatima , Khan Nabihah , Detloff Sally Jo , Sarathy Jayashree , Ronkainen Niina
Published: June 2021

Polyphenols are naturally occurring compounds in plants which are typically involved in defense against ultraviolet radiation or pathogens. Previous studies have shown that various polyphenols have anti‐proliferative properties in a variety of human cancers. The purpose of this study is to determine the effect of Quercetin (QC), Epigallocatechin‐3‐gallate (EGCG), Catechin and Resveratrol (RVL) on cell death and apoptosis in human colon carcinoma, T84, cells.

Cell death and apoptosis was measured by using Annexin V: FITC Apoptosis Detection Kit I and a flow cytometry. Short term (1 hour) and long term (overnight; O/N) effects were observed. Cells were grown in 6 well plates, serum starved O/N and treated with different concentrations of polyphenols for varying time. Linear Sweep Voltammetry (LSV) was used to electrochemically characterize the antioxidant capacity and to quantify active RVL and QC (the polyphenols acting as reducing agents) for the time frame of the study.

While EGCG and Catechin did not have an effect, RVL and QC showed an interesting dose and time‐dependent change in cell death and apoptosis. O/N exposure to 200 µM QC caused an increase in cell death and apoptosis (control: 5.5+ 1.0%; QC: 15.5+ 4.3%). However, 30 µM RVL increased cell death and apoptosis at 1 hr (control: 4.3+ 0.8; RVL: 13.9+ 3.2) and returned back to control levels with time. Based on LSV, QC has a higher antioxidant capacity than RVL. However, the antioxidant effect of RVL decreased over time. In summary, QC and RVL both have anti‐proliferative properties with QC having prolonged effects in human colon carcinoma cells. Thus, a combination of polyphenols might have a better effect in preventing colon cancer.
Gene Expression Profiling of Glycosaminoglycan Drived from G. bimaculatus in High Fat Dieted Rat
Authors: Ahn Mi Young , Hwang Jae Sam , Yun Eun Young
Published: June 2021

The glycosaminoglycan (GAG) from cricket (Gryllus bimaculatus) was studied as a potential health supplement. Antiatherosclerotic and antilipemic effects of the GAG (5 or 10 mg/kg) of G. bimaculatus (Gb) were investigated in 15 mo.‐aged Wistar rats treated with glycosaminoglycan for over a month. This GAG of Gb produced meaningful anti‐edema effect showing inhibition of C‐reactive protein (CRP). Also, the weight of abdominal and epididymidal fat was reduced in conjunction with a mild increase in body weight in the rats for over 1‐month. Furthermore, the sero‐biochemical parameters showing anti‐hyperlipdemia effect decreased the levels of Phospholipid, AST, ALT, total cholesterol, and glucose in dose dependent manner. In addition to plasma study, anticoagulant and antithrombotic effects in the GbGAG high fat Wistar rats, platelet, thrombin time, prothrombin time, and Factor I were more increased than control group or Statin (positive) group. Based on these results, GbGAG was shown to prevent or treat fatty liver or hyperlipidemia in high‐fat dieted rats. Furthermore, the GAG treated rat group (10 mg/kg) compared to control, showed that 588 genes (test/control ratio>2.0) including lipocalin 2 (Lcn2), alpha 2‐macroglobulin (A2m) and RT1 class Ib, and locus EC2 (RT1‐EC2) were up‐regulated; and 569 genes (test/control ratio>0.5) including stearoyl‐Coenzyme A desaturase 1 (Scd1), potassium intermediate/small conductance calcium‐activated channel, subfamily N, member 2 (Kcnn2) and ATPase, H+ transporting and lysosomal accessory protein 2 (Atp6ap2) were down‐regulated. Data suggest Lcn2 and A2m related ant‐high fat, were up‐regulated by GbGAG treatment which was indicated to be potential therapeutic markers for anti‐high fat disease.
Arfaptin‐1 Negatively Regulates Arl1‐mediated Retrograde Transport
Authors: Yu Chia‐Jung , Huang Lien‐Hung , Lee Wei‐Chung , You Shu‐Ting , Cheng Chia‐Chen
Published: June 2021

Thesmall GTPase Arf‐like protein 1 (Arl1) is well known for its role in intracellular vesicular transport at the trans‐Golgi network (TGN). In this study, we used differential affinity chromatography combined with mass spectrometry to identify Arf‐interacting protein 1b (arfaptin‐1b) as an Arl1‐interacting protein and characterized a novelfunction for arfaptin‐1 (including the arfaptin‐1a and 1b isoforms) in Arl1‐mediated retrograde transport. Using a Shiga‐toxin subunit B (STxB) transportation assay, we demonstrated that knockdown of arfaptin‐1 accelerated the retrograde transport of STxB from the endosome to the Golgi apparatus, whereas Arl1 knockdown inhibited STxB transport compared with control cells. Arfaptin‐1 overexpression, but not an Arl1 binding‐defective mutant (arfaptin‐1b‐F317A), consistently inhibited STxB transport. Exogenous arfaptin‐1 expression did not interfere with the localization of the Arl1‐interacting proteins golgin‐97 and golgin‐245 to the TGNand vice versa. Moreover, we found that the N‐terminal region of arfaptin‐1 was involved in the regulation of retrograde transport. Our results show that arfaptin‐1 acts as a negative regulator in Arl1‐mediated retrograde transport and suggest that different functional complexes containing Arl1 form in distinct microdomains and are responsible for different functions.
The Role of En­doplasmic Reticulum–Associated Degradation in the Regulation of a Potassium Channel Associated with Type II Bartter Syndrome
Authors: O'Donnell Brighid , Mackie Timothy , Brodsky Jeffrey
Published: June 2021

Type II Bartter Syndrome is an autosomal recessive disease caused by a loss of function mutation in the human KCNJ1 gene. This gene encodes the Renal Outer Medullary Potassium channel (ROMK), the primary route for K+ efflux within the nephron. To maintain K+ homeostasis, renal epithelial cells must regulate the apical surface density of ROMK. The molecular defects in ROMK channel trafficking and regulation that cause Bartter Syndrome are poorly defined, but we propose that some of the disease‐causing mutations lead to protein misfolding. Because misfolded membrane proteins are subject to ER quality control, we asked if some mutations target ROMK for En­doplasmic Reticulum–Associated Degradation (ERAD) in a yeast model. The ability of wild‐type and Bartter ROMK mutants to rescue low K+ sensitivity was investigated. Yeast lacking Trk1 and Trk2 K+ channel proteins were transformed with mutant ROMK, serially diluted, and plated onto media of various K+ concentrations. Growth was scored via imaging, and relative ROMK expression levels were assayed by Western blot. Wild type ROMK and Bartter mutant stability was analyzed by cycloheximide chase analysis. To half of each culture, MG132 was added to inhibit the proteosome, and protein translation was halted with cyclo­heximide. Aliquots were collected at various time points, and relative levels of ROMK were assessed by Western blot. We found that yeast expressing ROMK Bartter mutants exhibit growth defects on low K+ media, and the mutant proteins are less stable compared to wild‐type ROMK. Degradation is proteasome‐mediated. Bartter Syndrome mutants increase targeting for ERAD when compared to wild‐type ROMK.

NIH Grant 5T32DK071492‐09 and R01GM075061
Determining the Role of Lafora Disease Patient Mutations on Glycogen Dephosphorylation.
Authors: Turner Benjamin , Brewer Kathryn , Rathagala Madushi , Vander Kooi Craig , Gentry Matthew
Published: June 2021

Lafora Disease (LD) is fatal, congenital neurodegenerative epilepsy caused by mutations in the gene that encodes the protein laforin. These mutations disrupt glycogen metabolism in cells causing accumulations of malformed glycogen inclusions called Lafora Bodies (LB). LBs inhibit normal neuronal functions, leading to epilepsy, dementia, and death by age 25. Laforin contains a carbohydrate binding module (CBM) and a dual specificity phosphatase (DSP) domain. To date, more than 40 laforin point mutations have been identified in LD patients. However, how each of these mutations leads to LB formation and/or how they impact glycogen dephosphorylation is not known. Our lab recently determined the 3‐dimensional structure of laforin using X‐ray crystallography. The laforin X‐ray crystal structure reveals that these patient point mutations localize in four different regions within the DSP and CBM domains suggesting multiple possible disease mechanisms. To determine the role of each of these mutations on glycogen dephosphorylation, we employed a generic pNPP phosphatase activity assay and a biologically relevant assay utilizing glycogen to measure glycogen phosphatase activity. The glycogen phosphatase assay was very specific and faithfully reported both wildtype and mutant laforin phosphatase activity. Mutations that are located in the CBM‐DSP interface and the DSP‐DSP dimer interface resulted in 50‐80% decrease, suggesting the importance of these regions in optimal laforin activity. We found a 70‐90% decrease in glycogen phosphatase activity for mutations that are localized in the CBM binding site and the DSP active site.
The Hedgehog Signaling Pathway in Isodiametra Pulchra and Stenostomum Virginianum
Authors: Minor Marquet , Smith Julian
Published: June 2021

The purpose of this study is to compare the Hedgehog Signaling Pathway (Hh) in the acoelmorphan, Isodiametra pulchra (Ipul) and the flatworm Stenostomum virginianum (Svirg). The former occupies a primitive placement in the bilaterians, while the latter is place in a primitive position in the flatworms. Certain organisms are cilia‐dependent in their Hedgehog signaling, while others conduct signaling independently of cilia. Protostomes (Svirg), for example, have cilium‐independent signaling, whereas deuterostomes (Ipul) require cilia to conduct signaling. Proteins associated with the Hh pathway were identified from the unpublished (Univ. of Innsbruck) transcriptome for Ipul, including orthologues for Hedgehog and multiple possible orthologues for Patched. Primers have been designed and tested to verify the existence of the transcripts listed. The transcriptomic sequences of wild‐type animals (NC coast) displayed both synonymous and non‐synonymous mutations when compared to the transcriptome of the animals cultured in Innsbruck. Next, qPCR primers will be designed and tested to analyze Patched expression, since it is a marker for Hh signaling. Animals treated with cyclopamine, a Hh pathway inhibitor, should display a reduction in Patched expression; this technique will allow us to identify the correct Patched orthologue. The Hh pathway plays a critical role in development in many organisms, including humans. The importance of this study is to establish the relationship between the Hh pathway and cilia. This can ultimately lead to control of abnormalities associated with both cilia and the Hh pathway, and prevent disorders in humans. Funded by SC‐INBRE.
CG10126, A Calcium‐Binding Microtubule‐Associated Protein, Promotes Mitosis during Drosophila Development
Authors: Nie Qian , Spencer Susan
Published: June 2021

The Epidermal Growth Factor Receptor (EGFR) is a receptor tyrosine kinase that regulates signaling pathways critical for development and proliferation in Drosophila. EGFR activity is also highly upregulated in a number of human cancers, making identification of its downstream targets important for understanding both basic cellular processes and disease states. We have identified CG10126, a Drosophila homolog of the human protein Calcyphosine‐like, as a transcriptional target of EGFR signaling. Calcyphosine encodes a calcium‐binding protein regulated in several human cancers, but little is known of its function. Here we examine the role of CG10126 during Drosophila development. We expressed RNA interference constructs at various points in development. Reducing the level of CG10126 in eye imaginal discs produced adults with small eyes, while reducing its level ubiquitously in embryos was generally lethal. To further examine the role of CG10126, we used mass spectrometry to identify the binding partners of CG10126 in the presence or absence of calcium. Specifically in the presence of calcium, CG10126 bound α‐ and β‐tubulin and several spindle assembly proteins, suggesting that it may influence formation of the mitotic spindle. Consistent with this, embryos with reduced CG10126 show a diminished number of mitotic nuclei and abnormal spindle structures. Calcium is indispensable for cell cycle regulation; our results suggest that calcium waves present during mitosis may enable CG10126 to influence microtubule dynamics and spindle assembly. Together, our studies suggest that CG10126, a downstream target of EGFR signaling, is a microtubule‐associated protein that affects cell proliferation by regulating mitotic spindle formation.
Structure and Function of the Vibrio cholerae Master Virulence Regulator ToxT
Authors: Cruite Justin , Kovacikova Gabriela , Skorupski Karen , Taylor Ronald , Kull F. Jon
Published: June 2021

The epidemic diarrheal disease cholera is caused by the ingestion of food or water contaminated with the gram‐negative bacterium Vibrio cholerae. Cholera toxin (CT) and the toxin‐coregulated pilus (TCP) are the two primary virulence factors produced by V. cholerae. The expression of both CT and TCP is activated by the AraC/XylS family transcription regulator ToxT. Previously, the crystal structure of ToxT revealed the presence of the unsaturated fatty acid (UFA), palmitoleic acid, in the ligand binding pocket. Unsaturated fatty acids, such as palmitoleic acid, are components of bile and inhibit ToxT DNA binding activity, allowing virulence to be controlled in response to the host environment. In contrast to UFAs, bicarbonate has been shown to enhance ToxT DNA binding affinity and increase virulence gene expression. In a first step towards gaining further insight into 1) how ToxT binds to DNA and activates expression, and 2) how binding to UFAs leads to conformational changes that inactivate ToxT, ToxT has been crystallized in complex with the ToxT binding region of the tcpA promoter in the presence of bicarbonate.
Tau phosphosignatures discriminate Alzheimer Disease from other tauopathies
Authors: Seyfried Nicholas , Dammer Eric , Ren Rujing , Gearing Marla , Duong Duc , Lah James , Levey Allan
Published: June 2021

Neurofibrillary tangles (NFTs) comprised of highly phosphorylated tau are a core pathological feature of Alzheimer's disease (AD) and several other neurodegenerative diseases collectively termed tauopathies, which include progressive supranuclear palsy, corticobasal degeneration and certain frontotemporal dementias. There are six isoforms of human tau in brain and over 40 serine (S), threonine (T), and tyrosine (Y) tau phosphorylation sites have been identified. Phosphorylation of tau at specific S/T/Y sites is hypothesized to cause both functional deficits as well as gain‐of‐function toxicity that ultimately lead to NFT formation, synapse loss and cognitive dysfunction. Despite being clinically distinct diseases it remains unclear whether site‐specific tau phosphorylation differs between AD and other tauopathies. To address this question we employed immobilized metal affinity chromatography followed by liquid chromatography‐tandem mass spectrometry to identify and quantify tau phosphopeptides from individual AD, tauopathy and age‐matched control postmortem human brain tissues (n= 7 per group). In total 77 tau phosphopeptides representing 32 S/T/Y sites were analyzed by weighted correlation network analysis to delineate tau phosphosites co‐enriched in a disease specific manner. Our findings suggest that the mechanisms underlying site‐specific tau phosphorylation differ between AD and other tauopathies.
Arrest defective 1 regulates the oxidative stress response in human cells and mice by acetylating methionine sulfoxide reductase A
Authors: Shin Seung‐Hyun , Park Jong‐Wan
Published: June 2021

Methionine sulfoxide reductase A (MSRA) protects proteins from oxidation, and also helps remove reactive oxygen species (ROS) by recovering antioxidant enzymes inactivated by oxidation. Although its functions have been investigated extensively, little is known about the mechanism by which MSRA is regulated. Arrest defective 1 (ARD1) is an enzyme that catalyzes not only N‐terminal acetylation as a cotranslational modification but also lysine acetylation as a posttranslational modification. ARD1, which is expressed in most cell types, is believed to participate in diverse biological processes, but its roles are poorly understood. Given that MSRA was hunted in a yeast two‐hybrid screen with ARD1 as the bait, we here investigated whether ARD1 is a novel regulator of MSRA. ARD1 was shown to interact with and acetylate MSRA in both cells and test tubes. It specifically acetylated the K49 residue of MSRA, and by doing so repressed the enzymatic function of MSRA. ARD1 increased cellular levels of ROS, carbonylated proteins and DNA breaks under oxidative stress. Moreover, it promoted cell death induced by pro‐oxidants, which was attenuated in MSRA‐deficient cells. When mice were exposed to hyperoxic conditions for 2 days, their livers and kidneys were injured and protein carbonylation was increased. The oxidative tissue injury was more severe in ARD1 transgenic mice than in their wild‐type littermates. In conclusion, ARD1 has a crucial role in the cellular response to oxidative stress as a bona fide regulator of MSRA. ARD1 is a potential target for ameliorating oxidative injury or for potentiating ROS‐producing anticancer agents.
Molecular Crowding Effects on the CyaA Toxin RTX Domain: Implication for Toxin Secretion
Authors: Chenal Alexandre , Sotomayor Pérez Ana Cristina , Subrini Orso , Hessel Audrey , Ladant Daniel
Published: June 2021

Ligand‐induced disorder‐to‐order transition plays a key role in the biological functions of many proteins that contain intrinsically disordered domains. Here, we present data on an RTX (« Repeat in ToXin ») protein, RCL, an intrinsically disordered protein (IDP) that folds upon calcium binding. RTX motifs are calcium‐binding nonapeptide sequences that are found in more than 250 virulence factors secreted by Gram‐negative pathogenic bacteria. The RCL protein is a RTX region of the CyaA toxin, one of the major virulence factors secreted by Bordetella pertussis, the causative agent of the whooping cough. Using a combination of biophysical approaches, we showed that RCL exhibits the hallmarks of IDP in the absence of calcium. Calcium binding triggers a strong reduction of the mean net charge, dehydration and compaction, folding and stabilization of secondary and tertiary structures of RCL. We further showed that the crowding agent Ficoll70 did not affect the structural content of the apo‐state and holo‐state of RCL but increased protein affinity for calcium and stabilizes both states of RCL, increasing their half‐melting temperature (ΔTm), without affecting enthalpy changes. The power law dependence of the ΔTm increase on the volume fraction allowed the estimation of the Flory exponents of the thermally unfolded states. Altogether, our data suggest that, in the apo‐state as found in the crowded bacterial cytosol, RTX proteins adopt extended unfolded conformations that may facilitate protein export by the secretion machinery. Subsequently, both calcium gradient across bacterial cell wall and molecular crowding enhance the calcium‐dependent folding and stability of RTX proteins once secreted in the extracellular milieu.
Three‐dimensional Domain Swapping in the α Subunit of Tryptophan Synthase
Authors: Michalska Karolina , Bigelow Lance , Endres Michael , Joachimiak Andrzej
Published: June 2021

Tryptophan synthase is an enzyme participating in the last two steps of tryptophan synthesis in plants, fungi and bacteria. It consists of two protein chains, α and β, encoded by trpA and trpB genes, that function as an αββα complex. Structural and functional features of tryptophan synthase have been extensively studied, explaining roles of individual residues in catalysis and allosteric regulation of the two active sites. TrpA has become a model system to investigate folding mechanisms of larger proteins. In 1969, Jackson and Yanofsky observed that typically monomeric TrpA forms a small population of dimers. The dimerization was postulated to occur through an exchange of structural elements of the monomeric chains – a phenomenon currently known as 3D domain swapping. Nevertheless, structural details of the dimeric form of TrpA protein remained unknown. We determined the crystal structure of TrpA that for the first time illustrates domain swapping in a TIM barrel fold. The exchanging fragment represents the N‐terminal section and includes H0‐S1‐H1‐S2 elements. The hinge region corresponds to loop L2 linking S2 with H2'. S2 and L2 carry the catalytic residues, Glu and Asp. As the S2 element is part of a swapped domain, architecture of the catalytic apparatus in α2 is regenerated from the two protein chains. The dimer interface corresponds to the α‐β interface in the tryptophan synthase complex, suggesting that the observed α2 dimer may not form a functional complex with the β subunit.

This work was supported by the NIH grant GM094585 (MCSG) and the U.S. DOE, OBER, contract DE‐AC02‐06CH11357 (SBC).
Study The Role of Serotonin 2B Receptor in Osteoclastogenesis
Authors: Lin Shiuan‐Yea , Fu Yaw‐Syan , Lin Chih‐Lung , Cheng Tsung‐Lin
Published: June 2021

Osteoporosis is one of the most disease in menopausal women, middle‐aged and elderly people and it's second only to cardiovascular disease. Overexpression of osteoclastogenesis in the body resulting in outflow of sclerotic is one the main reason to cause osteoporosis. In recent years, clinical research has found persisting in taking anti‐depression drugs',Selective Serotonin Reuptake Inhibitor, SSRI, patients have higher ratio to suffer osteoporosis. It means serotonin, 5‐HT, should involve in the regulation of sclerotic metabolism. The researches demonstrated that there are many types of 5‐HT receptor, 5‐HTR, expressing in the human cell and regulate different physiological functions. In previous reports indicated both osteoblasts and osteoclasts expressing 5‐HT2BR. 5‐HT2BR can promote osteoblast proliferation and differentiation, but the effect on osteoclasts is unknown. In this study, we utilized receptor activator of nuclear factor kappa‐B ligand, RANKL to induce RAW264.7 cell differentiating to osteoclast as a model, and investigated how does the concentration of 5‐HT and 5‐HT2BR antagonist affect osteoclast differentiation. The result suggested that the degree of osteoclast differentiation was positive correlation with concentration of 5‐HT, and 5‐HT2BR antagonist treatment could reduce osteoclasts formation. In short, this investigation confirmed 5‐HT2BR involved in regulating osteoporosis. In the future, the further exploration will aim to intracellular signaling pathways. This research might provide another direction of prophylaxis and therapy for osteoporosis.
Transcriptional evidence for failed reinnervation in aging muscle
Authors: aare S , Gove T , Vuda M , Hepple R.T.
Published: June 2021

Muscle displays a marked accumulation of denervated myofibers at ages associated with an acceleration of atrophy and weakness. As muscle undergoes repeating cycles of denervation and reinnervation throughout adulthood, we hypothesize that the accumulation of denervated myofibers in advanced age is due to failed reinnervation. To address this issue, we quantified neuromuscular junction (NMJ) associated genes including five AChR subunits, MuSK, Rapsyn, Agrin, Lrp4 and APC in three different model systems. Firstly, we examined vastus lateralis (VL) muscle from young adult (YA) and very old (VO) Fisher 344xBrown Norway F1‐hybrid rats. Secondly, we examined biopsies from human VL in a young physically active group (23.7±2.7), an old active group (71.2±4.9), an old inactive group (64.8±3.1) and a very old inactive group (82.5±4.8). Lastly, we examined Extensor digitorum longus(EDL) & Tibialis anterior (TA) muscle from wild type and neurotrypsin over‐expressing (Sarco) mice as a model of unstable NMJs secondary to reduced MuSK activation. Interestingly, transcripts of MuSK (21 fold), Rapsyn, Agrin and AChR subunits α (68 fold), β, and the fetal isoform γ (47 fold) were upregulated in very old muscle of rat. In contrast, only Musk, Agrin and Rapsyn were increased in aging human muscle of very old inactive group compared with a young active group, consistent with the milder denervation phenotype by morphology. In contrast, in Sarco mice these NMJ transcripts were largely preserved despite a muscle atrophy phenotype and small angular fibers characteristic of long‐term denervation, which were frequent in VO rat and very old inactive human muscle, were rare in Sarco mice suggesting a high fidelity of reinnervation. We conclude that the dramatic changes in the NMJ transcriptional profile in aging muscle is indicative of a failed reinnervation response.
Analysis of 5' and 3' snoRNA termini maturation in Saccharomyces cerevisiae
Authors: Matuszek Zaneta , Szczepaniak Sylwia , Kufel Joanna
Published: June 2021

Small nucleolar RNAs (snoRNAs) are the best characterized non‐coding RNAs transcribed by RNA polymerase II. They are produced as precursors, whose extended 3' ends are trimmed exonucleolytically, whereas 5' ends either undergo combined endo‐ and exonucleolytic processing by Rnt1 and Rat1, respectively, or remain unchanged. In the latter case, the m7G cap becomes hypermethylated by Tgs1. [figure1]

cRT‐PCR analyses suggest that processing of snoRNA 3' and 5' termini is tightly coupled. Inhibition of 5' end maturation, in rnt1Δ strain, cause the accumulation of snoRNA precursors that don't possess mature 3' ends but carry polyadenylated extensions. This defect is further increased when components of the cap‐binding complex (CBC) or methyltransferase Tgs1 are missing. Although we observe an accumulation of pre‐snoRNAs in rnt1Δ strain, there are also mature snoRNAs present. These results, obtained in Northern blot analysis, suggest the existence of an alternative pathway of 5' end processing, that most likely involves pre‐snoRNA cap removal by Dcp1/Dcp2 complex. Interestingly most pre‐snoRNAs that accumulates in dcp2Δ strain, are not cleaved by Rnt1, which was confirmed by 5´ RACE analysis.

Obtained data strongly suggest the existence of a quality control mechanism that coordinates 5' and 3' end processing.

Research project was financially supported by by “Generacja Przyszłości” Ministry of Science and Higher Education Funding Programme.
Effects of Pre‐Workout Supplement on Strength, Anaerobic Power, and Body Composition
Authors: Jung Y , Cho M , O'Connor A , Chang C , Koozehchian M , Goodenough C , Barringer N , Ayadi F , Walker D , Simbo S , Galvan E , Dalton R , Levers K , Garcia E , Mitchell C , Rasmussen C , Greenwood M , Murano P , Earnest C , Kreider R
Published: June 2021

Pre‐workout supplements can increase energy availability and/or exercise capacity. We examined the effects of ingesting a pre‐workout supplement with and without synephrine for 8w in 78 healthy, resistance‐trained men (22 ± 3 y). Subjects were randomly assigned to treatments of: (1) a dextrose flavored placebo (PLA); (2) a supplement containing 3.0 g beta alanine, 2 g creatine nitrate, 2 g arginine AKG, 300 mg of N‐acetyl tyrosine, 270 mg caffeine, 15 mg of Mucuna pruriens (PWS) or (3) PWS + 20 mg of synephrine (PWS+S). Primary outcomes: one repetition maximum for the leg (LP) & bench press (BP). Secondary outcomes: a 30s Wingate test to measure peak (PP) and mean power (MP) and DXA body composition. Measures were assessed at 0, 4 & 8 wks. We used GLM statistical analyses covaried for age and respective baseline measures to determine mean (95% CI) change from baseline. We observed a significant increase in LP 1RM at 4wks for PWS (61.83 kg; 24.96, 98.75) and PWS+S (44.89 kg; 8.3, 81.5) and 8 wks for PWS (79.23 kg; 39.9, 118.5), PWS+S (89.53 kg; 50.6, 128.5) and PLA (43.28 kg, 4.1, 82.4). Similar patterns in BP 1RM were noted at 4 wks for PWS (6.94 kg; 0.6, 13.3), PWS+S (8.17 kg; 1.9, 14.5) and 8 wks for PWS (14.35 kg; 8.1, 20.6), PWS+S (13.83 kg; 7.6, 20.0) and PLA (7.18 kg; 1.0, 13.4). We observed a significant increase in PP for PWS+S and PLA at week 4; however, we observed no significant differences at 8 wks, between group differences throughout the study or an additive benefit from including synephrine. Results indicate that the pre‐workout supplement can promote strength and anaerobic power.
Effects of 8 Weeks Ingestion of a Pre‐Workout Supplement With and Without Synephrine on Cognitive Function, and Perceptions of Readiness to Perform
Authors: Jung Y , Cho M , O'Connor A , Chang C , Koozehchian M , Goodenough C , Barringer N , Ayadi F , Walker D , Simbo S , Galvan E , Dalton R , Levers K , Garcia E , Mitchell C , Rasmussen C , Greenwood M , Murano P , Earnest C , Kreider R
Published: June 2021

Pre‐workout supplements can increase energy availability and/or exercise capacity. We examined the effects of ingesting a pre‐workout supplement with and without synephrine for 8w in 78 healthy, resistance‐trained men (22 ± 3 y). Subjects were randomly assigned to treatments of: (1) a dextrose flavored placebo (PLA); (2) a supplement containing 3.0 g beta alanine, 2 g creatine nitrate, 2 g arginine AKG, 300 mg of N‐acetyl tyrosine, 270 mg caffeine, 15 mg of Mucuna pruriens (PWS) or (3) PWS + 20 mg of synephrine (PWS+S). Primary outcomes: Cognitive function (CF) via a Stroop test. Secondary outcome: Rate of readiness via a visual analogue scale (RTP‐VAS). Measures were assessed at 0, 4 & 8 w. We used GLM statistical analyses covaried for age and respective baseline measures to determine mean (95% CI) change from baseline. We observed a significant increase in CF for the word test at 4 wks for PWS+S (5.64 count; 2.09, 9.19) and PLA (3.9 count; 0.39, 7.45), and 8 wks for PWS (7.55 count; 4.14, 10.97), PWS+S (9.93 count; 6.48, 13.37) and PLA (6.74 count; 3.31, 10.16). Similar patterns in CF for the color test were noted at 4 wks for PWS (5.05 count; 2.72, 7.38), PWS+S (2.56 count; 0.24, 4.88), PLA (2.76 count; 0.43, 5.09) and 8 wks for PWS (8.3 count; 5.76, 10.89), PWS+S (5.07 count; 2.51, 7.63) and PLA (4.89 count; 2.32, 7.4). Decrease ratings of feelings about 'optimistic about future performance' were observed in all groups at 4 & 8 wks. Results indicate that the pre‐workout supplement used herein can improve cognitive function.
Changes in fMRI Hypothalamic Activity Via Intrinsic Oscillations After Intake of Regular Soda, Diet Soda or Water During a Meal
Authors: Bravo Stephen , Lowndes Joshua , Rippe James
Published: June 2021

The aim of this study was to examine hypothalamic changes after intake of different beverages at normal levels of consumption.

A total of 63 healthy, non‐diabetic, normotensive subjects with BMI 21‐29.9 kg/m2, between the ages of 20‐50 years took part in an fMRI scanning session. The protocol consisted of six runs of resting‐state‐fMRI. During the first two runs subjects were in the fasting state. Subsequently they ate a standardized meal for 30 minutes outside the scanner that included either a regular soda, a diet soda, or water, based on random allocation (n=21 for each group). After the meal, subjects were scanned four additional runs. For data analysis, standard image preprocessing was performed using FSL software. We used fractional amplitude of low‐frequency fluctuation (fALFF), a validated method that allows the detection of the regional intensity of spontaneous low fluctuations (0.01–0.08 Hz) in fMRI BOLD signal. Voxelwise fALFF maps were calculated for each participant in native space, and then transformed into the MNI152 standard brain space. Before statistical analyses, each individual fALFF map was Z‐transformed. A seed of 3 mm of hypothalamus based on a previous study was used to extract the Z values from these fALFF maps for group‐level parametric analyses. We modeled the data using ANOVA with group (regular soda, diet soda, water), and condition (fast, fed) as factors.

There was no effect of metabolic condition (F=1.99, p=0.16) or group (F=0.77, p=0.46). The interaction group x condition was also not significant (F=0.57, p=0.56). These results suggest that the previously identified differences may not apply to the case of SSBs under conditions that are more representative of normal daily intake levels and patterns.
Gastric Clearance Does Not Influence Glycemic Response to Cranberry Juice in Metabolic Syndrome Subjects.
Authors: Dahl Rachel , Mann Francis , Hooks Tisha , Wilson Ted
Published: June 2021

Acetaminophen (AC) may be used to estimate gastric clearance effects on glycemic response to a dietary challenge. Healthy (n=6) and metabolic syndrome (MSD) subjects (n=14; BMI >30) consumed reduced calorie cranberry juice (RCCBJ) (74 Cal/240 ml) or standard cranberry juice (CBJ) (110 Cal/240 ml) in single crossover fashion with 1 g AC. AC plasma concentration was measured pre‐challenge (0‐min) and 30, 60 and 120 min post‐prandially with HPLC. Plasma AC in healthy subjects after RCCBJ at 0, 30, 60 and 120 min was 0.14±0.20, 3.52±3.14, 16.07±8.54, 18.37±6.11 μg/ml. Plasma AC in healthy subjects after CBJ at 0, 30, 60 and 120 min was 0.33±0.37, 3.54±4.79, 19.80±15.03, 15.53±5.40 μg/ml. Plasma AC in MSD subjects after RCCBJ at 0, 30, 60 and 120 min was 0.11±0.25, 4.36±4.75, 10.64±6.60, 10.75±6.70 μg/ml. Plasma AC in MSD subjects after CBJ at 0, 30, 60 and 120 min was 0.16±0.28, 5.08±7.15, 12.86±10.15, 10.94±6.92 μg/ml. AC area under the curve (μg/ml·min) for healthy and MSD subjects to RCCBJ were 1382±523 and 934±569 respectively. AC area under the curve (μg/ml·min) for healthy and MSD subjects to CBJ were 1468±891 and 1062±644 respectively. No statistically significant differences in clearance were observed between RCCBJ and CBJ in either population, therefore in studies of cranberry juice glycemic response, gastric clearance is probably not a factor.