bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of a Highly Specific Met Antibody for Screening Met Overexpression in NSCLC
Authors: Chen Caiwei , Wei Haitao , Yuan Kehu , Ma Donghui , He Wei‐wu
Published: June 2021

Approximately 5%‐22% of non‐small cell lung cancer (NSCLC) patients with secondary resistance to EGFR TKIs had evidence of amplification of the Met oncogene. This dysregulation of Met signaling was implicated in the proliferation and eventual metastasis of NSCLC, and therefore represents an attractive target for anticancer drug development.

Currently, there are a number of clinical trials for Met targeted small molecule and monoclonal antibody therapies. Immunohistochemistry (IHC) analysis of the Met protein will be very important for these Met targeted therapies and requires a highly specific antibody to prevent false positives. To develop a highly specific monoclonal antibody against Met for IHC analysis in NSCLC, we generated 97 monoclonal antibodies. IHC evaluation showed that 8 of the antibodies had suitable performance in Met positive NSCLC patient tissues. The 8 Met monoclonal antibodies were further tested on a high density protein microarray chip containing 17,000 human proteins. The microarray analysis showed that clone 3G5 was highly specific as evidenced with it only reacting with Met on the microarray. This new 3G5 Met monoclonal antibody will be a promising candidate as a diagnostic reagent for detecting Met protein in NSCLC with minimal false positives.
FUSE Binding Protein1 Facilitates Persistent Hepatitis C Virus Replication in Hepatoma Cells by Regulating Tumor Suppressor p53 and p53‐Regulatory Factors
Authors: Dixit Updesh Kumar , Pandey Ashutosh , Mishra Priya
Published: June 2021

Hepatitis C virus (HCV) is a leading cause of chronic hepatitis C (CHC), liver cirrhosis (LC), and hepatocellular carcinoma (HCC). Immunohistochemistry of archived HCC tumors showed abundant FBP expression in HCC tumors with CHC background. Oncomine data analysis of normal versus HCC tumors with CHC background indicated a 4‐fold increase in FBP expression with a concomitant 2.5‐fold decrease in the expression of p53.We found that FBP promotes HCV replication by inhibiting p53, and regulating BCCIP and TCTP, which, respectively, are positive and negative regulators of p53. The severe inhibition of HCV replication in FBP‐knockdown Huh7.5 cells was restored to a normal level by downregulation of either p53 or BCCIP. Although p53 in Huh7.5 cells is transcriptionally inactive as a result of Y220C mutation, we found that activation and DNA binding ability of Y220C p53 were strongly suppressed by FBP, but significantly activated upon knockdown of FBP. Transient expression of FBP in FBP‐knockdown cells fully restored the control phenotype in which the DNA binding ability of p53 was strongly suppressed. Using EMSA and ITC, we found no significant difference in in‐vitro target DNA binding affinity of recombinant wild‐type p53 and its Y220C mutant p53. However, in the presence of recombinant FBP, DNA binding ability of p53 is strongly inhibited. We confirmed that FBP downregulates BCCIP, p21, and p53, and upregulates TCTP under radiation‐induced stress. Since FBP is overexpressed in most HCC tumors with HCV background, it may have a role in promoting persistent virus infection and tumorigenesis.
A marine fungal antibiotic inhibits tumor adaptation to hypoxia by deregulating HIF‐1
Authors: Choi Yong‐Joon , Park Jong‐Wan
Published: June 2021

Hypoxia‐inducible factor 1 (HIF‐1) is a master regulator of the transcriptional response to Hypoxia. HIF‐1 has been implicated in the regulation of genes implicated in angiogenesis, tumor growth and survival. To date, the roles of HIF‐1 have been extremely investigated and its overexpression is found in various organs. The expression of HIF‐1 in tumors are positively related with tumor aggressiveness and poor prognosis. Additionally, the effects of HIF‐1 on tumor growth and angiogenesis have been demonstrated in xenograft tumor models. Thus, there is considerable interest in identifying compound which target for HIF‐1 signaling pathway. We here searched for HIF‐1 inhibitors on the cell‐based screening platform using the Hep3B cell line that stably expresses luciferase depending on HIF‐1. Of 7,280 compounds in a chemical library, we finally picked up a compound that originates from a marine fungus parasitizing red alga. The fungal antibiotic at <10 nM concentration potently inhibited HIF‐1 expression and its transcriptional activity in cancer cells exposed to hypoxia. We also investigated the mechanism by which the antibiotic inhibits HIF‐1. Furthermore, the anti‐cancer and anti‐angiogenic effects of the antibiotic were demonstrated in anchorage‐independent colony formation and endothelial tube formation assays, respectively. Based on these results, we propose that marine fungal antibiotic be a potential HIF‐targeting anticancer agent.
Isolation of Human Glioblastoma Multiforme Primary Cells and analysis of CD133+
Authors: Yukselten Yunus , Akcora Yildiz Dilara , Demircan Kadir , Sunguroglu Asuman , Ugur Hasan
Published: June 2021

Objective

Glioblastoma multiforme (GBM) is an incurable and the most common type of lethal brain tumors. A transmembrane glycoprotein CD133 has been used as a marker in cancer stem cell analysis.

In this study, we aimed isolation of GBM Primary Cells from human GBM tumor tissue which is surgically resected and to determine amount of CD133+ in the primary cultures with using AC133 monoclonal antibody.

Material Method : Primer Cell culture

Tumor tissues taken from three patients who had GBM diagnosis were separated into 1 mm3 fractions with bistoury within fresh medium. 1ml of DMEM mixture were seeded into T‐25 cm2 culture flasks and incubated at 37°C with 5% CO2.

CD133+ staining

CD133+ staining was used as a marker of GBM stem cell marker. Cells were method according to cell surface staining.

Results

We demonstrated the GBM primary culture isolation of tumor tissues taken from four patients who had GBM diagnosis. We studied three GBM primary cultures such as P1, P2, and P3. We performed amount of CD133+ cells in GBM primary cell cultures by using the method of FCM analysis. According to the FCM analysis results in %1,5 ‐% 1,6 ‐%1,3 are founded respectively.

Conclusion

Finally, Isolated GBM primary cells provide an invaluable mediate to study how tumors form, progress, and relapse and also to elucidate the underlying deriving cellular mechanisms that eventually could provide understandings to therapeutic options. GBM Stem Cells (GSC) are responsible for tumor generation and treatment resistance in GBM. Identifying GSC cells is an important step in GBM therapy.
Glehnia littoralis Root Extracts Induces G1‐Phase Cell Cycle Arrest in MCF‐7 Human Breast Cancer Cells
Authors: dela Cruz Joseph , Jo Yura , Hong Heeok , Kim Dong‐Hwan , Hwang Seong‐Gu
Published: June 2021

Glehnia littoralis (GL) is widely used as an oriental medicine for cough, fever, stroke and other disease conditions. However, the anti‐cancer properties of GL have not been investigated. In order to elucidate the anti‐cancer properties and underlying cell death mechanism, MCF‐7, human breast cancer cells (5 X 104/well) were treated with Glehnia littoralis root extract at 0‐400 ug/ml. The hot water extract of GL root inhibited the proliferation of MCF‐7 cells in both dose‐dependent and time‐dependent manners. Analysis of the cell cycle after treatment of MCF‐7 cells with increasing concentrations of GL root extract for 24 hours showed a significant cell cycle arrest in the G1 phase. RT‐PCR and Western blot analysis both revealed that GL root extract significantly increased the expressions of P21, P27 and P38 while CDK2, CDK4 and Cyclin D1 expressions were down regulated. It is demonstrated that GL root extract arrested the proliferation of MCF‐7 cells at G1 phase through the inhibition of CDK2, CDK4 and Cyclin D1 via an increased induction of P21, P27 and P38. In summary, it is suggested that GL could have possible therapeutic potential against human breast cancer.
Our experiences with creating active learning environments for undergraduate Biomedical and Medical students at King's College London
Authors: Kiecker Clemens , Williams Darren
Published: June 2021

Growing evidence indicates that active learning improves student performance in science teaching (Freeman et al., 2014, Proc Natl Acad Sci U S A 111:8410‐5). Based on our experience that our Biomedical students tended to perform well in memorising taught facts, but often less well in the translational, synoptic aspects of using this knowledge (application, analysis and evaluation), we have implemented active learning scenarios on several 2nd and 3rd year modules. Their format ranges from reflective exercises for individual students to embodiment exercises for the entire class. Here I will discuss their feasability for different class sizes as well as potential pitfalls in their implementation. Our scenarios are frequently rated 'best feature of the course' in student feedback. Intriguingly, average exam results have not improved since their implementation‐‐most likely because we have also changed our assessment style which has now a stronger emphasis on testing application of knowledge, rather than memorised knowledge per se. Active learning scenarios pose different challenges for teachers compared to traditional teaching methods. Although their outcome tends to be less defined, they require more detailed preparation‐‐ideally writing of a script that helps choreograph the scenario and maintain its pace, and that contains contingency plans for failed exercises. Active learning scenarios benefit from having more than one teacher. In conclusion, active learning has improved student satisfaction and, arguably, performance on our courses and has made teaching more enjoyable.
Effects of Prolactin and Testosterone on MT1‐MMP mRNA Expression and Enzymatic Activity of MMP‐2 and MMP‐9 in the Rat Prostate
Authors: Muñoz David , Serrano Maria , Sampieri Clara , Cocotle Lourdes , Herrera Deissy , Rojas Fausto , Manzo Jorge , Hernandez Maria
Published: June 2021

Endocrine studies have shown prolactin (PRL) and testosterone (T) regulate prostate function. Both hormones are involved in the diseases of this gland as hyperplasia, dysplasia, and metastatic cancer. Since it has been reported metalloproteinases (MMPs) are underlying processes of metastasis and these are regulated by PRL and T. By histological studies we have detected PRL and T induce hyperplasia in many areas of the rat prostate within one month of treatment. In this work, we evaluated membrane type‐1 matrix metalloproteinase (MT1‐MMP) mRNA and enzymatic activity of MMP‐2 and MMP‐9 MMPs at the ventral (PV) and dorsolateral (PDL) prostate of the rat in response to treatments induced with PRL and T for one month. Our preliminary results show not differences of MT1‐MMP mRNA expression in all groups at PV and PDL. Although there is a trend of increased MT1‐MMP mRNA in groups T and PRL+T at PV, probably in response to an increase of androgen receptor. MMP‐2 and MMP‐9 were found. In all groups, inactive MMP‐2 was detected in all conditions and active MMP‐2 in the majority, although there were not differences in all groups. Inactive MMP‐9 was detected in almost all groups and active form was not detected, but there is a trend of increase of enzymatic activity in T group. In conclusion, we detected MT1‐MMP, MMP‐2 and MMP‐9 MMPs are involved in hyperplasia and T increases expression and enzymatic activity respectively of MMPs. Acknowledgements: CONACYT fellowship No. 290978 y Cuerpo Académico de Neuroquímica UV‐CA‐304.
An Investigation Into Student Perceptions of Peer Learning During a Medical Undergraduate Course
Authors: Parson Simon , Ezzat Ahmed , Foster Olivia , Moffat Mandy , Johnston Peter
Published: June 2021

Peer‐learning occurs at UK universities, but not previously at the University of Aberdeen. We piloted a peer‐assisted learning scheme (PALS) for medical students to determine student perceptions to peer‐learning. The project was conducted over one academic year. Fifth year students applied as PALS‐tutors and attended a Training the Trainer workshop. Tutorials were available to Year 3 students. Student opinion of peer learning & teaching was surveyed: (Likert scale) pre and post‐tutorial questionnaire. 52 students attended the tutorials, 80% had not previously experienced institutional peer‐led tutorials in the undergraduate course. There was a positive shift in attitude to peer teaching from pre to post‐tutorial surveys, with all students agreeing peer‐led teaching is a useful learning tool. Students found peer‐led more engaging than staff‐led teaching and that there were better opportunities to ask questions. Post‐tutorial, most agreed peer‐based learning was a better experience than lectures, and that peer‐based learning should be incorporated into the undergraduate curriculum; pre: 4 median (3‐4 interquartile range) and post‐ tutorial: 4 (4‐5). After the session all students agreed it was beneficial and more events should be organised. Focus groups revealed that students found student tutors easier to question, more focussed on their needs (especially regarding examinations) and less intimidating. They were less concerned about receiving incorrect information. From a staff perspective, peer teaching is most beneficial with a light academic touch. This can be challenging for faculty to accept. This pilot scheme suggests that more peer‐led teaching should be introduced into the medical curriculum.
Uncovering Novel Roles of Tandem BRCA1 C‐terminal (BRCT) Domains in DNA Repair, Genome Stability and Breast Cancer
Authors: Davis Jamaine
Published: June 2021

Pax transactivation domain‐interacting protein (PAXIP1/PTIP) has an important role in preserving genomic stability and is essential for cell survival following DNA damage, although the precise mechanism is undefined. PTIP is primarily a nuclear protein consisting of three pairs of tandem BRCA1 C‐terminal (tBRCT) domains. These BRCT domains are required for its assembly into multiple protein complexes. It is known that mutations that disrupt BRCT domain binding correlate with increased cancer rates. To determine its precise role in DNA repair, we profiled the expression of PTIP in breast cancer cell lines and observed differences in protein localization. We have recombinantly expressed the C‐terminal BRCT domains of PTIP, (BRCT36), which retains the ability to form nuclear foci. Using this construct, we set out to investigate how PTIP assembles into DNA repair complexes by conducting differential and ultra‐centrifugation experiments. The biophysical characterization of PTIP uncovers new oligomerization interfaces of higher order structures composed exclusively of BRCT36. These studies indicate that BRCT36 participates in the formation of multiple structurally and stoichiometrically diverse complexes during DNA repair.These studies suggest new and unusual functional roles of PTIP in DNA repair, genome stability and breast cancer.
The Structure and Mechanism of the ATPase that Powers Viral Genome Packaging
Authors: Hilbert Brendan , Hayes Janelle , Stone Nicholas , Duffy Caroline , Sankaran Banumathi , Kelch Brian
Published: June 2021

Many viruses actively package their genomes into preformed procapsids using a dedicated ATPase machine that is among the most powerful known biological motors. However, the structural mechanism by which this packaging motor couples ATP hydrolysis to DNA translocation is still unknown. Here we describe crystal structures of the packaging motor ATPase domain that exhibit nucleotide‐dependent conformational changes involving a 13° rotation of an entire subdomain. We also identify the arginine finger residue that catalyzes ATP hydrolysis in the ATPase domain of a neighboring motor subunit. The ATPase domain structures and identification of the arginine finger allow us to derive a structural model for the motor ring. We validate this model with a combination of small angle x‐ray scattering, ATPase measurements, DNA‐binding assays, and comparison with a previous cryo‐ electron microscopy reconstruction. Finally, we integrate our results to propose a mechanistic model for DNA translocation by this molecular machine.
Production of Fluorescent Oligosaccharide Substrates for the Neisseria meningitidis Serogroup W Capsule Polymerase
Authors: McCarthy Pumtiwitt , Williams Corshai , Ukaegbu Ophelia , Gordon‐Travers Tresha
Published: June 2021

Neisseria meningitidis is a Gram‐negative bacteria that causes most cases of bacterial meningitis. The six disease‐causing serogroups of N. meningitidis differ in capsular polysaccharide composition and linkage. Our long‐term goal is to develop a better understanding of the molecular mechanism of the serogroup W capsule polymerase. This enzyme synthesizes a heteropolysaccharide of galactose and sialic acid. We aim to develop a fluorescence‐based assay to aid in our studies. In this work, we describe our initial efforts to produce fluorescent substrates for the serogroup W capsule polymerase. Preliminary radioactivity assays indicate the enzyme has the ability to elongate serogroup W oligosaccharide fragments. The enzyme was more active with oligosaccharides than full length polysaccharide. These fragments, obtained by acid‐hydrolysis and heating, are the starting point for production of fluorescent substrates. Oligosaccharides were subjected to reaction with the fluorescent dye 1,2‐diamino‐4,5‐methylenedioxybenzene (DMB). Initial results indicate successful incorporation of the dye as assessed by HPLC with fluorescence detection. Alternately, copper‐catalyzed “click” chemistry is also being used to produce fluorescent oligosaccharides. Chemical reactions are being performed to add an azido group to oligosaccharides. These will be conjugated to an alkyne‐containing dye by 1,3‐cycloaddition “click” chemistry. Future work seeks to further characterize and optimize fluorescent substrates from both methods for testing with the serogroup W capsule polymerase.

Funded in part by ACS Project SEED (C.W).
Enzymogenesis of Zinc‐Dependent Alcohol Dehydrogenases and Aldehyde Dehydrogenases in Animals Show That Their Origin Predates Natural Availability of Ethanol from the Diet
Authors: Julián‐Sánchez Adriana , Hernández‐Tobías Aída , Vara‐Cisneros Paola , Gaona‐López Carlos , Alvarado‐Leaño Juan , Piña Enrique , Riveros‐Rosas Hector
Published: June 2021

In vertebrates, ethanol metabolism is performed through the concerted action of two enzymes: a Zn‐dependent alcohol dehydrogenase (Zn‐ADH) that oxidizes ethanol to acetaldehyde, and an aldehyde dehydrogenase (ALDH) that oxidizes acetaldehyde to acetate. The presence of these Zn‐ADHs and several ALDHs in animals has been assumed to be a consequence of chronic ethanol exposure from the diet. The main natural source of ethanol is fermentation of fruit sugars by yeast. The origin of angiosperms with fleshy fruits dates from the Lower Cretaceous (130‐140 million years ago). Because association between yeast and angiosperms dates also from the Cretaceous, dietary exposure of diverse frugivorous taxa to ethanol is similarly ancient. Since substrate availability must precede enzyme selection, then it can be expected that the enzymogenesis of the ADHs/ALDHs began after the appearance of fleshy fruits. Phylogenetic analyses reveal that all different Zn‐ADHs (seven classes) in animals appeared before land plants, independently of ethanol availability. With respect to aldehyde dehydrogenases (ALDH), phylogenetic analyses showed 13 different ALDH families in animals, and all they appeared before land plants. Because all these enzymes are not induced by ethanol and possess a high activity with non‐ethanol endogenous substrates, it can be concluded that Zn‐ADH and ALDH participation in ethanol metabolism can be considered as incidental, and not adaptive.

Supported by DGAPA‐UNAM grant IN216513.
Investigating the Nucleotide Selection Mechanism of Bacillus Stearothermophilus DNA Polymerase I
Authors: Christian Thomas , Konigsberg William
Published: June 2021

The movement of the fingers subdomain of Bacillus stearothermophilus DNA polymerase I Large Fragment relative to a DNA substrate was monitored via Fӧrster Resonance Energy Transfer (FRET) during nucleotide (dNTP) selection. Changes in FRET were monitored in bulk solution and at the single molecule level in the presence of a variety of substrates and cofactors. The presence of the 3'‐hydroxyl at the primer terminus strongly influenced the ability of the polymerase to discriminate between correct and incorrect dNTPs. Using a deoxy‐terminated primer in Ca2+, the ground state dissociation constant (KdG) obtained for the correct dNTP was nearly 10‐fold lower than the KdG obtained for an incorrect dNTP. When a dideoxy‐terminated primer was used the difference in the KdGs was reduced to only 2‐fold. Using a deoxy‐terminated primer in Ca2+ with the D653A mutant, where the ability to bind the B‐metal ion is compromised, there was a complete loss of discrimination between correct and incorrect dNTPs. Using a dideoxy‐terminated primer in Mg2+, we observed a 10‐fold difference in the KdGs. Non‐hyrolyzable nucleotide analogs will be used to determine the difference in the KdGs using a deoxy‐terminated primer in Mg2+. When Mn2+ and a dideoxy‐terminated primer were used a 20‐fold difference in KdGs was observed favoring incorrect substrates over correct substrates. The changes in FRET efficiencies suggest the subdomain may adopt different substrate and cofactor dependent conformations. These results indicate that the 3'‐hydroxyl and the metal ions that coordinate the dNTP are integral to the fidelity of this polymerase and influence the way in which it interacts with its substrates. NIH Grant GM 063276−09
Reconstructing Membrane‐Anchored Mitochondrial AAA+ Proteases For In Vitro Analysis
Authors: Glynn Steven , Rampello Anthony , Shi Hui
Published: June 2021

Mitochondrial AAA proteases harness energy from ATP hydrolysis to remove and degrade damaged protein components of the mitochondrial inner membrane. Transmembrane domains anchor the proteases in the inner membrane and drive assembly of the active hexameric form of the enzymes. To date, the requirement for these transmembrane domains has hampered detailed analysis of the substrate binding and processing activities of these important enzymes. The objective of this study is to develop a system for reconstituting membrane‐anchored ATP‐dependent proteases in a soluble, assembled, and active form that can be used to elucidate the molecular mechanisms of these important enzymes.

We report a novel method for reconstructing soluble, assembled and active proteases by replacing the insoluble transmembrane domain with a soluble analogue. In combination with a series of solution biochemical assays, we demonstrate that the human mitochondrial AAA protease, YME1L, is a capable of mechanically unfolding stable protein substrates, requires substrates to contain a recognition sequence of defined sequence, and consumes substrates by processive unfolding and translocation. Together these results clarify a number of open questions in the field and provide a framework for the study of the wider family of membrane‐anchored AAA+ molecular machines.

This research was supported by Stony Brook University.
Endothelial Glycocalyx Health Plays Critical Role in Nanoparticle Uptake
Authors: Cheng Ming , Homayoni Homa , Kumar Rajiv , Sridhar Srinivas , Webster Thomas , Ebong Eno
Published: June 2021

The endothelial cells in the blood vessels are lined with a meshwork of polysaccharides called the glycocalyx (GCX). There is evidence this protective layer is dysfunctional in patients suffering from various diseases including diabetes, atherosclerosis, and metastatic cancer. GCX dysfunction may impair or amplify the effect of drugs delivered to treat disease, and particularly drugs delivered using nanoparticle vehicles that are becoming very popular. However, there is currently no in vitro process of screening nanoparticle‐based drug delivery that tests the role of GCX health in delivery of the nanoparticles or drug itself. To address this issue, we are using ultra‐small PEGylated gold nanoparticles (NP) to visualize the effects of GCX on nanoparticle uptake by rat fat pad endothelial cells. We are examining the effect of intact GCX. In addition, two types of GCX dysfunction are modeled: degradation due to the addition of heparinase III enzyme that degrades the heparan sulfate component of the glycocalyx, and collapse due to culturing the cells in low serum media. In another model, we induce GCX regeneration through addition of heparan sulfate after heparinase III treatment. The cells are then incubated with nanoparticles overnight, fixed and stained, then imaged through confocal microscopy. Both cell cultures with damaged GCX showed significantly higher NP uptake compared to the cultures with intact or regenerated GCX. This work indicates that the GCX integrity and composition does influence NP uptake for the endothelial cells.​
A novel Visualization Table by Sectra was used to render cadaver CT/MRI scans into 3‐D images which were manipulated to enhance functional healthcare anatomy
Authors: Horn David , Benninger Brion
Published: June 2021

INTRODUCTION. Sectra Visualization Table (SVT) is a large interactive screen with an image display system that enables interaction with 3D images rendered from any CT/MRI in minutes. This allows one to easily manipulate 3D imaging, aiding baccalaureate, medical students, residents. Previously CT or MRI of live patients have been viewed on the machine and rendered in 3D via the SVT. Using donor cadaver patients (DCP) CT/MRI scans in the SVT would be an extremely beneficial teaching tool for healthcare students in anatomy courses. The objective of this study was to assess whether CT/MRI scans from DCP could be rendered in 3D for teaching. METHODS. Literature search was conducted regarding the SVT and donor cadaver CT/MRI. Prior to the first year anatomy course the DCP used for dissection by the medical students had full body CT and selective MRI. The CT/MRI from the DCP were rendered into 3D by the SVT and reviewed by medical students and faculty. Likert scale questionnaire was conducted regarding SVT utility. SUMMARY. Literature search revealed no known studies relating DCP and SVT. DCP CT/MRI was successfully converted into 3D images. The SVT and DCP 3D images were well received, both subjectively and objectively as per the Likert scale score. A significant percent of medical care has evolved into multiple relatively short investigative procedures which require the healthcare provider to have 3D understanding of the anatomical landscape to ensure success. An SVT allows a trainee to supplement their dissection anatomy to enhance their 3D understanding. CONCLUSION. This study successfully revealed that 3D images can be rendered from CT/MRI scans of DCP for functional anatomy education.
Cytosolic Cathepsin Inhibitor Stefin B (Cystatin B) Regulate Caspase‐11 Expression
Authors: Kopitar‐Jerala Nataša , Maher Katarina , Jeri[ccaron]‐Kokelj Barbara , Butinar Miha , Mikhaylov Georgy , Man[ccaron]ek Keber Mateja , Stoka Veronika , Vasilieva Olga , Turk Boris , Grigoryev Sergei
Published: June 2021

Stefin B (cystatin B) is an endogenous cysteine cathepsin inhibitor, and the loss‐of‐function mutations in the stefin B gene were reported in patients with Unverricht‐Lundborg disease (EPM1). In this study we demonstrated that stefin B‐deficient (StB KO) mice were significantly more sensitive to the lethal LPS‐induced sepsis and secreted higher amounts of pro‐inflammatory cytokines IL‐1β and IL‐18 in the serum. We further showed that increased caspase‐11 gene expression and better pro‐inflammatory caspase‐1 and ‐11 activation determined in StB KO bone marrow‐derived macrophages resulted in enhanced IL‐1β processing. Pretreatment of macrophages with the cathepsin inhibitor E‐64d did not affect secretion of IL‐1β, suggesting that the increased cathepsin activity determined in StB KO bone marrow‐derived macrophages is not essential for inflammasome activation. Upon LPS stimulation, stefin B was targeted into the mitochondria, and the lack of stefin B resulted in the increased destabilization of mitochondrial membrane potential and mitochondrial superoxide generation. Collectively, our study demonstrates that the LPS‐induced sepsis in StB KO mice is dependent on caspase‐11 and mitochondrial reactive oxygen species but is not associated with the lysosomal destabilization and increased cathepsin activity in the cytosol.
Dynein Activity Induced by a Kunitz‐type Molecule Acting on the Proteasome
Authors: Pacheco Mario , Sciani Juliana , Morais Katia , Andrade Sonia , Chudzinski‐Tavassi Ana Marisa
Published: June 2021

Methods

Cell culture: Mia PaCa‐2 and SK‐Mel‐28 tumor cells. Western blotting: Proteins were analyzed via chemiluminescence. Confocal microscopy: Live or fixed cells were analyzed using labeled Amblyomin‐X or specific antibodies. Proteasome assay: Trypsin and chymotrypsin‐like activities were accessed using chromogenic substrates. Co‐IP: Cell lysates were submitted to Co‐IP and injected into a mass spectrometer.

Results

Proteasome inhibition by Amblyomin‐X and its cellular uptake was abolished when the cells were pretreated with a dynein inhibitor. Amblyomin‐X increased Rab11A protein levels and induced its co‐localization with dynein. Several proteins were found bound to dynein, such as receptors, signaling transducers, early and recycling endosome components, proteasome, aggresome, ubiquitin, cell cycle, apoptosis and extracellular matrix components.

Conclusions

Dynein was required for Amblyomin‐X uptake and may transport the endosome towards the endocytic recycling compartment, because Rab11A was overexpressed, co‐localized with dynein; and Rab11A and Rab20 but not late endosome markers were bound to dynein. In addition, dynein was required for proteasome inhibition induced by Amblyomin‐X, thus suggesting its transportation to the molecular target. Some proteins, such as Bag3 and Raptor were found bound to dynein and this data corroborates with the aggresome formation and autophagy inhibition induced by Amblyomin‐X, as previously reported. Furthermore, dynein ligands indicated a possible involvement of the lipid rafts, intracellular pathways, transcription factors and membrane receptors. Those findings distinguish this molecule from proteasome inhibitors, such as bortezomib and carlfizomib. Supported by FAPESP.
Astrocytes of ABCD1 deficient mouse, adrenoleukodystrophy model have functional deficits but not respective brain mitochondria
Authors: Reiser Georg , Schoenfeld Peter
Published: June 2021

The neurodegenerative X‐ALD (X‐linked adrenoleukodystrophy) has symptoms caused by VLCFA. ABCD1 dysfunction results in VLCFA accumulation in patients. VLCFA accumulation is considered the X‐ALD pathogenic factor. We aim to understand the molecular mechanism underlying X‐ALD pathogenesis. A mouse model for X‐ALD, the Abcd1‐knockout mouse, displays biochemical abnormalities, reduced VLCFA ß‐oxidation and accumulation of VLCFA. We thank Aurora Pujol (Barcelona, Spain) for the Abcd1‐/‐ animals used here. Isolated brain mitochondria from Abcd1‐/‐ and wild‐type mice respond similarly to VLCFA, indicated as rise in ROS generation, impaired oxidative ATP synthesis and diminished Ca2+ uptake capacity. Remarkably, in mitochondria from adult wild‐type and one‐year‐old Abcd1‐/‐ deficient mice, the response of ATP synthesis to VLCFA was comparable. However, astrocytes from wild‐type and Abcd1‐knockout mice, exposed to supraphysiological concentrations of the C22:0, C24:0 and C26:0 exhibit impairments of energy metabolism. Long‐term exposure to VLCFA enhances ROS generation, in situ depolarization of mitochondria. Moreover, the VLCFA‐induced intracellular Ca2+ response is diminished in Abcd1‐/‐ astrocytes. VLCFA promote cell death in astrocytes. Important for understanding the X‐ALD pathogenic mechanism is the finding that in Abcd1‐/‐ astrocytes, the capability to revert oxidized pyridine nucleotides to NAD(P)H is diminished. Defective ABCD1 exerts no adaptive pressure on mitochondria. We suggest that detrimental activities of VLCFA in astrocytes involve defective functions besides mitochondria. Astrocytes from Abcd1‐/‐ mice respond more sensitively to VLCFA than from wild‐type mice. The antioxidative defense is diminished in Abcd1‐/‐.
Compared Craniometry in Dry Bones of the Anomalous Cranium.
Authors: Camara Eduardo , Tavares Marcelo , Guimarães Matheus , Fazan Valéria
Published: June 2021

The increase in the cranium can be caused by conditions like hydrocephalus, aneurism, hemorrhagic and ischemic stroke; however, these cause a secondary increase in the skull only in the childhood. Quantitative and morphological disproportions of anatomic structures are considered anatomical variations and have forensic importance. Therefore, the aim of this study is to characterize the craniometry and cranioscopy of the referred atypical part as well as enumerate the wormian bones seen with the naked eye on a dry cranium found at the Anatomical Center II of José do Rosário Vellano University ‐ Alfenas, Brazil. The object of study is in good handling conditions, fully dry and treated accordingly to the local procedure. Full cranial measurements were carried out, especially the measurement of the circumference, anteroposterior and latero‐lateral diameter, using an analog caliper rule, Castro Viejo compass and tape‐measure. We found a measurement of 60.2 cm for the circumference, 27.4 cm for the latero‐lateral diameter and 33.0 cm for the anteroposterior diameter. Furthermore, there were 60 wormian bones in the skull and a hole that, probably, indicated the absence of one more suture bone. Pathological investigation was performed by means of magnetic resonance and x‐ray to search for complications inside the cranium, as well as highlight the mastoid cells. Given the fact, it is concluded that the cranium is an anatomical variation rarely found, both by the size of the diameter and circumference, and the number of sutural bones, constituting a forensic data for future studies.
Chemical Tools to Study the Biology of Protein Arginine Phosphorylation
Authors: Fuhrmann Jakob , Subramanian Venkataraman , Thompson Paul
Published: June 2021

Reversible protein phosphorylation is a central mechanism of cellular signaling in both eukaryotes and prokaryotes. The major phosphoacceptor sites comprise serine, threonine and tyrosine residues. Recently, however, phosphorylation of arginine residues was shown to occur in bacterial proteins under stress conditions. There, the protein arginine kinase McsB mediates arginine phosphorylation, while the protein phosphatase YwlE efficiently hydrolyses the generated phosphoramidate (P‐N) bond. The aim of the current study was to generate chemical tools to better understand the physiological regulation of protein arginine phosphorylation. To evaluate the cellular regulation of YwlE we designed and synthesized a diverse set of non‐hydrolyzable phosphoarginine mimetics. The most potent inhibitors contain a sulfonate‐amidine (SO3‐amidine) group and inhibit YwlE with a Ki of ~390 nM. We adapted these inhibitors to synthesize SO3‐amidine based photo‐probes. Biochemical and mass spectrometric analyses revealed that these probes specifically recognize the active (reduced) form of YwlE, whereas they do not label the inactive (oxidized) form. Given the high specificity for the active enzyme we utilized this probe to identify the redox regulation of cellular YwlE and thereby derive a mechanistic model of the regulation of arginine phosphorylation under oxidative stress conditions. These findings will stimulate further investigations to characterize the function and regulation of protein arginine phosphorylation under different stress conditions.
Ribosomal Protein L5 Plays a Moonlighting Role in HAC1 mRNA
Authors: Ashish Anshu Fnu , Dey Madhusudan
Published: June 2021

The unfolded protein response (UPR) pathway is a conserved pathway from yeast to human, which maintains cellular protein homeostasis. An endoplasmic reticulum (ER)‐resident RNase Ire1 initiates the UPR in yeast Saccharomyces cerevisiae by cleaving out an inhibitory intron from the HAC1 mRNA, thus generating a new open reading frame that yields Hac1 protein. Hac1 is a transcription factor that activates the expression of the UPR genes. For effective cleavage and processing, HAC1 mRNA assembles around the Ire1 protein, which requires a 3'‐bipartite element (3'BE) located at the 3'‐untranslated leader. To understand the inner mechanisms of the 3'BE‐mediated HAC1‐mRNAIre1 protein assembly, we expressed in vivo a 3'BE‐RMB mini RNA consisting of 48 nucleotides of the BE mRNA and 43 nucleotides of an RNA aptamer mimicking biotin (RNA mimic of biotin, RMB). Uracils of the 3'BE‐RMB mini RNA were labeled with a thiol group while growing yeast cells in the presence of 4‐thiouridine (4sU). The 4sU‐labeled RNA and bound proteins were photo‐crosslinked and immobilized on streptavidin‐agarose. The 3'BE‐RMB mini RNA bound proteins were identified by SDS‐PAGE followed by LC/MALDI mass spectrophotometry. We identified two S1 RNA‐binding domain‐containing proteins: the ribosomal protein L5 (RpL5) and the a‐subunit of initiation factor eIF2 (eIF2a). An in vitro electrophoretic mobility shift assay showed that RpL5, but not eIF2a, specifically bound to the synthetic 3'BE RNA. Furthermore, we found that targeted mutations in the RPL5 gene reduced the normal yeast growth, but increased the ER stress resistance. These results suggest that, apart from ribosome biogenesis, RpL5 plays a moonlighting role in the UPR signaling while regulating HAC1 mRNA processing.
Structural Basis of HIV‐1 Genome Recognition via Viral RNA‐Protein Interaction
Authors: Florwick Alyssa , Carter Greg , Keane Sarah , Summers Michael
Published: June 2021

According to the World Health Organization, more than 35 million people worldwide are currently living with Human Immunodeficiency Virus (HIV). There are 31 antiretroviral drugs to date approved for treatment, yet there is no cure for infection. To inform new drug development strategies, we study the mechanism of genome packaging, a crucial step in viral proliferation. Packaging is mediated via the highly specific selection of the dimeric genome by the nucleocapsid (NC) domain of viral Gag polyprotein. Previous studies have identified the Core Encapsidation Signal (CES), the minimal area of the 5' untranslated region required to direct genome packaging. Using NMR spectroscopy, we have determined the 3‐D structure of CES. This structure contains tandem three‐way junctions that sequester the splice donor site. We propose that the tandem three‐way junctions, along with the psi hairpin, both of which containing a number of exposed or weakly base‐paired guanosines, have a large number of NC binding sites. To identify NC binding sites, we used site‐directed mutagenesis to mutate specific guanosines (G) to adenosines (A), which should inhibit NC binding. Using isothermal titration calorimetry, we performed binding studies with RNA constructs containing clusters of G to A mutations at the Psi hairpin and junctions. These data confirm the existence of NC binding sites at junction and hairpin Gs. Understanding the determinants of RNA‐protein interaction between CES and NC further elucidates the mechanism of genome selection. Better understanding of the structural mechanism of HIV‐1 genome recognition provides new possibilities for late‐phase viral inhibition.

This research was funded by NIH/NIGMS grant 1P50GM103297.
Gender and Age Differences in the Effectiveness of the Medifast 4 & 2 & 1 Meal Replacement (MR) Plan for Weight Loss in Overweight and Obese Adults: A Retrospective Chart Review of 21 Medifast Weight Control Centers
Authors: Coleman Chris , Kiel Jessica , Davis Kevin , Mitola Andrea , Arterburn Linda
Published: June 2021

The 4 & 2 & 1 Plan is a calorie controlled meal plan (1000‐1200 kcals/day) rich in high quality protein and includes MRs and regular foods. Body weight (wt) and body composition (BC) were collected at multiple times through 24 wks; the primary endpoint was wt change at 12 wks. Subgroup analyses by gender and age (< and 蠅65 yrs) were performed. Random effect regression models were used to examine the effects of age and gender on wt loss through 12 wks, controlling for baseline wt and time. 310 charts (42.9% male, 28.4% 蠅65 yrs) were assessed. Adults following the 4 & 2 & 1 Plan lost on average 24.4 lbs at 12 wks and 35.7 lbs at 24 wks (p<0.0001). Both gender and age subgroups lost significant wt at all times (p<0.0001) with favorable changes in BC; fat mass (FM) loss exceeded lean mass (LM) loss by >3.5X after 12 wks in all subgroups. Loss of LM was similar (蠄5%) for all subgroups. Loss of FM was similar across age groups while males lost more FM than females. Controlling for baseline wt and time, male gender and age <65 yrs significantly predicted wt loss at 12 wks. Males and adults <65 yrs lost more than females and adults age 蠅65 yrs respectively: males lost 3 lbs more than females (p<0.0001) and adults <65 yrs lost 1.4 lbs more than those 蠅65 yrs (p<0.0001) over 12 wks. The Medifast 4 & 2 & 1 Plan was effective for wt loss in both genders and in adults of both age groups. Wt loss was slightly greater for males and adults <65 yrs. Regardless of gender or age, LM was preserved relative to FM, of importance given concerns over age‐related sarcopenia during wt loss. Medifast, Inc. supported this research.
A Cluster RCT Evaluating the Effect of Iodized Salt on Infant Development in Amhara Region of Ethiopia
Authors: Mohammed Husein , Marquis Grace , Aboud Frances , Bougma Karim , Samuel Aregash
Published: June 2021

Iodized has been associated with the physical and mental development of children. We examined the effects of iodized salt on the growth and mental development of infant. Forty‐four communities in the Amhara region were randomly assigned and had access to iodized salt early (intervention) or later through market forces (control). A total of 1220 pregnant women were recruited and their health and micronutrient status were assessed at enrollment. Data were collected on infants (2 to 13mo), including SES, diet, anthropometry, Hb, home stimulation and mental development (Bayley scales, Cognition, Expressive language, Receptive language and Fine motor scales). The median UI excretion was higher in the intervention vs controls, for both mothers during pregnancy (161 vs 121 µg/L, p<0.0001) and infants (141 vs 110 µg/L, p<0.02). Additionally, (45 vs 60%, p<0.05) of mothers and (37 vs 44%, p<0.05) of infants were iodine deficient. The intervention had a tendency to improve cognition with an effect size of 0.09, p=0.06, but did not affect the scores for the other three bayley scales. The intervention significantly improved home score (delta=0.45, p=0.04). Iodized salt intake improved iodine status of both pregnant women and their infants, and could lead to improved infant development.

Funded by Micronutrients Initiative, Canada
Sasa quelpaertensis leaf extract inhibits colon cancer by suppressing colon cancer stemness in vitro and in vivo
Authors: Kim Eunju , Min Soo Jin , Lim Ji Ye , Kim Yoo‐Sun , Kim Kyung‐Mi , Ko Hee‐Chul , Kim Se‐Jae , Kim Yuri
Published: June 2021

Colon cancer is a leading cause of cancer death in worldwide. Most colon cancer patients develop recurrence suggesting that colon cancer is driven by a subpopulation of self‐renewing cancer cells, termed cancer stem cell (CSCs), which may be responsible for tumor relapse and resistance against chemotherapy. Targeting CSCs to treat colon cancer may be an important therapeutic strategy. Sasa quelpaertensis Nakai is a bamboo grass that only grows in Jeju Island in South Korea. Although the anti‐obesity and anti‐inflammatory effects of Sasa leaves have been reported, the anti‐cancer stem cell effects of Sasa leaves remain unknown. The aim of this study was to investigate the effects of Sasa quelpaertensis leaves extracts (SQE) on suppressing cancer stemness of colon cancer cell. To obtain CSCs, we isolated CD133+/CD44+ population of HT29 colon cancer cells using Flow Cytometry Cell Sorting (FACS). We found that SQE significantly suppressed CD133+ and CD44+ population using FACS analysis. SQE inhibited colony formation and nonadherent sphere formation which are characteristics of cancer stem cells. SQE down‐regulated expressions of CSC markers, including SOX‐2, Notch1, DLK1, and b‐catenin. Furthermore, SQE down‐regulated the expression of HIF‐1α, and its down‐stream gene, VEGF. In conclusion, SQE has an important role in regulating colon cancer stemness by suppressing self‐renewal potential and CSC markers, thereby contributes to the overall inhibition of cancer cell growth.
Compensatory Mechanisms In Mouse Offspring With Inherently Weak Bones Are Suggesting A Gene‐By‐Environment Interaction In Utero
Authors: Raygorodskaya‎ Maria , Torchinsky‎ Arkady , Gabet Yankel , Shochat Chen , Karasik David
Published: June 2021

Exposure of mouse embryo to different environmental factors, including sub‐threshold teratogens, can detrimentally affect bones of the offspring later in life. We investigated to what extent the genetic differences translate into variable response to teratogen 5‐deoxy‐2'‐cytidine (5‐AZA). We used mice of 2 inbred strains, C3H/HeJ (with inherently stronger bones) and C57Bl/6J (with weaker bones). Mothers were injected with 5‐AZA or saline (control) at day 10 of pregnancy. Five offspring of mice from each strain/sex groups (C3H or C57, males and females), treated and untreated, were tested at age 3 mo (adolescent) and 6 mo (adult). Femora from 80 offspring were analyzed by Scanco μCT50. At the age of 3‐mo, treated females (C57) had significant decrease (p<0.05) of bone mineral density (BMD) or loss of trabecular thickness (C3H); treated mice demonstrated loss of cortical thickness (p<0.05). In 6‐mo‐old, BMD was slightly lower in treated C3H males and females, while treated C57 had increased trabecular thickness (p<0.05). Treated C3H males demonstrated loss of cortical thickness, had lower trabecular parameters (p<0.05). This study demonstrates that the exposure to low‐dose 5‐AZA during embryonic life affects bone structure in adult offspring. In 3‐mo‐old offspring, teratogenic exposure caused bone loss in females of both strains. In 6‐mo‐old, bone loss was observed in offspring of strong‐bones strain (C3H), but not in the offspring of weak‐bones‐strain (C57). In mice with inherently weaker bones the effect of adverse intrauterine environment triggered some compensatory mechanisms.
Blueberry Supplementation Attenuates Inflammation and Oxidative Stress and Increases Brain‐Derived Neurotrophic Factor in the Brains of Middle‐Aged Mice Consuming a High Fat Diet
Authors: Carey Amanda , Gildawie Kelsea , Gilman Casey , Rovnak Abigail , Shukitt‐Hale Barbara
Published: June 2021

Previously it was demonstrated that 9 month old mice fed a high fat diet (HFD, Harlan, TD.06414, 60% calories from lard fat) for five months had impaired memory; however supplementation of the HFD with 4% blueberry (BB, freeze‐dried extract, U.S. Highbush Blueberry Council) reduced these memory deficits. The brains of these mice were dissected, homogenized, and assayed with ELISA for brain‐derived neurotrophic factor (BDNF), the inflammatory cytokine tumor necrosis factor (TNF)‐alpha, and were evaluated for oxidative stress by examining 4‐Hydroxynonenal (4‐HNE). There was a significant increase in hippocampal BDNF in the animals fed the HFD + BB (273±56 pg/ml/per mg of tissue) in comparison with the low fat diet (LFD, TD.08806, 10% calories from fat) and HFD‐fed animals without BB supplementation (44±3 and 46±2 pg/ml, respectively). There was also a trend suggesting that animals fed a LFD + BB had elevated BDNF levels (101±57 pg/ml). Greater levels of TNF‐alpha were found in the cerebral cortex of mice fed a HFD compared to mice fed a LFD, and there was a partial reduction of TNF‐alpha in mice fed HFD + BB. 4‐HNE was found to be elevated in the cortex of mice fed a HFD compared to the mice that had BB supplementation in the HFD. This study attempts to determine the pathways and molecules that HFD consumption modulates that may underlie the previously demonstrated memory impairment, and also to begin to elucidate the mechanisms by which blueberry supplementation mediates these changes. Supported by U.S. Highbush Blueberry Council