bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of Histone Chaperone APLF in Mammalian Embryo Development
Authors: Varghese Pallavi C. , Rajam Sruthy M. , Mukherjee Ananda , Dutta Debasree 
Published: May 2022

Embryonic development involves orchestrated events regulated by a plethora of factors. Epigenetic regulation is one such key aspect regulating embryonic development. We are investigating the role played by Aprataxin and PNK‐like factor (APLF), a histone chaperone, in regulating cellular transitions in the context of embryonic development. APLF is documented as a DNA damage‐specific histone chaperone and has already been studied in our lab in the context of the generation of induced pluripotent stem cells and breast cancer metastasis as a regulator of epithelial to mesenchymal transition.We are looking into the expression profile, functional role, and mechanism of action of APLF in embryonic development. The expression study in pre‐and post‐implantation mouse embryos and mouse trophoblast stem cells (TS) through different days of differentiation showed a dynamic expression pattern of APLF, clearly indicating prospective functional significance. The localization of APLF in mouse embryos was enhanced in trophectoderm (TE) and lineages derived from TE, which contributes to the extraembryonic lineage crucial for developing a proper maternal‐fetal connection. Functional studies by sh RNA mediated knockdown (KD) of Aplf in mouse embryos by microinjection induced the hatching of embryos in vitro at E3.75, with a significant increase in Cadherin 1 (Cdh1) and Caudal‐type homeobox 2 (Cdx2) expression. Hatching is a crucial event whereby the embryo breaks out of the protective sheath called zona pellucida and prepares itself to attach itself to the uterine wall by the process of implantation. The Aplf KD embryos failed to implant in vivo. Rescue experiments neutralized the effects of AplfKD both in vitro and in vivo. We used differentiated TS cells as our model system to understand the mechanism behind the observed phenotype in Aplf KD embryos. Reduced expression of Snail Family Transcriptional Repressor 2 (Snai2) and TEA Domain Transcription Factor 4 (Tead4), and the gain in Cdh1 level, marked the differentiation of AplfKD TS cells. Embryo implantation is one of the pioneer developmental stages where EMT comes into action. Significantly high level of epithelial marker Cdh1 and downregulation of mesenchymal marker Snai2 in Aplf KD TS cells would have impaired the crucial EMT process operational during implantation.Further studies are being conducted to unravel the underlying mechanism completely. Hence, our findings suggest a novel role for APLF during the implantation and post‐implantation development of mouse embryos. As we observed these phenotypes in the mouse embryo, we checked APLF expression in the human placenta, and interestingly, our preliminary studies showed its expression in cytotrophoblast and syncytiotrophoblast cells. As we have seen that a delicate balance of APLF is crucial to achieve implantation and attain proper pregnancy, we anticipate that it would contribute to establishing a felicitous maternal‐fetal connection.
Investigating the miRNA regulatory landscape of OGT and OGA via the 3’UTR and 5’UTR regions utilizing the miRFluR high‐throughput platform
Authors: Chu Thu 
Published: May 2022

The dynamic post‐translational modification of serine or threonine residues by O‐linked N‐acetyl‐beta‐D‐glucosamine (O‐GlcNAc) contributes to diverse cellular processes including epigenetic modifications, transcription, metabolism, and cell signaling that play significant roles in development and normal physiology. O‐GlcNAcylation is catalyzed by O‐GlcNAc transferase and the modification is removed by O‐GlcNAcase (OGA). These genes are highly regulated at multiple levels, but little is known about their regulation by microRNAs (miRs). miRs are small non‐coding RNAs that fine‐tune protein expression through binding to messenger RNA (mRNA). In this work, we built a comprehensive dataset of OGT and OGA regulation via both their 3’UTR and 5’UTRs. Downregulation was almost exclusively mediated through binding to the 3’UTR. We observed independent regulation of OGT and OGA by the majority of regulatory miRs, which did not overlap. However, we did see significant co‐regulation of OGT and OGA by a subset of miRs. This is in keeping with the known transcriptional regulation of these genes.In summary, this work provides a better understanding of OGT and OGA regulation through miRNA binding via both the 3’UTR and 5’UTR regions.
Serial crystallography and kinetics reveal how the FimH bacterial lectin tweaks between mono‐ and multivalent binding of high‐mannose N‐glycans
Authors: Bouckaert Julie M. J. 
Published: May 2022

BackgroundThe lectin domain of the fimbrial adhesin FimH from Escherichia coli recognizes with the highest affinity and in seemingly monovalent fashion oligomannosides‐3 and ‐5 N‐glycans. This happens with a high affinity (Kd=20 nM) because the alpha1,3‐linked arm remains free from any further alpha1,2‐mannose substitution. The latter causes an at least 10‐fold affinity loss in creating the oligomannose‐6 N‐glycan (1).MethodsThe binding of FimH to pauci‐ and oligomannoside‐containing N‐glycans and similarly glycosylated proteins, as well as its dimannoside endings Manalpha1,2, Manalpha1,3 and Manalpha1,6, have been profiled using the FimH Lectprofile kit (GlycoDiag, Applied Glycomics, France) and a newly designed paucimannose‐containing glycan array. The kinetics of binding, ka and kd, were analysed using surface plasmon resonance and compared with earlier incomplete data (2).ResultsMono‐ and multivalent binding were found in co‐crystal structures of the FimH lectin domain with alpha1,6‐fucosylated oligomannose‐3 and with oligoannose‐6, respectively. The kinetics of binding indicate that once alpha1,2‐linked mannose comes into play, the complexes are not so stable, as previously also measured using calorimetry (3), and accompanied with a high dissociation constant. Molecular dynamics simulations further support that it is not the total interaction energy that determines the final retained complex (metastable complexes), but the kinetics of interaction with the three possible dimannoside endings.ConclusionsKinetics and stability of the complexes drive the most favoured arrangement, via either a unique or multivalent interaction, between the N‐glycan and the FimH protein in the crystal.References :(1) Bouckaert, J., Mackenzie, J., de Paz, J. L., Chipwaza, B., Choudhury, D., Zavialov, A., Mannerstedt, K., Anderson, J., Pierard, D., Wyns, L., Seeberger, P. H., Oscarson, S., De Greve, H., Knight, S. D. (2006) The affinity of the FimH fimbrial adhesin is receptor‐driven and quasi‐independent of Escherichia colipathotypes. Molecular Microbiology 61, 1556‐1568(2) Sauer, M. M., Jakob, R. P., Luber, T., Canonica, F., Navarra, G., Ernst, B., Maier, T., Glockshuber, R. (2019) Binding of the bacterial adhesin FimH to its natural, multivalent high‐mannose type glycan targets. J Am Chem Soc 141, 936‐944(3) Dumych, T., Bridot, C., Gouin, S. G., Paryzhak, S., Szunerits, S., Bilyy, R., Bouckaert, J., (2018) A novel integrated way for deciphering the glycan code for the FimH lectin. Molecules 23, 2794
Codon Optimization of saCas9 Gene Enhances Protein Expression in Human Kidney Cells to Compensate for Difficulties in Delivery Vectors to Boost CRISPR‐Cas9 in vivo Therapeutic Application
Authors: Cheng Benjamin , Cooper Eli , Krishnan Anoushka , Groshong Tyler , Madejski Irene , Sundaresan Avirath , Holmes Grace , Gupta Ishir , Leschly Emma , Hauser Paul , De Luke 
Published: May 2022

The novel gene editing technology CRISPR‐Cas9 shows great promise in revolutionizing treatments of genetic disorders; however, currently the only practical treatments are ex vivo, limited to blood diseases. In vivo treatment can substantially extend the clinical reach of the technology, but is limited by a challenging delivery process. The large size of the Cas9 gene (~4500bp) inhibits delivery by the small viral vectors and lipid nanoparticles used. We hypothesize that a Cas9 gene codon optimized for human cells will support significantly higher protein expression than the presently used genes; this can compensate for the low delivery efficiency and has great potential to improve the viability of in vivo CRISPR‐Cas9 therapies.To investigate our hypothesis, we selected a commonly used parent saCas9 Addgene’s public plasmid database. We engineered a novel, codon‐optimized gene sequence from the parent using GenScript’s OptimumGene™ algorithm. We will compare protein expression via ELISA assay and Western Blot analysis. In this poster we will present the data gleaned from these assays.
Hypoxia Re‐Oxygenation Modelling Using Cancer Cells Expressing Cell Cycle and Cell Death Probes to Understand the Dynamics of Resistance Mechanisms
Authors: Tiwari Shivanshu Kumar , Kumar T. R. Santhosh 
Published: May 2022

BackgroundMost solid tumors are difficult to treat with conventional cancer drugs. Non‐responsiveness to drugs and the recurrences even after successful treatment are serious problems. Although, extensive studies have been performed to understand the mechanisms of resistance and tumor recurrences, the complex and variable nature of microenvironmental factors that contribute this remains as a challenging topic.Hypoxia is also one of the key factors that play a crucial role in tumor progression. Hypoxia‐Induced Factor 1 α (HIF 1‐α) are involved in the resistance of hypoxic tumors to radiation therapy as well as conventional drugs. However, the growing tumor is often exposed to hypoxia and re‐oxygenation because of the tumor driven hypoxia and occasional angiogenic response that primarily affect the cell cycle and cell death.HypothesisWe hypothesize that the defined models of cell cycle and cell death in the setting of hypoxia‐reoxygenation will help us to understand the hypoxia‐mediated cell signaling and resistance mechanisms.MethodologyWe have employed cancer cells expressing cell cycle probe FUCCI to study the dynamics of cell cycle progression during hypoxia and reoxygenation using real‐time imaging. Similarly, cancer cells were expressed with real‐time probes for cell death, FRET‐based caspase sensor to track their long‐time fate. These innovative strategies allowed us to visualize both cell cycle and cell death events in hypoxic and re‐oxygenation condition and to track the long‐time fate of cancer cells.Results & ConclusionWe have shown that the hypoxic exposures alter the cells’ ability to undergo apoptosis dynamically after its recovery under normoxia. Initially, the cells adapt to hypoxia through mitophagy dependent manner that alters its cell cycle progression both in normoxia and hypoxia. Despite adaptive mitophagy, hypoxic cells remain sensitive to re‐oxygenation and also to cancer drugs owing to the oxidation damage to organelles.However, rare cells survive during these two events progresses towards expansion with a highly variable mitochondrial density that governs differing functional traits of cell cycle, cell death response, ability to invade and migrate. This functional heterogeneity appears to play a key role in temporally regulated tumor resistance and recurrences.
Reference chart for transverse cerebellar diameter in developing fetuses according to their gestational age in Indian population
Authors: GHATAK SURAJIT , MISHRA SANJAY , AGARWAL DUSHYANT , SINGH PRATIBHA , GARG PAVAN K. 
Published: May 2022

ObjectivesThe objective of the study was to develop reference chart and to evaluate the efficacy of transverse cerebellar diameter (TCD) as one of the fetal biometric parameters used in ultrasonography to determine gestational age in the Indian population. Also, in this study we compared our findings with similar studies done on different ethnic populations to check effect of different ethnicity, environmental and nutritional factors in development of TCD.DesignA retrospective cross‐sectional study.MethodIn this study routine antenatal check‐ups of 300 singleton pregnant women were done after obtaining ethical clearance from Institute Ethical Committee. Ultrasonographic measurements of various fetal biometric parameters were taken which included, biparietal diameter (cm), head circumference (cm), abdominal circumference (cm), femur length (cm) and transverse cerebellar diameter (cm). Reference chart with mean TCD for corresponding gestational age (GA) in weeks was developed and compared with results of similar other studies.ResultsStatistically significant correlation found between TCD and gestational age (R2=0.92, p=0.0006). Gradually increasing linear pattern obtained between the development of TCD and corresponding gestational age of the fetus. Therefore, it is more reliable parameter to be used in regression formulae used in USG machine to predict accurate gestational age of fetus. When reference charts for TCD is compared with findings in other studies in different ethnic population, it is found that there is statistically significant difference exists.ConclusionsIn normally developing fetuses the TCD has linear correlation with advancing gestational age. Therefore, along with other fetal biometric parameters measurement TCD can be used in determination of gestational age for more accuracy. Thus by preventing over or under estimation of gestational age will help in reducing no judicious use of health resources and medical interventions. A separate reference chart is required for every different population because ethnicity, nutrition and environmental factors can have impact on normal TCD values. This will help to avoid misinterpretation of data to determine gestational age in various ethnic populations.
Melanoma Impairs Mouse Heart Function Which Short‐Term Exercise Cannot Restore
Authors: Uurasmaa Tytti‐Maria , Streng Tomi , Alkio Milla , Karikoski Marika , Heinonen Ilkka , Anttila Katja 
Published: May 2022

The aim of this study was to investigate whether melanoma impairs intrinsic heart function in mice and whether short‐term voluntary running wheel exercise could reduce the possible negative effects. Additionally, we investigated whether changes in cell size, capillary density, calcium channel levels, metabolic enzyme activities or oxidative stress could explain the possible changes in heart function. Advanced melanoma has been shown to cause cardiac muscle wasting and influence heart function in vivo, we hypothesized that melanoma would also impair intrinsic heart function, which has not been investigated previously. We also hypothesized that voluntary short‐term exercise could reduce the effects of melanoma on cardiac function since exercise training is known to improve cardiovascular function and reduce the negative effects of some cancers.Male mice were divided into untrained tumor‐free group (control) and untrained melanoma group and trained melanoma group. The mice did voluntary running‐wheel exercise until predetermined tumor size after which their hearts were isolated. The cardiac function was measured in retrograde perfusion at a constant pressure in multiple oxygen levels using Langendorff apparatus. The molecular and tissue level markers were measured afterwards.The melanoma animals were not cachectic as indicated lack of body weight loss. The rate of pressure production, pressure amplitude and rate of pressure decline were all significantly lower in the isolated hearts of the melanoma animals as compared to tumor‐free animals. However, the heart function did not differ between the untrained and trained melanoma groups. Furthermore, there were no differences between the groups in the calcium channel levels, reactive oxygen species, catalase activity, lipid peroxidation or citrate synthase and lactate dehydrogenase activity. However, mice from both melanoma groups had significantly lower superoxide dismutase activity as compared to tumor‐free animals, which might reduce the heart’s ability to respond to possible changes in oxidative stress. Exercise trained animals had higher capillary density, but their cell size and heart weights did not significantly differ from the untrained groups. Running wheel exercise did not affect the final tumor growth either even though there was tendency at the beginning of the experiment that running wheel exercise could slow down the tumor growth. One reason for this could be that when melanoma proceeded, the mouse running activity reduced significantly.In conclusion, melanoma is an aggressive cancer that impairs intrinsic heart function even when no cachexia is present. The aggressiveness of melanoma also prevented the short‐term voluntary exercise from alleviating the changes caused by melanoma. Future studies should combine the training to medical cancer treatment in order to estimate whether exercise training could be beneficial as adjunct therapy in melanoma treatment.
Polarized Macrophages As Potential Anti‐Endometrioid agent
Authors: Artemova Daria , Vishnyakova Polina , Fomina Mariia , Lokhonina Anastasia , Elchaninov Andrey , Fatkhudinov Timur 
Published: May 2022

Endometriosis is an estrogen‐dependent chronic inflammatory disease characterized by the presence of endometrial stroma and glands outside the endometrium. The gold standard in the treatment of endometriosis is laparoscopy but after that, the relapse rate is about 50%. Thus, the search for new effective non‐invasive methods of endometriosis therapy is an actual problem. Endometriosis has many properties similar to tumors including the fact that macrophages with an anti‐inflammatory phenotype (M2) prevail in the foci of endometriosis. It is already known that activated macrophages with a pro‐inflammatory phenotype (M1) have an antitumor effect. In this regard, we hypothesized that increasing the proportion of M1 macrophages could help in the treatment of endometriosis. The objectives of our work were (1) to assess the expression profile of macrophages in the foci of endometriosis in mice model of endometriosis; (2) to test the efficacy of administration of reprogrammed M1 macrophages as a possible anti‐endometrioid agent in the mouse.We studied the phenotypic profile of macrophages in the foci of endometriosis and the anti‐endometrioid activity of M1 macrophages on in vivoallogeneic model of mouse endometriosis induced in ovariectomized mice after intraperitoneal transplantation of the mouse uterus accompanied by 17β‐estradiol therapy. Analysis of the phenotypic profile of macrophages in the foci of endometriosis was carried out using immunohistochemical staining, western blot analysis, and qPCR. We obtained a reproducible model with lesions that had characteristic morphology. It was shown that macrophages with an anti‐inflammatory arginase1+ phenotype prevail in the foci of endometriosis compared with the intact endometrium. After verifying endometriosis development in mice, the experimental group was injected with M1‐polarized macrophages, while the control group was injected with unpolarized macrophages, and then the animals were monitored for two weeks. Macrophage cell line RAW264 was incubated with lipopolysaccharide (100 ng/ml) for 24 h to obtain M1‐polarized macrophages. M1‐polarization was verified by significant up‐regulation of MARCO, iNOS markers using western blot and qPCR analysis. After treatment of animals with M1 macrophages, we found that the number of foci and their sizes significantly decreased (p<0.05) in comparison with control animals.In our work, we proved the predominance of anti‐inflammatory macrophages in the foci of endometriosis and obtained the first results of a positive effect of M1 macrophages on the regression of foci of endometriosis.
Phenotypic profiling of mGlu7 overexpressing mice and its implications for neurodevelopmental disorders such as Rett syndrome
Authors: Buch Aditi , Fisher Nicole , Frietas Geanne , Vermudez Sheryl , Weiss Kelly , Gogliotti Rocco , Niswender Colleen 
Published: May 2022

Neurodevelopmental disorders (NDDs) are characterized by deficits in communication, cognition, attention, social behavior and/or motor control. Previous studies have pointed to the involvement of genes that regulate synaptic structure and function in the pathogenesis of these disorders. One such gene, GRM7, encodes the metabotropic glutamate receptor 7 (mGlu7), a G protein‐coupled receptor that regulates presynaptic neurotransmitter release. Mutations and polymorphisms in GRM7 have been associated with (NDDs) in clinical populations; however, limited preclinical studies have evaluated mGlu7 in the context of this specific disease class. Rett syndrome (RTT) is an NDD caused by mutations in the Methyl‐CpG Binding Protein 2 (MECP2) gene. The cognitive deficits seen in RTT model mice have been previously correlated with a deficit in long‐term potentiation (LTP) at Schaffer collateral‐CA1 (SC‐CA1) synapses in the hippocampus. mGlu7 is abundantly expressed presynaptically at SC‐CA1 synapses and its role in the regulation of GABA release is imperative for the induction of LTP. Previous studies from our lab have shown that mGlu7expression is reduced in autopsy samples from patients diagnosed with RTT and in mouse models of RTT. Our lab has also shown that abnormal phenotypes in RTT mice can be corrected with mGlu7 positive allosteric modulators (PAMs). Despite the beneficial effects of mGlu7 potentiation, it is important to understand how an increase in mGlu7expression affects phenotypes seen in NDDs like Rett syndrome.Here, we hypothesize that an excess of either of the two mGlu7 isoforms, mGlu7a and mGlu7b, in mice is sufficient to alter phenotypes. mGlu7 was globally overexpressed in mice by crossing a Grm7(a/b) Flx/+male with a CMV‐Cre+/‐ female. Starting at 6 weeks of age, behavioral assays were conducted for motor, cognitive, and stereotypic phenotypes. After the behavioral phenotyping, tissue was collected and processed for expression studies. Our data indicate abnormal phenotypes in motor, cognitive, and stereotypic assays. In particular, mGlu7 over‐expressing mice, regardless of isoform, display hyperactivity in a locomotor assay, associative memory deficits in a contextual fear conditioning assay, and increased repetitive behaviors as assessed by hindlimb clasping.In conclusion, our study indicates that overexpressing mGlu7 in mice leads to abnormal phenotypes. We are now assessing synaptic plasticity phenotypes, including abnormal long‐term potentiation in mGlu7 overexpressing mice, and determining the effect of mGlu7 allosteric modulators in affecting these cognitive aberrations. Overall, these studies aim to capitalize on previous studies demonstrating the role of mGlu7 in the manifestation of phenotypes associated with NDDs.