bluuebunny/crossref_metadata_2025 huggingface dataset:
Temporal Distribution of EPAC‐1 and EPAC‐2 on Normal Skin and Cutaneous Wound Healing of Chronically Stressed Mice
Authors: Romana‐Souza Bruna , dos Santos Jeanine Salles , Schmidt Martina , Monte‐Alto‐Costa Andréa , Porto Luis Cristóvão
Published: November 2023

Psychological stress‐induced catecholamines impair cutaneous wound healing through engagement of different pathways. Recently, it was demonstrated that beta‐adrenoreceptor driven generation of cyclic adenosine monophosphate (cAMP) signals via adenylyl cyclase and exchange protein directly activated by cAMP (EPAC) in skin wound healing. However, it has not been shown yet if chronic stress‐induced high catecholamine levels could alter the expression of EPAC‐1 and EPAC‐1 in cutaneous wound healing. Thus, our aim was to observe the temporal distribution of the EPAC‐1 and EPAC‐2 in normal skin and full‐thickness excisional wounds of chronically stressed mice. Adult male mice (n=15) were submitted to a rotational stress which psychological stress model applied in rodents. For this, animals were daily spun at 115 rpm for 15 min every hour during 24 h, until euthanasia. Other animals (n=15) did not submit to stressful environment. Three days after stress protocol, two full‐thickness cutaneous lesions (8‐mm diameter) were created in the animals' dorsum. The wound contraction was macroscopically measured. Lesions were collected 3, 7 and 10 days after wounding. This study was approved by the Ethical Committee for Animal Use of the State University of Rio de Janeiro (n° 027/2014). The macroscopic analysis showed that chronic stress delayed wound contraction when compared to control group 3, 7 and 10 days after wounding. The protein EPAC‐1 expression was greater in the normal skin of stressed mice than in the normal skin of non‐stressed mice. In the wound area, protein EPAC‐1 expression was diminished stress group 7 and 10 days after wounding when compared to control group. The protein EPAC‐2 expression was decreased in the normal skin and wound area of stressed mice after 7 and 10 days when compared to non‐stressed mice. In conclusion, stress alters the protein expression of EPAC‐1 and EPAC‐2 on normal skin and cutaneous wound healing of mice.

Support or Funding Information

The Coordination for the Improvement of Higher Education Personnel (CAPES) and the National Council of Research (CNPq)
Regeneration of Rats Mental Nerve with Mesenchymal Stem Cells Obtained from Dental Pulp of Human Exfoliated Deciduous Teeth
Authors: Sasaki Robson Tetsuo , Saez Daniel Martinez , Nicoliche Tiago , Monteiro Bábyla Geraldes , Almeida Sonia Regina Yokomizo , Kerkis Irina , Silva Marcelo Cavenaghi Pereira
Published: November 2023

The aim of this study was to evaluate the regeneration of mental nerve after injury by compression and treatment with mesenchymal cells obtained from dental pulp of human exfoliated deciduous teeth (SHED). Dental pulp from human exfoliated deciduous teeth were obtained from children aged 5–7 years old. Thirty‐six male Wistar rats, divided into 3 study groups (n=12): Positive Control (PC) – no lesion, Negative Control (NC) ‐ lesion by compression; and Experimental (SHED) ‐ lesion by compression and treatment with SHED. After the compression of the nerves, groups NC and SHED received application of media culture and media culture associated with SHED, respectively. Euthanasia was performed at 3, 7, 14 and 21 days for immunohistochemistry and transmission electron microscopy analysis. The immunohistochemical reaction was performed for nerve growth factor (NGF) detection. Ultra‐thin sections with 0.3μm were contrasted and observed under transmission electron microscope with 40,000× and 100,000× magnifications. NGF expression was present in all groups and time span studies, but less expressed in group NC while the staining was stronger in group SHED at 21 days. The ultrastructural analysis showed after 14 days of treatment the group SHED showed similar morphological aspects when compared with the PC group. It can be concluded that the use of mesenchymal cells obtained from dental pulp of human exfoliated deciduous teeth for the regeneration of rats mental nerve is efficient in a single dose of application after two weeks of treatment.

Support or Funding Information

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Premature ovarian failure patient‐specific induced pluripotent stem (iPS) cells show compromised potential to develop to germ cells and granulosa cells
Authors: Lu Chen‐Yu , Chen Yu‐An , Wu Fang‐Chun , Ho Hong‐Nerng , Chen Hsin‐Fu
Published: November 2023

Premature ovarian failure (POF; also called primary ovarian insufficiency, POI), is a common endocrine disorder in females, accounting for 1–2% in reproductive‐age women. Patients with the disease experience spontaneous menopause before the age of 40 years, and frequently encounter significant difficulties in overcoming the problems of infertility and menopausal syndrome. However, the mechanisms underlying POF are difficult to study due to the lack of a suitable disease model. Recently pluripotent stem cells, including human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells, are becoming useful cell sources to study and dissect the molecular mechanisms underlying in vitro differentiation into specialized cell types. We and other researchers have previously shown that pluripotent stem cells including both ES cells and iPS cells are capable of developing into primordial germ cells (PGCs) and ovarian granulosa‐like cells. In this study therefore, we aimed to derive human iPS cells from POF patients and further differentiate the stem cells to germ cell‐related lineages. Now we have successfully derived and characterize patient‐specific iPS cell lines from five POF patients and two control subjects by transduction of skin fibroblasts or peripheral blood mononuclear cells (PBMCs) with four defined factors (OCT3/4, SOX2, KLF4 and c‐Myc) using Sendai virus reprogramming method. The resultant human iPS cells were then induced by using the cytokine cocktails towards PGCs, which expressed germ cell markers VASA, GDF9 and meiosis marker SCP3, and also towards functional granulosa‐like cells, which expressed FOXL2, FSHR, AMHR2, AMH and CYP19A1. We also observed that the POF‐patient iPS cells derivatives showed differential and reduced expression levels of the granulosa cell markers (AMH, AMHR2, CYP19A1 etc.) and germ cell marker VASA, when compared to control iPS and ES cells. The present data thus supported that we could produce POF‐specific iPS cells and induce the stem cells in vitro to PGCs and granulosa cells. Considering the altered phenotypes of the stem cell derivatives, the POF patient‐specific iPS cells derived in this study thus represent a potentially useful disease model for further studies into the disease mechanisms and to screen drugs that may be useful for treating and arresting POI.

Support or Funding Information

National Science Council of the Republic of China (NSC) and National Taiwan University Hospital (NTUH), Taiwan
Molecular Mechanisms of Mutagenesis Induced by DNA Repair
Authors: Shen Birong , Chapman Joseph , Furano Anthony V.
Published: November 2023

Cancer is a complicated outcome of multiple somatic mutations, however, the molecular mechanisms underlying most of the mutations are unknown. Previous work in our lab using a shuttle vector system showed that repair of mismatched base pairs and other lesions induced mutations in flanking DNA. Most of these mutations exhibited a mutational spectrum typical of a cytidine deaminase, APOBEC3B (A3B). We also showed that both base excision repair and mismatch repair (BER & MMR) are required and the amount of APOBEC bound to the shuttle vector is proportionate to the extent of mutagenesis. We therefore postulated that the single‐stranded DNA substrate of the APOBEC deaminase can be generated during repair (Chen et al eLife 2014;3:e02001).

Recent studies reported the involvement of A3B in generating mutations in some cancers, an example of the so‐called “mutator phenotype” long‐known to typify many cancers. We are currently using our shuttle vector to elucidate the mechanism of APOBEC‐mediated mutations in breast cancer cell lines. We found that repair of pre‐formed mismatches inserted into the shuttle vector can induce APOBEC‐mediated mutations, indicating that this system can monitor and reflect the mutational environment in tumor cells. We showed that two cancer cell lines, which contained comparable amounts of A3B, exhibited dramatically different repair‐induced mutation rates. Therefore, we postulated that the mutation rate is related to the amount of the repair‐generated APOBEC single‐stranded DNA substrate in each cell line, perhaps due to dysregulation of their DNA repair enzymes. Quantification of these enzymes showed that the relative level of some BER genes was positively correlated with repair‐induced mutagenesis, and preliminary results showed that knock down of one such gene reduced mutagenesis. Thus, our shuttle vector system could provide a tractable experimental system to explore the mechanism of the cancer cell mutator phenotype.
Functional Analysis of the Role of Phosphorylated PCNA in Cancer Development and its Potential in Cancer Therapy
Authors: Peng Bo , Jiang Qingwen , Ortega Janice , Gu Liya , Dai Junbiao , Li Guo‐Min
Published: November 2023

Proliferating cell nuclear antigen (PCNA), a ring‐shaped homotrimer, is an essential factor in DNA metabolism including DNA replication and repair. PCNA functions in these reactions appear to be regulated by posttranslational modifications, such as ubiquitylation, sumoylation and phosphorylation. Recently, we have shown that PCNA phosphorylation by EGFR at tyrosine 211 (Y211) disrupts PCNA interactions with mismatch recognition proteins, thereby inhibiting DNA mismatch repair (MMR). In addition, phosphorylated PCNA promotes misincorporation during DNA replication. However, it is unknown whether one, two or all three phosphorylated subunits are responsible for MMR inhibition and error‐prone DNA synthesis. To answer this question, we created a series of phosphorylation‐mimicking and non‐phosphorylation‐mimicking PCNA molecules with variously phosphorylated subunits, and examined their roles in MMR and DNA synthesis in vitro and in vivo. Our biochemical experiments reveal that all forms of phosphorylated PCNA inhibit MMR, with the inhibition activity proportional to the increased levels of phosphorylation; all forms of phosphorylated PCNA slow down the speed of DNA synthesis while conducting error‐prone DNA synthesis. Our in vivo data show that high‐levels of PCNA phosphorylation induce cell death. This study therefore provides novel mechanisms for phosphorylated PCNA in cancer development and its potential role in cancer therapy.

Support or Funding Information

This work is supported in part by grants from National Natural Science Foundation of China (31370766), by Tsinghua‐Peking Joint Center for Life Sciences, and by National Institutes of Health of the United States (GM089684).
Using Transcriptome Analysis to Solve the Puzzle of DNA Repair in Bdelloid Rotifers
Authors: Huber Christa Caye , Glazko Galina , Rahmatallah Yasir , MacLeod Stewart , Boerma Marjan , Schurko Andrew
Published: November 2023

Bdelloid rotifers are microscopic aquatic animals that have apparently survived for more than 40 million years without sex, males, or meiosis. Bdelloids have an efficient DNA repair system that gives them the ability to recover from levels of ionizing radiation that are lethal to other eukaryotes. In bdelloids, DNA double strand breaks induced by high doses (>1000 Gray) of ionizing radiation are repaired without significant loss of viability. The objective of this project is to gain insight into the mechanisms of DNA repair in bdelloids by identifying genes involved in this process. This was done using transcriptome sequencing (RNA‐seq) to characterize genes that are differentially expressed during DNA repair in the bdelloid Adineta vaga. To induce DNA double‐strand breaks, bdelloid cultures were irradiated with a total dose of 280 Gray of ionizing radiation using a cesium‐137 source. RNA was then isolated from these cultures at three time points (0, 30 and 60 minutes) post‐irradiation and also from non‐irradiated controls. RNA‐seq was done using the TruSeq RNA Library Preparation Kit and the Illumina HiSeq 2500 platform. From this data, over 500 genes were differentially expressed during DNA repair. Real‐time PCR was done to validate some differentially expressed genes from RNA‐seq. Gene ontology enrichment analysis was carried out to identify biological processes and pathways that were differentially expressed in irradiated bdelloids. Many upregulated genes were related to DNA repair functions (e.g. ligation, non‐homologous end‐joining, DNA polymerases) while the majority of downregulated genes were associated with transcription, translation and cell cycle regulation. Interestingly, over 20% of differentially expressed genes are apparent “bdelloid‐specific genes,” meaning that they lack homologs in other eukaryotes. However, in some cases these bdelloid‐specific genes represent inadequately annotated gene models; for example, the Random Amplification of cDNA Ends (RACE) method was used to identify a previously unannotated map kinase‐activated protein kinase 2 homolog among these genes. Conversely, other differentially expressed genes appear to be genuine bdelloid‐specific genes since further investigation using computational and molecular methods failed to identify their homolog. Overall, this ongoing gene expression analysis has identified several candidate genes for having functions in DNA repair, which will provide further insight into the mechanism of DNA repair in bdelloid rotifers.

Support or Funding Information

This work was supported by grants from the National Center for Research Resources (5P20RR016460) & National Institute of General Medical Sciences (8P20GM103429) from the NIH.
An Investigation of Protein Function During DNA Repair in Bdelloid Rotifers
Authors: McReynolds Jeanita Cherise , Boerma Marjan , Byrum Stephanie D , Orr Lisa M , Tackett Alan J , Schurko Andrew
Published: November 2023

Bdelloid rotifers are a class of aquatic microinvertebrates that have seemingly flourished for over 40 million years without sex, meiosis, or males. The long‐term success of this ancient ameiotic lineage is attributed, in part, to the extraordinary ability of bdelloids to repair DNA double strand breaks induced by desiccation. Our objective is to characterize proteins and their interactions during DNA repair in bdelloid rotifers. We first targeted meiotic proteins as a group of candidate DNA repair proteins. Despite the longstanding absence of meiosis in bdelloids, their genomes contain four genes that code for proteins (SPO11, HOP1, MSH4, MSH5) specific to meiosis in other eukaryotes. Western blot assays using custom antibodies for SPO11, HOP1, MSH4 and MSH5 were done to confirm that their corresponding genes were expressing proteins in the bdelloid Adineta vaga. To characterize binding partners of these meiotic proteins, protein pull‐down assays were then done. We generated 6×histidine‐tagged copies of each meiotic protein (and also of DNA repair proteins RAD51 and MRE11) and these were used in pull‐down assays with Dynabeads and protein lysates from A. vaga. SDS‐PAGE and mass spectrometry were used to identify potential binding partners. Next, to further our investigation into DNA repair proteins, we also identified other candidate DNA repair proteins using a whole proteome analysis of irradiated and non‐irradiated cultures of A. vaga with high‐resolution mass spectrometry. To induce DNA double‐strand breaks, bdelloid cultures were irradiated with a total dose of 280 Gray of ionizing radiation using a cesium‐137 source. Proteins were then isolated from these bdelloids and identified using tandem mass tag labeling and high‐resolution mass spectrometric analysis with a Thermo Velos Orbitrap mass spectrometer. These proteins will the targeted for future analyses using pull‐downs, yeast two‐hybrid and other techniques to further examine their role in DNA repair. Overall, this study aims to identify candidate proteins and characterize their roles in DNA repair in bdelloids, which will further our understanding of this process in rotifers and other eukaryotes.

Support or Funding Information

This work was supported by grants from the National Center for Research Resources (5P20RR016460) & National Institute of General Medical Sciences (8P20GM103429) from the NIH.
Re‐emergence of chloramphenicol resistance and associated genetic background in Vibrio cholerae O1
Authors: Kumar Pramod , Yadav Priti , Nema Akansha , Goel Ajay K , Yadava Pramod K
Published: November 2023

Acquisition of antibiotic resistance in Vibrio cholerae is a growing concern for management of the disease. Here, we aimed to explore re‐emergence of evolution of chloramphenicol resistance in clinical strains of V. cholerae. A total of 120 strains originating from clinical sources (2004 to 2016) in different parts of India were subjected for antimicrobial susceptibility testing using disc diffusion and broth microdilution with reference to chloramphenicol. Further, the strains were screened using PCR for the presence of chloramphenicol resistance gene floR and the catB9 gene cassette. The floR and catB9 genes were further located in chromosomes followed by DNA sequence and prediction of promoters, higher order structures in mRNA and proteins. All clinical strains carried catB9 gene cassettes associated with super integrons (SI) on chromosome‐II. The floR gene was detected in 90% of the clinical strains and located on Variable Region‐III of Integrated and Conjugative Elements (ICE) on chromosome‐I. A strong positive correlation of floR gene and catB9 with higher chloramphenicol MICs were observed as compared to isolates harbouring catB9 alone. It was observed that floR gene is only responsible for significant increase in MICs but the strains were grouped as reduced susceptible as per CLSI breakpoints. catB9 gene cassette located downstream to a distal promoter (Pc) in SI was associated with very low‐level of resistance to the chloramphenicol. We have observed change in gene localization, change in 5′‐UTR (a ribo‐regulation) and point mutations in coding regions of floR associated with of differential resistance pattern. Acquisition of catB9 (permanently integrated) and floR genes are sequential events and their differential expression is tightly controlled in two different chromosomes. The Screening of floR gene can serve as the basis of V. cholerae classification as resistant
Sensitive and Specific Detection of Ligands Using Engineered Riboswitches
Authors: Laney Jessilyn P , Morse Daniel P
Published: November 2023

Riboswitches are RNA elements found in non‐coding regions of messenger RNAs that regulate gene expression through a ligand‐triggered conformational change (for review, see ref. 1). In, B. subtilis and other gram positive bacteria, purine metabolism is regulated by riboswitches that bind to guanine (2). The best studied of the guanine riboswitches regulates the transcription of the B. subtilis xpt‐pbuX operon. Guanine binding to this riboswitch inhibits transcription by stabilizing an alternative conformation that allows formation of a premature transcriptional terminator. In order to study guanine‐triggered structure switching, we have developed a fluorescence quenching assay for the function of the guanine riboswitch. Our system consists of the guanine‐binding domain (aptamer domain) of the riboswitch (xpt RNA), and two DNA oligonucleotides that are related to the 5′ and 3′ halves of the terminator stem (5′T and 3′T). xpt RNA is labeled at its 3′ end with a fluorophore and 5′T is labeled at its 5′ end with a fluorescence quencher. When 5′T is annealed to a complementary sequence at the 3′ end of xpt RNA, fluorescence is quenched. Guanine binding causes 5′T to dissociate from xpt RNA and anneal to 3′T. This strand‐exchange reaction causes an increase in fluorescence intensity as the quencher moves away from the fluorophore. Using this assay, we reproducibly detected as little as 5 nM guanine. We are currently using the same strategy to adapt a 2′‐deoxyguanosine riboswitch (3) for use as a probe that can detect its ligand in vitro. In addition, we have used our strand‐exchange reaction to develop a method for in vitro selection of variant riboswitches with altered ligand specificity. The results of our latest experiments will be discussed.

Support or Funding Information

Defense Threat Reduction Agency (DTRA), Office of Naval Research (ONR), Chemistry Department, U.S, Naval Academy
SOX18 transcription factor interactome: Protein‐Protein interaction a new road for anti‐cancer drug discovery
Authors: Moustaqil Mehdi Denis , Fontaine Frank , Overman Jeroen , Giles Nichole Denis , Bhumkar Akshay Denis , O'Caroll Ailis Denis , Francois Mathias , Gambin Yann Denis , Sierecki Emma Denis
Published: November 2023

Deciphering how transcription factors (TFs) coordinate genes expression is fundamental to understand biological processes and further develop new therapeutic approaches. A single transcription factor has a multiple transcriptional effects that are context‐dependent. This versatility of activity is thought to be mediated by different protein‐protein interactions (PPIs). These PPIs offer a new avenue for the selective pharmacological modulation of transcription factor activity, which has proven to be a challenging endeavour using standard drug discovery approaches centred on blocking protein/DNA binding or interfering with post‐translational modifications.

In this study we show that SOX18, a transcription factor a molecular switch of embryonic lymphatic vascular development and neo‐lymphangiogenesis during cancer metastasis, interacts with multiple protein partners specific to endothelial cells. Using a systematic truncation analysis of SOX18 domains, we propose a comprehensive map of the protein binding domains, revealing the existence of different interacting regions specific to subsets of SOX18 interactors. Using mutant forms of SOX18 protein associated with human rare disease Hypotrichosis‐Lymphedema‐Telangiectasia (HLT) we validate domains essential to drive specific SOX18‐dependent PPIs and correlate these variations with phenotypic variations observed in HLT.

Using a focused library of small molecules, we are able to show that distinct SOX18 protein partner recruitment can be disrupted. On this basis we propose an initial Structure‐Activity Relationship study, paving the way to the development of specific inhibitors to SOX18‐dependent protein‐protein interactions. This approach has enabled us to better understand the mode of action of a compound currently used in clinic to manage HLT condition. Finally, we show that one of our newly identified SOX18 small molecule inhibitor is able to prevent metastasis in a pre‐clinical model of breast cancer, further validating the value of targeting SOX18‐dependent transcription factor complexes.

This work opens up a new molecular strategy to target transcription factor activity and reposition this class of protein as viable drug target.
Selective Regulation of Hypoxia Inducible Factors by Endogenous Ligands
Authors: Romero Claudia Moreno , Wang Hanzhi , Bruick Richard
Published: November 2023

Hypoxia inducible factors (HIFs) are transcription factors that regulate hundreds of genes that are important in a multitude of pathways under low oxygen conditions. HIFs are heterodimers composed of an alpha subunit (HIF‐1a, HIF‐2a, or HIF‐3a) and a beta subunit, also known as the aryl hydrocarbon nuclear translocator (ARNT). The alpha subunit is stabilized under hypoxic conditions and regulates a number of genes involved in oxygen adaption and recovery. Once oxygen levels are normalized, the protein is ubiquitinated and degraded. However, in many cancers there is a defect in HIF regulation such as the inability to be inactivated, therefore HIF activity levels are always high which promotes tumor progression. Two members of the HIF family, HIF‐1 and HIF‐2, have been the focus of our research. Though these two factors are very similar, they activate certain unique genes, such as PGK1 and EPO, respectively. It is now evident that there are key structural differences among these two HIF isomers, most notably the presence of a significantly larger cavity within the Per‐ARNT‐Sim (PAS) protein‐protein interaction domain of HIF‐2a. It is hypothesized that this cavity could confer the ability of an endogenous ligand(s) to bind and regulate HIFs differentially, though the nature of those ligands remains unknown. In order to test the hypothesis, we first need to develop a model system in cultured cells which will allow us to explore the different regulation of the HIF isomers. In doing so, we will identify the HIF‐1 and HIF‐2 expression profile of different cell lines. We are specifically looking for cell lines that express both HIFs, with each having unique target genes that can serve as diagnostic reporters. Currently, we are looking at Hep3B cells, SW156, and SV582 to see if they meet these criteria. Once we identify a suitable cell line, we will attempt to introduce mutations via state of the art manipulation techniques such as the CRISPR/Cas‐9 system, in order to interfere with the ligand binding activity of these factors.

Support or Funding Information

Supported by the Cancer Prevention and Research Institute of Texas (RP130513)
Probing the Role of Dynamics in Allosteric Signal Transduction in the CO‐Sensing Transcription Factor CooA Using Site‐Directed Spin Label EPR Spectroscopy
Authors: Dent Matthew R. , Hines Judy P , Stevens Daniel J. , Burstyn Judith N
Published: November 2023

CooA (CO oxidation activator) is a CO‐sensing transcription factor that is a member of the cyclic AMP receptor protein/fumarate and nitrate reductase regulatory protein (CRP/FNR) superfamily. The CO‐sensing function of CooA is carried out by a heme cofactor: the heme can be reduced from Fe(III) to Fe(II) under anaerobic conditions, and binding of CO to Fe(II) heme results in allosteric activation of DNA binding activity. While crystal structures of CooA in both active and inactive states exist, there is no clear consensus on how the signal of CO binding propagates from the regulatory domain to the DNA binding domain. Growing evidence supports the notion that dynamics, in addition to structure, plays a crucial role in allosteric regulation. In CRP, NMR experiments highlighted the importance of entropy‐driven dynamics in effector‐mediated allostery. To understand the role of dynamics in propagating the effector‐binding signal in CooA, we employed site‐directed spin label electron paramagnetic resonance (SDSL‐EPR) spectroscopy. Through site‐directed mutagenesis of a fully active protein missing four native Cys residues, we reintroduced Cys residues in key locations for attachment of spin labels: the surface of the heme domain (K26C), the top of the 4/5 loop (E60C), the domain‐linking hinge (F132C, D134C) and the end of the DNA‐binding helix (S175C). All variants exhibited heme characteristics, and most exhibited CO‐dependent DNA binding affinities, nearly identical to those of wild‐type CooA. The variant F132C, missing a phenyl ring believed to make a key allosteric contact with the DNA‐binding helix, exhibits weak, constitutive DNA binding. Multicomponent signals were observed in EPR spectra of all Fe(III) variants labelled with the nitroxide probes MTSL and MAL‐6. EPR spectra of these probes are sensitive to variations on the nanosecond timescale, consistent with large‐scale motions of entire protein domains, and report the same multiplicity of dynamic populations. The presence of multicomponent signals that are responsive to osmolytes implicate multiple dynamic populations that arise from different local conformational states. The relative intensities of the component signals varied with the site of label attachment, demonstrating that local dynamics vary as a function of position in the CooA protein. These observations suggest that interconversion among different dynamic populations may play a role in the allosteric mechanism of signal transduction in CooA.

Support or Funding Information

National Science Foundation supported these studies under CHE‐1213739 (to J.N.B.)
Characterization of Transcriptional Changes in a Vimentin Knock‐out Mouse Model Following Influenza A Infection Using RNAseq
Authors: Koch Clarissa M , Davis Jennifer M , Abdala‐Valencia Hiam , Misharin Alexander V , Ridge Karen M
Published: November 2023

Introduction/Rationale

Our group has previously demonstrated that vimentin, an intermediate filament, is required for the assembly and activation of the NLRP3 inflammasome in an infection model of Influenza A virus (IAV). To further investigate the role of vimentin in the response to IAV, our aim was to compare wild‐type and vimentin‐null mice and study changes in specific cell populations of the lung following IAV infection using RNAseq as an unbiased approach to study transcriptional changes.

Methods

Wild‐type and vimentin‐null 129S mice were infected with 200pfu of Influenza A/WSN/33 virus. Lungs were collected on days 0, 2, 3, 4, and 6 post‐infection (p.i.) and tissue was processed into single‐cell suspension. AM, MoDC, and ATII cells were flow‐sorted and total RNA was subsequently isolated using an RNeasy Plus Mini extraction kit (Qiagen). Following mRNA‐enrichment, libraries were prepared and sequenced using Illumina NextSeq 500.

Results

RNA sequencing data demonstrated significant differences in gene expression between the wild‐type and vimentin‐null ATII, AM, and MoDC cells post‐IAV infection in three cell sub‐populations of the lung. Differential gene expression analysis and pairwise comparison between wild‐type and vimentin‐null cells identified a significant number of differentially expressed genes between the two groups. The newly identified gene clusters were grouped into multiple GO processes involved in the innate immune response and cellular structure.

Conclusions

Our RNAseq data demonstrated that following IAV infection, vimentin‐null mice have a strikingly different response compared to the wild‐type animals, which was evident in the alveolar macrophages (AM), monocyte‐derived cells (MoDC), and to a lesser extent in the alveolar type II (ATII) cell subpopulations and their respective differential gene analyses.

Support or Funding Information

Funding: HL128194 and HL071643
Developing a Tagged CRISPR‐Cas9 Enhancer Pull‐Down Assay
Authors: Yang Sophia , Rose Robert
Published: November 2023

The discovery of the CRISPR‐Cas system as an adaptive immune system for bacteria and archaea has stimulated much excitement in the scientific press. In type two CRISPR‐Cas systems, Cas9 is the RNA‐mediated endonuclease. Using a FLAG tagged Cas9, we propose to utilize the CRISPR‐Cas9 system to excise and pull down an enhancer to study associated proteins. We are first developing the technique using guide sequences targeting a GFP plasmid in mammalian cell culture. Cas9 is a unique single turnover enzyme and must undergo significant conformational changes during catalysis. In vitro studies indicate Cas9 does not release cleaved DNA after cutting. We propose that there is an ordered release of the products in vivo after Cas9 mediated cleavage of DNA, and that the off‐rate is slow. We are determining how Cas9 releases its products through a combination of different guide orientations for pull downs as well as performing atomic‐force microscopy studies. We will crystallize the product form of the Cas9/DNA complex to characterize the structural rearrangement. Knowing the structure of the product complex will improve our understanding of how Cas9 releases DNA post‐cleavage during genome editing. Ultimately, we will utilize this Cas9 pull down technique to excise and study proteins associated with the insulin enhancer.
Cas9: The Gateway to Genome Engineering
Authors: Schroeder Alexandra , Mendoza Naomi , Mahesh Rhea , Lim Shulammite , Howard Ashley
Published: November 2023

Bacterial defense mechanisms employ an adaptive immune system encoded by clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR‐associated (cas) genes to acquire immunity against phage infection and plasmid transfer. A CRISPR array is a series of repeated segments separated by unique spacer sequences. Immunity is gained after a short sequence of foreign DNA is inserted in a CRISPR locus as a new spacer. The entire CRISPR locus is transcribed into a long precursor‐CRISPR RNA (pre‐crRNA), which is processed into a mature form containing a spacer region and portions of the CRISPR repeat sequence. The crRNA serves as a template with which complementary sequences in foreign DNA are recognized and cut by Cas proteins, allowing bacteria to fend off invaders. In nature, the CRISPR‐Cas system makes site‐specific double‐strand breaks in target DNA to prevent viral infection, a function that is being repurposed as a powerful means of genome editing among researchers. There are six types of CRISPR‐Cas systems, each employing a unique set of Cas proteins. Type II‐A CRISPR systems use a single DNA endonuclease, Cas9, for the targeting and degradation of target DNA. Streptococcus pyogenes Cas9 (SpyCas9) is a 1368‐amino acid DNA endonuclease which has two distinct lobes: the alpha‐helical recognition lobe (REC), which is responsible for holding the DNA in place, and the nuclease lobe (NUC) containing the HNH and RuvC nuclease domains that snip the target double‐stranded DNA (dsDNA). When Cas9 is assembled with guide RNA, either a native CRISPR RNA (crRNA) with trans‐activating crRNA (tracrRNA) or a synthetic single guide RNA (sgRNA) in which the 3′ end of the crRNA is linked to the 5′ end of the tracrRNA, it begins DNA interrogation. Upon finding a proper protospacer adjacent motif (PAM), a three‐nucleotide sequence present only in targets and not in the host CRISPR array, Cas9 checks the flanking DNA for possible guide RNA complementarity. The 20‐nucleotide spacer sequence of the sgRNA can be changed easily, allowing scientists to make double‐stranded breaks to sequences of interest for editing. Although the CRISPR‐Cas9 system remains susceptible to error and the reasons behind undesired off‐target cleavage are not yet fully understood, the high specificity of Cas9 makes it a powerful genome editing tool. Our literature review and structural analysis using PyMol software and 3D printing of a physical model reveal how the structure of the Cas9 protein allows it to function as a programmable, RNA‐guided DNA endonuclease.

Support or Funding Information

This is a SMART Team project supported through the contributions of Dr. Megan Hochstrasser of The University of California, Berkeley, the Milwaukee School of Engineering, and the Olathe Medical Professions 21st Century Academy
Postmortem study on psychiatric disorders
Authors: Bao Ai‐Min
Published: November 2023

With the postmortem brain material obtained from the Netherlands Brain Bank, we have carried on a series of studies with the aim to elucidate the specific changes in the stress‐related brain systems in relation to special subtypes of mood disorders. Our final destination is to set up tailor‐made treatment for psychiatric disorder patients on the basis of his/her developmental history, genetic and epigenetic background, the vulnerability in particular neurobiological systems.

Support or Funding Information

AAA Symposium: Progress of Human Brain Banking in China: Construction and Research. Supported by AAA (American Association of Anatomists) and CSAS (Chinese Society for Anatomical Sciences), and by the National Natural Science Foundation of China (91332102 and 31271130).
Establishment of fetal brain atlases during the early second trimester
Authors: Liu Shu‐Wei
Published: November 2023

Objective

The use of spatiotemporal atlases can significantly improve the results and efficiency of automated analysis of fetal brain MRI data. The current study aims to establish age‐specific fetal brain atlases of the early second trimester (15–22 gestational weeks).

Materials and Methods

The thirty‐four postmortem fetal specimens of 15–22 weeks GW were collected and performed in a 7.0T Micro‐MRI. Both the data conversion and bias field correction were implemented as Pipeline workflows developed by Laboratory of Neuroimaging of University of Southern California. Manual removal of the non‐brain tissue was performed using BrainSuite software. For each cortex, 4 complementary global shape metrics were computed using LONI ShapeTools pipeline library – volume, surface area, shape index and curvedness. The script buildtemplateparallel.sh of Advanced Normalization Tools (ANTs) developed by University of Pennsylvania was run in each week to build the average template of each week. Three‐Dimension surface reconstruction of these templates were performed by BrainSuite. The general template was built from these weekly templates.

Results

From 15 to 22 gestational weeks, growth trajectories of brain area and volume is fitted well by linear regression model. The whole brain increased in volume by approximately 4‐fold and about 2.5‐fold in area. To reduce the bias effects of different demographic distributions of different numbers within each week, we built the templates each week first. After 3D surface reconstruction of these templates, we found that the 15th week still has some trails of neural folding in the early stages. The brain surface of 22nd week still looks smooth, but the whole brain's general shape looks more mature. The aggregated general template was then built based on these weekly templates. Four layers of lamination structures are displayed, from outer to inner, these layers are: cortical plate, subplate zone, intermediate zone, ventricular zone. The composed parts of basal ganglia could also be distinguished.

Conclusion

Our study first established the spatial‐temporal atlas of the early second trimester. And we also obtained the general developmental trajectories of the early fetal brain development. These atlases based on the high‐field MRI data provide good resolution and contrast, which will enable to characterize the dynamic anatomical changes of fetal brain development.

Support or Funding Information

The National Natural Science Foundation of China(No. 31071050, No. 31571237)
Formation of Dystrophic Neurites in Alzheimer's Disease
Authors: Yan Xiao‐Xin
Published: November 2023

Dystrophic neurites (DNs) are abnormal neuronal processes characterized microscopically by aberrant sprouting, dystrophic expansion, and accumulation of various cellular organelles and cytoskeletal/signaling proteins. DNs occur around neuritic plaques in the brains of subjects with Alzheimer's disease (AD) and various other neurological conditions. Despite decades of research, the mechanism underlying the development of DNs remains unclear. This talk will discuss the understanding of DNs in historic perspectives, with a focus on the relationship between the formation of DNs and beta‐amyloid deposition according to recent studies using postmortem human brains and AD animal models.

Support or Funding Information

AAA Symposium: Progress of Human Brain Banking in China: Construction and Research. Supported by AAA (American Association of Anatomists) and CSAS (Chinese Society for Anatomical Sciences).
Genetic Delivery of RNA Molecules to Alter Expression of EGFR in Brain Cancer Cells
Authors: Sivetz Nicole , Falotico Sarah C. , Nekrasov Peter , Hicks Martin J.
Published: November 2023

Glioblastoma multiforme (GBM) is an incurable and aggressive type of brain tumor. It is the most common central nervous system malignancy with a median survival of only 14 months. GBM is characterized by increased activation of one or more types of cell membrane receptors that drive the proliferation of tumor cells. One receptor in particular, epidermal growth factor receptor (EGFR), is dysregulated in about 60% of GBM tumors. The amplification and overexpression of the EGFR gene makes it a primary target for therapy. Although a great deal is known about the biology of EGFR‐activated GBM tumors, therapies against its biologic processes are limited by the blood‐brain barrier. Therapeutic strategies targeting splicing of the pre‐mRNA transcript, before the mRNA is translated into an active peptide, show great promise. In our current approach, we have designed and cloned a therapeutic gene that encodes a pre‐trans‐splicing RNA (PTR). Using anti‐sense targeting, the therapeutic PTR delivers an early stop codon and polyadenylation signal into the EGFR pre‐mRNA transcript, leading to a short, non‐membrane bound soluble peptide. To optimize the PTR we are testing alternative antisense binding sequences, as well as distinct splicing elements along the EGFR transcript. We make use of multiple GBM cell lines and RT‐PCR to measure changes in expression of the EGFR transcript. In addition, we are developing a novel approach using a fluorescent PTR (fPTR) to quickly measure changes in expression. Results show that our vectors were expressed at high levels with subsequent changes in EGFR mRNA expression. Our approach to deliver the DNA encoding anti‐EGFR PTRs to alter pre‐mRNA splicing of the EGFR mRNA transcript in GBM cells has the potential to work from the inside‐out, bypassing systemic barriers.

Support or Funding Information

Monmouth University School of Science Research Program, Bristol‐Myers Squibb, Independent College Fund of New Jersey, Johnson & Johnson
Understanding how two Similar RNA Binding Domains Mediate Different Protein‐Protein Interactions.
Authors: Marshall Collin
Published: November 2023

The process of alternative splicing is partly regulated by RNA binding proteins that bind to cis‐elements on the pre‐mRNA transcript and influence the assembly of a functional spliceosome at adjacent splice sites. Many RNA binding proteins occur as gene families with members sharing high sequence identity and domain arrangement yet have non‐overlapping expression patterns and different splicing activity. Polypyrimidine Tract Binding Protein 1 (PTBP1) and Polypyrimidine Tract Binding Protein 2 (PTBP2) are paralogous RNA binding proteins that share 74% sequence identity and a similar domain arrangement, which consists of four RNA binding domains connected by 3 linker regions. The two proteins differ in their splicing activity, where both proteins repress certain target exons well while others are more strongly repressed by PTBP1. Recent studies conducted to identify regions of primary structure that contribute to the different repression activity highlighted that RNA binding domain 2 (RRM2) of PTBP1 confers higher splicing repression compared to PTBP2. The RRM2 region of PTBP1 and PTBP2 share 87% sequence identity and have identical RNA interacting residues. PTBP1 RRM2 binds strongly to co‐repressor Raver 1. PTBP2 RRM2 has counterparts of most of the residues in the Raver interacting motif, however interacts weakly with Raver 1. These results indicate that the two proteins engage in different protein‐protein interactions albeit the high sequence identity in the RRM2 region. Thus we hypothesize that the RRM2 region is post‐translationally modified and plays a role in protein‐protein interactions, which lead to differences in splicing repression activity. To test this, we have over‐expressed Flag‐tagged PTBP1 RRM2 and PTBP2‐RRM2 in mouse neuro 2A cells, purified via Flag‐immunoprecipitation and analyzed for modifications via Mass Spectrometry. Preliminary results indicate that the region is modified in the two proteins. We are currently conducting site‐directed mutagenesis to identify the role of modifications on protein‐protein interactions and splicing repression activity.

Support or Funding Information

Howard Hughes Medical Institute (HHMI)
Biochemical Investigation into Novel Activities of Yeast SPOUT‐Superfamily RNA Methyltransferases
Authors: Mast Hannah , Hoskins Aaron
Published: November 2023

The SPOUT superfamily of S‐adenosyl‐L‐methionine (SAM) dependent methyltransferases are involved in post‐transcriptional RNA modifications. Nep1, a SPOUT superfamily member, methylates rRNA at the N1 position of a post‐transcriptionally incorporated pseudouridine in the 18S rRNA of Saccharomyces cerevisiae (yeast) and in humans is implicated in cases of Diamond‐Blackfan anemia. We found that Nep1 can bind and methylate a specific pseudouridine found in the U2 snRNA in vitro, suggesting that Nep1 may also play a role in splicing regulation. Given this finding, other SPOUT proteins may also be able to modify snRNAs and be involved in splicing regulation. YGR283C and YMR310C are two other yeast SPOUT methyl transferases whose specific biological functions are not known but are known to interact with the U2 snRNP yeast protein, Lea1, and the yeast homolog of dyskerin, a protein involved in pseudouridylation of the U2 snRNA. We have expressed yeast YGR283C in E. coli and purified the protein for biochemical and structural characterization. Fluorescence polarization assays and electrophoretic mobility shift assays will be conducted to quantify YGR283C binding affinities for snRNAs. Filter binding assays will be used to quantify [3H]‐methyl incorporation from SAM into snRNA substrates. Together these studies will elucidate the function of these previously uncharacterized SPOUT superfamily members.
The Sbp1p Paralog Rnp1p Has A Role In P‐body Assembly Without Affecting Translational Repression
Authors: Segal Scott P , Chanhthee Alvin , Mackay Katherine
Published: November 2023

Global translational repression occurs in response to many physiological stresses, including glucose deprivation, heat stress and genotoxic stress. Upon exit from the polysomes, mRNA can localize to either stress granules to be stored or P‐bodies to possibly be degraded. In yeast, a number of proteins are involved in translational repression in response to physiological stress. Among these proteins is Sbp1p, which contains two RRM domains that flank a central RGG box. Sbp1p, in part, acts to repress translation by binding eIF4G through its RGG box. Additionally, Sbp1p can also localize to both P‐bodies and stress granules. Since Sbp1p has the ability to interact with the translation initiation machinery, this suggests that this protein may have a greater role in mRNA storage in stress granules.

Rnp1p is an Sbp1p paralog and structurally shares the two RRM domains, but does not contain the central RGG box. Interestingly, Rnp1p appears to have no effect on translational repression in response to genotoxic stress. However, overexpression of Rnp1p can drive assembly of P‐bodies, but not stress granules. Moreover, strains lacking Rnp1p are deficient in P‐body assembly in response to physiological stresses that result primarily in P‐body assembly, but not assembly of stress granules. Taken together, these data suggest that Rnp1p may have a role in the assembly of P‐bodies. Further work will ascertain whether Rnp1p can localize to P‐bodies and with which proteins Rnp1p interacts.
Characterization of Macromolecular Interactions that Influence Pichia pastoris Vac8p Function in Microautophagy
Authors: Bellmore Jason T. , Potokar Elijah S. , Morales Joseph A. , Patel Shreyas D. , Fry Michelle R.
Published: November 2023

Previous work in our laboratory has demonstrated that PpVac8p, a protein required for microautophagy in Pichia pastoris, is an armadillo repeat protein that contains putative fatty acylation sites at the amino terminus. Furthermore, we had demonstrated that PpVac8p armadillo repeat domains were essential for protein function in the vacuolar sequestration steps of microautophagy. We have employed immunoblot analysis and fluorescence microscopy to investigate a putative interaction between PpVac8‐GFP and actin, a cytoskeletal protein. The results of these studies will help elucidate protein‐protein and protein‐membrane interactions that influence the behavior and function of PpVac8p.