bluuebunny/crossref_metadata_2025 huggingface dataset:
Landmark‐Based Shape Analysis of the Brain: An Error Study
Authors: Chollet Madeleine B , Aldridge Kristina , Pangborn Nicole , Weinberg Seth , Nopoulos Peg , DeLeon Valerie B
Published: June 2021

Neuroanatomic phenotypes are often assessed using volumetric analysis. Although powerful and versatile, this approach is unable to 1) determine whether changes in size are global or local, 2) quantify changes in shape, or 3) describe how regions are interrelated. Statistical shape analysis is the preferred method for testing these aspects of phenotype. Coordinate data from biologically relevant landmarks are one commonly used way to quantify “shape” of an object. To date, approximately fifty landmarks have been used to study brain shape. Of the studies that used landmark‐based statistical shape analysis on the brain, most have not published the protocols they used for landmark identification or the results of reliability studies on these landmarks. The primary aims of this study were two‐fold: (1) to collaboratively develop detailed protocols for a set of brain landmarks, and (2) to complete an inter‐observer (n = 4) validation study of the set of landmarks on 10 MR scans of three trials each. Detailed protocols were developed for 29 cortical and subcortical landmarks. MANOVA revealed that this set of landmarks was able to accurately distinguish between individuals (p < 0.01). Precision varied between 0.71 – 6.50 mm. This was the first study to quantitatively assess landmarks for use in statistical shape analysis of the brain and documents the utility of landmark data for studies of neuroanatomic phenotypes.

Grant Funding Source: NIDCR 1F31DE021302‐01
True Membranous Labyrinth in Human being
Authors: TANIOKA HISAYA , KAGA KIMITAKA
Published: June 2021

BACKGROUD

The standard for studying temporal bone anatomy has been histopathological sectioning by microtome following chemical fixation and deossification of the temporal bone specimen. No one can observe the membranous labyrinth in a living body by any methods.

PURPOSE

Direct synthesize the membranous labyrinth from the conventional CT exam.

SUBJECTS and METHODS

A total 10 normal ears were studies. All CT images were acquired on a single row CT scanner (ProSpeed AI; GE). Scanning parameters were 120kV and 120mA. Scanning was performed using collimation and pitch values of 1.00mm and 0.50 respectively, and exposure duration of 22 sec. For high‐resolution reconstructions, we used 1mm slice thickness; the reconstructed interval was 0.50mm. The field of view was reduced 9.8cm, using a 512 matrix. The axial data were applied to create 3D images on the CT work station for post processing with the standard distributed software.

RESULTS

3D visualization of the different structures of the membranous labyrinth allowed display of the correct anatomy by high detail in all cases. We could see cochlear duct, ductus reunions, saccule, utricle and ampullae.

DISCUSSION

The microimaging techniques we have employed produce images that are similar in appearance to those acquired in the multiplanar atlas of preserved cadaver temporal bone specimens acquired with CT and MRI. But, the shape of saccule and utricle in human being was different from the cadaver temporal bone specimens. The normal shape of saccule was a semicylindrical shape, not balloon shape. The utricle was seen by a solid heart‐shaped form. These findings were inconsistent with previous text books. The main cause of these results was due to living or not living subjects.
Microanatomy Of The Lateral Femoral Cutaneous Nerve In Relation To Inguinal Ligament
Authors: Chakravarthy Marx Sadacharan , D'Souza Antony Sylvan , Anitha Marx Chakravarthy
Published: June 2021

Aim

Meralgia paresthetica is a painful mononeuropathy resulting from compression or focal entrapment of lateral femoral cutaneous nerve (LFCN) as it passes deep to or through the inguinal ligament (IL). There are no reports available on cross‐sectional microanatomy of LFCN. The aim of the study was an attempt to find the changes in non‐fascicular components (Adipose (FAT) and non‐adipose tissue (connective tissue) (nFAT)) of LFCN just above IL (LFCN‐1), at IL (LFCN‐2) and below IL (LFCN‐3).

Methods

We studied 21 human cadaveric LFCN‐1, LFCN‐2 and LFCN‐3 samples. Paraffin sections of these nerves were processed by Masson's trichrome stain for connective tissue. FAT and nFAT areas were calculated from the non‐fascicular areas of these nerves.

Results

Fascicles was found to be more in LFCN‐3 when compared to LFCN‐1 and LFCN‐2. There was more nFAT in LFCN‐2 when compared to LFCN‐1 and LFCN‐3. The comparison of the nFAT and FAT between LFCN‐1 and LFCN‐2 (p<0.001), LFCN‐1 and LFCN‐3 (p<0.001), and LFCN‐2 and LFCN‐3 (p<0.001) were statistically significant.

Conclusions

More nFAT when compared to FAT was found in LFCN‐2. These results of the study might help to explain Meralgia paresthetica resulting from compression or focal entrapment of the LFCN as it passes deep to or through the IL. Microanatomical knowledge of the LFCN is essential for decompression and neurolysis of the nerve in the treatment of meralgia paresthetica.
Congenital solitary kidney with three renal arteries. An MDCT angiographyc study
Authors: MICLAUS Gratian D. , PLES Horia , MATUSZ Petru L.
Published: June 2021

Congenital solitay kidney (CSK) is one of the most rare renal malformations. CSK has usually a single renal artery. The MDCT angiography of 1700 clinical cases revealed 4 with CSK. Among these only one case with right CSK (male) presented besides the main renal artery also two additional arteries. The main renal artery with origin in the abdominal part of aorta (APA) at the level of L1‐L2 intervertebral disc, with a diameter at origin of 0.53cm, a length of 3.51cm gives rise at three segmentat arteries (SA) (superior, anterior‐superior and posterior superior). The first additional artery originated in the APA at the level of 1/3 lower of L2 vertebra, with a diameter at origin of 0.34cm, and a length of 3.70cm gives rise to two SA (anterior‐inferior and inferior). The second additional renal artery originated in the APA at the level of 1/3 lower of L3 vertebra, with a diameter at origin of 0.32cm and a length of 4.77cm gives rise to a single SA (posterior‐inferior). This association of CSK with the presence of the additional renal arteries involves two distinctive embryologic mechanisms: non‐development of the nephrogenic tissue in the left part, with the persistence of the mesonephric arteries in the right. The knowlwdge of these aspects is important when performing partial kidney resections. (Supported by Grant CNMP 42097/2008).
Dab2 in early skeletal muscle development
Authors: SHANG Na , Samuel Mok C. , ZHAO Hui , CHAN Wood Yee
Published: June 2021

Dab2 is an intracellular adaptor protein and a potential tumor suppressor. In mouse embryos, our study indicated that Dab2 was first expressed in the medial aspect of the dermomyotome at E9.5, and then co‐localized with the early muscle markers Pax3 and Myf5 at the ventrolateral lip of the dermomyotome at E10.5. It has also been known that Dab2 is involved in the MAPK signaling pathway, which is crucial to the muscle development. These observations suggested the potential role of Dab2 in the early skeletal muscle development. To further prove this role, Xenopus laevis embryos and C2C12 myoblasts were employed as in vivo and in vitro models in this study. In situ hybridization results showed that XDab2 was expressed in somites of Xenopus embryos and co‐localized with the muscle markers Pax3 and MyoD. Knockdown of XDab2 expression with antisense morpholinos in somites down regulated the expression of these two muscle markers. When C2C12 myoblasts were induced to differentiate into myotubes, Dab2 expression was simultaneously increased. Suppression of Dab2 expression with miRNAs during myotube formation resulted in reduced myoblast fusion and decreased numbers of myotubes. Our results indicated that Dab2 plays an essential role in the early development of skeletal muscle.
Syndecan peptides alter cell migration and fibronectin fibrillogenesis of syndecan 1 null dermal fibroblasts
Authors: Palsen Sarah , Parthasarathy Arpitha , Tadvalkar Gauri , Pal‐Ghosh Sonali , Pal‐Ghosh Sonali , Pal‐Ghosh Sonali , Pal‐Ghosh Sonali , Stepp Mary Ann
Published: June 2021

Syndecan 1 (Sdc1) is a transmembrane proteoglycan known to regulate integrin activity at the cell surface. The influence of sdc1 on integrin activity can be assessed by evaluating the effect of peptides derived from syndecan core proteins on cell migration rates and extracellular matrix assembly. If syndecan peptides activate integrins, cell migration rates will be reduced because activated integrins have a higher affinity for adhesion to substrates, which increases the frictional force needed to allow cells to migrate. In addition, sdc peptide‐induced integrin activation would enhance fibronectin (FN) fibrillogenesis. To determine whether the core proteins of sdc1 and sdc4 affect cell migration and FN fibrillogenesis, sdc1 and sdc4 ectodomain peptides tagged with GST were produced in transformed bacteria. Peptides were added to wild‐type and sdc1‐null tertiary passage dermal fibroblasts. Cells were tracked by time‐lapse microscopy and were also fixed and stained to reveal the organization state of the fibronectin matrix. Results show that sdc1 and sdc4 peptides had no statistically significant impact on wt cell migration rates but the sd4 peptide eliminated the difference between wt and sdc1 null cell migration rates whereas the sdc1 peptide had no affect. Sdc1 null dermal fibroblasts had less FN present within their matrix. Preliminary studies show that sdc4 peptides increased FN fibrillogenesis in sdc1 null fibroblasts. These data suggest that sdc peptides alter integrin activity in cells that lack endogenous sdc1.
CAG/CTG hairpin removal by coordinated actions of EXO1 and FEN‐1
Authors: Zhang Tianyi , Huang Jian , Gu Liya , Li Guo‐min
Published: June 2021

Instability of CAG/CTG trinucleotide repeats in humans is associated with many neurological and neurodegenerative diseases, including Huntington's disease. Increasing evidence suggests that formation of a stable DNA hairpin structure within the CAG/CTG repeats during DNA metabolism leads to their instability. Recent biochemical studies have revealed that human nuclear extracts can accurately remove CAG/CTG hairpins in a manner dependent on structure‐specific endo‐ and exonucleases, followed by DNA resynthesis and ligation. However, none of the components involved in this hairpin removal pathway has been identified. Based on the fact that exonuclease 1 (EXO1) and flap endonuclease 1(FEN‐1) play important roles in maintaining CAG/CTG repeat stability, we tested FEN‐1 and EXO1 for their roles in removing a CAG/CTG hairpin located in the nicked strand of a circular heteroduplex in a defined in vitro hairpin repair system. We demonstrate here that EXO1, but not FEN‐1, can initiate the excision reaction at the site of the strand break. However, the EXO1 excision terminates when the enzyme reaches the base of the hairpin, leaving the hairpin as an intact structure. Interestingly, while FEN‐1 cannot initiate the hairpin repair reaction, it can remove the hairpin structure when it is incubated with the excision intermediates generated by EXO1. These results suggest that the processing of the CAG/CTG hairpin heteroduplex by EXO1 allows the formation of a 5′ flap structure containing the hairpin, which can be removed by the flap endonuclease activity of FEN‐1. This activity does not require mismatch proteins MutSα, MutSβ, and MutLα, nor do these proteins interfere with the removal activity. Therefore, our data indicate that CAT/CTG hairpins located in a nicked DNA strand can be removed by the coordinated actions of EXO1 and FEN‐1, followed by DNA resynthesis and ligation.
Identification of critical residues in DNA polymerase III alpha through protein engineering
Authors: Parasuram Ramya , Sharma Rajal , Sefcikova Jana , Beuning Penny J , Ondrechen Mary Jo
Published: June 2021

The process of replication of DNA is carried out with high efficiency and accuracy by DNA polymerases. The replicative polymerase in E. coli is DNA Pol III, which is a 10‐subunit complex that coordinates simultaneous replication on the leading and lagging strands. The 1160‐residue alpha subunit copies DNA accurately, making one error per 105 nucleotide incorporations. The goal of my research is to understand the residues that contribute to this high fidelity and activity of polymerase subunit. Homology modelingand the methods of Theoretical Microscopic Anomalous Titration Curve Shapes (THEMATICS) and Partial Order Optimum Likelihood (POOL)have been used to predict functionally important amino acid residues. Site‐directed mutagenesis and primer extension assays have been used to validate the predictions. In addition to the three known catalytic Aspartates, to date we have identified five more residues that are critical for the polymerase activity. Work supported by NSF‐MCB‐0843603, NSF‐MCB‐0845033 and Research Corporation for Science Advancement.
Understanding radiation resistance using resistance‐promoting RecA mutants
Authors: Piechura Joseph R. , Petrova Vessela , Cox Michael M.
Published: June 2021

Multiple bacterial species exist that can survive the extensive DNA damage caused by ionizing radiation, yet the exact mechanisms of this resistance are not entirely understood. Using a directed evolution protocol, our lab has generated several strains of E. coli that are extremely radiation‐resistant. The genomes of multiple isolates from the evolved populations have been sequenced to identify mutations responsible for this phenotype. Two alleles were identified which encode mutations in RecA (A289S and D276A), a central protein in DNA repair. Inserting these alleles into wildtype E. coli resulted in small increases in survival after exposure to radiation. Thus, understanding the altered activity of these mutants can provide insight into DNA repair mechanisms and how an organism can survive ionizing radiation.

RecA D276A displays an increased ability to bind to DNA coated with the single stranded DNA binding protein (SSB). This suggests that faster activation of RecA may aid in cell survival after DNA damage. Conversely, the RecA A289S protein has a diminished capability to bind to SSB‐coated DNA, and thus, the RecF, RecO, and RecR loading proteins may have a greater role in facilitating RecA binding to SSB‐coated DNA with this mutant. Also, RecA A289S filaments disassemble faster when exposed to the inhibitory RecX protein. This data suggests that tighter regulation of RecA A289S activity may produce a filament form that enhances certain types of RecA‐mediated recombination events. This work was supported by a grant from the National Institutes of Health, GM32335.
Motif Q and I of SWI2/SNF2 family proteins are not involved in ATP binding but are required for hydrolysis
Authors: Gupta Meghna , Nongkhlaw Macmillan , Komath Sneha Sudha , Muthuswami Rohini
Published: June 2021

Objective

Deciphering the role of motif Q and I in the catalysis by Active DNA‐dependent ATPase A Domain (ADAAD).

Methods

Site‐directed Mutagenesis, Deletion Mutagenesis, Fluorescence Spectroscopy, Circular Dichroism Spectroscopy, and ATPase assays.

Results

ADAAD a representative member of SWI2/SNF2 family of proteins possesses the conserved helicase motifs including motif Q that are a characteristic feature of this family. It hydrolyzes ATP in the presence of stem‐loop DNA which mimics the double‐strand to single‐strand transition regions of DNA in a cell. In this study we have investigated the role of motif Q and motif I in ATP hydrolysis in ADAAD. The motif Q directs the catalytic rate while motif I is absolutely necessary for mediating catalysis. But neither of these motifs is required for ATP and DNA binding. The interaction of the protein with ATP appears to be mediated via adenine ring alone both in the absence and presence of DNA. The γphosphate of ATP is required only for inducing ATPase‐competent conformation.

Conclusion

The function of both motif Q and I appear to be at variance from other helicases.

Source of research support: Defense Research and Development Organization, Council of Scientific and Industrial Research, Department of Biotechnology.
Identification of Novel Domains within the Ioc2 and Ioc3 Subunits of the ISW1 Chromatin Remodeling Complex That Explicitly Distinguishes the Catalytic Activity of ISW1a and ISW1b
Authors: Olufemi Lola , Bartholomew Blaine
Published: June 2021

In S.cerevisiae, ISW1 forms two distinct chromatin remodeling complexes, ISW1a and ISW1b. ISW1a is composed of Isw1 and Ioc3 while, ISW1b is comprised of Isw1, Ioc2, and Ioc4. Although these complexes share the same ATPase subunit, both exhibit catalytic activity that is distinctive. ISW1a remodels with directionality preferences, spaces nucleosomes, and adapts active/inactive conformational changes dependent on linker DNA length. Meanwhile, ISW1b has substrate preference for short nucleosome arrays, lacks directional remodeling and spacing activity. The differences exhibited in these complexes are attributed to the associated subunits of each complex. Ioc2 has an atypical PHD motif that is often found in chromatin associated proteins and Ioc4 has a PWWP motif, a putative DNA binding domain. Ioc3 has no conserved domains that have been previously characterized, neither does it bear any similarity to proteins which their function has been defined. No work has been done to identify the domain organization or the contribution of these subunits to the activity of the ISW1 complexes. The goals of this project was to characterize the domain architecture of the Ioc2 and Ioc3 subunits of the ISW1 chromatin remodeling complexes, and also identify the key defining features of these two complexes. Using mutational analysis, this work explicitly identifies regions within the subunits that are required for the maintenance of complex integrity, modulation of nucleosome interaction, directional remodeling, and spacing activity. Further characterization of these domains revealed that their deletion results in remodeling and spacing activity reminiscent of its sister complex, suggesting that these domains serve as the distinguishing features that define the ISW1a and ISW1b complexes. This work was supported by Public Health Service grant GM 70864 from the National Institute of Health.
Direct interaction between Ku70/86 and BRG1 are required for nuclear receptor‐dependent transcriptional activation in vivo
Authors: Trotter Kevin W. , Archer Trevor K
Published: June 2021

The mammalian BRG1 chromatin remodeling complex has been shown to regulate gene expression by altering local chromatin structure and recruiting transcription factors to sequence‐specific DNA. BRG1, the central ATPase of the SWI/SNF complex, is critical for the functional activity of nuclear receptor complexes. Analysis using BRG1 mutants suggests the remodeling protein contains functional motifs outside of the ATPase domain that are important for transcription. To further examine these conserved domains we performed Y2H‐screens using baits spanning the BRG1 N‐terminus to identify proteins that may be required for SWI/SNF activity. Several proteins were found to associate with the N‐terminus of BRG1, including proteins involved in DNA repair, replication, transcription, and modulation of chromatin architecture. Interestingly, Ku70 was found to associate with the BRG1 region encompassing the conserved HSA and BRK domains. Ku70 functions as a single‐stranded DNA‐dependent ATP‐dependent helicase and plays a key role in multiple nuclear processes such as DNA repair and V(D)J recombination. Transcription assays and chromatin immunoprecipitation studies suggest Ku and components of the TopoIIâ/PARP1 complex may be necessary components of the SWI/SNF complex required for nuclear‐receptor mediated transcriptional activation in vivo.
Homodimerization of LDB1 is necessary for activation of the mouse β‐globin gene in vivo
Authors: Krivega Ivan , Dean Ann
Published: June 2021

A multiprotein complex that includes LDB1, TAL1, GATA‐1 and LMO2 proteins participates in long‐distant interaction in the β‐globin locus. The complex occupies the locus control region and the β‐globin promoter and mediates their proximity. Reduction of LDB1 using RNAi disrupts long‐distant interaction and blocks expression of the β‐globin gene. LDB1 protein has two defined domains that may participate in this process. The C‐terminal LIM‐interaction domain (LID) of LDB1 interacts with LMO2 which provides association of LDB1 with chromatin through DNA‐binding partners GATA‐1 and TAL1. It is known that the N‐terminal dimerisation domain (DD) of LDB1 participates in homodimerisation of the protein in vitro. We proposed that the DD domain of LDB1 plays a key role in long‐distant interaction between the LCR and β‐major gene promoter in vivo. Several deletions of LDB1 and a fusion protein that contains LMO2 and the DD domain of LDB1 were expressed in the background of endogenous LDB1 knock down MEL cells. These experiments demonstrated an important role for the LDB1 DD domain in β‐globin gene activation and LDB1 protein stability. Co‐immunoprecipitation of a truncated LDB1 protein consisting of only the DD domain with endogenous LDB1 confirms that homodimerisation of LDB1 in vivo occurs by interaction between DD domains. These results confirm the necessity of homodimerisation of LDB1 in the gene regulation processes.
Transcriptional Regulation by the Ccr4/Not4 Complex
Authors: Mehrotra Swati , Moon John , Vancura Ales
Published: June 2021

The CCR4/NOT complex is a global regulator of gene expression that affects expression both positively and negatively. The complex, conserved from yeast to human, is composed of at least nine subunits. The Ccr4 subunit functions in the control of mRNA deadenylation and transcriptional initiation/elongation. The NOT proteins are involved in transcriptional repression by restricting access of TFIID to promoters. The CCR4‐NOT complex also plays a key role in sensing nutrients and stress. We observed increased expression of stress responsive genes HSP12, HSP26 and CTT1 in ccr4Δ and not4 Δ strains. The aim of this study was to elucidate the mechanism of transcriptional regulation of the stress responsive genes in ccr4Δ and not4Δ strains. Western blot and chromatin immunoprecipitation revealed that ccr4Δ and not4Δ cells display a decreased level of histone acetylation that is not confined to specific loci but is global in distribution. The increased expression of stress responsive genes HSP12, HSP26 and CTT1 in ccr4Δ and not4Δ strains can be partially suppressed by mutations in histone deacetylases HDA1 and RPD3. The results implicate Ccr4/Not complex in regulation of histone acetylation and are consistent with a model in which Ccr4/Not complex‐supported histone acetylation is required for repression of stress‐inducible genes.
Histone deacetylase 9 regulates choline acetyltransferase gene expression in NG108‐15 cells
Authors: Aizawa Shu , Yamamuro Yutaka
Published: June 2021

Histone deacetylases (HDACs) catalyze the removal of acetyl groups from N‐terminal lysine residues of histone tails, resulting in chromatin condensation and transcriptional repression. We have previously shown that the inhibition of HDAC activity by a general and specific inhibitor of HDACs, trichostatin A, markedly increased the gene expression of choline acetyltransferase (ChAT), the synthetic enzyme for neurotransmitter acetylcholine and a specific marker for cholinergic phenotype, in NG108‐15 cells as a model of cholinergic neurons. In the present study, we examined which HDACs are involved in the regulation of ChAT gene. In experiments using class‐specific HDAC inhibitors, class IIa HDACs were a strong candidate for involvement in the regulation of ChAT gene in NG108‐15 cells. In addition, the expression of HDAC9, a class IIa enzyme, was dramatically decreased at the level of mRNA and protein during dibutyryl cyclic‐AMP‐induced cholinergic differentiation, whereas class I and other class IIa HDACs were not. Furthermore, the knockdown of HDAC9 by siRNA induced ChAT gene expression in proliferating cells. These results suggest that HDAC9 plays a key regulatory role in the acquisition of cholinergic phenotypes in NG108‐15 cells. This study was supported, in part, by a Research Fellowship of the Japan Society for the Promotion of Science to SA.
MassSQUIRM: an assay for quantitative measurement of lysine demethylase activity
Authors: Avaritt Nathan L. , Blair Lauren P. , Tackett Alan J.
Published: June 2021

In eukaryotes, DNA is wrapped around proteins called histones and condensed into chromatin. Lysine residues of the N‐terminal tails of histones can be mono‐, di‐ or tri‐methylated and these varying methylation states have been associated with different levels of gene expression. Enzymes that can add and remove these modifications are of particular interest due to their role in regulating transcription. The first demethylase discovered, LSD1 has been implicated in human cancers and contributes to DNA damage response pathways. Currently, there are limited methods for accurately studying the activity of demethylases in vitro or in vivo. In this work, we present MassSQUIRM: (Mass Spectrometric Quantitation Using Isotopic Reductive Methylation), a quantitative method for studying the activity of demethylases capable of removing mono‐ and di‐methyl marks from lysine residues. We focus specifically on LSD1 due to its potential as a prime therapeutic target for human disease. Our results show this quantitative approach will enable better characterization of the activity of LSD1 and other chromatin modifying enzymes in vitro, in vivo, or in response to inhibitors. This work was supported by the following National Institutes of Health grants: NIH R01 Roadmap DA025755 (AJT), NIH INBRE P20RR016460, and NIH COBRE P20RR015569.
Real‐time imaging and quantification of p300/CBP acetyltransferase inhibition using a FRET‐based reporter in living mammalian cells
Authors: Dancy Beverley Marie , Yoshida Minoru , Zhang Jin , Boeke Jef D. , Cole Philip A.
Published: June 2021

p300/CBP activate transcription by acetylating histones and other proteins and recruiting cofactors. p300/CBP act in response to a variety of signals, and their regulation of transcription controls major cellular pathways. Mutation of p300/CBP can cause Rubinstein‐Taybi syndrome, acute myeloid leukemia, and solid tumors. We have previously developed C646, a small molecule inhibitor of p300/CBP that is potent and specific in vitro. Our goal is to evaluate the pharmacodynamics of C646. Using a FRET‐based reporter in living cells, we observed that C646 is cell‐permeable, fast‐acting, and has a profound effect on histone acetylation; this effect is dose‐dependent and influenced by changes to the C646 structure. Furthermore, we found that cells with more active p300/CBP, generated either by deacetylase inhibition or p300 overexpression, respond more dramatically to C646, in contrast to cells with knocked down CBP, which do not respond to C646. We found that human melanoma cell lines are highly sensitive to C646, lending support to the view that p300/CBP are upregulated in this cancer. Thus, our work sheds light on the mechanism of oncogenesis in melanoma, as well as providing a potential therapeutic strategy. Technology Centers for Networks and Pathways, National Institutes of Health
Interactions Between HP1 Chromodomain and H3 Trimethyllysine9 Histone Tail
Authors: Eisert Robyn J , Waters Marcey
Published: June 2021

Recent findings suggest that a variety of post‐translational modifications (PTMs) on N‐terminal tails of histones are intricately involved in DNA packaging and directly control levels of gene expression. One such modification is the methylation of lysine9 on the H3 histone tail, which recruits heterochromatin protein 1 (HP1) via its chromodomain and results in silenced gene expression. The HP1 chromodomain binds the H3 K9Me3 with a KD of 17 μM with an aromatic cage surrounding the trimethyllysine. The histone tail is also inserted between two β‐strands of the chromodomain to form a 3‐stranded β‐sheet. Here, we are studying the sequence selectivity provided by the β‐sheet interactions and how those interactions compare to other model systems. Residue Thr6 of the histone tail forms cross‐strand interactions with Ala25 and Asp 62 of the chromodomain. Each of these three residues was systematically substituted for amino acids known to form favorable interactions in β‐sheets including hydrophobic, hydrogen‐bonding, and aromatic interactions. These studies were able to provide insights into the sequence selectivity of the chromodomain for the histone tail and demonstrated the applicability of information gleaned from model systems and statistical studies to protein‐protein recognition.
Differential histone modifications induced by chronic methamphetamine exposure in the rat striatum
Authors: Jayanthi Subramaniam , McCoy Michael T , Ladenheim Bruce , Martin Tracey , Beauvais Genevieve , Krasnova Irina N. , Hodges Amber B.
Published: June 2021

Methamphetamine (METH) is a very addictive drug. Repeated METH injections cause increases in spine density in striatal medium spiny neurons that receive inputs from corticostriatal glutamatergic projections. We thus undertook the present study to test the possibility that repeated injections of METH might influence the expression of AMPA glutamatergic receptors and of scaffolding proteins. We also measured the effects of METH on the enrichment of acetylated histone 4 on the promoters of AMPA receptors and Homer1. Repeated injections of increasing doses of METH caused significant decreases in the expression of AMPA glutamate receptors (GluA1 and GluA2). There were also METH‐induced decreases in the mRNAs that code for Homer1 and PSD95, proteins that participate in the regulation of the functional effects of glutamate receptors. ChIP‐PCR experiments using antibodies against histone H4 acetylated at lysine 5 (H4K5ac) or lysine 16 (H4K16ac) documented chronic METH‐mediated decreases in the enrichment of H4K5ac and H4K16ac proteins on the promoters of AMPA GluA1 and GluA2 receptors, and on the Homer1 promoter. These results suggest that dynamic regulation of histone H4 acetylation might be involved in the regulation of GluA1 and GluA2. These findings suggest that changes in AMPA receptors might underlie METH‐induced changes in striatal spine density.

This work is supported by DHHS/NIH/NIDA/IRP.
Heterochromatin is retained during lens fiber cell differentiation
Authors: Sheehan Robert Patrick , Duncan Melinda K
Published: June 2021

The lens of the eye contains both epithelial and fiber cells, with fibers cells deriving from the equatorial epithelium. When these cells differentiate, the nucleus and cellular organelles are broken down to facilitate transparency. However, small fragments of DNA can remain in fully mature lens fiber cells, although their structure is unknown. We found that histone H3, trimethylated on Lysine 9, as well as 5‐Methylcytosine, which are both associated with compact heterochromatin, co‐localize with these DNA fragments. In contrast, the DNA remnants are not associated with either histone H3 Lysine 9 acetylation or histone H4 lysine 8 acetylation, which are predominant in the more accessible euchromatin. This data suggests that the DNA fragments persisting in mature lens fiber cells are entirely composed of heterochromatin, likely because their highly compacted state prevented access of the nucleases responsible for DNA degradation during lens fiber cell differentiation. This led to the further hypothesis, which is currently being tested, that these fragments predominantly contain transcriptionally silent genes, which are likely to be found in heterochromatin. Supported by Howard Hughes Medical Institute and the National Eye Institute.
Structural insights into the regulation of histone methyltransferase SmyD3: hinge motion control of posttranslational activation
Authors: Sirinupong Nualpun , Brunzelle Joseph S , Doko Ernada , Yang Zhe
Published: June 2021

SmyD family represents a new class of chromatin regulators that play an important role in heart and skeletal muscle development, but the critical questions regarding how they are regulated posttranslationally remain unknown. Here we report the crystal structure of SmyD3 in complex with methyltransferase inhibitor sinefungin at 1.7 Å. The structure reveals that SmyD3 has a two‐lobed structure with the substrate binding cleft located at the bottom of a 15 Å deep crevice formed between the two lobes. Comparison of SmyD3 and SmyD1 clearly suggests that the CTD domain is able to undergo a large hinge bending motion that defines two distinct forms: SmyD3 adopts a closed conformation with the CTD domain acting like a lid and partially blocking the substrate binding cleft; in contrast, SmyD1 appears to represent an open form, where the CTD domain swings out by 12 Å from the N‐terminal lobe, forming a deep open cleft with the active site completely exposed. These findings provide novel structural insights into the autoinhibition mechanism that modulates the histone methyltransferase activity of the SmyD proteins, and support the observation that a posttranslational activation by Hsp90, is required to potentiate the activity of the proteins. Such knowledge would then be valuable for the development of new anticancer drugs targeting SmyD3 since this protein is essential for transcriptional regulation in human tumorigenesis.
Alternative Splicing of chrng gene generates Novel Isoforms with different N‐terminals
Authors: Azim Shafquat , Banday Abdul Rouf , Tabish Mohammad
Published: June 2021

The γ‐subunit of nicotinic acetylcholine receptor is encoded by chrng gene of mouse genome. It plays an important role in maturation of the receptor. The gene contains 12 exons and 11 introns. Two transcripts of chrng have already been reported that arise due to the presence or absence of 5th exon. The aim of this study is to analyze the gene structure and identify any other transcript that may produce different isoforms.

A combination of bioinformatics tools like gene finders, blast searches, alignment tools, etc. were used to study chrng gene. Using these methods three new coding exons namely N1, N2, and N3 were identified from 5′ UTR and 1st intron of chrng. Each one of these is capable of alternatively replacing the 1st exon of the reported transcript making three new transcripts differing at the 5′ end. Est database searches also revealed the presence of an Est corresponding to N3 exon. To confirm the presence of these transcripts RT‐PCR followed by semi‐nested PCR of RNA isolated from mouse brain was performed. Sequencing of the products further confirmed the results.

Thus, using bioinformatics and molecular biology techniques we have identified three new transcripts of chrng encoding γ‐subunit and having different N‐terminal sequences. The sequence encoded by N3 exon is predicted to have a signal peptide, phosphorylation and glycosylation site which lack in all other isoforms. Funding from CSIR, India is acknowledged.
Analysis of 5′Untranslated Region in Trans‐Sialidase Annoted Sequences in the T.cruzi Genomes
Authors: Galdino Tainah Silva , Brandão Adeílton Alves
Published: June 2021

Trans‐sialidase (TS) is a membrane glycoprotein belonging to a family of genes from multiple copies involved in the process of cell invasion of the vertebrate host by the Trypanosoma cruzi (T. cruzi). Previous studies have shown that 5′ untranslated region is responsible for controlling gene regulation. In this work, we evaluated segments from TS cDNAs deposited in the Genbank‐dbEST such as the trans‐splicing additional or multiple sites, and their respective signals. We also investigated the compositional variation and the size of the sequences among T. cruzi epimastigotes strains: Dm28c (TcI), Y and CL‐Brener (TcII) and 4167 (Zymodeme 3). To corroborate these theoretical sequences, we acquired new test sequences based on RT‐PCR and qPCR. The fragments obtained from the different T. cruzi strains were cloned, sequenced and compositionally analyzed. CLUSTAL X was used to edit and align both theoretical and obtained sequences. All the TS theoretical sequences were searched against the dbEST database using BLAST program, which resulted in 735 cDNAs sequences. We also estimated the size of the mini‐exon‐containing fragments as 312 base pairs found in Z3, 209 in TcI and 218 in TcII. No mutations in the gene duplication presented in the trans‐splicing site were observed in the different strains. The qPCR analysis detected no differences in TS genes expression, although these genes are involved in host infectivity.
Developmentally regulated alternative splicing of anti‐apoptotic proteins during adipogenesis
Authors: Patel Niketa A. , Apostolatos Andre , Yoder Sean , Ghansah Tomar , Watson James E. , Cooper Denise R. , Li Pengfei
Published: June 2021

Novel therapies to treat obesity and insulin resistance will benefit millions of people. 3T3‐L1 cell differentiation provides a model parallel to mouse adipogenesis. Pre‐adipocytes undergo apoptosis while mature adipocytes don't undergo apoptosis. The basis for this is poorly understood. Analysis of the differentiation of pre‐adipocytes using RT‐PCR, Western analysis and microarrays from days (D) 0 to 10 showed substantial changes in the expression of alternatively spliced proteins involved in apoptosis: PKCδ, caspase‐9 and Bcl‐x, suggesting post‐transcriptional control of adipogenesis. PKCδ is a crucial signaling kinase affecting downstream apoptotic pathways. On D0, cells expressed the apoptotic splice variants PKCδI, caspase 9a and Bcl‐xS. By D6, cells started expressing the anti‐apoptotic proteins PKCδII, caspase 9b and Bcl‐xL with a concurrent decrease in pro‐apoptotic proteins. Expression of PKCβII, pBAD, pPTEN, pAKT and Bcl2 involved in proliferation and survival, also increased by D6. We observed that the switch in splice variants was accompanied by increased expression of SR splicing factors which modulate alternative splicing. Hence, the programmed splicing of anti‐apoptotic proteins is a pivotal switch in differentiation that commits cells to a pro‐survival pathway. It may be possible to modulate adipogenesis for therapeutic treatments of obesity. Supported by NIDDK 054393 and VA.
Mutation in the βB2‐crystallin gene leads to cataracts, epithelial mesenchymal transition, and upregulation of integrins
Authors: Roop Victoria H. , Mamuya Fahmy , Duncan Melinda K.
Published: June 2021

The most abundant protein in the lens fiber cells is βB2‐crystallin (Crybb2). Mutations in the Crybb2 gene have been shown to cause cataracts in both mice and humans. A twelve nucleotide deletion in the lens epithelial cells, known as the Crybb2Phil mutation, leads to epithelial mesenchymal transition (EMT) during development. However, the mechanism behind this phenotype is poorly understood. The aim of this project is to understand the causes and effects of mutations in βB2‐crystallin. Tissues obtained from Crybb2Phil mutant adult mice were immunostained with αV, α2, α3, α5, α6, β1 integrin subunits, and phospo‐SMAD3. Compared to the wild type lens, β1, αV, and α5 were upregulated in the Crybb2Phil mutant. However, no changes were seen in α2, α3, and α6 integrin subunits. Phospho‐SMAD3 and smooth muscle actin (SMA) levels were also upregulated in Crybb2Philmice. Upregulation of SMA signifies that the Crybb2phil mice undergo EMT during development. Furthermore, over expression of β1 and αV integrin along with SMAD3 in these mutants suggest that the TGFβ‐SMAD interacting pathway may be involved. This project is funded by National Eye Institute grant EY015279. VR is a Charles Peter White Scholar.
APPLICATION OF HIGH HYDROSTATIC PRESSURE FOR RECOVERY OF BIOLOGICALLY ACTIVE CRUZAIN FROM RECOMBINANT INCLUSION BODIES
Authors: Chambi Rosa Maria Chura , Lemke Laura Simone , Vallejo Natália Malavasi , Neto Luiz Juliano , Morganti Ligia
Published: June 2021

Aggregation is a serious obstacle to recovery of biologically active proteins from inclusion bodies (IBs) produced by E. coli. High hydrostatic pressure was described as an interesting tool to solubilize and refold aggregated proteins. In order to obtain the refolding of aggregated and insoluble protein, cruzain IBs were subjected to compression (2 kbar) for 16 h and the effects of changes in ratio and concentration of oxido‐shuffling reagents and concentration of guanidine hydrochlorid were assayed. A 50% yield in refolding of the native cruzain was obtained using optimal conditions of the refolding buffer (Tris‐HCl buffer, pH 8.0, 0.75 M Gdn‐HCl, 2 mM EDTA). Additives in the refolding buffer did not further improve cruzain refolding yields. The presence of Bis‐ANS, a hydrophobic extrinsic fluorescent dye, which has been previously described to be able to suppress protein aggregation, surprisingly abolished completely cruzain refolding. The refolded cruzain presents enzymatic activity hydrolyzing a specific substrate (Z‐FR‐MCA). We demonstrated that high pressure can successfully refold cruzain with biological activity from insoluble aggregates in inclusion bodies.
Structure‐function differentiation of beta‐edge negative‐design variants of hemolysin A using size‐exclusion chromatography
Authors: McDermott James Joyce , Weaver Todd Michael
Published: June 2021

Hemolysin A (HpmA) from Proteus mirabilis is secreted via a Two‐Partner Secretion (TPS) pathway, which is utilized by Gram‐negative bacteria as an energy‐independent mechanism for secretion of virulence factors. Truncated HpmA (HpmA265) has been shown to activate full‐length HpmA in a template‐assisted fashion. Crystallographic analysis of HpmA265 revealed a right‐handed β‐helical structure and a dry dimeric interface between exposed carboxy‐terminal β‐edges. A series of carboxy‐terminal mutants of HpmA265 were engineered using negative‐design theory, where a positively charged lysine residue was substituted at the non‐polar dry, dimer interface. The replacements included phenylalanine 241 (F241K), methionine 245 (M245K), and leucine 263 (L263K). Recent circular dichroism and hemolysis studies reported decomposition of the classic beta signal and loss in hemolytic activity for M245K and L263K. To further investigate the relationship between HpmA265 structure and function, quantitative hemolytic activity was mapped to the resultant size‐exclusion chromatogram. Template‐assisted hemolysis of HpmA265 after size‐exclusion chromatography (SEC) showed hemolytic activity corresponding to a molecular weight of 27 kDa. This is close to the monomeric weight of 25 kDa for HpmA265, which suggest a monomeric form of HpmA265 initiates the folding of full‐length HpmA during hemolysis. Template‐assisted hemolytic activity of HpmA265 variants, F241K, L263K, and M245K, had activity concentrated around a molecular weight of 21 kDa, which again is indicative of monomeric activation of hemolysis. The following awards supported the research: NSF‐RUI (0744754) to TMW and ASBMB‐UAN Undergraduate Research Award to JJM.
Calnexin levels are regulated by the ER localized ubiquitin ligase Nixin
Authors: Neutzner Albert , Neutzner Melanie , Benischke Anne‐Sophie , Ryu Seung‐Wook , Youle Richard J , Karbowski Mariusz
Published: June 2021

To identify novel regulators of ER linked protein degradation and ER function, we determined the entire inventory of membrane spanning RING finger proteins localized to the ER. We identified twenty four ER membrane anchored ubiquitin ligases and found Nixin to be central for the regulation of calnexin turnover. Using fluorescence protease protection and hydrophobicity plotting, we established that Nixin spans the ER membrane once, thus exposing its RING domain to the cytosol.

Ectopic expression of wild type Nixin induced a strong downregulation of the ER localized chaperone calnexin that was prevented by inactivation of the Nixin RING domain as well as by proteasomal inhibition. Furthermore, Nixin physically interacts with calnexin in a glycosylation independent manner and enhances calnexin ubiquitination.

Taken together, the ER membrane‐anchored ubiquitin ligase Nixin regulates calnexin levels through proteasomal degradation and thus might play a role during ER stress recovery when calnexin levels are adjusted to pre‐stress levels.
The ATPase cycle of the RNA helicase protein NS3 from hepatitis C virus
Authors: Bradley Michael , Henn Arnon , Ding Steve , Pyle Anna , De La Cruz Enrique
Published: June 2021

The hepatitis C virus (HCV) NS3 protein possesses both RNA helicase and serine protease activities that are vital to HCV replication in infected cells. NS3 is also a more effective RNA helicase with the protease domain intact. NS3 is a member of the DExD/H‐box RNA helicase family, which recognize and remodel short structured stretches of RNA during every stage of RNA metabolism from synthesis to degradation. However, NS3 plays an important role in unwinding the + and − strands of the HCV genome during viral replication. This role requires ATPase activity that is tightly coupled with rapid helicase translocation and high processivity in concert with the viral RNA polymerase. To characterize the coupling of ATPase with helicase translocation activity of NS3, we utilized equilibrium binding and kinetic biophysical methods. By comparing a series of RNA substrates, we show that maximal RNA stimulation of ATPase activity occurs with very long RNAs, which affect both KM,RNA and kcat. The rate of ATPase cycling and helicase translocation along the RNA may be partially limited by rebinding NS3 to RNA following dissociation from the nucleic acid lattice during the ATPase cycle. The intact protease domain affords more productive engagement with the RNA lattice, which increases the ATPase‐translocase coupling efficiency (ATP consumed per RNA nt/bp travelled). An NSF CAREER Award (MCB‐0546353 to E.D.L.C.) supports this research.
Assembling a Molecular Switch: Using Protein Folding Mechanisms to Guide the Design of Novel Regulatory Elements
Authors: Wilson Corey J.
Published: June 2021

The quaternary‐structure of oligomeric proteins has far reaching implications. Namely, the ability to form dimeric or higher‐order oligomers dramatically enhances the biological activity of many proteins; this is of crucial importance for DNA‐binding proteins. From the vantage point of evolution, it may be simpler to create complex biological machinery from smaller protein subunits rather than from a massive single chain system. Likewise, understanding the relative stability and dynamic inter‐conversion during the protein folding process of oligomeric systems can provide insight into the energetics underlying biological regulatory mechanisms and assembly, in addition to the physicochemical properties that result in protein misfolding associated with disease. In turn, well‐defined folding‐assembly mechanisms can be used as a framework to create novel protein assemblies that are functional; this collection of engineered molecular switches can be used in a range of bio‐technological applications.