bluuebunny/crossref_metadata_2025 huggingface dataset:
Anatomical substract of the trapezius muscle myofascial trigger points
Authors: Akamatsu Flávia Emi , Saleh Samir , Andrade Mauro , Jácomo Alfredo
Published: June 2021

Myofascial pain syndrome (MPS) is a common cause of chronic musculoskeletal pain and is characterized by myofascial trigger points(MTP), limited range of motion in joints, referred pain and local twitch response during mechanical stimulation of MTP. MTP are clinically identified by palpation of a taut band of muscle or fascia. Physiopathology of MPS remains unclear and it has been suggested that MTP occur when a nociceptor and a muscular motor endplate coincide. Seven different MTP are clinically described for the trapezius muscle, but anatomical correlation is still lacking. Trapezius muscles from two adult male cadavers were carefully dissected from their origins in order to observe the exact point where branches of the spinal accessory nerve entered the muscle belly. Four points were identified: (1) Mid portion of the superior margin, into the vertical fibers that reach the clavicle, (2) Caudal and lateral to the first point, located into the transverse fibers of the muscle; (3) Medial fibers near to the inferior margin of the muscle; (4) Central part of the muscle belly, between C7‐D3 levels. These locations correspond to the clinically described MTP. Anatomical correlation of the MTP may be useful for a better understanding of the physiopathology of these disorders and provide basis for their treatment.
Sternalis muscle: Importance of its awareness in chest imaging and clinical significance
Authors: Pinhal‐Enfield Grace , Varricchio Paolo , Vasan Cheryl , DeFouw David , Vasan Nagaswami
Published: June 2021

The sternalis is an uncommon, variant muscle of the anterior thoracic wall that is estimated to be present in 8% of the human population. Students in a medical gross anatomy course were fortunate to discover a right, unilateral sternalis muscle during dissection of a 76‐year‐old female Caucasian cadaver. The sternalis appeared as a ribbon‐like strap and measured 15 cm in length with an average width of 2.5 cm. It was oriented on the anterior thoracic wall along the right margin of the sternum and medial to the sternocostal head of the right pectoralis major muscle. Despite its rarity, radiologists must be aware of the possibility of encountering the sternalis during thoracic imaging (CT scans, mammography, MRI) because of the risk for its misdiagnosis as a tumor. Further, risk for surgical complications such as damage to this muscle during breast surgery must be considered. Thus, although it may be difficult to perform a proper anatomic analysis of the infrequently observed sternalis muscle, it is important that students and clinicians be aware of its existence because of its potentially significant impact on clinical diagnosis and patient management. Therefore, anatomic studies through prosections, illustrations, photographs, diagnostic images, and detailed descriptions are warranted to increase awareness of the sternalis muscle and its variations among clinicians (especially radiologists and surgeons).
Comparative anatomy, ontogeny, evolution and phylogeny of primates, with special attention to the phylogenetic position of Tarsius, the relationships of hominoids, and the muscular variations of modern humans
Authors: Diogo Rui Pedro Boliqueime Martins , Wood Bernard
Published: June 2021

In this presentation we report the results of the first comprehensive cladistic analysis based on muscular characters that addresses the relationships among the major groups of living primates and taxa such as tree‐shrews and colugos (‘flying lemurs'). This analysis is based on information obtained from a review of the literature and from dissections of more than 50 specimens of various primate and non‐primate taxa. The genera included in the analysis were: Rattus, Tupaia, Cynocephalus, Lemur, Propithecus, Loris, Nycticebus, Tarsius, Callithrix, Pithecia, Aotus, Saimiri, Macaca, Papio, Cercopithecus, Colobus, Hylobates, Pongo, Gorilla, Pan and Homo. The cladistic analysis was based on 208 characters concerning the head, neck, pectoral and upper limb muscles of these taxa, using Nona & Winclada 2002. The relationships among the taxa in the most parsimonious trees obtained (L=382; RI=0.53; CI=0.65) largely conform to those supported by recent molecular studies: the Primates, Strepsirrhini, Lemuriformes, New World monkeys, Old World monkeys, the subfamily Cercopithecinae and the Hominoidea are monophyletic groups, and there is strong support for the following relationships among hominoids: [Hylobates, [Pongo, [Gorilla, [Pan, Homo]]]]. Our study revealed that, regarding its muscles, Tarsius is one of the most peculiar primates, displaying some plesiomorphic (e.g., the presence of a distinct muscle spinotrapezius) and derived (e.g., the presence of two series of contrahentes in the hand and of the facial muscle zygomatico‐auricularis) features that are rarely seen in other living primates. We will examine the phylogenetic, evolutionary and medical implications of our data, and discuss the use of myological, molecular and osteological characters in phylogenetic reconstructions.

Grant Funding Source: Mathers Foundation, GW Signature Program
Histological and lectin histochemical characterization of the epididymal duct in dromedary camels (Camelus dromedarius)
Authors: Hafez Shireen Abdelgawad , El‐Shafey Samir , Caceci Thomas
Published: June 2021

Regional microscopic anatomy of the epididymal duct of the dromedary camel was studied using H&E and a combination of Masson's trichrome and Verhoeff's staining. The initial segment of the epididymis was lined with high pseudostratified columnar epithelium with stereocilia; and with varying height levels from place to place giving the duct a “festooned” appearance. The nuclei of principal and basal cells were located in the basal one‐third of the epithelial cells. The duct was surrounded by a layer of circularly oriented smooth muscle cells; this peritubular smooth muscle thickened toward the termination of the epididymal duct. The middle and terminal segments of the epididymal duct were lined with the same type epithelium with no noticeable variations in height. The nuclei were located in the basal one‐half of the cells and spermatozoa were more evident in the lumen of the middle and terminal segments. Histochemical characteristics of epithelial cells were investigated using a panel of different lectins: Con‐A, DBA, MAA, SBA, SNA‐1, and WGA. These showed selective regional binding to different portions of the epididymal duct and varying degrees of binding to different cellular compartments. These regional variations in carbohydrate composition are discussed; they indicate a great deal of functional compartmentalization in the epididymal duct.

Grant Funding Source: professional development
Anatomical variants of the puboprostatic ligaments: Are they relevant to outcomes of radical prostatectomy?
Authors: Kim Michael , Johnson Marjorie , Carreno Alfonso Fernandez , Pautler Stephen E.
Published: June 2021

Prostate cancer affects men worldwide and is frequently treated by radical prostatectomy. The puboprostatic ligaments anchor the prostate to the pubis and must be manipulated for successful prostate removal. Previous studies have identified these ligaments but have not characterized variations typically observed. This study aims to classify the variants and investigate possible relationships between ligament type and post‐operative outcomes. Videos of 300 robot‐assisted laparoscopic radical prostatectomies (RARP) were viewed and images of the puboprostatic ligaments were isolated. The ligaments were classified based on morphology into a four‐class system: Fused, V‐shaped, Inverted V‐shape, Parallel. Initial results show that the majority of ligaments are parallel (37%) while fused ligaments are least common (2.7%). V‐shaped ligaments and inverted V‐shaped ligaments were identified in 19.7% and 11% of the population, respectively. This study proposes a puboprostatic ligament classification system that could be adopted universally, allowing better prediction of post‐operative outcomes. Further validation of our proposed classification system and investigation into its clinical significance is ongoing.

Grant Funding Source: Internal
MEF2A regulation of a miRNA mega‐cluster is required for proper skeletal muscle regeneration
Authors: Naya Frank J , Snyder Christine M , Held Aaron , Ewen Elizabeth P , Kielbasa Ondra M , Kandarian Susan C
Published: June 2021

Skeletal muscle has the ability to regenerate, in response to disease or injury, through the activation of muscle stem cells and their subsequent differentiation into myofibers. This process is controlled by a hierarchy of just a few transcription factors. The role of the MEF2 transcription factor family in skeletal muscle regeneration has not been studied. We performed RNAi‐mediated knockdown of MEF2A in C2C12 cells, an established line of proliferating myoblasts isolated from injured muscle. Unlike developmental myogenesis, MEF2A is the first MEF2 factor expressed in this model. Knockdown of MEF2A in myoblasts resulted in markedly impaired differentiation. Given the source of C2C12 myoblasts we investigated the potential function of MEF2A in regenerative myogenesis in vivo. Cardiotoxin‐induced muscle injury in MEF2A knockout mice resulted in widespread necrosis and reduced myofiber cross‐sectional area indicating defective regeneration. Mechanistically, microarray analysis revealed a downregulation of more than 40 microRNAs (miRNAs); all of which are located in a large miRNA gene cluster. These miRNAs are not related to any of the well‐characterized skeletal muscle‐specific miRNAs. We demonstrate that a single, upstream promoter is bound and activated by MEF2A, suggesting its ability to regulate the entire miRNA domain. Furthermore, miRNA target gene prediction analysis revealed that a cohort of these miRNAs targets inhibitors of Wnt signaling, a key pathway promoting skeletal muscle regeneration. Consistent with the miRNA downregulation these Wnt inhibitors are massively upregulated in injured MEF2A‐deficient muscle. Thus, miRNA‐mediated modulation of Wnt signaling by MEF2A is a requisite step for proper muscle regeneration. Supported by NIH grant HL73304 to FJN.
Tubulobulbar complexes in Sertoli cell primary cultures
Authors: Du Min , Chan Alex , De Asis Marc , Cipollone Jane , Roskelley Calvin , Vogl Wayne
Published: June 2021

In the seminiferous epithelium, unique double membrane structures termed tubulobulbar complexes (TBCs) develop in association with basal junction complexes between Sertoli cells. These actin‐related structures are thought to internalize intercellular junctions during the movement of spermatocytes from basal to adluminal compartments of the epithelium. We are establishing a primary Sertoli cell culture system to study the role of basal TBCs in junction turnover. We confirm that Sertoli cells isolated from 20 day‐old rat pups morphologically differentiate when grown on Matrigel in Transwells. The cells are high cuboidal to columnar in shape with basal nuclei, and with basal junction complexes similar to the appearance of junction complexes between Sertoli cells in vivo. Significantly, TBCs and TBC‐like structures develop in association with intercellular junctions. Double‐membrane coated‐pits, together with double‐membrane tubular structures capped by coated‐pits are observed in regions related to ectoplasmic specializations. When immunostained for integral junction proteins, “rod‐like” structures are observed to extend into the cell from the periphery. These observations indicate that TBCs are able to develop in differentiated Sertoli cells in culture, making further functional analyses of TBCs possible in vitro. Supported by an NSERC Discovery Grant to AWV.

Grant Funding Source: NSERC
Spectrin and plectin are present at apical tubulobulbar complexes in the seminiferous epithelium
Authors: de Asis Marc Aristaeus , Vogl A. Wayne
Published: June 2021

Tubulobulbar complexes (TBCs) are actin‐related double membrane structures that are suspected to internalize intercellular junctions, a process that is crucial for junction turnover and sperm release. At apical sites of adhesion between spermatids and Sertoli cells, each TBC consists of a tubulobulbar projection of the spermatid plasma membrane, the surrounding attached plasma membrane of the adjacent Sertoli cell, and an actin network cuff. In other systems, spectrin is part of the membrane skeleton that links actin filaments to the plasma membrane. Others previously have reported that the protein is present at differentiating germ cells and epididymal spermatozoa. Plectin links cytoskeletal elements to each other and to the plasma membrane. We probed the seminiferous epithelium for spectrin, plectin and actin on fixed frozen sections of rat testis and fixed epithelial fragments. Both in sections and in epithelial fragments, beta‐spectrin II and plectin are localized to apical actin‐related tubulobulbar. Neither protein is concentrated at actin‐related intercellular adhesion junctions (ectoplasmic specializations). Our results demonstrate that both spectrin and plectin are present in Sertoli cells at tubulobulbar complexes. Supported by an NSERC Discovery Grant to AWV.

Grant Funding Source: NSERC
The C‐terminus of Decorin Regulates Class II Small Leucine Rich Proteoglycans (SLRPs) in Corneal Stromal Matrix Assembly
Authors: Chen Shoujun , Kao Winston , Birk David
Published: June 2021

The purpose of this work is to study the regulatory role of the decorin in corneal stromal matrix assembly. In human congenital stromal corneal dystrophy mutant decorin is expressed with a truncated C‐terminus. A transgenic mouse model of the disease was created in a decorin wild type background. Both wild type and mutant decorin were expressed in mutant mice. Corneal opacities developed throughout the stroma in mutant mice, but were more severe in posterior stroma. The orthogonal lamellar structure was disrupted, with relatively normal lamellae separated by abnormal zones. In these abnormal zones, collagen fibrils were heterogeneous in diameter with increased and irregular inter‐fibril spacing. The aberrant zone was more obvious near keratocyte suggested an area differential dysfunctional matrix assembly. Expression of all class II stromal SLRPs was altered in the mutant mouse. Both lumican and keratocan were down‐regulated, and fibromodulin was up‐regulated. The C‐terminus of decorin is implicated in maintenance of protein core conformation and ligand binding. The data suggest that loss of the C‐terminus altered decorin binding directly or indirectly through other extracellular matrix components to keratocytes, changing the interactions between decorin and other SLRPs. These interactions are important for the rigid control of fibrillogenesis and fibril organization in cornea stromal matrix assembly.

Grant Funding Source: NIH grants EY05129, AR 44745 (DEB), 2010 postdoctoral fellowship from American Association of Anatomists (SC), 2010 postdoctoral fellowship from Fight for Sight (SC).
Coupling dTTP hydrolysis with DNA unwinding by the helicase of bacteriophage T7
Authors: Satapathy Ajit Kumar , Kulczyk Arkadiusz W , Ghosh Sharmistha , Richardson Charles C
Published: June 2021

The functional form of the DNA helicase encoded by bacteriophage T7 is a hexamer of six identical subunits. The helicase couples the hydrolysis of dTTP to unidirectional translocation and unwinding of DNA. The nucleotide binding site of the helicase is located at the subunit interface. In a crystallographic structure of the T7 helicase with bound nucleoside 5′‐triphosphate, Phe523 positioned in a beta‐hairpin loop at the sub‐unit interface is buried within the interface. However, in the unbound state, Phe523 is more exposed towards the outer surface of the helicase. This different orientation suggests that Phe523 and the associated beta‐hairpin loop may undergo a conformational change coupled with nucleotide hydrolysis. Replacement of Phe523 with alanine or valine abolishes the ability of the helicase to unwind DNA and to stimulate T7 polymerase activity on double‐stranded DNA. Genetic and biochemical experiments indicate that a hydrophobic residue with longer side chains is essential at this position. In addition, Phe523 appears to play a critical role in coupling the hydrolysis of dTTP to DNA unwinding. We postulate that upon dTTP hydrolysis, Phe523 moves from within the sub‐unit interface to a more exposed position where it may intercalate into double stranded DNA to facilitate DNA unwinding by the helicase. Results from this study support the strand exclusion mechanism of DNA unwinding by ring helicases.
A new role for SPO11 during mouse spermatogenesis
Authors: Boateng Kingsley A , Bellani Marina A , Pratto Florencia , Camerini‐Otero Rafael Daniel
Published: June 2021

Synapsis of the paired homologs and recombination are prerequisites for accuratechromosome segregation in meiosis. It is not clear whether there is a homology dependent mechanism that brings homologs together before double‐strand breaks (DSBs) initiate recombination in prophase. We are currently investigating the existence of homologous chromosomal pairing in mammalian premeiotic (spermatogonia) and meiotic S‐phase. Our data so far suggest that a significant level of pairing occurs in cells in meiotic S–phase (preleptotene mouse spermatocytes) as assessed by both interstitial and sub‐telomeric chromosome specific FISH probes. Taking advantage of a mouse expressing a catalytic mutant of SPO11, we show that this pairing requires SPO11, but is independent of its DSB activity, consistent with a previous report in budding yeast (Cha et al. (2000) Genes and Development 14: 493). This is the first report of such potential early functions of SPO11 in mammals. We also demonstrate that early pairing may require the nuclear membrane protein SUN1, important for telomere attachment to the nuclear membrane and gametogenesis (Ding X. et. al. (2007) Dev Cell 12:863–872; Chi Y. et. al. (2009) Development 136:965–973 ) and that SUN1 may directly interact with SPO11. Although the generation of DSB is normal in Sun1−/− mice, pairing and synapsis are impaired during meiotic prophase. Our observation that preleptotene homologous pairing is also disrupted in Sun1−/− mice suggests that this phenomenon is not required for the generation of SPO11 mediated DSBs.
Genome‐wide Analysis Reveals Novel Molecular Features of Mouse Recombination
Authors: Brick Kevin , Gregoretti Ivan , Smagulova Fatima , Khil Pavel , Camerini‐Otero Rafael Daniel , Petukhova Galina
Published: June 2021

Meiotic recombination occurs at discrete genomic loci known as recombination hotspots. To date, the lack of scalable, genome wide assays has limited studies of hotspot formation.

Recombination begins with the formation of DNA double‐stranded breaks (DSBs) by the meiosis specific SPO11. Strand resection from the DSB exposes single stranded 3′ overhangs which are bound by recombinases (RAD51, DMC1). We performed ChIP against DMC1 & RAD51, followed by Solexa sequencing (ChIP‐Seq) to generate the first direct map of DSB hotspots in a mammalian genome. We identified 9,874 DSB hotspots at high resolution, including a large cluster in the pseudo‐autosomal region, the site of an obligate recombination event.

Mouse hotspots are over‐represented in genes, have a distinct DNA skew pattern and hotspot DNA encodes a preference for nucleosome binding which is manifested in‐vivo. A consensus motif, found at 73% of hotspots is highly similar to the predicted binding site for PRDM9, a histone methyltransferase implicated in hotspot formation. The motif quality also correlates with hotspot strength. ChIP‐Seq against H3K4me3 showed that testis‐specific H3K4me3 peaks are co‐centered with 94% of hotspots, alluding to a central role for this motif, for PRDM9 and for H3K4me3 in DSB formation.

The Intramural Research Program of the NIDDK, NIH, supported this research.
Cucurbitacin B Suppresses The Transactivation Activity of RelA/p65
Authors: Jin Hongri , Jin Xuejun , Dat Nguyen Tien , Lee Jung Joon
Published: June 2021

Cucurbitacin B, a natural triterpenoid is well known for its strong anticancer activity, and recent studies showed that cucurbitacicn B inhibits JAK/STAT3 pathway. In this study, we demonstrate for the first time that cucurbitacin B is also a potent inhibitor of NF‐κB activation. Our results showed that cucurbitacin B inhibited TNF‐α‐induced expression of NF‐κB reporter gene and NF‐κB target genes in a dose‐dependent manner, however, cucurbitacin B did not prevent either stimuli‐induced degradation of IκBα or nuclear translocation and DNA‐binding activity of NF‐κB. On the other hand, cucurbitacin B dose‐dependently suppressed not only NF‐κB activation induced by overexpression of RelA/p65 but also transactivation activity of RelA/p65 subunit of NF‐κB. Consistently, treatment of HeLa cells with cucurbitacin B significantly suppressed TNF‐α‐induced activation of Akt and phosphorylation of Ser536 in RelA/p65, which is required for transactivation activity. Consequently, cucurbitacin B inhibited TNF‐α‐induced expression of NF‐κB dependent anti‐apoptotic proteins such as c‐IAP1, c‐IAP2, XIAP, TRAF1, and TRAF2 and sensitized TNF‐α‐induced cell death. Taken together, our results demonstrated that cucurbitacin B could be served as a valuable candidate for the intervention of NF‐κB‐dependent pathological condition such as cancer.
Novel Alternative and Multistranded DNA Microarrays: Z‐DNA, Triplex DNA and Quadruplex DNA
Authors: Gagna Claude Eugene , Dannibale Robert , Beheshti Kayvan , Demorcy Ashley , Lui Pearl , Yadiapalli Divya , Pasquarella Anthony V , Rehmat Shabia , John Akaz , Okereke Gloria , Chiang Goretti , Marler Shanacy , Nanda Priya , Syed Sarah , Kohli Sahil , Malik Shehryar , Rahman Sabreen , Patel Aney , Joy Job , Lambert W Clark
Published: June 2021

At the ends of chromosomes are segments of DNA called telomeres, which permit cells to replicate, protect DNA, and repair DNA. Multistranded and alternative DNA structures play important roles in gene expression. Novel alternative, multistranded, and helical transitional nucleic acid microarrays have been constructed, viz., Z‐DNA, triplex DNA and quadruplex DNA. These microarrays allow for characterization of the structure and function of Z‐DNA, triplex DNA and quadruplex DNA under different conditions. They allow for identification of drugs that bind to multistranded nucleic acids and for characterization of genomic elements that contain triplex DNA and telomeres. The novel microarrays will allow for a new approach towards studying gene expression and drug discovery. The new microarrays go beyond the limitations of conventional DNA microarrays, which focus on the primary structure of nucleic acids (i.e., base pairs) and ignore DNA secondary structure. These microarrays can be used for aging, cancer and infectious disease (e.g., tuberculosis, malaria and AIDS) research. The microarrays can also be employed for enhancing or inhibiting of gene expression. With these next generation DNA microarrays, scientists will have access to all of the uncharted structures that control gene expression, and aid in developing new classes of drugs for disease. Supported by a 2010 NYIT ISRC grant.
The adenovirus E4orf4 protein targets PP2A to the ACF chromatin remodeling factor and controls cell death through different SNF2h‐containing complexes
Authors: Kleinberger Tamar , Brestovitsky Anna , Sharf Rakefet
Published: June 2021

The adenovirus E4orf4 protein regulates the progression of viral infection and when expressed individually it induces non‐classical apoptosis in transformed cells. Here we show that E4orf4 associates with the ATP‐dependent chromatin remodeling factor ACF that consists of a SNF2h ATPase and an Acf1 regulatory subunit. Furthermore, E4orf4 targets protein phosphatase 2A (PP2A) to this complex and to chromatin. Obstruction of SNF2h activity inhibits E4orf4‐induced cell death, whereas knockdown of Acf1 results in enhanced E4orf4‐induced toxicity in both mammalian and yeast cells, and Acf1 overexpression inhibits E4orf4's ability to downregulate early adenovirus gene expression in the context of viral infection. Knockdown of the Acf1 homologue, WSTF, inhibits E4orf4‐induced cell death. Based on these results we suggest that the E4orf4‐PP2A complex inhibits ACF and facilitates enhanced chromatin remodeling activities of other SNF2h‐containing complexes, such as WSTF‐SNF2h. The resulting switch in chromatin remodeling determines life vs. death decisions and contributes to E4orf4 functions during adenovirus infection.
A novel role of Rad26 in dynamic chromatin disassembly during transcriptional elonagtion in vivo
Authors: Malik Shivani , Chaurasia Priyasri , Bhaumik Sukesh R
Published: June 2021

Cockayne Syndrome group B (CSB) is a DNA dependent ATPase in human, and plays an important role in transcription as well as transcription‐coupled repair (TCR). Mutations in CSB gene lead to cockayne syndrome (CS) charaterised by many traits reminiscient of normal aging. Deciphering the molecular roles of CSB is, thus, essential for disease management and understanding the causes of general aging and related ailments. However, the precise in vivo modes of action of CSB in regulation of transcription and TCR are not yet clearly understood. In this study, we show that the yeast homologue (Rad26) of CSB specifically associates with the coding regions of transcriptionally active genes, and promotes association of elongating RNA polymerase II (RNAPII). However, Rad26 does not control the recruitment of initiating RNAPII or formation of the pre‐initiation complex at the promoter. Intriguingly, we also find that Rad26 enhances the eviction of histone H2A‐H2B dimer, but not histone H3‐H4 tetramer, from the coding regions of active genes. These results, together, demonstrate for the first time the distinct function of Rad26 in promoting specific eviction of histone H2A‐H2B dimer during transcriptional elongation. This work was supported by Mallinckrodt Foundation.
Association of mRNA export factor Yra1 with the C‐terminal Domain of RNA Polymerase II: a mechanism for cotranscriptional recruitment
Authors: MacKellar April Lee , Greenleaf Arno L
Published: June 2021

The unique C‐terminal domain (CTD) of RNA polymerase II (Pol II) is composed of tandem heptad repeats of the consensus sequence YSPTSPS. This domain is subject to differential phosphorylation throughout the transcription cycle, mainly on serines 2 and 5 of the heptad. RNA processing factors have been shown to bind the CTD to localize to nascent pre‐mRNA during transcription. In S. cerevisiae, the mRNA export protein Yra1 (ALY/REF in metazoa) cotranscriptionally associates with mRNA and delivers it to the nuclear pore complex for export to the cytoplasm. Here we report that Yra1 directly binds in vitro to the elongating or hyperphosphorylated form of the CTD. Using truncated versions of Yra1, we have determined that amino acids 18–184, which includes its RNA Recognition Motif (RRM), are involved in phosphoCTD binding. Although the RRM is involved in CTD binding, RRM point mutations that produce defects in RNA binding and mRNA export do not affect CTD binding. In addition, posttranslational modifications in the N‐ and C‐termini of Yra1 abrogate RNA binding but not CTD binding. Using Yra1 truncations and point mutants, the coordinated recruitment of mRNA packaging and export factors in yeast will be investigated in vivo. This study was supported by NIH grant GM040505 to ALG.
Double knockdown of the Rheb gene in mammalian cells using RNA interference
Authors: Bonneau Ashley Rebecca , Parmar Nitika S
Published: June 2021

The insulin/mTOR pathway has been implicated in a variety of cancers and activation of mTOR (mammalian Target of Rapamycin) is regulated by an upstream G‐protein, Rheb (Ras Homolog Enriched in Brain). In mammalian systems two Rheb genes exist – Rheb1 and Rheb2 (RhebL1). Although significant sequence homology is shared, we hypothesize that Rheb2 may have distinct functions of its own. To elucidate the function of Rheb2, RNA interference was utilized to silence the expression of the Rheb genes in vitro. Rheb1 and Rheb2 were expressed individually as GFP‐fused proteins in mammalian cell lines and co‐transfected with Rheb specific siRNAs. Endogenous Rheb1/2 was silenced either individually or in a dual manner. Effect of silencing on growth profiles of cells was analyzed via MTT cell viability assays and the mTOR activation status was studied using immunoblotting and probing for phophorylation status of downstream effectors. This study shows for the first time that silencing Rheb2 impacts the mTOR pathway in a different way than Rheb1 and reinforces our hypothesis that Rheb2 may have specific roles not attributed to Rheb1. This is also the first report to simultaneously study the endogenous versions of Rheb1 and Rheb2 rather than over‐expressed forms of the same and provides a true profile of natural signaling conditions within a cell. This work was supported by a CSUPERB‐Doris A. Howell Research Award to AB.
MicroRNAs during Murine Molar Tooth Development
Authors: Osmundsen Harald , Sehic Amer , Jevnaker Anne‐Marthe
Published: June 2021

MicroRNAs (miRNAs) may have essential functions as regards regulation of gene expression, and are likely involved also during development. We have mapped miRNAs during development of the murine first molar tooth germ. The miRNA expression profile was found highly dynamic. During late odontogenesis miR‐214 is highly expressed, and was selected for further studies using antagmomir‐214. Antagomir‐214 was given as mandibular injections (0–100 pmol) at postnatal day 1 (P0). First molar tooth germs were removed at various times. Levels of miRNAs and mRNAs were measured using microarrays. A dosage of 50 pg was found to be optimal. At 24 h after injection the level of miR‐214 was about 0.02‐fold of control level, the level of expression returning close to the control at 72 h. Using a dosage of antagomir‐214 of 50 pmol about 1200 mRNAs and 8 miRNAs were differentially expressed in the tooth germ 24 hrs after injection of antagomir‐214. The microarray results were validated using RT‐PCR. Western‐blotting data was in agreement with changes mRNA levels. Differentially expressed genes were subjected to bioinformatic analysis, suggesting that cellular functions and signaling pathways were significantly altered. We conclude that miR‐214 may have significant regulatory functions during late odontogenesis. Project funding from Dental Faculty, University of Oslo.
Tissue tectonics: towards a quantitative understanding of morphogenesis
Authors: Adams Richard
Published: June 2021

The morphogenesis of embryos involves substantial reorganisation of many hundreds or thousands of cells. We know much of how individual cells move but understand far less of how tissues are able to attain their shape. We approached this problem by developing a framework with which we can measure and analyse the fundamental morphogenetic signatures of cell and tissue behaviour. We begin with a record of traced and tracked 3D cell shapes and locations, for many hundreds of cells. From this raw data we derive high resolution measures of the rate and direction of tissue deformation, cell shape change and cell rearrangements. When mapped in time and space across the embryo this gives a complete record of morphogenesis. Statistical methods campare maps from wild type and experimental embryos, showing where and when the dynamics of development become altered. We are using this approach to study the development of the vertebrate central nervous system. Dissection of forebrain and spinal development in these ways shows that complex tissue morphogenesis can be achieved using a small number of conserved developmental mechanisms.
Reconstructing multiscale dynamics in vertebrate morphogenesis
Authors: Peyrieras Nadine
Published: June 2021

A complete understanding of early morphogenetic processes has been an elusive goal. For most of the last century quantifying cellular behavior and motion patterns presented a formidable technical challenge. More recently, the availability of modern optics, ingenious software and reasonably priced computers has begun to revolutionize embryology. We designed a framework for imaging and reconstructing deep focus images of whole zebrafish embryos during early morphogenetic stages with minimal experimental manipulation. Our approach records time and distance measurements along with cell lineage analysis at micrometer spatial resolution and minute‐level temporal accuracy. The automated image processing methodology extracts membrane geometry and cell positional fate information while monitoring mitosis synchrony. The analyses are conducted on entire embryos and the resulting data provide a realistic quantitative multi‐scale ensemble view of morphogenetic mechanisms. Our global approach to dynamical understanding of embryogenesis establishes rigorous standards of measurement precision and the accuracy of automated segmentation and tracking procedures. It is expected that these in vivo data will provide a foundation for further analysis of the interplay between molecular, cellular and tissue level dynamics during formation of vertebrate embryos.
Automated inference of 4‐D cell polarization fields in an agent‐based model of early vertebrate embryogenesis
Authors: Delile Julien , Kowaliw Taras , Peyriéras Nadine , Doursat René
Published: June 2021

We present a theoretical model of animal morphogenesis construed as a self‐organized phenomenon emerging from a complex system made of a myriad of individual cell behaviors. It is implemented in an agent‐based simulation centered on the mechanic‐chemical coupling between cellular and genetic dynamics. The goal is to integrate the collective motion of cells and the dynamics of their gene expression underlying the patterning of morphogenetic fields. Within this larger framework, we examine here cell intercalation, a generic process underlying tissue elongation in vertebrate embryogenesis, in particular the convergence and extension movements shaping the embryonic axes during gastrulation. Cell intercalation is thought to arise from polarized cell movements. In our model, we include monopolar and bipolar protrusive activity of polarized cells to investigate the causal bottom‐up link from local cell behavior to global tissue deformation. Conversely, this model also allows reverse inference, e.g., by evolutionary optimization, of the local quantitative features of cell adhesion and polarization from a global 4 D (3 D + time) computational reconstruction of the cell lineage tree, itself based on in toto imaging data of developing zebrafish embryos.
Annotation and fluorescent assays of Anabaena Group I ribozyme variants
Authors: Koaches Hannah C. , Rohlman Christopher E.
Published: June 2021

Our research focuses on the molecular architecture of biomolecules to obtain insight into the folding and catalytic behavior of the Anabaena group I intron, in order to correlate its self‐splicing function with its structure. Group I introns are RNA sequences found in many organisms and are capable of both self‐splicing and removal from neighboring RNA sequences. The Anabaena group I intron itself comes from a cyanobacteria. To study the Anabaena ribozyme we are using both chemical methods coupled with computer bioinformatics. The folding and catalytic behavior of the ribozyme can be studied using fluorescent‐based assays to observe structural changes in the ribozyme. JalView sequence alignment software has also been adapted to analyze and annotate specific sequences of this Group I catalytic RNA. We can use this to map both chemical and genetic studies of this RNA molecule. As the structure of Anabaena itself has not yet been determined, it is our goal to analyze kinetic interactions identified through fluorescence‐based assays, while using computation software to develop to an accurate three‐dimensional representation of the Anabaena ribozyme. This research was supported by the Albion College Foundation for Undergraduate Research, Scholarly and Creative Activity.
Clearance of Aggresome‐Like Inclusion Bodies from SK‐N‐SH Human Neuroblastoma Cells through Increasing Autophagic Flux
Authors: Patton Wayne Forrest , Shen Dee , Adams Jeffrey K. , Sheppard Paul W.
Published: June 2021

Pathological inclusion bodies containing aggregated proteins are characteristic of various neurodegenerative diseases, including Parkinson's, Alzheimer's and Huntington's disease. Aggresomes and related inclusion bodies may serve as storage depots for the disposal of toxic protein aggregates by the autophagy pathway. One strategy for reducing cytotoxicity associated with aggregated protein accumulation is by enhancing their clearance through activation of autophagy. The SK‐N‐SH human neuroblastoma cell line was induced to form inclusion bodies by incubation with amyloid beta 1–42 peptide, thus mimicking the accumulation of â‐amyloid within neurons in Alzheimer's disease. Members of a focused compound library, containing well‐characterized small molecules with defined modes of action relevant to autophagy, were screened for their impact upon inclusion body accumulation using a fluorogenic dye that selectively highlights aggregated protein cargo. Certain compounds, such as chloroquine, promoted aggregate accumulation. Others, like SMER28, an inducer of autophagy that acts via an mTOR‐independent mechanism to increase autophagosome synthesis and enhance autophagic flux, reduced accumulation of â‐amyloid peptide within the cells. The described cell‐based screening assay facilitates identification of activators of autophagic flux that promote clearance of aggregated protein cargo.
In vitro reconstitution of substrate dislocation from the endoplasmic reticulum using Perfringolysin O
Authors: Sanyal Sumana
Published: June 2021

Secretory proteins unable to assemble into their native states in the endoplasmic reticulum (ER) are transported back or “dislocated” into the cytosol for ER‐associated degradation (ERAD). To examine different roles of components in ERAD we have taken a biochemical approach to reconstitute the process of dislocation of misfolded substrates. Semi‐intact cells are prepared by treating with perfringolysin O (PFO), a pore forming toxin, for selective removal of the cytoplasm. We use a truncated version of ribophorin (RI332) as model dislocation substrate. Cells expressing RI332 are pulse‐labeled with 35S; following treatment with PFO, cytosol is selectively removed from these cells, leaving the ER and other organelles intact. We find that RI332 is quantitatively released from the ER and appears in the cytosolic fraction as the deglycosylated form due to activity of cytosolic PNGase F. Using this in vitro strategy we have been able to test various mutants of ER and cytosolic proteins to determine what affects dislocation of misfolded substrates. Yod1 is a deubiquitinating enzyme that has been shown to be involved in the dislocation of RI332. With a catalytically inactive mutant of Yod1, we find that the entire pool of RI332 remains in the ER whereas with wild‐type Yod1, the deglycosylated version of RI332 starts to appear in the cytosolic fraction in a time‐dependent manner.
Two types of oligomeric structures of DegP in protein quality control
Authors: Sui Sen‐Fang , Jiang Jian‐sen , Shen Qing‐tao , Bai Xiao‐chen
Published: June 2021

This study was conducted in attempt to clarify the structural status of DegP functioning as active protease and chaperone. Our combined cryo‐EM and biochemistry studies revealed that DegP exhibit distinct assembly states in solution and on membranes. In solution, the DegP hexamers effectively convert to cage‐like oligomers (12‐mers and 24‐mers) via the pairwise PDZ1‐PDZ2 interactions between neighboring trimeric units upon binding to substrate proteins, and such conversion is a crucial step for activating both protease and chaperone activity. On lipid membranes, however, DegP forms a range of bowl‐shaped oligomeric structures, independent of substrate proteins, each with a 4‐ or 5‐fold symmetry and all with a DegP trimer as the structural unit. These bowl‐shaped membrane‐bound DegP assemblies have the capacity to recruit and process substrates in the bowl chamber and exhibit higher proteolytic and lower chaperone‐like activities than DegP in solution. Our findings imply that DegP might regulate its dual roles during protein quality control, depending on its assembly state in the narrow bacterial envelope. This research was supported by National Natural Science Foundation of China.
Epidermal Growth Factor (EGF) Suppresses Chaperone‐Mediated Autophagy (CMA) Via FoxO1 Transcription Factors in Kidney Cells
Authors: Franch Harold A. , Ding Changlin , Zoromsky Sara
Published: June 2021

Diabetic kidney growth suppresses CMA via Akt, but oxidative stress (OS), which increases CMA, also increases Akt signaling. To examine how Akt suppresses CMA when OS activates Akt, we looked at isoform differences in EGF and OS Akt‐mediated phosphorylation (P) of FoxOs 1,3a, and 4. At 48 hrs in NRK‐52E tubular cells, 10 nM EGF reduced proteolysis, increased CMA marker protein pax2 (2.7 fold), and decreased the CMA receptor, LAMP2a (54%, p<0.05, n=4), consistent with reduced CMA. OS (H2O2 100μM) did not reduce proteolysis, increase pax2, or decrease LAMP2a. EGF and OS both increased Akt P equally and, using a FoxO response element luciferase reporter, reduced FoxO transcription. Akt regulates FoxO abundance and thus activity by P on T and S sites. Using phospho‐specific antibodies, EGF for one hour induced FOXO1 T34 P(255%, p<0.05) and FoxO3a T24 P(>10‐fold), while OS had little effect (40% & −4%, NS, n=3). OS increased FoxO4 S193 P much more than EGF(>10 fold vs 220% p<0.05). EGF decreased FoxO1 and FoxO4 abundance by 40% and 31% (p<0.05) at one hour, but did not change FoxO3a. OS decreased FoxO4 abundance by 69% (p<0.05), but not FoxO3a or FoxO1. Adenoviral expression of constitutively active (CA) FoxO1 activated FoxO luciferase 3‐fold, reversed EGF‐induced Pax2 and GAPDH accumulation, and increased the expression of the LAMP2a consistent with increased CMA. CA FoxO1 accelerated lysosomal proteolysis by 21 & −4% (n=12, p<0.01) and FoxO1 DN reduced proteolysis. siRNA against FoxO1 or 3a reduced these proteins by >60% or >80% and FoxO luciferase activity 50% or 20% respectively. EGF or FoxO1 siRNA, but not OS, scrambled or FoxO3a siRNA, reduced CMA as measured by peri‐nuclear LAMP2a aggregates. In conclusion, EGF reduces FoxO1 abundance which regulates CMA, while OS reduces FoxO3a and FoxO4 abundance. FoxO isoform specificity may allow for growth factor suppression of CMA in the presence of OS during diabetes.
Influence of the DnaK chaperone system on ClpB‐substrate interactions
Authors: Doyle Shannon M , Hoskins Joel R , Wickner Sue
Published: June 2021

ClpB is a Clp/Hsp100 molecular chaperone belonging to the AAA+ (ATPases Associated with various cellular Activities) superfamily. Following severe stress, ClpB promotes cell survival by disaggregating and reactivating damaged proteins. ClpB forms a hexamer as its active conformation, which is stabilized in the presence of ATP and substrate. ClpB uses the energy of ATP hydrolysis to unfold and translocate substrate polypeptides through the central pore of the hexamer. To disaggregate insoluble aggregates, ClpB functions with the DnaK chaperone system although it can act alone on specific substrates. We are interested in determining how the substrate translocation process is linked to substrate binding in the ClpB hexamer and how the DnaK system may influence this process. To this end, we mutated ClpB in regions known to interact with substrate, including highly conserved tyrosines located in loops in the central channel of the hexamer. Our data from protein remodeling experiments suggest that each ClpB pore tyrosine acts independently on substrates requiring DnaK, but more cooperatively when ClpB works alone. These data indicate that DnaK influences the function of the ClpB pore residues, possibly by stabilizing ClpB‐substrate interactions.
Functional Characterization of the Interactions of the 70‐kD Heat Shock Proteins (Hsp70s) with Lipids
Authors: Nikolaidis Nikolas , McCallister Chelsea Marie , Donnelly Gabrielle , Chaudhari Sunil
Published: June 2021

Hsp70s are essential for cellular homeostasis and survival. Apart from their functions as molecular chaperones, specific Hsp70s interact with lipids. It has been proposed that this interaction allows Hsp70s to stabilize biological membranes and be integral parts of signaling and trafficking pathways. Despite their importance, the Hsp70‐lipid interactions remain largely undefined, since it is unknown which Hsp70s bind to lipids and what types of lipids they bind. To this end, we generated recombinant proteins of three mouse Hsp70s, and by using protein lipid overlay and vesicle sedimentation assays we determined that these proteins bind to phospholipids with significantly different affinities and specificities. To further characterize the Hsp70‐lipid interactions and facilitate future mutagenesis analysis, we determined the precise location of the Hsp70‐lipid binding by generating overlapping fragments of the Hsp70 protein. Lipid‐binding assays revealed the presence of at least two regions on the Hsp70 molecule responsible for binding to lipids. Our data strongly suggest that Hsp70s are functionally differentiated with respect to their lipid‐binding properties. Moreover, the high specificity of the interaction of specific Hsp70s to particular lipids suggests that these interactions could facilitate their translocation, anchorage, and function at cellular membranes.

This project was supported by funds from CSUPERB and CSUF to NN
Engineering of an Idealized WD Repeat as NanoBuilding Block for β‐propeller Protein Scaffolds
Authors: Nlkkhah Maryam , Paoli Massimo
Published: June 2021

The great diversity in the structure and function of proteins has stimulated interest in using them for potential applications in nanosystems and nanodevices. This diversity has been further enhanced by protein engineering, which provides the capability to modify existing molecules or create entirely new ones. By using tandem repeated sequences that form the structural building blocks in a group of β‐propeller proteins; we have used a consensus design approach and structural considerations to engineer modular and relatively large scaffolds. An idealised WD repeat was designed from a structure‐based sequence alignment and considering a set of structural guidelines. Using a plasmid sequential ligation strategy, artificial concatemeric genes with up to 10 copies of idealised repeat were then constructed. Corresponding proteins with 4 through to 10 WD repeats were found to be soluble when over‐expressed in E. coli. The designed modular proteins obtained could be further evolved by random mutagenesis similarly to those proteins in nature which have arisen through events of gene duplication and fusion, followed by divergence of sequence repeats. The results show that the β‐propeller scaffold proves to be an attractive platform for future engineering work, particularly in experiments in which directed evolution techniques might improve the stability of the molecules and/or tailor them for a specific function.