bluuebunny/crossref_metadata_2025 huggingface dataset:
New Tools for Teaching Energy Transfer in Biology ‐ Effects of a Randomized, Controlled Trial in Progress
Authors: Batiza Ann , Gruhl Mary , Herman Tim , Nelson Dave , Zhang Bo
Published: June 2021

We have begun a randomized, controlled trial of the effectiveness of a new way of teaching photosynthesis and cellular respiration. Key innovations include: 1) The use of a hydrogen fuel cell to illustrate four key principles: the relative tendencies of substances to give away electrons, the electron donor, the electron acceptor, and the fact that moving electrons can do work; 2) Comparing the hydrogen fuel cell and how living things are powered using those principles and rapid‐prototyped model components in the context of nested trays; 3) An animation that toggles between the hydrogen fuel cell and cellular respiration; and 4) A mechanical model of the ATP synthase. Teachers who attended a workshop increased their content understanding as reported at the NARST 2010 Conference. Here we focus on changes in classroom practice documented through videotaped focus groups, online reporting, and classroom observations of treatment and control groups. These data will inform future hierarchical linear modeling of content understanding effects in students. Implications for undergraduate education will be discussed. This work is supported by the US Dept of Education's Institute of Education Sciences.
Qualitative Research to Improve Teaching of Experimental, Ethical and Quantitative (TEEQ) Biology
Authors: Neroorkar Samiksha , Gundlach Ellen , Pelaez Nancy
Published: June 2021

Qualitative research is very useful in undergraduate biology education. As we are dealing with adult students, we can constructively use their input on how to make improvements to course instructional systems. In a freshman biology course, we implemented a Calibrated Peer Review system to introduce students to the experimental, ethical and quantitative aspects of biology. Students were involved in writing assignments, followed by grading peer work based on criteria pre‐determined by the instructors. At the end of the course, students were interviewed in focus groups to take their feedback on aspects of CPR like benefits, improvements, challenges, relevance to their educational goals and their learning about ethical and quantitative aspects of biology. Upon data analysis, the need for improvements in the qualitative focus group methodology became apparent. To encourage use of qualitative research to improve undergraduate biology education this project shows the need to consider concise, unbiased and probing questions, and trained interviewers. (Funding from NSF CCLI #0837229)
An Unusual Branching Pattern of the Axillary Artery
Authors: Singh Keerti , Rao Ramachandra
Published: June 2021

During routine dissection of a female cadaver, an unusual branching pattern of right axillary artery was observed. It was seen that second part of axillary artery gave only one branch i.e. the thoracoacromial artery. While its third part gave two branches namely, the anterior circumflex humeral and subscapular. The subscapular artery (with its usual branches) also gave origin to the lateral thoracic and the posterior circumflex humeral branch. It furthur continued as brachial artery from which arose anterior and posterior descending (radial collateral) arteries (branches of profunda), which coursed through the lower triangular space along with the radial nerve.

Grant Funding Source: University
The bilateral nature and origin of the lateral circumflex femoral artery
Authors: Ahamed Tarnima , Abramowitz Jason , Pagano Anthony Santino , Marquez Samuel
Published: June 2021

The lateral circumflex femoral artery (LCFA) gives rise to the ascending, transverse, and descending branches before anastamosing with the medial circumflex femoral artery (MCFA). Aside from its great importance in this proximal femoral anastomotic network, the descending branch of the LCFA is often used in a range of reconstructive vascular surgeries. Thus variation in LCFA branch origins is of great clinical relevance. The LCFA is typically described as arising from the deep artery of the thigh close to the femoral artery. However, few have investigated the distribution of LCFA branches. This study surveys 14 lateral circumflex femoral arteries from seven donor bodies representing adults from an urban population. Results showed a large amount of variation in the origin of the three contributing branches of the LCFA. In 50% of the sample, all three LCFA branches arise from one point. The descending and ascending arteries will each arise from separate locations in 22% and 14% of cases, respectively. In 14% of cases, each branch arose from different locations on the LCFA while 7% showed all branches arising from the deep femoral artery with no LCFA present. Approximately 43% of the individuals showed complete bilateral asymmetry, underscoring the highly variable nature of LCFA branching patterns. These findings reveal a great amount of diversity in vascular anatomy both between and within individuals.

Grant Funding Source: Downstate Medical Center
Morphological contribution of the pectoralis major muscle to the stability of the pectoral girdle
Authors: Kumka Myroslava
Published: June 2021

Methods

This study utilized 14 embalmed cadavers using traditional anatomical dissections.

Results

The clavicular head (CH) of the pectoralis major muscle (PMM) enhances the fibrous capsule (FC) of the sternoclavicular joint (SCJ) attaching at the periosteum of the clavicle. The sternocostal head and the abdominal part of the PMM are formed by seven segments. They attach to the upper seven costal cartilages, the FC of the SCJ and sternocostal joint, the sternum, the external oblique aponeurosis and the rectus sheath. Each segment is invested into the deep pectoral fascia which continues with the deep cervical, thoracolumbar, axillary and brachial fasciae. The biliminar tendon of PMM's distal attachment is formed by anterior lamina (AL) and posterior lamina (PL). There is continuity between the AL and PL at their humeral attachment. The AL is prolonged into the brachial fascia, FC of the glenohumeral joint (GHJ), deltoid tendon and deltoid tuberosity. Some fibers of PL continue into the FC of GHJ, while others invest the long head of biceps brachii, or fuse with the tendon of latissimus dorsi.

Conclusion

The myofascial continuity of the PMM, the connections with axial and appendicular skeleton, the pectoral girdle joint's fibrous capsules and tendons of other muscles may be important in the stability of the pectoral girdle. They should be considered in the training of athletes, and diagnosis and management of injury.
Fixing the all Variable of Tendineous Insertion in the long Abductor Muscle of the Thumb. Dissection. Dissecting room. Medical School. National University San Agustin. Arequipa Peru. 1998–2002
Authors: Mayta‐coaguila Hector‐juan
Published: June 2021

Because of its clinical, surgical and in particular macroscopic anatomy we decided to perform this research work. We worked on 32 cadavers (a total of 64 upper limbs) in the labs of anatomy in the Medical school at the San Agustin National University in Arequipa Perú. We used a descriptive, comparative method and dissected all cadavers. We studied them from January 1998 to January 2002 year.

We show that 21.87% of all cases present a variable tendineous insertion in the long abductor muscle of the thumb, with a predominance of male gender (85.71%) over female gender (14.28%). Also, most of the cases of the tendon long abductor muscle of the thumb show a division in two segments (57.14%), also it splits into three, four or even in six tendons with minor percentages. The average of tendons in the male sex was 3,16 and the female sex 2,0, the women did not have 3 or more divisions of the tendon of insertion of this muscle
The role of the transverse carpal ligament in carpal stability: an in vitro study
Authors: Bortier Hilde Elisa , Tengrootenhuysen Mike , van Riet Roger , Pimontel Paul , Van Glabbeek Francis
Published: June 2021

A biomechanical in vitro study was performed on 16 fresh frozen cadaver forearms to investigate the role of the transverse carpal ligament (TCL) in carpal stability.

The distance between the scaphoid and hamate was measured, as a reference for the length of the TCL. Distances were recorded in both loaded and unloaded conditions after gradual sectioning of the transverse carpal ligament, the palmar scapholunate, long radiolunate ligament and radioscapholunate ligament.

The largest increase in spread of the carpal bones (55% of total spread) was noted after loading with the ligament intact. Thereafter, sectioning of the TCL resulted in a further 32.9% increase in the distance between the scaphoid and the hamate.

We conclude that the intact carpal bones‐ligament complex displays some elasticity. Progressive sectioning of the TCL ligament under loading further opens the palmar arch.

Nevertheless it appears that the carpal arch will still retain reasonable intrinsic stability even without an intact TCL.
The maxillary bone and its dental and soft tissue interfaces in newborn primates
Authors: Smith Timothy D , DeLeon Valerie B
Published: June 2021

Midfacial morphology is strikingly different between higher taxonomic groups of primates. Compared to strepsirrhines (e.g., lemurs), haplorhines (e.g., monkeys, tarsiers) possess a greater magnitude of orbital convergence relating to evolutionary trends in the special senses. To understand the development of this dichotomous morphology, we examined serially sectioned newborn heads of a monkey (Saguinus sp), tarsier (Tarsius sp) and lemur (Eulemur sp). We have characterized the spatial arrangement of the dental sacs and orbitonasal soft tissue matrices relative to the maxilla, and assessed bone cell distribution to identify resorptive and depositional surfaces of the adjacent bone. Position of the dental sacs, infraorbital canal, nasolacrimal duct, and paranasal cavities differs among species. For example, deciduous premolars are positioned relatively laterally in Saguinus compared to other taxa. Regionally, this corresponds to resorption of the medial alveolar wall, a shared boundary with the maxillary sinus. Differences in spatial association of the nasolacrimal canal, infraorbital canal, and maxilla corresponds to species‐specific patterns of bone cell distribution. These phylogenetic variations ultimately relate to major evolutionary trends of soft tissues (e.g., special sense organs) and dentition, and offer unique natural experiments to understand mechanisms of midfacial growth.
THE ESTIMATION OF SEX FROM LUMBAR SPINE
Authors: Jelaca‐tavakoli Maria , Lewis Mary e.
Published: June 2021

The estimation of the sex of an unknown individual forms an essential part of biological identification in forensic anthropology, but is often hindered by poor preservation of the skull or pelvis. This study explores the relationship between the spinal dimensions of the lumbar spine and biological sex through means of osteometry. Osteometric data from a total of 3645 dry lumbar vertebra (N=385 and N=278 male and female skeletons respectively) were collected from four anatomical collections of known age and sex individuals located in Africa and North America. The Maximum Lumbar Length (ML) was determined for each of the five vertebrae and the measurements were then combined to provide an average for each individual. Of the single vertebrae, the ML dimensions of L1 provided the greatest accuracy when comparing the estimated sex against the known sex of the individual (80.3%). However, maximum lumbar length proved more accurate when the measurements for the five vertebrae were combined (82%). This study proposes possible usages of the sexually dimorphic features of the lumbar spine of Homo sapiens in challenging clinical, anatomical, forensic and osteoanthropometric cases in which either one or combination of factors such as decomposition, scavenging, burning and dismembering of human bodies provide no other feasible clues for identification of sex. Further examination and detailed studies of sexually dimorphism in the spine are needed to fully develop this method of forensic use, and may also have potential applications in evolutionary biology, possibly providing new information on sex specific bipedal adaptations.

Grant Funding Source: AAA
Patterns of strain in the primate pelvis: implications for theoretical models of pelvic biomechanics
Authors: Lewton Kristi Lynn
Published: June 2021

Until recently, the relationship between pelvic anatomy and stress resistance during loading had not been investigated in non‐human primates. This study seeks to test a theoretical model of pelvic mechanics to 1) determine whether the pelvis reacts similarly to applied load throughout primates (or whether multiple models are necessary to describe variation within primates) and 2) inform hypotheses of adaptation to locomotion.

Strain gages were applied to cadaveric pelvic specimens (Macaca, Papio, and Hylobates) and realistic loads (based on in vivo data) were applied unilaterally throughout a range of hindlimb angular excursions using a materials testing system. Directions and magnitudes of principal strains and peak shear strains were compared to predictions generated by the cylinder model of pelvic mechanics. Principal strains were highest in the pubic region during angular excursions of the hindlimb that correspond approximately to mid‐stance and toe‐off. Patterns of compressive and tensile strains provide preliminary support for dorsoventral and mediolateral 1) bending and 2) compression as dominant loading regimes. The results indicate that the pubis plays a pivotal role in stress resistance of the primate pelvis and that its morphology should be directly related to locomotor function.

Grant Funding Source: National Science Foundation, Leakey Foundation, Sigma Xi, GPSA
COINCINDENT ACTIVATION OF THE WNT AND SHH PATHWAYS BY FGF IN THE POSTERIOR LIMB BUD
Authors: Van Arsdale Jonathan M , Feenstra Jennifer M , Pira Charmaine U , Oberg Kerby C
Published: June 2021

Shh is critical to limb patterning during development and regeneration. The apical ectodermal ridge secretes Fgfs that induce limb outgrowth and maintain Shh expression in the zone of polarizing activity through a reciprocal Fgf‐Shh loop. Gene arrays performed following FGF2 application to the posterior region of the chick limb bud demonstrate elevated levels of WNT5A, FZD4, DKK1, and SHH. We hypothesized that the Wnt pathway might participate in the Fgf‐induced up‐regulation of Shh.

Thus, we analyzed the expression time course of WNT5A, FZD4, and DKK1 during FGF‐induced SHH up‐regulation. FGF2 soaked‐beads were implanted into the posterior aspect of stage 23 chick wing buds; the chicks were then harvested after 3, 6, 12, 18, and 24 hr of incubation and performed in situ hybridization for SHH and the Wnt‐associated genes.

SHH was up‐regulated by FGF2 within 3 hr of application. DKK1 was the first of the Wnt‐associated genes to be up‐regulated by FGF2, 3 hr after application followed by WNT5A at 12 hr, and then FZD4 at 18 hr.

This sequence of expression was unexpected. It was anticipated that up‐regulation of WNT5A by FGF2 would regulate SHH. However, up‐regulation of SHH precedes WNT5A. It is possible that the Wnt pathway is activated by FGF2 in a coincident, but separate pathway from SHH up‐regulation. It is also feasible that DKK1 could up‐regulate SHH and further studies are needed to investigate this possibility.
The Postnatal Development of the Feline Distal Phalanx
Authors: Bragulla Hermann H. , Homberger Dominique G.
Published: June 2021

In adult cats, the distal phalanx consists of (1) a proximal articular base articulating with the middle phalanx and bearing a palmar flexor tubercle and a dorsal extensor process for the attachment of tendons; (2) a distal unguicular process supporting the cornified claw sheath; and (3) an unguicular hood cupping the proximal part of the unguicular process and anchoring the cornified claw sheath. The postnatal development of this complex was analyzed with light microscopy, SEM, and x‐ray CT. The tomography data were reconstructed with OsiriX. In newborn cats, the distal phalanx consists only of a cartilaginous articular base, a peri‐ and enchondrally ossifying unguicular process, and a growth plate between them. At this stage, the unguicular hood consists only of fibrous connective tissue. At about six months, the hood starts to ossify at its base on either side of the articular base. At about twelve months, the hood is almost completely ossified, except for a small proximodorsal gap. By 22–24 months, the hood is fully ossified. The delayed desmal ossification of the hood allows the cartilaginous dorsal extensor process to complete its growth while the dorsally overarching hood is still expandable.
An EGFP reporter system to visualize homologous recombination in vivo
Authors: Jackson Michelle R Sukup , Jonnalagadda Vidya , Matsuguchi Tetsuya , Engelward Bevin P
Published: June 2021

The ability of cells to repair damaged DNA via homologous recombination (HR) is crucial to maintaining the fidelity of genomic information. While generally beneficial, HR can cause tumorgenic sequence rearrangements.

Our laboratory has developed a method for measuring HR frequency in the mouse. The Fluorescent Yellow Direct Repeat (FYDR) mice harbor incomplete repeat copies of the EYFP coding sequence. In the event of HR repair at the substrate, a complete EYFP sequence can result, leading to a fluorescent cell visible via flow cytometry.

In the FYDR mice, the EYFP substrate is not adequately expressed in all tissue types, preventing study of HR. To study previously inaccessible tissues, we have developed a new vector targeted at the ubiquitously expressed Rosa26 locus. We describe a new targeting vector consisting of tandem EGFP cassettes lacking essential sequences and initial characterization of the model.

We will use this model for many studies, specifically genomic stability in tissues susceptible to inflammation‐induced cancer. Knowledge of the relationship between exposures and HR will help us identify key factors modulating cancer susceptibility. NSF GRF, NIH# P01‐CA26731, R33‐CA112151‐01A2

Grant Funding Source: R33‐Ca112151‐01A2; NSF GRF, NIH Grant# P01‐CA26731
Coexisting Chiari malformation (CM) and thoracic outlet syndrome (TOS) in patients presenting with migraine: MRI/MRA/MRV
Authors: Saxton Ernestina H , Collins James D , Ahn Samuel S , Gelabert Hugh , Carnes Alfred
Published: June 2021

In Chiari malformation the cerebellar tonsils are displaced below the foramen magnum impressing the medulla, with altered cerebrospinal fluid (CSF) flow. Clinically CM mimics other neurologic disorders including migraine and headache disorders; increased intracranial pressure; lower cranial nerve palsies, and spinal cord syndromes, this last in syringomyelia. Thoracic outlet syndrome is misdiagnosed as CM when low‐lying cerebellar tonsils are incidentally found on MRI. Patients undergo decompression surgery without resolution of TOS symptoms because of post‐surgical immobilization; increased sling/erector muscle laxity; round shoulders, and costoclavicular compression. Bilateral MRI/MRA/MRV displays sites of costoclavicular compression of bicuspid valves within lymphatics and draining veins of the neck and supraclavicular fossae and of subclavian and axillary arteries with binding nerve roots (JNMA 1999; 91:333–41). Monitored multiplanar images with abduction external rotation and 2D Time of Flight MRA/MRV without contrast were acquired on a 1.5 Tesla GE Signa LX unit, 44 cm field of view, 512 × 256 matrix and saline water bags to enhance signal to noise ratio. Two patients with both diagnoses are presented whose TOS symptoms worsened after CM decompression, one with syringomyelia, the other a teenage baseball player. Bilateral MRI/MRA/MRV confirmed both diagnoses.
Chromatin‐Associated HMG‐17 Is a Major Regulator of Transcription Factor Activity Modulated by Wnt/beta‐catenin Signaling
Authors: Amendt Brad A , Wang Jianbo
Published: June 2021

The chromatin‐associated high mobility group protein (HMG‐17) is associated with transcriptionally active chromatin, however its role in regulating gene expression is unclear. This report reveals a unique strategy in which, HMG‐17 acts a molecular switch regulating the transcriptional activity of several transcription factors. The switch utilizes the Wnt/beta‐catenin signaling pathway and adds to the diverse functions of beta‐catenin. A high affinity HMG‐17 interaction with the PITX2 homeodomain protein, LEF‐1 high mobility group protein, and FoxJ1 forkhead factor inhibits their DNA binding activity. The HMG‐17/PITX2 inactive complex is concentrated to specific nuclear regions primed for active transcription. Beta‐catenin forms a ternary complex with PITX2/HMG‐17 to switch it from a repressor to an activator complex. Without beta‐catenin, HMG‐17 can physically remove PITX2 from DNA to inhibit its transcriptional activity. shRNA to HMG‐17 decreases HMG‐17 expression and relieves the inhibitory action of HMG‐17. The PITX2/HMG‐17 regulatory complex acts independently of promoter targets and is a general mechanism for the control of transcriptional activity. Chromatin IP (ChIP) assays reveal acetylated Histone H4 and H3 interaction with the PITX2/HMG‐17 complex. HMG‐17 is developmentally regulated and its unique role during embryogenesis is revealed by the early embryonic lethality of HMG‐17 homozygous mice. This is a novel mechanism regulating transcriptional activity during development in which HMG‐17 recruits factors to chromatin and acts as a molecular switch. Support for this research was provided from grants DE13941 and DE18885 to BAA from the National Institute of Dental and Craniofacial Research.
The regulation of SWI/SNF chromatin remodeling complexes on human genes under heat shock and IFN‐gamma treatment
Authors: CHENG MO‐BIN , Zhang Yi , Zhang Ye , Shen Yu‐fei
Published: June 2021

The ATP dependent chromatin remodeling complex can repost nucleosomes on the genome DNA, which is crucial for the regulation of human genes. The ATPase subunits of the human SWI/SNF chromatin remodeling complexes induce ATP release for chromatin remodeling at the promoter of its target genes either independently or in coordination. Here, we explore how an IFN‐γ activating sequences in the promoter can mediate chromatin remodeling and enhance gene activation in human leukemic cells under non‐lethal heat shock or IFN‐γ treatment. Our data were obtained mainly from real‐time RT‐PCR, chromatin immunoprecipitation, promoter activity assay, endonuclease accessibility, immunoprecipitation and domain‐specific interactions. We show a substitution of one SWI/SNF chromatin remodeling complex by its counterpart in the promoter of the genes is a requisite in the activation of hsp90α and two immune genes under heat shock. However, a similar change under IFN‐γ treatment in the SWI/SNF chromatin remodeling complexes to activate a gene occurs only on certain specific genes and cell types. We present a novel example showing the similarity and difference at the chromatin level in regulating activation of human genes under heat shock and IFNγ induction.

This work is funded by NSFC #30721063 & #90408007
Roles for epigenetic modifications at proinflammatory genes in tumor metastatasis
Authors: Truax Agnieszka Dorota , Greer Susanna Fletcher
Published: June 2021

Epigenetic regulation is an essential mechanism by which cells regulate accessibility to chromatic DNA, hence allowing critical processes such as gene transcription to occur. The contribution of epigenetics to specific human cancers remains unknown. In order to achieve metastatic ability, tumors alter gene expression to escape host immunosurveilance. Major histocompatibility class II, MHC II, molecules are glycoproteins that present tumor derived antigens to CD4+ T cells and activate anti tumor immune responses. Thus, MHC II molecules are crucial for initiation and maintenance of the anti tumor response. MHC II molecules are regulated at the level of transcription by the Class II Transactivator, CIITA, whose association with the MHC II promoter is necessary to initiate MHC II transcription. To further understand the role played by epigenetics in regulating CIITA and MHC II expression in metastatic tumors, we have investigated histone modifications to CIITA and MHC II promoters in three variants of breast cancer line MDA MB435. We show that MHC II and CIITA expression are significantly down regulated in the highly metastatic variant which correlates with elevated levels of H3K27 trimethylation and decreased levels of H3 acetylation at the inducible CIITA promoter. Epigenetic changes are now thought to play important roles in the cancer progression. Research supported by GCC, ACS, GSU MBD.
Kinetic mechanism of a novel HAT‐histone chaperone complex, Rtt109‐Vps75
Authors: Albaugh Brittany Nicole , Denu John M
Published: June 2021

Rtt109 is a novel histone acetyltransferase (HAT) involved in promoting genomic stability, DNA repair and transcriptional regulation. A histone chaperone, either Vps75 or Asf1, is required for efficient acetyltransferase activity toward H3 histone substrates. In complex with Vps75, Rtt109 acetylates lysine 56 within the H3 globular domain, as well as N‐terminal tail residues on H3 peptide and H3‐H4 tetramer substrates. The ability of histone chaperones to activate and direct substrate targets is unique among HAT enzymes. Here we explore the molecular mechanism of histone acetylation and report a detailed kinetic investigation of the Rtt109‐Vps75 complex. A steady‐state kinetic analysis reveals evidence for a sequential kinetic mechanism whereby Rtt109‐Vps75 complex, acetyl‐CoA (Km= 0.27 μM) and H31–20 peptide substrates (Km=146 μM) form a complex prior to chemical catalysis (kcat =0.11 s−1). Product inhibition studies demonstrate that CoA competitively inhibits acetyl‐CoA, while thermodynamic binding measurements demonstrate that Rtt109‐Vps75, in the absence of H3 substrate, binds acetyl‐CoA and CoA with a Kd value of 1.41 μM and 43.29 μM, respectively. Additionally, pre‐steady state kinetic analysis suggests the chemical attack of substrate lysine on the bound acetyl‐CoA is the rate‐limiting step of catalysis. The pH profile of kcat reveals a crucial ionization of pKa=8.5, possibly representing the deprotonation of the substrate lysine on H3. Together, these data support a mechanism in which acetyl‐CoA binds first, and following H3 binding, the ε‐amino group of lysine directly attacks acetyl‐CoA, transferring the acetyl group to the targeted lysine residue. This research is funded by grants from the National Institutes of Health.
Enrichment of Short interspersed transposable elements (SINEs) in hypomethylated genic regions in embryonic stem cells
Authors: Muramoto Hiroki , Yagi Shintaro , Hirabayashi Keiji , Sato Shinya , Ohgane Jun , Tanaka Satoshi , Shiota Kunio
Published: June 2021

Genome‐wide studies of DNA methylation modification of embryonic stem cells (ESCs) and somatic cells have revealed that there are numerous tissue‐dependently and differentially methylated regions (T‐DMRs). Combination of methylation status of each T‐DMR, called DNA methylation profile, is specific to ESCs. Transposable elements (TEs) occupy about the half of the mammalian genome. In general, DNA methylation modification repressed transposition of TEs. In this study, we examined whether there exist genomic sequence features, focusing on TE distributions, around genes that have T‐DMRs differentially methylated between ESCs and somatic cells. We first identified T‐DMRs hypo/hypermethylated in ESCs in promoter regions by comparing genome‐wide methylation status of ESCs with liver, and explored the TE proportions in flanking region over 100 kb. We found that genes with hypomethylated T‐DMRs in ESCs showed significantly higher SINE and lower LINE proportions. The enriched SINEs had a CpG‐rich property and were of specific subfamilies. These results demonstrated that SINE enrichment in the flanking regions is a distinctive characteristics of genes with hypomethylated T‐DMRs and further suggest that SINEs play a role in the formation of ESC DNA mehtylation profile.

This work was supported by the grants from NEDO, NIBIO, and JSPS Research Fellowship for Young Scientists.
Estradiol (E2) Regulation And ERα, C/EBPβ And SP1 Interactions Are Essential For Human Prolactin Receptor (hPRLR) Gene Transcription In a Live Cell‐Based System
Authors: Kang Jung‐Hoon , Tsai‐Morris Chon‐Hwa , Dufau Maria L.
Published: June 2021

E2/ERα, independent of a DNA estrogen response element, regulates hPRLR transcription. Direct interaction of the DNA binding domain of ERα with the leucine zipper (LZ) of C/EBPβ, and the third zinc finger motif (ZnF) of Sp1 bound to the promoter, were essential for PRLR transcription and E2 promoted the association. Interaction between the LZ of C/EBPβ and Sp1 ZnFs (1st & 3rd) was also observed. In this study, formation of complexes in live cells by bioluminescence resonance energy transfer (BRET) assays revealed constitutive ERα homodimers and ERα/C/EBPβ, and ERα/SP1 heterodimers in COS1 and HEK293. The BRET signal was increased by E2, and the relative affinity of ERα homodimer was enhanced by E2 (4‐fold). BRET saturation analysis revealed formation of specific C/EBPβ/Sp1 heterodimers, which could lead to the apparent promoter activity found in ERα negative breast cancer cells. BRET with bimolecular fluorescence complementation assay (BiFC) applied to ERα revealed assembly of dimerized ERα with C/EBPβ, which was further stimulated by E2. These studies indicate that E2 is required for activation/stabilization of constitutive complexes of ERα dimer with ‐C/EBPβ, ‐SP1 and ERα/SP1/C/EBPβ association for the transcriptional activation of the hPRLR promoter. E2/ERα‐induced association with Sp1 and C/EBPβ could be of relevance to breast tumor progression in post‐menopausal women.
Mechanism and Inhibition of the Dihydroxynaphthoyl‐CoA Synthase MenB from Mycobacterium Tuberculosis
Authors: Liu Nina , Li Xiaokai , Tonge Peter J
Published: June 2021

The lipid soluble redox cofactor menaquinone is an essential component of the respiratory chain in many bacteria including the pathogen Mycobacterium tuberculosis. Enzymes involved in menaquinone biosynthesis are thought to be promising drug targets since the pathway is absent in humans and also because compounds that affect respiration may be active against latent populations of M. tuberculosis. Currently we are comparing the mechanism of the M. tuberculosis MenB (mtMenB) with that from Eschericia coli (ecMenB). MenB, which is a member of the crotonase superfamily, catalyzes an intramolecular Claisen condensation leading to the formation of dihydroxynaphthoyl‐CoA from O‐succinylbenzoyl‐CoA. While most active site residues are conserved between mtMenB and ecMenB, mycobacterial MenBs contain an active site aspartate (D185) which is replaced with glycine in MenBs from other organisms. D185 is structurally homologous to E164, a conserved base in the active site of enoyl‐CoA hydratase, the prototypical member of the crotonase superfamily. Kinetic and structural studies suggest a fundamental difference in the catalytic mechanisms of mtMenB and ecMenB due to the presence or absence of D185. Inhibitor discovery efforts are also in progress both as mechanistic probes and also as leads for the development of potent MenB inhibitors
Mechanism of Epac Activation:Structural and Functional Analyses of Epac2 Hinge Mutants
Authors: Tsalkova Tamara N , Mei Fang C , White Mark , Cheng Xiaodong , Blumenthal Donald
Published: June 2021

Epac2 is a guanine nucleotide exchange factor which directly activated by cAMP. According to the model of Epac activation, a localized “hinge” motion is a major change in the Epac structure upon cAMP binding. In this study, we test the functional importance of hinge bending for Epac activation by targeted mutagenesis. We show that substitution of the conserved residue phenylalanine 435 by glycine facilitates the hinge bending. As a result, Epac2‐F435G mutant is constitutively active and stimulates nucleotide exchange in the absence of cAMP. In contrast, substitution of the same residue with a bulkier side chain, tryptophan, impedes the hinge motion and results in a dramatic decrease in Epac2 catalytic activity. Structural parameters for wild type Epac and two of its mutants determined by small‐angle X‐ray scattering (SAXS) further confirms the importance of hinge motion in Epac activation. Our study also suggests that the side‐chain size of the amino acid at the position 435 is a key to Epac functioning. It seems that phenylalanine at this position has the optimal size to prevent “hinge” bending and keep Epac closed and inactive in the absence of cAMP while still allowing the proper hinge motion for full Epac activation in the presence of cAMP.
Mechanistic Insights into the Function of the α‐Helical Tail in Haemophilus influenzae 3‐Deoxy‐D‐manno‐Octulosonate 8‐Phosphate Phosphatase
Authors: Briski Laurence Michael , Hanuszkiewicz Anna , Woodard Ronald W.
Published: June 2021

The eight carbon sugar, 3‐deoxy‐d‐manno‐octulosonate (KDO), serves as a fundamental link between lipid A and core oligosaccharides in the outer membrane of the Gram‐negative bacterial cell wall. Inhibition of KDO biosynthesis is lethal to most bacteria, implicating this pathway as an ideal target for novel antimicrobials. KdsC, which converts KDO 8‐phosphate to KDO, is one of four key enzymes in this pathway. In Haemophilus influenzae, KdsC is a homotetrameric protein with an α‐helical C‐terminal tail in each subunit. A truncated derivative of H. influenzae KdsC was constructed by cleavage of the last 18 C‐terminal amino acid residues through site‐directed mutagenesis to investigate the role of the α‐helical tail. Both native and truncated enzymes were cloned, overexpressed, and purified to apparent homogeneity. Preliminary data from crude protein extracts showed that the activity of the native enzyme was 33‐fold greater than that of the truncated derivative, as determined by a malachite green assay to assess inorganic phosphate release, providing further insight into KdsC as a potential target to inhibit KDO biosynthesis. This work was funded by the National Science Foundation.
Glycogen synthase kinase 3 beta (GSK3β) forms multiple protein complexes
Authors: Hamer Sabine , Gerlach Martin , Naguib Marian , Reinartz Michael , Gödecke Axel
Published: June 2021

GSK3β is a serine/threonine protein kinase, which is part of the PI3‐kinase/Akt‐, Wnt‐ and Hedgehog‐pathway and is involved in the regulation of gene transcription, protein synthesis and apoptosis.

To systematically analyze GSK3β dependent signal transduction we aimed to identify GSK3β associated protein complexes via Tandem Affinity Purification (TAP) and fused a TAP‐Tag consisting of a Flag/Strep II Tag at the C‐terminal end. This enables us to perform a two‐step purification of the recombinant GSK3β and its interacting partners under native conditions.

We found a similar behaviour of the recombinant GSK3β‐TAP‐Tag compared to the endogenously expressed GSK3β concerning subcellular localization, phosphorylation and enzymatic activity. MS‐based analysis of copurified proteins revealed that Acetyl‐CoA‐carboxylase, ATP‐citrate synthase and Axin, which are known to be substrates of GSK3β, form stable complexes with GSK3β. Moreover we identified numerous new interaction partners e.g. Alanyl‐tRNA synthetase, Annexin A2, Elongation factor 1, Filamin.

Conclusion

GSK3β mediates its signal transduction by extensive formation of stable complexes with other proteins.
A new mechanism explaining the sustained effect of Bone Morphogenetic Protein‐2 (rhBMP‐2) during reconstruction of segmental bone defects
Authors: El‐Awady Ahmed Rifaat , Hussein Khaled Mohamed , Zakhary Ibrahim , Emam Hany , Elsalanty Mohammed
Published: June 2021

This study investigates the role of the soft tissue bed in rhBMP‐2‐induced bone formation in large segmental bone defects. We hypothesized that delivery of rhBMP2 triggers endogenous BMP2 release by the soft tissue bed of the defect. Thirty‐five millimeter defects were created in the mandibles of five foxhound male dogs and immediately reconstructed with 8ccs of rhBMP‐2 (0.2 mg/ml) infused into absorbable collagen sponge (ACS). Soft tissue samples were collected 12 weeks after reconstruction. Gene expressions of endogenous BMP2 as well as 28 related genes were measured in the collected samples using real‐time PCR (qrt‐PCR) arrays. Analysis of the mRNA at treated sites revealed significant up‐regulation in the expressions of several genes related to BMP2 and inflammatory pathways. The highest significant up‐regulation detected in COL1A1 (+10.6 folds), BMP2 (+9.5 folds). Protein extracted from the periosteal tissue at the regenerate sides showed statistically significant higher concentration of BMP2 than contralateral sides. These results suggest that rhBMP‐2 grafting stimulates the production of endogenous BMP‐2 in the tissue pad surrounding the defect.

This study was supported in part by Medtronic, inc.
NAD+‐dependent deacetylase SIRT3 regulates mitochondrial protein synthesis by deacetylation of mitochondrial ribosomal protein L10
Authors: Koc Emine C , Yang Yongjie , Cimen Huseyin , Han Min‐Joon , Tong Shi , Deng Jian‐Hong , Koc Hasan , Palacios Orsolya M , Montier Laura , Bai Yidong
Published: June 2021

Reversible acetylation regulates many biological processes including mitochondrial energy metabolism, cell survival and longevity. A member of the sirtuin family of NAD+‐dependent deacetylases, SIRT3, is important for regulation of reversible acetylation in mammalian mitochondria. In this study, we identified ribosomal MRPL10 as one of the major acetylated proteins in the mitochondrial ribosome. Ribosome associated SIRT3 was found to be responsible for deacetylation of MRPL10 in an NAD+‐dependent manner. We mapped the acetylated Lys residues by tandem mass spectrometry and determined the role of these residues in acetylation of MRPL10 by site‐directed mutagenesis. Furthermore, we observed that the increased acetylation of MRPL10 lead to an increase in translational activity of mitochondrial ribosomes in SIRT3−/− mice. In a similar manner, ectopic expression and knock‐down of SIRT3 in C2C12 cells resulted in the suppression and enhancement of mitochondrial protein synthesis, respectively. Our findings constitute the first evidence for the regulation of mitochondrial protein synthesis by the reversible acetylation of the mitochondrial ribosome and characterize MRPL10 as a novel substrate of the NAD+‐ dependent deacetylase, SIRT3.

Supported by NIH R01s to ECK (GM071034), YB (AG025223), and QT (DK075978).
Analysis of flanking nucleotide contributions to translation start codon selection in yeast
Authors: Pittman Yvette Renee , Dever Thomas E.
Published: June 2021

Nearly all protein synthesis occurring in organisms from bacteria to humans initiates at an AUG start codon. Interestingly, the nucleotides flanking the AUG start codon are not random. In mammals, there is a strong preference for A or G at position ‐3 and G at position +4 relative to the A(+1) of the AUG codon. Our analysis of the yeast S. cerevisiae genome revealed a clear bias for A at positions −4 to −1 preceding the AUG codon, and a bias against C at position +4. Studies of mammalian translation established a start codon consensus sequence, (A/G)‐C‐C‐AUG‐G, and mutating this optimal sequence drastically impairs translation. However, previous studies concluded that flanking nucleotides are not important for AUG codon selection in yeast. Using a reporter assay based on leaky scanning of an upstream AUG codon in the GCN4 mRNA, we systematically re‐examined the impact of flanking nucleotides on start codon selection in yeast. Similar to mammals, substituting U at position ‐3 had the most severe impact on translation. In addition, we found that replacing the optimal context A‐A‐A‐AUG‐G with the least preferred U‐U‐U‐AUG‐C context reduced translation over 100‐fold. We are exploiting this strong dependence on flanking nucleotides by using yeast genetic analyses to identify factors that influence start site selection and to reveal the mechanism of how flanking nucleotides govern AUG selection.
ACSVL3 depletion in U87 glioma cells affects both fatty acid and cholesterol homeostasis
Authors: Watkins Paul , Kolar Elizabeth , Pei Zhengtong
Published: June 2021

Human malignant gliomas overexpress ACSVL3, a member of the very long‐chain fatty acyl‐CoA synthetase family. Depletion of ACSVL3 in cultured human U87 glioma cells by RNA interference decreased both their malignant growth properties in vitro and their ability to produce tumors in mice. To understand how ACSVL3 knockdown (KD) in U87 cells promotes these beneficial effects, we investigated fatty acid and cholesterol homeostasis. Incorporation of [14C]C16:0 into several lipids, including phosphatidylinositol, phosphatidylethanolamine, and non‐hydroxy ceramide, was reduced in KD U87 cells. Although the mass of triacylglycerol (TAG) in U87 cells was barely detectable, [14C]C16:0 incorporation into TAG was robust, and was decreased in KD glioma cells. Surprisingly, the mass of free cholesterol was reduced by ~40% in ACSVL3 KD cells. This was due, in part, to decreased de novo cholesterol synthesis, as measured by [14C]acetate incorporation. Acetate incorporation into free fatty acids was also reduced, indicating that ACSVL3 depletion also affects de novo fatty acid synthesis. We hypothesize that ACSVL3 depletion affects fatty acid and cholesterol homeostasis via a common mechanism. Studies to elucidate this mechanism are in progress. Supported by NIH grant NS062043 and a grant from the National Brain Tumor Society.
Structural and Functional Analysis of Phospholipid‐Specific Nucleotide Pyrophosphatase/ Phosphodiesterase Isoforms
Authors: Baker Daniel Lee , Wanjala Irene , Pham Truc Chi , Howard Angela , Cox Jayne , Parrill Abby
Published: June 2021

Lysophosphatidic acid (LPA) is an important mediator in development and disease. Despite roles in human health, little is known about its biosynthesis and regulation. In 2002, autotaxin (NPP2) was shown to produce LPA in plasma. We are using computational, biochemical, and biophysical approaches to examine the structure and function of NPP2/6/7. Previous work suggested that these three isoforms show differences in regioselectivity for hydrolysis of lysophosphatidyl choline (LPC). Likewise, these isoforms are differentially sensitive to the removal and subsequent replacement of divalent cations. Recombinant proteins were expressed in insect cells and were purified by affinity chromatography. The isoforms showed similar ability to utilize LPC as substrate, but different abilities to hydrolyze other substrates. EDTA inhibited all three isoforms with maximal effect at 16h and 120‐fold excess chelator. Following EDTA treatment, Zn2+ was able to restore activity to all three isoforms, whereas Co2+ differentially restored activity. Significant effects on secondary structure or thermal stability was not seen for either treatment (CD). Homology models based on the Xac NPP crystal structure (2gsu) have been used to guide site‐directed mutagenesis. Mutation of the reported catalytic residues to alanine, gives catalytically dead proteins. Characterization of additional mutations is ongoing.
Crystal structure and acyl chain selectivity of Francisella novicida LpxA, the first enzyme in lipid A biosynthesis
Authors: Joo Sang Hoon , Raetz Christian R. H.
Published: June 2021

The first step of lipid A biosynthesis in Escherichia coli is catalyzed by LpxA (EcLpxA), an acyltransferase selective for UDP‐GlcNAc and R‐3‐hydroxymyristoyl‐acyl carrier protein (ACP). While EcLpxA is selective for R‐3‐hydroxymyristoyl‐acyl chain (C14), Francisella novicida LpxA (FnLpxA) is thought to be selective for longer acyl chain (C18) in vivo. Here we report the 2.06 Å crystal structure of the FnLpxA. The N‐terminal parallel beta‐helix (LbetaH) and C‐terminal alpha‐helical domain are similar to the structures of LpxA's solved so far. The preference for the longer acyl chain is thought to be derived from the L180 ‐ G188 region which takes a wider turn with two extra residues compared to EcLpxA, providing a flexible pocket to accommodate longer acyl chain. While purified FnLpxA prefers R,S‐hydroxypalmitoyl‐ACP over R,S‐3‐hydroxymyristoyl‐ACP under standard in vitro assay conditions, complementation of an LpxA knockout Escherichia coli strain with wild‐type FnLpxA generates lipid A species mainly of the usual R‐3‐hydroxymyristate (C14) at positions 2 and 2′, with smaller population of one or two longer hydroxy fatty acids (C16), as judged by mass spectrometry. Our results indicate the acyl chain length of lipid A is determined in several levels including the acyl carrier protein pool, LpxA specificity, and beyond.
Quantification of prostaglandin D synthase peptides in human cerebrospinal fluid by selective reaction monitoring
Authors: Fonteh Alfred Nji , Fisher Danielle , Biringer Roger , Huhmer Andreas , Rush John , Harrington Michael
Published: June 2021

Objective

To quantify prostaglandin D synthase (PTGDS) in human cerebrospinal fluid (CSF) using multiple stable isotope peptides.

Methods

Eight tryptic peptides of PTGDS were synthesized containing stable isotopes ([13C]/[15N]) of amino acids. Experimental tandem mass spectrometry was used to obtain the best fragments and selective reaction monitoring (SRM) parameters were optimized using a triple quadrupole mass spectrometer. CSF or recombinant PTGDS was spiked with 50 pmol stable isotopes before digestion with trypsin. Liquid chromatography tandem mass spectrometry (LCMS) with SRM of multiple transitions and charged states for each peptide was used to quantify PTGDS in CSF.

Results

Peptide infusions reveal major b and y ions that can be used for experimental SRM. Standard curves are linear (0–100 pmol) with a peptide dependent on‐column limit of detection of 0.1–1 pmol. We detect all eight PTGDS peptides, and stable isotope SRM quantification reveals levels of PTGDS in CSF for normal subjects to be 15.2 ± 6.2 mg/L (n=14). CSF levels in subjects with migraine differ from those without, and average quantification using all peptides confirms a significant increase of PTGDS in migraine.

Conclusions

Experimental SRM can be used to accurately quantify proteins in biological samples. This approach can be useful in discovering biomarkers of diseases when multiple peptides of biological pathways are monitored.
Increased ceramide accumulation correlates with downregulation of the autophagy protein ATG‐7 in MCF‐7 cells sensitized to photodamage
Authors: Separovic Duska , Kelekar Ameeta , Nayak Arun , Tarca Adi , Bielawski Jacek
Published: June 2021

The purpose of this study was to determine the sphingolipid (SL) profile in cells defective in autophagy protein ATG‐7 and overall cell death after photodynamic therapy (PDT) with the photosensitizer Pc 4. MCF‐7 human breast cancer cells with downregulated ATG‐7 and their scrambled controls (Scr) were used. Exposure of ATG‐7 knockdown cells to PDT led to increased cell killing. PDT evoked an early (2 h) greater global increase in ceramides in ATG‐7 defective cells compared to Scr cells. The increases in dihydroceramide (DHceramide) were distinctly higher at 2 h than at 24 h in both cell types post‐PDT. The levels of sphingosine‐1‐phosphate (S1P) and sphingosine were decreased below resting levels at 2 h irrespective of the cell type. The data imply that ceramide might be a marker of ATG‐7 deficiency in cells sensitized to PDT.