bluuebunny/crossref_metadata_2025 huggingface dataset:
Essential roles of zinc finger factor epiprofin in tooth development
Authors: Nakamura Takashi , Yuasa Kenji , Jimenez Lucia , Unda Fernando , Koyama Eiki , Fukumoto Satoshi , Yamada Yoshihiko
Published: June 2021

The precise reciprocal interaction between the dental epithelium and dental mesenchyme during tooth development is essential to the formation of teeth that are functional in shape and size. Epiprofin (Epfn), a transcription factor belonging to the Sp family, regulates dental epithelial cell proliferation and differentiation into ameloblasts. Epfn deficiency results in the formation of an excess number of teeth. To study the mechanism of epiprofin functions in tooth morphogenesis, we have used Epfn‐transfection in cell culture and K5‐Epfn‐transgenic mice, which expressed Epfn under the control of the epithelial cell‐specific promoter, keratin 5 (K5‐Epfn). Transient expression of Epfn stimulated the proliferation of dental epithelial progenitor cell line SF2, while stable expression of Epfn blocked cell growth and elevated the expression of ameloblast marker genes such as ameloblastin. We found that K5‐Epfn mice had fewer molars and developed an ectopic enamel layer in the incisors. Thus, the tooth phenotypes are opposite in Epfn‐deficient and K5‐Epfn mice. In K5‐Epfn mice, both proliferation and differentiation of the dental epithelium were accelerated. Taken together, these results suggest that Epfn functions to maintain the balance of dental epithelial cell proliferation and cytodifferentiation in tooth development.

Grant Funding Source: Intramural Program of the NIDCR, NIH, and the Ministry of Education, Culture, Sports, Science and Technology of Japan
Developing Animal Models to Study Paranasal Sinus Function
Authors: McCullough Stephen Andrew , Reidenberg Joy S. , Laitman Jeffrey T.
Published: June 2021

This study describes anatomic and volumetric differences of paranasal sinuses (PS) as a first step in developing in vivo models for PS function. Three species were used (N=4/ea): rats, Rattus norvegicus; guinea pigs, Cavia porcellus; and rabbits, Oryctolagus cuniculus.

Methods included

postmortem dissection of each maxillary sinus (MS); measurement of hard palate length (HPL) to standardize for splanchnocranial differences; and measurement of MS volume (MSV) by filling with 70% ethanol after lateral wall perforation with a syringe.

Anatomic results

1) rat MS ostium is identifiable upon nasal septum removal, while in guinea pigs and rabbits, MS ostia are covered by the middle concha; 2) MS of rats and rabbits have thin lateral walls, and are easily accessible upon syringe perforation, whereas the guinea pig MS is considerably smaller with a thicker lateral wall, making entry more difficult.

Volumetric results

1) MSV correlates with HPL (r2=0.7307, N=3/species) across species; 2) species vary in mean MSV/HPL ratio significantly (p<0.0001) as expected from splanchnocranial differences.

Discussion

Although differences exist, each species can model PS function depending on the approach. Data suggest MSV is related to cranial size and, by extension, physiologic demand of the animal. This phase provides foundation for selecting models to test hypotheses of PS function and implication in disease.

Grant Funding Source: Mount Sinai Research Fellowship
Appearance of Parasagittal Suture and Bregmatic Bone after Surgical Intervention for Craniosynostosis
Authors: Cray James , Kinsella Christopher R , Cooper Gregory M , Pollack Ian F , Losee Joseph E
Published: June 2021

Here we report on the appearance of a sagittal suture in the parasagittal plane and of a bregmatic bony ossicle in a patient after surgical intervention for nonsyndromic sagittal synostosis. This case study gives insight into the role of biomechanical forces on cranial suture location and orientation with respect to secondary ossification centers. We suggest the possibility that the surgical intervention adjusted biomechanical forces and allowed the activation of ossification at the bregmatic point within the coronal suture. We also suggest that the formation of a sagittal suture where there was not one previously may be due to unequal biomechanical forces acting on the cranial vault and allowing the formation of a suture in a region not associated with the underlying dura mater that induced the original synostosis.
Identifying the transverse tarsal joint(s) of the foot
Authors: Greiner Thomas M. , Ball Kevin A.
Published: June 2021

The transverse tarsal joint is traditionally equated with “Chopart's Joint,” which separates the calcaneus and talus from the more distal aspects of the foot. This line of demarcation is frequently used to separate the foot into two functional units: the hindfoot and forefoot. However, Chopart's Joint was not identified based upon intertarsal joint function, but was instead borrowed from a surgical procedure that amputates the foot along this division. As a result, many biomechanical studies make use of a hindfoot/forefoot divide that lacks a functional basis. This study is based upon 3D kinematic evaluations of the intertarsal joints in response to plantarflexion/dorsiflexion, inversion/eversion and medial/lateral rotation movement cycles. Results show that the array of intertarsal joints on the medial and lateral side of the foot respond differently, and in different joint combinations, to each of these driving motions. As a result, we suggest that the simplification of a single “transverse tarsal joint” may not be justified in many biomechanical foot models. These models instead should adopt a fuller appreciation of the interplay between the intertarsal joints.
Chronic exercise does not alter limb bone morphology or microstructure in the American alligator
Authors: Owerkowicz Tomasz , Tsai Henry P. , Sanchez Linda , Felbinger Krista , Andrade Fernando , Blank Jason M. , Eme John , Gwalthney June , Hicks James W.
Published: June 2021

In contrast to mammals and birds, effects of exercise on bone microstructure in reptiles have received scant attention. We investigated the effects of long‐term exercise on a treadmill or in a flume on limb bones of the American alligator. Juvenile alligators were run or swum to exhaustion every other day for 17 months, and received fluorescent dye injections to determine bone deposition rates. We found no significant differences in whole bone morphology and cortical bone deposition rates in the alligator humeral or femoral midshaft, regardless of exercise regimen. Similarly, we found no effects of either exercise regimen on trabecular number, thickness or density in the distal femur. In addition, bone mineral content was similar across exercise groups. Altogether, this suggests that long‐term exercise has no discernible effect on bone microstructure in alligators. These results stand in contrast to studies on endothermic vertebrates. The disparity could be due to metabolic differences between ectothermic and endothermic vertebrates. An alternative explanation is that alligator limb bones are subjected to insufficient strain levels or load cycles during short exercise bouts, which may account for lack of bone remodelling in response to exercise in alligators. Funded by the NSF IOB 04445680 to JWH.
Influence of different types of diets and a protocol of aerobic exercise on the mechanical properties of bones
Authors: Okubo Rodrigo , Leonardi Daphne Santoro , Fernandes Cleverson Rodrigues , Falcai Maurício José , Frajacomo Fernando Tadeu Trevisan , Fazan Valéria Paula , Shimano Antônio Carlos , Volpon José Batista , Jordão Alceu Afonso , Garcia Sérgio Britto
Published: June 2021

The purpose of this study was to evaluate the effects of different types of diets and the swimming exercise in tibias of rats. This study was approved by Ethical Committee in Animals Experiment. Sixty three‐month‐old male Wistar rats were randomly assigned to six groups (n=10): (G1) control AIN‐93 diet sedentary, (G2) control AIN‐93 diet aerobic training, (G3) high‐fat diet sedentary, (G4) high‐fat diet aerobic training, (G5) high‐fat‐protein diet sedentary, (G6) high‐fat‐protein diet aerobic training. Control diet is AIN‐93. The total caloric values of control, high‐fat and high‐fat‐protein diet is respectively 394.8 kcal, 609.8 Kcal, 609.8 Kcal for 100g of diet. Physical exercise consisted of swimming on a large aquarium. Rats swam 5 days a week, 60 minutes, during 8 weeks. Tibias were dissected and tested in a universal testing machine. Data were analyzed using specific tests; level of significance was set at 5%. Mean values of maximum load for groups: G1 (40.5±5.6)N, G2 (44.8±7.4)N, G3 (64.2±4.1)N, G4 (53.3±9.4)N, G5 (48.5±10.8)N and G6 (47.4±7.1)N. Data from our study suggest that a diet programs may influence bone resistance however changing its body weight.

Grant Funding Source: FAPESP
Bears May Maintain Bone Strength During Hibernation by Reducing Osteoblast Response to Apoptosis
Authors: Bradford Rachel Marie , Gray Sarah , Heiden David , Henriksen Kim , Gundberg Caren , Tsai Chung‐Jui , Donahue Seth
Published: June 2021

Despite annual periods of disuse lasting up to 6 months (hibernation), black bears (Ursus americanus) maintain bone mechanical strength throughout life. Our objective is to determine the mechanism responsible for this maintenance.

METHODS

Serum from 6 captive black bears was collected every 10 days from the fall active (October) through the spring active season (April), fully encompassing hibernation. Osteoblasts were cultured in 2% bear serum, and cell responses were quantified.

RESULTS

Compared to active seasons, hibernation serum exhibited a 57% decrease in bone specific alkaline phosphatase (p<0.0001) and a 29% decrease in Tartrate Resistant Acid Phosphatase (p<0.0001). Compared to osteoblasts cultured in active serum, those cultured in hibernation serum exhibited reduced gene expression of CyclinD1 (p=0.0244) and had 36% less caspase‐3/7 activity after serum starvation (p<0.0001). The caspase response (R2adj=0.34) and CyclinD1 expression (R2adj=0.32) were negatively and positively correlated to serum creatinine, respectively.

CONCLUSION

Serum markers suggest a decrease in bone turnover during hibernation. Furthermore, serum from hibernating bears seems to decrease osteoblast apoptosis.
Evidence that the mutation responsible for osteopetrosis in the op rat is not contained within the coding region of the Tbl3 gene
Authors: Dobbins David E.
Published: June 2021

The spontaneously occurring mutation in op rats produces an animal with severe osteopetrosis. The op mutation has been localized to a 0.51 cM portion of rat chromosome 10. Lying within the genetic critical region for op is the Tbl3 (transducin beta‐like protein 3) gene. The Tbl3 protein resembles the transducin superfamily of proteins that are known to regulate cellular functions such as cell division, cell‐fate determination, gene transcription, transmembrane signalling, mRNA modification and vesicle fusion. In this study, we tested if this gene could be responsible for the osteopetrosis seen in the op rat. M‐13 tailed primer sets were designed from rat genomic sequence to cover the 22 exons of the rat Tbl3 gene. These primer sets were amplified by PCR against genomic DNA from affected mutant animals and the two parental strains, BN and LEW. Mutational analysis of the Tbl3 sequences from the three strains failed to reveal significant differences between affected mutant animals and the two parental strains although single nucleotide polymorphisms (SNPs) between BN and the other two strains were identified. These data indicate that the osteopetrosis seen in the op rat is not the result of a mutation within the coding region of the Tbl3 gene. Analysis of additional functional and positional candidate genes will be required to identify the causative mutation in this animal model of failed bone homeostasis.
Molecular Variation of Mandibular Periosteum
Authors: Tee Boon Ching , Sun Zongyang
Published: June 2021

This study investigated the molecular features of pig mandibular periosteum related to the characteristic posterior‐apposition and anterior‐resorption of the ramus during growth. Periosteal tissue (for direct RNA extraction) and cells (for culturing) were obtained from growing pig anterior and posterior mandibular‐ramal surfaces. Cells were cultured in osteogenic media and loaded by cyclic tension (magnitude 0, 5%, 10%), followed by RNA extraction. The expression of molecules involved in osteogenic differentiation (osteocalcin, OCN; runt‐related transcription factor 2, RUNX2) and bone formation/resorption regulation (osteoprotegerin, OPG; receptor‐activator for nuclear factor κ‐b ligand, RANKL) was quantified by real time RT‐PCR. We found that both tissue and unloaded cell samples had stronger OPG expression at the posterior location (p<0.05), while RANKL, RUNX2 and OCN expression was similar between locations. Coinciding with bone apposition, the posterior cells had lower RANKL/OPG ratio than the anterior. With tensile strain, the expression of RANKL, RUNX2 and OCN tended to be upregulated in anterior cells but downregulated in posterior cells. These results indicate that regional difference of mandibular periosteal apposition/resorption may be related to the variation of RANKL/OPG expression and reaction to mechanical strain. Funded by American Association of Orthodontists Foundation.
Anabolic Steroids Modulate the NPYergic Circuitry of the Hypothalamus in Adolescent Rats: A Molecular and Behavioral Approach
Authors: Ramos‐Pratts Keyla Mariel , Santiago‐Gascot Maria Elisa , Parrilla Jeffrey , Barreto‐Estrada Jennifer , Roig‐Lopez Jose Luis
Published: June 2021

The abuse of anabolic steroids has increased at alarming rates among adolescents, however, little is known about their behavioral consequences in this population. Given that neuropeptide Y (NPY) and the androgen receptor (AR) are highly expressed in the hypothalamus, we aimed to assess the effects of 17a‐methyltestosterone (17a‐meT) in the behavioral and molecular aspects of the NPYergic circuit in this brain region. Male pubertal rats were chronically exposed to 17a‐meT. Animals were sacrificed and brain punches were done at the mPOA, VMN, and the extra‐hypothalamic BNST. NPY protein levels and mRNA expression for NPY and its receptors were determined by RIA and Real Time‐PCR, respectively. An increase in NPY was observed in the VMN, a region associated with male sexual motivation, while no changes were observed in the mPOA. Preliminary results showed a 3‐fold increase of NPY mRNA in the mPOA in AAS‐treated rats, and an 8‐fold decrease in the VMN. This effect was accompanied by a decrease in mRNA expression of receptors NPY Y2, Y5, and AR in the VMN. In the BNST, a 3‐fold decrease in NPY mRNA, and an increase in NPY Y1, Y2, and Y5 was observed. At the behavioral level, AAS‐treated rats increased the incentive sexual motivation. Our results suggest an interaction between synthetic androgens and neuropeptide systems to control behavior. NIH‐NCRR (2P20RR016470‐09), NIH‐EARDA (G11HD046326), RCMI (G12RR030551).
Non‐myeloablative autologous HSCT for relapsing remitting multiple sclerosis (RRMS) and new onset type 1 diabetes mellitus (TIDM)
Authors: Burt Richard
Published: June 2021

Autologous non‐myeloablative hematopoietic stem cell transplantation (HSCT) is a method of delivering intense immune suppression. We, therefore, evaluated the safety and clinical outcome of autologous non‐myeloablative HSCT in 2 autoimmune diseases relapsing‐remitting multiple sclerosis (RRMS) failing interferon therapy and new onset Type 1 Diabetes mellitus. Peripheral blood hematopoietic stem cells (HSC) were mobilized with cyclophosphamide (2.0 g/m2) and granulocyte colony stimulating factor (10 ug/kg/day). HSCT conditioning regimen was cyclophosphamide (200 mg/kg) and rabbit anti‐thymocyte globulin (either 6 mg/kg or 4.5 mg/kg).

Eligible patients with RRMS had, despite interferon beta, 2 corticosteroid treated relapses within the prior 12 months, or 1 such relapse and gadolinium enhancing lesion(s) on magnetic resonance imaging (MRI) demonstrated on an occasion distinct from the relapse. All patients are surviving, 17 patients (81%) improved by at least 1.0 or more EDSS points, and five patients (24%) relapsed but achieved another remission after further immunosuppression. After a mean of 3 years, progression‐free survival is 100%, and relapse‐free survival is 76%. Significant improvement occurred in neurologic disability determined by extended disability status scale (EDSS), neurologic rating score (NRS), paced auditory serial addition test (PASAT), and 25 foot walk, as well as quality of life as measured by the short form‐36 (SF‐36). Non‐myeloablative autologous HSCT in RRMS appears capable of reversing neurologic deficits.

Eligible patients for HSCT of T1DM were diagnosed in the previous 6 weeks by clinical findings and hyperglycemia and confirmed with positive antibodies against glutamic acid decarboxylase. 23 type 1 diabetic patients (aged between 13 and 31 years) were treated. During a 7‐to 58‐month follow‐up (mean 29.8) 20 patients without previous ketoacidosis and not receiving steroids during the preparative regimen became insulin‐free; 12 patients maintained this status for 14 to 52 months (mean 31) while 8 patients relapsed and resumed insulin use at low dose (0.1 to 0.3 IU/kg). In the continuous insulin‐independent group, HbA1c levels were always <7.0%, continuous glucose monitoring was within normal levels and mean AUC of C‐peptide levels increased significantly from 225.0 ng/mL pre‐transplantation to 785.4 ng/mL at +24 months (P <0.001) and to 728.1 ng/mL at +36 months (P = 0.03). In the transient insulin‐independent group mean AUC of C‐peptide levels also increased from 148.9 ng/ml before transplantation to 546.8 ng/mL at +36 months (P = 0.001) that was sustained at + 48 months In this group, two patients regained insulin independence after treatment with sitagliptin which was associated with increase in C‐peptide levels. HSCT induces insulin and drug free remission for up to 5 years post HSCT and appears to preserve or allow regeneration of islet cell mass with maximal C‐peptide levels reached 2–3 years after HSCT.
Somatostatin modulates mast cell‐induced responses in murine spinal sensory neurons and satellite cells
Authors: Van Op den Bosch Joeri , Van Nassauw Luc , Van Marck Eric , Adriaensen Dirk , Timmermans Jean‐Pierre
Published: June 2021

The expression of somatostatin (SOM) receptors (SSTRs) in mucosal mast cells and sensory nerve fibers suggests a modulatory effect for this peptide in the mast cell‐neuron communication. Therefore, the modulatory effects of SOM on the short‐term and long‐term effects induced by histamine (HIS) and serotonin (5‐HT) on sensory neuronal characteristics were assessed using murine spinal neuronal cultures.

ELISA and live cell imaging demonstrated acute HIS‐ and 5‐HT‐induced neuronal CGRP release and calcium‐mediated activation of both neurons and non‐neuronal cells. Both of these effects were strongly reduced by preincubating the spinal cultures with SOM. Long‐term incubation of the spinal cultures with HIS and 5‐HT significantly elevated neuronal expression of substance P, CGRP and SOM as demonstrated by qPCR and quantitative analysis. Also these long‐term effects of HIS and 5‐HT were partly inhibited by chronic SOM exposure. Immunocytochemistry and RT‐PCR revealed the involvement of SSTR1 and SSTR2A in these SOM‐dependent effects.

The data demonstrate an interfering effect of SOM on the mast cell‐neuron communication, implying a major role for this peptide in intestinal inflammatory responses. Since SOM affects HIS‐ and 5‐HT‐induced responses in non‐neuronal cells as well, SOM could indirectly modulate sensory neuronal characteristics through modulation of the surrounding non‐neuronal cells.
Hypoxic preconditioning by HIF‐1 accumulation not angiogenesis provides neuroprotection in rats following MCAO
Authors: Darlington Timothy Ryan , Xu Kui , LaManna Joseph C
Published: June 2021

Hypoxic preconditioning has been shown to be neuroprotective in ischemic models. We have previously reported an accumulation of HIF‐1α and its target genes at 3 days of hypoxic exposure in rats whereas at 3 weeks of exposure vascular adaptation is successfully completed. To distinguish the relative contributions from the two mechanisms to neuroprotection, we compared the effects of 3‐day and 21‐day hypoxic preconditioning on infarction of brain tissue following permanent middle cerebral artery occlusion (MCAO) in rats. Male Wistar rats were exposed to hypobaric hypoxia (0.5 ATM) for 3 or 21 days before undergoing permanent MCAO. Infarct volumes (% of contralateral hemisphere) were determined at 24 hours post‐occlusion in each rat. The 3‐day hypoxic preconditioned rats had significantly lower (ANOVA, p < 0.05) infarct volumes compared to the normoxic control animals following MCAO. Control animals (n = 7) produced an average infarct of 50 ± 4% (mean ± SEM) whereas an average infarct of only 32 ± 2% was observed in 3‐day hypoxic preconditioned rats (n = 9). The 21‐day hypoxic preconditioned rats did not show significantly decreased infarct volumes with an average of 50 ± 4% (n = 7). In conclusion, in permanent MCAO rat model the 3‐day hypoxic preconditioning appears to reduce infarct volumes compared to 21‐day hypoxic preconditioning. This suggests that the neuroprotective contribution from the accumulation of HIF‐1α and its target genes is greater than increased capillary density in the permanent focal ischemia model. Study supported by NIH grant 38632

Grant Funding Source: NIH
QUALITATIVE AND QUANTITATIVE STUDY OF THE SYNAPSES IN THE HIPPOCAMPUS OF PATIENTS WITH ALZHEIMER's DISEASE
Authors: Shaibah Hassan , Elbarrany W.G.
Published: June 2021

Human brains were obtained at postmortem examination from 5 control male individuals aged from 56 to 78 years and with no history of Alzheimer's disease (Group I). Also from 7 patients aged between 59 to 80 years and with history of Alzheimer's disease (Group II). Tissues were obtained from the same area of the hippocampus in both groups within 24 hours postmortem and processed for electron microscopic study.

Two main types of synapses were encountered in the control Group I, symmetric and asymmetric synapses. The former had wide pre‐ and postsynaptic densities. The dendritic and axon terminals contained clear and dense‐cored vesicles in both types of synapses. There was no obvious difference between the width of the synaptic cleft of the two types of synapses. The mean surface density of the symmetric synapses of the control group was 990/mm2 and that of asymmetric synapses was 2164/mm2. The mean lengths of the synaptic opposition was 0.100 μm in the symmetric synapses and 0.121 μm in the asymmetric ones. In Group II with Alzheimer’ disease, vacuolations appeared within the dendrites and in the axon terminals. The myelinated nerve axons showed splitting of the lamellae of its myelin sheaths by intramyelinic vacuoles. The mean surface density of the symmetric synapses was 940/mm2 and that of asymmetric synapses was 1178/mm2. The mean length of the synaptic opposition was 0.271 μm in the symmetric synapses and 0.280 μm in the asymmetric ones.

It could be concluded that the hippocampus of individuals with Alzheimer's disease showed vacuolations at the synaptic junctions and in the myelin sheaths. The number of synapses decreased in these individual with a compensatory increase in the synaptic lengths.
Inflammatory and angiogenic potential of primary and hTERT‐transduced human coronary artery endothelial cells
Authors: Baumer Yvonne , Ivanov Svetlana , Schlosshauer Burkhard
Published: June 2021

In vascular research the use of endothelial cell (EC) lines would be desirable because of often high priced primary EC which display donor diversity. However, endothelial cell lines are often insufficiently characterized or represent irrelevant models because of cellular dedifferentiation like the reduced inflammatory response and permeability properties or deregulated protein expression. We established a human coronary artery EC line (HCAEC) by overexpression of human telomerase reverse transcriptase (hTERT). In comparative analyses we showed that primary and hTERT transduced HCAEC displayed comparable characteristics. These studies included: 1) detection of 9 endothelial and nonendothelial proteins by fluorescence‐microscopy (IF), western blot‐ and fluorescence activated cell sorting (FACS)‐analysis, 2) uptake of ac‐LDL by IF and FACS, 3) upregulation of ICAM‐1 after pro‐inflammatory stimulation, 4) analysis of changes in endothelial barrier function by measuring transendothelial electrical resistance and 5) detection of angiogenic properties by analyzing migration potential, Matrigel™ tube formation‐ and a fibroblast co‐culture‐assays. We conclude that hTERT overexpression did not alter typical endothelial characteristics. hTERT immortalized HCAEC are a valuable tool for endothelial research.

Supported by the German Ministry of Education and Research (BMBF) # 01GU0727.
FAK mediates the activation of cardiac fibroblasts induced by mechanical stress through regulation of mTOR complex
Authors: Dalla Costa Ana Paula , Clemente Carolina F.M.Z , Carvalho Hernandes Faustino , Carvalheira José B. , Franchini Kleber G.
Published: June 2021

We evaluated whether focal adhesion kinase (FAK) plays a role in the proliferation and differentiation of cardiac fibroblasts into myofibroblasts in response to cyclic stretch (CS). Neonatal (NF‐ 3rd passage, 80% confluence) and adult (AF– 1st passage, 80% confluence) rat cardiac fibroblasts were exposed to CS (biaxial, 1Hz), which enhanced phospho‐FAKY397. Proliferation (anti‐Ki67 nuclear), differentiation into myofibroblasts (expression of α‐smooth muscle actin –α‐SMA) and the activity of matrix metalloproteinase‐2 (MMP‐2) were equally enhanced in stretched NF‐P3/80 and AF‐P1/80. Assays with the integrin inhibitor RGD peptide increased apoptosis (TUNEL) in non‐stretched and stretched NF‐P3/80, while FAK knockdown induced by siRNA modestly enhanced apoptosis only in stretched cells. RGD peptide or FAK silencing suppressed the activation of NF‐P3/80 invoked by CS. NF‐P3/80 depleted of FAK were defective in phospho‐AKTS473, TSC‐2T1462, and S6 kinaseT389 induced by CS. The activation of NF‐P3/80 invoked by CS was prevented by pre‐treatment with rapamycin, the mammalian target of rapamycin (mTOR) inhibitor, whereas treatment with leucine, activated S6K and rescued the stretch‐induced activation of NF‐P3/80 depleted of FAK. These findings demonstrate a critical role for the mTOR complex, FAK downstream, in mediating the activation of cardiac fibroblasts in response to mechanical stress.
Does Tip60 Regulate the Cardiomyocyte Cell‐cycle?
Authors: Fisher Joseph Bernard , Kim Min‐Su , Lough John
Published: June 2021

Tat‐Interactive Protein 60 (Tip60), a putative tumor suppressor, is a member of the MYST family of histone acetyltransferases (HATs). Tip60 function may include DNA repair, transcriptional co‐activation/repression, apoptosis, and cell‐cycle regulation. Alternative splicing of Tip60 mRNA creates α (60kD) and β (53kD) isoproteins, both of which are expressed in embryonic hearts. However, Tip60α becomes extinguished in neonatal hearts as proliferation subsides, leaving Tip60β as the sole isoprotein in adult heart. The role of Tip60 is unknown in adult cardiomyocytes, wherein we hypothesize that Tip60β maintains the G0 cell‐cycle state while promoting apoptosis. We targeted the Tip60 gene, observing that although homozygous deletion causes embryonic lethality, heterozygous deletion has no adult phenotype in the absence of stress. Therefore, two approaches, both of which induce hypertrophy, were used to stress Tip60+/− adult hearts: (i) physical stress via aortic banding and (ii) genetic stress via c‐Myc over‐expression. Both stressors caused the predicted outcomes of increased myocardial cell‐cycle activity and reduced apoptosis. These results support the hypothesis that Tip60 inhibits the cell‐cycle in adult cardiomyocytes, a possibility to be investigated using conditionally‐targeted mice that should enable reduction of Tip60β in myocardium to haploinsufficient levels.

Grant Funding Source: American Heart Association; Grant Number: 0810108Z
In vivo analysis of the specificity of DNA adenine methyltransferase mutants R116A and N126A
Authors: Castillo Ana , Ghozalli Irene , Gharzeddine Jenna , Peterson Stacey
Published: June 2021

DNA adenine methyltransferase (Dam) methylates GATC sites and is important for mismatch repair and the expression of pathogenic genes in Escherichia coli (E. coli). The three base pairs flanking each GATC site influence the methylation efficiency of Dam for that site. In vitro and in vivo studies have demonstrated that methylation of GATC sites with A/T rich flanking sequences are less efficiently methylated (“bad” sites) than those with G/C rich flanking sequences (“good” sites). Our goal is to determine how the structure of Dam contributes to this preference for certain GATC sites and what residues are involved. R116 and N126 are conserved Dam residues that lie within close proximity to the GATC flanking sequences. These residues were independently mutated to alanine, forming Dam mutants R116A and N126A. We hypothesize that disruption of the hydrogen bonds between the GATC flanking sequences and the amino acid side chains of R116 or N126 will affect the methylation efficiency of Dam at “good” and “bad” GATC sites. An in vivo analysis of methylation efficiency of the mutants at various GATC sites was performed. Our preliminary results reveal that both mutants show a difference in GATC site preference compared to the wildtype, suggesting the importance of these residues in specificity. Analysis of the mutants will illuminate an alternate route for the control of bacterial DNA methylation that is crucial for pathogenesis.
Dynamic Effects of Cofactors and DNA on the Oligomeric State of the Human Mitochondrial DNA Helicase
Authors: Makowska‐Grzyska Magdalena M , Ziebarth Tawn D , Kaguni Laurie S
Published: June 2021

Mitochondria are the essential energy‐producing organelle in eukaryotes, and represent the only organelle in animals that contains its own genome. Maintenance of the mitochondrial DNA (mtDNA) genome is dependent on myriad nuclear‐encoded proteins including the mtDNA helicase, which is an essential component of the replicative machinery. Human mtDNA helicase shares a high degree of sequence similarity with the bacteriophage T7 primase‐helicase gene 4 protein, and catalyzes duplex unwinding in the 5’‐3’ direction. We have shown that it is an oligomer in solution that is destabilized under low salt conditions. We present here the effects of cofactors on its stability and oligomerization state. We show that the low salt sensitivity of the mtDNA helicase is alleviated by the presence of magnesium, nucleotide and increased temperature. Electron microscopic and glutaraldehyde cross‐linking analyses provide the first evidence of a heptameric oligomer, and its interconversion from a hexameric form. Limited proteolysis by trypsin shows that binding of nucleoside triphosphate produces a conformational change that is distinct from the conformation observed in the presence of nucleotide diphosphate. We find that single‐stranded DNA binding occurs in the absence of cofactors, and renders the mtDNA helicase more susceptible to proteolytic digestion. Experiments to examine oligomeric state upon DNA binding are underway to evaluate the functional form of the enzyme, and its dynamic changes along the reaction pathway. This research was supported by NIH grant GM45295.
Identification of hTERT gene regulators by RNAi approach
Authors: Chen Chi‐Yuan , Wang Hsin‐Tse , Huang Pei‐Rong , Yeh Yuan‐Ming , Wang Tzu‐Chien
Published: June 2021

Telomerase directs the synthesis of telomeric repeats (TTAGGG in human) at chromosomal ends and is thought to play an important role in the maintenance of telomere length and cellular senescence. The up‐regulated expression of human telomerase reverse transcriptase (hTERT) is a hallmark of tumorigenesis. As yet, little is known about the signaling pathways used by the various tumor cells to differentially activate hTERT transcription. In this study, we employed a large‐scale RNAi approach to identify kinases and phosphatases that may participate in the regulation of hTERT expression in a human nasopharyngeal cancer cell line NPC‐pRevh3.4k GFP, which contains an hTERT promoter‐driven GFP reporter. A total of 119 shRNA sequences corresponding to 76 candidate genes were identified that either increased or decreased the expression of GFP. Seven of these candidate genes (ERCC8, NF2, AKT3, TRIM24, DGKZ, PPP6C and PI3C3) were further evaluated. Our preliminary results indicated that knockdown of TRIM24 increased the hTERT promoter activity, while knockdown of AKT3 decreased the hTERT promoter activity. Furthermore, ectopic expression of AKT3 in a telomerase‐negative U2OS cell was found to reactivate hTERT expression. These results suggest that TRIM24 is a negative regulator and AKT3 is a positive regulator for hTERT gene expression. Studies are underway to investigate the signaling pathways that may be targeted by these two genes. Supported by Chang Gung Medical Research Grant CMRPD 170541 of Taiwan.
NFIA controls progenitor cell differentiation through repression of the Notch effector Hes1
Authors: Piper Michael , Barry Guy , Hawkins John , Mason Sharon , Lindwall Charlotta , Little Erica , Moldrich Randal X. , Boyle Glen M. , Gronostajski Richard M. , Bailey Timothy L. , Richards Linda
Published: June 2021

Notch signaling plays a central role in regulating the self‐renewal of progenitors. The transcription factor Nuclear Factor I A (Nfia) has been implicated downstream of Notch in mid‐gestation telencephalic development, regulating the switch towards astrocytic differentiation from radial progenitors. However, a crucial step in this process is the simultaneous repression of Notch signaling which enables subsequent differentiation to occur. How this is regulated is unknown. Here we demonstrate that, in addition to regulating astrocyte‐specific genes, Nfia represses expression of the Notch effector Hes1. During hippocampal development, we find that Nfia‐deficient mice exhibit delays in both neuronal and glial development. Hes1 is significantly up‐regulated in Nfia mutant hippocampi, and is a direct transcriptional target of Nfia as shown by bioinformatic approaches, chromatin immunoprecipitation analyses and in vitro transcriptional assays. Thus, Nfia promotes differentiation of progenitors via complementary mechanisms, through activation of neuronal‐ and glial‐specific genes and via repression of Notch signaling.
Effect of single base bulges on oxidative DNA‐protein crosslinking
Authors: Abramchayeva Marina , Madison Amanda , Perez Zitadel Anne , Stemp Eric
Published: June 2021

Oxidative DNA damage contributes to aging and diseases such as Alzheimer's. Guanine is the most easily oxidized DNA base and therefore is most susceptible to oxidative damage. Bulges in DNA arise from replication and recombination errors and if left unrepaired, cause disease. Here, we are studying the effects of bulges on DNA‐ protein crosslinks. A DNA duplex was assembled using the strand 5’‐TATAGATATGGATATGATAT‐3’, which was fluorescently labeled with Alexafluor 546, and a complementary strand containing a single base bulge, 3’‐ TATACTATACYCTATACTATA‐ 5’, where Y = C, G, A or T. A control strand with no bulge was also synthesized. The GG sequence across from the single DNA base bulge localized the guanine radical at that bulge location. The flash quench technique was used to generate guanine radicals and DNA samples were analyzed by the gel shift assay. DNA bulges containing purines lead to an increase in DNA‐ protein crosslinking when compared to the well‐matched DNA. Thus, structural perturbations such as bulges, when near a guanine base, can influence the amount of oxidative DNA‐protein crosslinking.
Use of trifluoromethylated nucleotide analogs as 19F‐NMR probes of DNA structure
Authors: Wright Justin , Stoub Darren
Published: June 2021

The dynamics of conformational changes in DNA and RNA are important in many biological processes. Fluorine NMR has been shown to be a valuable structural tool for studying RNA and DNA and their interactions with other compounds. Unfortunately, many of the fluorinated nucleosides utilized are either unstable, expensive, or change nucleic acid structure. We report our efforts toward the synthesis of the phosphoramidites of 8‐aza‐7‐deaza‐7‐trifluoromethyl guanosine, 8‐aza‐7‐deaza‐7‐trifluoromethyl adenosine, 8‐trifluoromethyl guanosine and 8‐trifluoromethyl adenosine ribonucleosides. We also report our efforts in incorporating the analogs into DNA, the impact of the analogs on DNA stability using thermal denaturation, and our initial NMR experimental results.
Activation of Innate Immune Signaling by Viral RNAs
Authors: Gehrke Lee , Uzri Dina
Published: June 2021

RIG‐I is a host pattern recognition receptor (PRR) that is critical for innate immune signaling during RNA virus infections. RIG‐I binds to pathogen associated molecular pattern (PAMP) RNAs by recognizing the 5’ triphosphate present on uncapped viral RNAs in addition to a polyU/UC domain that is present in the hepatitis C virus (HCV) RNA 3’ untranslated region. Following RNA recognition, RIG‐I is ubiquitinylated and binds to IPS‐1, a protein localized to the outer mitochondrial membrane. The RIG‐I/IPS‐1 interaction then activates a signal transduction cascade leading to interferon expression, thus initiating an antiviral state. Our research is aimed at understanding the RNA nucleotide sequence and structural determinants for RIG‐I binding and activation. Poly U/UC RNAs containing 2’‐fluoro‐ deoxyuridine or pseudouridine substituted for uridine bind the RIG‐I protein; however, these RNAs do not activate downstream interferon expression. Short homopolymeric U or A RNAs (50 nucleotides in length) are not activators of RIG‐I signaling; however, insertion of a single C or G nucleotide, respectively, increases the RNA activation potential. These data suggest that RNAs containing interrupted U or A homopolymer sequences are targets for RIG‐I binding. Current experiments focus on using the modified RNAs to define the mechanism of RIG‐I activation that leads to innate immune signaling.
Roles of HDV‐like self‐cleaving ribozymes in retrotransposition
Authors: Ruminski Dana , Webb Chiu‐Ho Ts , Riccitelli Nathan J , Luptak Andrej
Published: June 2021

Using structure‐based bioinformatics, we recently discovered self‐cleaving ribozymes of the Hepatitis Delta Virus (HDV) family in another virus, a bacterium, and many eukaryotes. The genomic locations of some of these ribozymes suggest that they play a variety of biological roles. HDV‐like ribozymes from several organisms are associated with putative reverse transcriptase genes and retrotransposons, pointing to a possible function in retrotransposition. The specific roles of HDV‐like ribozymes in retrotransposition are being investigated using a variety of in vitro and in vivo approaches.
Hypoxia Induces Global Protein Relocalization in Yeast
Authors: Dastidar Ranita Ghosh , Henke R. Michael , Shah Ajit N. , Zhang Li
Published: June 2021

Oxygen is critical for the survival and development of many living organisms, particularly eukaryotes. Living organisms ranging from yeast to humans have developed sophisticated mechanisms to respond to the changes of oxygen level in the environment. In the yeast Saccharomyces cerevisiae, our previous genome‐wide experimental and computational studies have suggested that a complex network of regulators likely mediate hypoxia responses. To identify potential global regulators mediating oxygen regulation and hypoxia responses, we initiated a live cell imaging screen to identify proteins whose cellular locations are altered under hypoxia. From a GFP‐tagged yeast library with over 4400 strains, we identified over 200 proteins whose cellular locations were changed under hypoxia. The large majority of the proteins are nuclear proteins with important functions in gene regulation and chromatin remodeling, and mitochondrial proteins with functions in energy metabolism. Notably, a group of nuclear pore and mitochondrial proteins with protein targeting and import functions are relocalized under hypoxia. The relocalization of such proteins may be important for global protein relocalization under hypoxia. Experiments are underway to further ascertain the roles of protein relocalization in oxygen regulation and hypoxia responses. GM062246
Molecular mechanisms underlying AMPK‐induced inhibition of glucose uptake in primary rat adipocytes
Authors: Ceddia Rolando B. , Perry Robert , Gaidhu Mandeep P.
Published: June 2021

This study investigated the molecular mechanisms by which AMP‐kinase (AMPK) activation inhibits basal and insulin‐stimulated glucose uptake in primary adipocytes. Rat epididymal adipocytes were exposed to AICAR for 1h. Subsequently, glucose uptake and the phosphorylation states of AMPK, ACC, Akt, and the Akt substrate of 160kDa (AS160) were determined in primary adipocytes either expressing LacZ (control) or a kinase dead AMPKα1 mutant (KD‐AMPKα1). AICAR increased AMPK and ACC phopshorylation, without affecting basal and insulin‐stimulated Akt phosphorylation. However, AMPK activation suppressed AS160 phosphorylation by 80% and 50% and glucose uptake by 35% and 50% under basal and insulin‐stimulated conditions, respectively. Expression of the KD‐AMPKα1 mutant fully prevented the suppression of AS160 phosphorylation as well as the inhibitory effect of AICAR‐induced AMPK activation on basal and insulin‐stimulated glucose uptake. This study provides novel evidence that suppression of AS160 phosphorylation by AICAR‐induced AMPK activation leads to inhibition of basal and insulin‐stimulated glucose uptake in primary rat adipocytes. Disruption of AMPKα1 signaling fully prevented these effects, indicating that insulin‐signaling steps that are common to white adipose tissue and skeletal muscle regulation of glucose uptake are distinctly affected by AMPK activation in these tissues.
Cocoa polyphenols inhibit sucrose hydrolysis in Caco‐2 cells
Authors: Ellam Samantha Lee , Williamson Gary
Published: June 2021

Disaccharidase enzymes bound to the microvilli of intestinal epithelial cells are essential in the liberation of glucose from dietary carbohydrates. Glucose uptake from the lumen of the small intestine is facilitated by transporters located in the apical membrane of enterocytes; subsequent diffusion into capillary blood occurs at the basolateral surface similarly facilitated by transporters.

Attenuation of enzyme and transporter activity may decrease post‐prandial blood glucose levels thereby providing a more uniform supply of glucose to the cells rather than an initial peak followed by rapid decline. This may have positive consequences not only for healthy subjects but also for individuals suffering from impaired glucose tolerance and type‐2 diabetes.

Using Caco‐2 cells, passages 36 to 40, the rate of sucrose hydrolysis has been determined in the absence and presence of cocoa extracts and various polyphenols present in chocolate and cocoa. Using sucrose as the substrate, kinetic parameters were: a maximum reaction rate (Vmax) of 4.5 μmol min−1 and a Michaelis‐Menten constant (Km) of 33 mM.

The addition of cocoa extract inhibited sucrose hydrolysis by Caco‐2 cells. (−)‐Epicatechin, (+)‐catechin and procyanidin B2, major components of cocoa, also diminished sucrose hydrolysis in Caco‐2 cells substantially.

These results show that intestinal hydrolysis of sucrose by alpha‐glucosidase enzymes may be inhibited by cocoa polyphenols thereby reducing the rate of liberation of glucose. This would subsequently limit the uptake of glucose into the blood thereby reducing post‐prandial blood glucose levels.

This work was funded by a BBSRC industrial case award to Nestlé, York UK.
Characterizing a coq‐3 mutation that extends life span in C. elegans
Authors: Gomez Fernando , Saiki Ryoichi , Chin Randall , Srinivasan Chandra , Clarke Catherine F.
Published: June 2021

Coenzyme Q (referred to simply as Q) is a lipophilic component of the electron transport chain in mitochondria. The biosynthesis of Q is known to include several steps, including two O‐methyltransferase reactions catalyzed by COQ‐3. Expression of C. elegans COQ‐3 restores respiratory growth and Q biosynthesis in an E. coli ubiG mutant. In C. elegans, coq‐3 mutants demonstrate altered fertility and survival. One mutant, coq‐3(qm188), is sterile. Another mutant, coq‐3(ok506), is fertile. Both mutant strains are sterile when fed the Q‐deficient bacteria strain GD‐1. Western blot analyses with a COQ‐3 protein antibody indicate that both mutants also lack the wild‐type protein. The coq‐3 gene is situated in a three‐gene operon. QT‐PCR analysis shows that transcript levels of the upstream gene, nuo‐3, in coq‐3(qm188) mutants are widely variant compared to wild‐type and coq‐3(ok506) mutants. This erratic expression pattern indicates that coq‐3(qm188) is not a representative coq‐3 mutant. Mutant coq‐3(ok506) raised on the Q‐less E. coli strain GD1 possess small, thin and severely underdeveloped gonads, and live 125% longer than wild‐type worms raised on a this diet. Mutants raised on OP50 have a 40% shorter life span than wild type worms and an asymmetrical uterine anatomy. Since uterine physiology affects life span, our anatomical data may help explain why coq‐3(ok506) homozygotes raised on GD1 live longer,
Metabolomics and Gene Discoveries Reveal Interrelationships between Glucose, Phosphate and NAD Metabolism
Authors: Brenner Charles
Published: June 2021

Early models that sought to explain how calorie restriction may alter NAD metabolism were formulated prior to the identification of all of the NAD metabolites and the enzymes that interconvert these metabolites. We have recently identified nicotinamide riboside and nicotinic acid riboside as metabolites and have identified roles for nicotinamide riboside kinase, uridine hydrolase, purine nucleoside phosphorylase, and two multifunctional 5′‐nucleotidases in yeast NAD metabolism. A metabolomic method has quantified the yeast NAD metabolome. Data from yeast cells whose lifespan is extended with vitamin provision or glucose restriction indicate that, while increasing net NAD synthesis is a mechanism for lifespan extension, it is not the mechanism that applies for glucose restriction, nor is a change in the NAD:nicotinamide or NAD:NADH ratio. Some of the same pathways that respond to glucose restriction are also regulated by phosphate restriction and the availability of vitamins. Thus, the calorie restricted metabolome appears to involve a large number of small changes that effect multiple targets rather than a small number of large changes that exclusively target sirtuins.