bluuebunny/crossref_metadata_2025 huggingface dataset:
Circulating Myostatin Decreases with Aerobic Exercise Training in Pre‐Diabetic but not Diabetic Human Subjects
Authors: HITTEL DUSTIN , Atkins Kerry , Kraus William E , Sigal Ronald J
Published: June 2021

Myostatin is a potent secreted inhibitor of skeletal muscle growth. We recently demonstrated that plasma myostatin decreased with aerobic exercise and a correlation with insulin sensitivity. We subsequently quantified myostatin in plasma samples from two exercise‐training cohorts to determine the effects of both endurance and resistance exercise on circulating myostatin levels in both insulin resistant (STRRIDE, n=100) and type 2 diabetic (DARE, n=360) human subjects. In both studies, fasting myostatin levels increased significantly (P<0.05) whilst markers of insulin resistance (Si and HOMA‐IR) worsened in the control subjects (~ 6 months sedentary). In the STRRIDE cohort the most intense aerobic exercise group (65–80% VO2, 2200 kcal/week) showed the most significant decrease in plasma myostatin. There were no significant decreases in plasma myostatin in the aerobic and combined exercise training groups in the DARE cohort, however, there was a small but significant decrease in resistance training group. These findings confirm that plasma myostatin levels are responsive to exercise training in both insulin resistant and diabetic patients albeit to different exercise modalities. These findings support our hypothesis that myostatin is in the causal pathway of acquired insulin resistance with physical inactivity.
Integrative approach reveals specific disturbances in β‐adrenergic signaling in a porcine model of post‐myocardial infarction remodeling
Authors: Kuster Diederik W , Verhoeven Adrie J. , Merkus Daphne , van der Velden Jolanda , Duncker Dirk J.
Published: June 2021

Loss of myocardial tissue after myocardial infarction (MI) leads to neurohumoral activation that is aimed at restoring cardiac pump function, but eventually leads to cardiac remodeling, which is an independent risk factor for the development of heart failure.

We studied the process of post‐MI remodeling in the pig, a physiologically relevant animal model, at the organismal, cellular and molecular level.

At 3 weeks after MI, β‐adrenergic sensitivity of the heart was reduced and left ventricular remodeling and dysfunction were apparent. At the cardiomyocyte level, force development was reduced and Ca‐sensitivity increased. Microarray analysis revealed alterations in the β‐adrenergic signaling pathway. Protein analysis showed increased PKA regulatory subunit while PKA catalytic subunit was decreased, which were normalized upon β‐blocker treatment. The β‐agonist induced increase in phosphorylation of the PKA target protein troponin I was attenuated.

This integrative approach allowed us to link the blunted response to exercise and the disturbed myofilament function to changes in expression of PKA subunits. β‐blocker treatment restored functional parameters to control levels. These observations are in line with medical practice that treatment with β‐blockers protects MI patients from developing heart failure.

Supported by a grant from Netherlands Heart Foundation (NHS2005B234)
Neonatal hyperoxia increases airway reactivity in response to respiratory syncytial virus (RSV) infection or a second hyperoxic exposure in rat pups
Authors: Jafri Anjum , Ali Nuzhat KM , Martin Richard J , Zaidi Syed I. A.
Published: June 2021

Increased airway reactivity is a well recognized complication of neonatal lung injury. Our prior studies have demonstrated that neonatal hyperoxia enhances contractility and impairs relaxation in lung parenchyma. In this study, we tested the hypothesis that neonatal hyperoxia predisposes rat pups to increased airway reactivity in response to infection or a second hyperoxic exposure. P0 rat pups were exposed to 50% O2 or room air for 7 days. In one protocol pups (n=12/group) were then infected with RSV at day 7, or control, kept in room air for 5 days and sacrificed. In another protocol pups (n=8/group) were recovered from hyperoxia for 7 days, and were then exposed either to 100% O2 or room air for 24 h and sacrificed. Graded electrical field stimulus (EFS) was applied to lung parenchymal strips in order to study their contractile or relaxant responses. RSV infection in hyperoxic pups significantly increased EFS‐induced contractile responses of strips (p<0.05) but had no effect on room air exposed animals. RSV infection also impaired EFS‐induced relaxation that was greater in hyperoxic than in room air groups (p<0.05). A later hyperoxic exposure resulted in greater contractile responses in pups exposed to prior hyperoxia versus controls. The results show that neonatal hyperoxia predisposes pups to increased airway reactivity in response to RSV infection or to a second hyperoxic exposure. Supported by NIH grant HL‐056470
Dynamic Imaging of Airways During Bronchoconstriction and Deep Inspiration in Intact Rats
Authors: Rapalo Gabriel , Sinclair Scott E. , Makena Patrudu , Molthen Robert C. , Waters Christopher M.
Published: June 2021

Airways are continuously subjected to cyclic mechanical stretch during normal respiratory movements, and large distensions occur periodically with deep sighs. However, there is limited information on the levels of mechanical stretch in vivo and how these might change in hyper‐responsive airways. Using a unique microfocal X‐ray imaging system to visualize airways coated with tantalum dust, we imaged dynamic changes in dimensions of rat airways in vivo following exposure to methacholine during mechanical ventilation. Circumferential strain and longitudinal strain were measured based upon changes in diameter and length of segments during a breath cycle relative to dimensions at end expiration. We simultaneously performed measurements of lung resistance and compliance using the Flexivent system. We found significant changes in the diameters of airways of all sizes (~0.2 mm to 2 mm), but there was substantially more heterogeneity in smaller airways. We also found that a deep inspiration caused significant relaxation of airways circumferentially, but deep inspiration had little effect on longitudinal dimensions. These measurements allow us to quantify changes in the levels of mechanical stretch in airways during bronchoconstriction and to assess the time‐dependent response to a deep inspiration.

Supported by NIH grant HL094366.
Bronchoconstriction induced by hyperventilation with humidified warm air in ovalbumin‐sensitized Brown Norway rats
Authors: Hsu Chun‐Chun , Martin Chayse B , Lee Lu‐Yuan
Published: June 2021

Previous investigators reported that hyperventilation with hot humid air caused bronchoconstriction in asthmatic patients (Am Rev Resp Dis 131: 357–361, 1985). This study was carried out to further investigate this response in an animal model of allergic asthma. Our results showed: 1) In Brown‐Norway rats actively sensitized by chronic inhalation of ovalbumin (Ova) aerosol, isocapnic hyperventilation with humidified warm air (HWA) for 2 min induced an increase in tracheal temperature (Ttr) to a peak of 40.6 ± 0.3°C and an immediate and sustained (> 10 min) increase in RL (from 0.12 ± 0.01 to 0.21 ± 0.02 cmH2O/ml/s; n=7, P<0.01). In sharp contrast, the HWA challenge produced the same increase in Ttr, but did not generate any increase in RL in matching control rats (n=7, P>0.05). 2) The responses in RL were reproducible in both groups when the same HWA challenge was repeated 60–90 min later (n=4). 3) This bronchoconstrictive effect was temperature dependent; a smaller increase in peak Ttr (39.9 ± 0.2°C) generated a smaller, but significant increase in RL in sensitized rats: from 0.12 ± 0.01 to 0.19 ± 0.03 cmH2O/ml/s (n=3, P<0.05). In conclusion, an increase in airway temperature within the normal physiological range triggered bronchoconstriction in sensitized rats, but not in control rats. Chronic airway inflammation in sensitized animals is likely a major contributing factor in causing this response. (NIH grant HL96914, fellowship NSC98‐2917‐I‐038‐101)
Hyperoxia enhances intracellular calcium in human fetal airway smooth muscle via cyclic nucleotide regulation of brain‐derived neurotrophic factor release
Authors: Thompson Michael A , Martin Richard J , Zaidi Syed I , Pandya Hitesh , Pabelick Christina M , Prakash Y S
Published: June 2021

Supplemental oxygen places the immature airway at risk for bronchopulmonary dysplasia and asthma. Hyperoxia enhances intracellular Ca2+ ([Ca2+]i) and contractility of airway smooth muscle (ASM), and impairs relaxation (potentially by altering cyclic nucleotide levels). The neurotrophin brain‐derived neurotrophic factor (BDNF) enhances [Ca2+]i in adult ASM. We tested the hypothesis that hyperoxia‐induced airway hyperreactivity involves cyclic nucleotide‐mediated enhancement of BDNF release in human fetal (18–22 weeks) ASM cells exposed to 21% (normoxia) or 80% oxygen in the presence/absence of 1mM dibutyryl cAMP (DBcAMP) or 8‐Br‐cGMP. BDNF release (ELISA) was increased by hyperoxia (143% of normoxia). DBcAMP enhanced BDNF release with normoxia (242% of baseline) and even more with hyperoxia (280%), while 8‐Br‐cGMP blunted BDNF release in normoxia (57% of baseline) but to a lesser extent with hyperoxia (68% of baseline). Measurements of [Ca2+]i in fetal ASM cells showed DBcAMP increasing [Ca2+]i responses to histamine or ACh (effects enhanced with hyperoxia). Further, chelation of released BDNF using chimeric TrkB‐Fc (1?g/ml) reversed DBcAMP effects and blunted [Ca2+]i responses to below controls. We conclude that an imbalance between cAMP vs. cGMP enhances BDNF release in the developing airway, resulting in greater airway reactivity with hyperoxia. Supported by NIH grants HL056470 and HL088029
Phrenic Nerve Morphometry in Short Term and Long Term Experimental Diabetes
Authors: Alcântara Adriana Cristina Licursi , Tanaka Natália Massumi , Filho Omar Andrade Rodrigues , Fazan Valéria Paula Sassoli
Published: June 2021

The aim of the present study was to investigate the morphometric parameters of the phrenic nerve in short term and long term experimental diabetes. Male Wistar rats received a single intravenous injection of streptozotocin (40mg/Kg) 15 days (N=7) or 12 weeks (N=10) before the experiments. Controls rats (N=7 and N=4, respectively) received citrate buffer (vehicle). On the experimental day, rats were perfused with glutaraldehyde 2.5%, had the right and left phrenic nerves dissected and the proximal and distal segments prepared for light microscopy study as described previously (Rodrigues Filho & Fazan, 2006). Morphometry was performed with the aid of computer software and took into consideration the fascicle area and diameter, number of myelinated fibers and their density. Comparison was made between segments, sides and groups and differences were considered significant when p < 0.05. Our results show that the myelinated fiber number is similar between control groups but larger on both diabetic groups, compared to controls, particularly on the distal segments. This is suggestive of a “dying back” neuropathy, with signs of nerve regeneration.

Support: CNPq, CAPES, FAPESP and FAEPA

Grant Funding Source: CNPq, CAPES, FAPESP and FAEPA
Phrenic Nerve Morphology in Normotensive and Spontaneously Hypertensive Rats with chronically induced diabetes
Authors: da Silva Ferreira Renata , Sanada Luciana Sayuri , Salgado Hélio Cesar , Fazan Valéria Paula Sassoli
Published: June 2021

The aim of this study was to investigate morphometric alterations in phrenic nerves of spontaneously hypertensive rats (SHR), compared to normotensive rats (WR), both with chronically induced diabetes (SHR+STZ and WR+STZ, respectively). WR+STZ and SHR+STZ received a single intravenous injection of streptozotocin (STZ) while WR and SHR received vehicle. Twelve weeks after injection, rats were anesthetized and had their blood pressure and heart rate recorded. After intracardiac perfusion, right and left phrenic nerves were prepared for light microscopy. Morphometric analysis was performed with the aid of computer software, and took into consideration the fascicle area and diameter, as well as myelinated fiber number, density, area and diameter. No differences in fascicular morphology were observed between groups and the phrenic nerves were statistically symmetrical longitudinally and laterally in all groups studied. However, both diabetic groups showed alteration of the myelinated fibers. Despite little alteration on phrenic nerve morphology in SHR+STZ, this study provides a morphological basis for future neuropathy studies in these models of human diseases.

Support: CNPQ, CAPES, FAPESP and FAEPA
Effects of noxious stimulation during neonatal phase in the sensory‐motor system of adult male Wistar rats
Authors: Machado Nathalia Leilane Berto , de Cássia do Carmo Elisabete , Sanada Luciana Sayuri , Fazan Valéria Paula Sassoli
Published: June 2021

Studies report that exposure of newborn to pain is predictive of global responses to pain in adults. The aim of the present study was to analyze the effects of noxious stimulation in neonatal phase in the sensory‐motor system of adult rats. Twelve male Wistar rats with 120 days of life were separated in 2 groups: pain group (n=6) and control group (n=6). The pain group was stimulated with a needle on the right paw, twice a day for 15 days. The control group was stimulated with a cotton swab on the right paw, twice a day for 15 days. To evaluate the hyperalgesia in both groups, we applied Von Frey monofilaments and a specific calibrated forceps; the evaluation of muscle strength of the hind paws was conducted through a special device (Grip Strength Meter). This device measures the grip strength of the hind paw in rats. Specific statistic tests were applied, and p value was considered significant when p less than 0.05. We observed that the pain group presented a hyperalgesia in the right hind paw and a decrease in muscle strength when compared to the control group. Our results suggest that noxious stimulation in the neonatal phase alters the sensorimotor system in the long term.
Spread of injected dye after ultrasound‐guided transversus abdominis plane block in cadavers
Authors: Morris Laura Ruth , Johnson Marjorie , Ganapathy Sugantha
Published: June 2021

Transverse abdominis plane (TAP) block is done before abdominal surgery to relieve post surgery pain. A TAP block deposits local anaesthetic between transversus abdominis and internal oblique muscles, anesthetising the thoracolumbar nerves. Difficulty encountered when performing this procedure is consistency reaching T7‐L1 nerves. In previous TAP block studies, only some of these nerves were covered by dye, mimicking anaesthetic migration, in embalmed cadavers. The goal of this study is to explore techniques that enable TAP blocking of the T7 to L1 nerves. Different injected substances tested were methylene blue dye, green paint, red latex solution, gelatine and paint solution, and methylcellulose and paint solution in embalmed cadavers using ultrasound as a guide. Using methylcellulose and paint solution, T7 to L1 nerves were reached in embalmed cadavers with 20cc of methylcellulose/paint solution injected rostral and with 10 cc of methylcellulose/paint solution injected caudal to the umbilicus at the anterior axillary line. These clinical findings may provide anaesthesiologists with information to more consistently relieve pain and decrease recovery time.

Grant Funding Source: Deparmental
Supra and infralevator autonomic‐somatic communications: Computer‐assisted anatomic dissection in male and female foetuses
Authors: alsaid bayan , MOSZKOWICZ David , PESCHAUD Frederique , BESSEDE Thomas , ZAITOUNA Mazen , KARAM Ibrahim , BENOIT Gérard , DROUPY Stéphane
Published: June 2021

Objective

to identify the location and the nature of communications between supra and infralevator pathways.

Materials and Methods

6 male and 6 female human fetal pelvic specimens, 15 to 32 wk of gestation, were studied using the 3D computer‐assisted anatomic dissection (CAAD) with neural, nitrergic and myelin sheath markers.

Results

Supra and infra levator neural pathways cross each other distally in spatial X shape area. They yield dual innervation in 5 directions: 1‐anal sphincter, 2‐levator ani muscle, 3‐urethral sphincter, 4‐corpus spongiosum and perineal muscles, and 5‐corpora cavernosa. The communications between the two pathways exist in 3 levels: proximal supralevator, intermediary intralevator and distal infralevator. Distal communications exist when autonomic nitrergic cavernous nerves interact with some somatic nitrergic branches of the dorsal penis/clitoris nerve and carried the nitrergic activity in its distal portion.

Conclusions

The pelvic structures responsible for sphincters continence and sexual function receive a dual innervation from the autonomic supralevator and the somatic infralevator pathways. Distal communication takes over the nitrergic activity to the distal part of cavernous bodies in both gender and represents an important structure which may impact erectile function, especially during reconstructive surgery.

Grant Funding Source: non
Anti‐Fertility Effects of Amlodipine in Adult Male Wistar Rats
Authors: Akinlolu Adelaja Abdulazeez , Ghazali Olaide , Adefule Adebayo Kehinde
Published: June 2021

Amlodipine is a long‐acting calcium channel blocker used in the treatment of hypertension and angina. In adult Man, the treatment regimen is 5 or 10mg daily. This study tested the hypothesis that prolonged oral administration of Amlodipine impairs fertility in adult male rats. Thirty‐three normotensive adult male wistar rats (140 – 250g) comprising of seven groups were employed in the study; Control Groups I and II received 4mls of Normal Saline while experimental Groups III – VII received 5, 10, 15, 20 and 40mg/kg BW of Amlodipine respectively for 56 – 65 days. Gross morphology, testicular histology, testosterone levels, sperm cells morphology, count and motility were examined in all rats. Gross morphology evaluations showed reduced bodyweight, hair loss and loss of locomotion and balance in rats treated with Amlodipine in weeks 4 – 9 in a dose‐dependent manner. No adverse effects were observed on testosterone levels in all rats. Dose ‐ dependent anomalies of sperm morphology such as sperms with no head and double tail as well as significantly reduced sperm cells' count and motility were observed in rats of the experimental group compared to rats of the control group. Histological analyses showed scanty seminiferous tubules, wider tubular lumen and reduced interstitial cells in rats of the experimental groups. This study observed impaired fertility in adult male rats treated with 5mg/kg BW or higher doses of Amlodipine.

Grant Funding Source: UNIVERSITY OF ILORIN, NIGERIA
Fine structure of tubular myelin from mammalian lungs
Authors: Meek Bill , Beju Delia , Palmer Tiffany
Published: June 2021

Tubular myelin and intracellular surfactant images are presented from human, rabbit, rat, and dog lung. The samples represent a long‐term study of normal as well as cystic fibrosis specimens. Tubular myelin is seen in its typical lattice‐like form as it is found on the extracellular surface. Surfactant within lamellar bodies is also shown with a unique relationship to mitochondria. Images from lung parenchyma and rabbit lung washes reveal the complex architecture of tubular myelin. Specimens are fixed with glutaraldehyde and secondary fix common to electron microscopy protocols. Comparative study of cystic fibrosis samples and normal lung did not reveal any differences in tubular myelin pattern. It is hoped that an analysis of these specimens will yield further information regarding cellular surfactant production, its arrangement, and its appearance on the cell surface in both the human normal and disease condition.
The spectrin cytoskeleton is an integral target and crucial component of adherent, invasive triggering and invasive zippering bacterial diseases
Authors: Ruetz Tyson Joel , Cornick Steve , Guttman Julian Andrew
Published: June 2021

Entropathogenic Escherichia coli (EPEC), Salmonella Typhimurium (S.Typhimurium), and Listeria monocytogenes (L. monocytogenes) all manipulate the host cell cytoskeleton at crucial stages of their disease processes. Although the actin cytoskeleton has been previously identified as a ubiquitous target of these pathogens, the examination of the spectrin cytoskeleton has been largely overlooked. Here we show that spectrin and the spectrin‐associated proteins adducin and protein 4.1 are recruited to sites of EPEC pedestals as well as sites of S. Typhimurium and L. monocytogenes invasion. Individual siRNA knockdowns of each spectrin cytoskeletal protein significantly impeded the ability of EPEC to form pedestals and inhibited the abilities of both S.Typhimurium and L.monocytogenes to invade host cells. Further studies identified spectrin cytoskeleton recruitment to later time‐points of S.Typhimurium and L. monocytogenes infections during the intercellular stage of pathogenesis, revealing novel cytoplasmic roles for the spectrin cytoskeleton. This work demonstrates that the spectrin cytoskeletal system is crucial for bacterial pathogenesis and represents a potential target for therapeutic treatment for a broad list of infections.

Grant Funding Source: CIHR
Beta1 integrin plays a role in lens fiber cell structure and interaction with the cytoskeleton
Authors: Scheiblin David A. , Simirskii Vladimir N. , Czymmek Kirk J. , Duncan Melinda K.
Published: June 2021

A conditional knockout for β1 integrin of lens fiber cells was produced. Scanning electron microscopy was used to assess the ultrastructure. Immunofluorescence of sectioned and whole lens was used to determine the expression of β1 integrin and its crosstalk with different cytoskeletal components.

At 14.5 dpc (days post coitum) expression of β1 integrin is lost in lens fiber cells. Mice lacking β1 integrin in lens fiber cells have variable phenotypes of clear and cataract lens. The ultrastructure of the lenses was analyzed from two different areas. Cortical fiber cells are newly differentiated cells that contain a highly regular structurally repeated ball and socket pattern along the membranes of fiber cells in wildtype mice. Lens lacking β1 integrin have ball and socket structures that are disorganized exhibiting variable width and extension from the cell. Nuclear fiber cells are older cells that contain ball and sockets as well as a random arrangement of membrane furrows in wildtype mice. Lens lacking β1 integrin have a membrane furrow pattern that is highly organized into a lattice like geometric pattern, no ball and sockets and a defect of Aquaporin‐0. Lens lacking β1 integrin also have a modified f‐actin network. The lack of the f‐actin network disrupts the hexagonal packing arrangement and radial cell columns of these fiber cells.

β1 integrin is needed for proper structure of lens fiber cells. Funded by NEI EY015279.

Grant Funding Source: NEI EY015279
Multi‐tasking focal adhesion protein zyxin is present at tubulobulbar complexes
Authors: Young J'Nelle Sarah , Vogl Wayne
Published: June 2021

Tubulobulbar complexes (TBCs) are actin‐related double‐membrane projections that form at intercellular junctions in the seminiferous epithelium of the mammalian testis. They form at basal junction complexes between neighboring Sertoli cells and apical junctions between Sertoli cells and germ cells. They are proposed to internalize intact junctions during sperm release and during the translocation of spermatocytes through basal junction complexes between neighboring Sertoli cells. Although TBCs are testis‐specific, they morphologically resemble podosomes in osteoclasts. Previously, we have reported that a complex of N‐WASp, Arp2/3, cortactin and dynamin that is found at podosomes also is present at TBCs. Here we demonstrate that zyxin, a focal adhesion protein known to be present at podosomes also is present at apical TBCs. A rabbit polyclonal anti‐zyxin antibody (B71) [generous gift from M.C. Beckerle, Huntsman Cancer Institute] was used to label fixed fragments and frozen sections of testis. Zyxin staining was intense in Sertoli cell regions known to contain tubular regions of TBCs. Zyxin is comprised of a proline rich N‐terminal domain and three LIM domains in its C‐terminal half. Although known to interact with the cytoplasmic domain of integrins, zyxin may be part of the mechanism of tubulobulbar complex formation. Supported by an NSERC Discovery Grant to AWV.

Grant Funding Source: NSERC
Disruption of mitochondrial DNA polymerase γ replication by the aldehyde adduct of DNA, M1dG
Authors: Cline Susan D. , Marnett Lawrence J. , Copeland William C.
Published: June 2021

M1dG is a mutagenic adduct generated in human nuclear and mitochondrial DNA (mtDNA) by reaction of the cellular peroxidation products, malondialdehyde and base propenal, with guanine. Since M1dG is known to interfere with DNA synthesis by replicative and Y‐family DNA polymerases, we have utilized recombinant exonuclease‐deficient human DNA polymerase γ (exo− pol γ) with or without its accessory subunit p55 to investigate the effects of the adduct on mtDNA synthesis. Using primer‐templates that required exo−pol γ to extend the primer by insertion opposite the adduct position, we found that M1dG decreased exo− pol γ primer extension by 50% and prevented synthesis of a fully‐extended 35‐mer primer. A dAMP was inserted preferentially opposite M1dG, but extension of the primer past the adduct was supported only by dCMP incorporation. The insertion efficiency for exo− pol γ with either dCTP or dATP as substrates was substantially reduced by M1dG, and p55 had no effect on exo−pol γ nucleotide selection or insertion kinetics at the adduct. Since human mitochondria have no known mechanism for repair of M1dG, which is a target of nucleotide excision repair in the nucleus, our data indicate that the endogenous adduct poses a significant threat to mtDNA stability by disrupting DNA pol γ replication. (Supported by National Institutes of Health Grants GM087681 to S.D.C. and CA087819 to L.J.M, and NIH intramural funding to W.C.C.)
Elevated embryonic expression of chromatin architectural protein HMGN5 alters higher order chromatin organization and leads to postnatal cardiac malfunction
Authors: Rochman Mark , Furusawa Takashi , Bustin Michael
Published: June 2021

HMGN5 is a chromatin architectural protein that specifically binds to nucleosomes, unfolds chromatin and affects transcription. We report that HMGN5 is highly and ubiquitously expressed at preimplantation stages of embryonic development but is significantly down regulated in the embryo during implantation. To assess the importance of developmentally regulated HMGN5 expression, we generated conditional transgenic mice and induced exogenous HMGN5 expression either in the whole embryo or only in the embryonic heart. We found that upregulation of HMGN5 did not alter embryonic development and the mice were born normal. However, transgenic animals remained small in size and died within 2–3 months after birth. Pathological examination revealed significant alteration in the nuclear size and chromatin architecture of cardiomyocytes evident by reorganization of chromocenters. In addition, biochemical analysis showed upregulation of genes associated with cardiac stress. MRI tests indicated severe cardiac malfunction and hypertrophy. Our studies provide a direct link between global structural organization of chromatin fiber in the heart and a phenotypic outcome evident at the level of an entire organism.
A nonsense mutation of the bHLH transcription factor TWIST2 found in Setleis Syndrome patients causes dysregulation of the human periostin gene
Authors: Franco Hector L , Casasnovas Jose J , Leon Ruth G , Friesel Robert E , Cadilla Carmen L
Published: June 2021

Setleis Syndrome (SS, MIM #227260) is a rare autosomal recessive disorder characterized by abnormal facial and skin development. Recently, we have shown that nonsense mutations in the basic helix‐loop‐helix (bHLH) transcription factor TWIST2 cause Setleis Syndrome. Microarray analysis suggests that periostin (POSTN), a cell adhesion protein involved in connective tissue development and maintenance, is down regulated in Setleis Syndrome patient fibroblasts. Overexpression and knockdown of TWIST2 resulted in increased and decreased expression of POSTN mRNA, respectively. Functional analysis of the Q119X TWIST2 mutant form revealed that it maintains the ability to localize to the nucleus, form homo and heterodimers with the ubiquitous bHLH protein E12 and shows binding to dsDNA. Chromatin immunoprecipitation assays suggest that both wild‐type TWIST2 and its mutant form can bind the POSTN regulatory region, however only wild‐type TWIST2 is associated with higher levels of acetylated histone H3 of the POSTN promoter. Reporter gene assays using deletion constructs of the POSTN regulatory region also reveal that wild‐type TWIST2 can activate this gene, but not the Q119X TWIST2 mutant. This data suggests that the C‐terminal domain of TWIST2, which is missing in Q119X TWIST2, is responsible for proper transactivation of POSTN. This research is supported by MBRS RISE Grant R25GM061838 and RCMI Grant G12RR03051.
A comparative analysis of native neutrophil gelatinase‐associated lipocalin (NGAL) protein isolated from various human source matrices
Authors: Rae Tracey Douglas , Rupprecht Kevin , Bonn Ryan , Stamenova Svetoslava , Zhao Cheng , Ramsay Carol , Fishpaugh Jeffrey
Published: June 2021

Elevated levels of Neutrophil Gelatinase Associated Lipocalin (NGAL) in the urine have been established as a promising diagnostic indicator for acute kidney injury (AKI). Although significant structural information on the human NGAL protein has been revealed from work with various recombinant expression systems, less has been revealed from native NGAL protein species produced in human leukocytes and secreted in the urine. Development of a quantitative diagnostic immunoassay for NGAL in urine prompts an understanding of the native protein analyte and its structural similarity to a biosynthetic recombinant protein used to calibrate the assay. Here we present a comparative analysis of human NGAL protein isolated from both leukocytes and urine sources. Multiple analytical techniques were applied to assess differences in protein charge, oligomeric state, molecular mass and post‐translational modifications. Affinity‐purified NGAL protein from both human leukocytes and a human urine specimen pool was also found to display significant visible spectrum absorbance indicative of an iron siderophore‐like substrate as speculated by previous work. This apparent substrate is shown to be tightly bound to the protein, as the protein retains the visible spectrum absorptivity through HPLC chromatographic separation. The purified protein also was found to contain a significant amount of iron by ICP‐MS. Implications from the observed structural differences and substrate characteristics on the protein function and behavior in clinical immunoassays are discussed.
Identification of novel long non‐coding RNA candidates in male germ cell development
Authors: LEE TIN‐LAP , Xiao Amy , Chan Wai‐Yee , Rennert Owen M
Published: June 2021

Male factor accounts for at least 40% of infertility cases. A large number of cases are attributed to a failure of spermatogenesis, a complex developmental process that gives rise to mature spermatozoa. Despite advances in genomics and molecular biology, previous attempts to delineate the developmental programs in spermatogenesis have had limited successes. This is largely because the focus is limited to protein‐coding genes. A significant portion of genome actually encodes many transcripts known as non‐coding RNAs (ncRNAs) of unknown functions. Long ncRNAs (lncRNAs) are generally classified as ncRNAs with more than 200 base pairs. They have been recently demonstrated to involve in developmental regulations in stem cell, brain and cancer. The lncRNA candidates and its functional importance in male germ cell development are not known. Based on our previous established Serial Analysis of Gene Expression (SAGE) libraries for male germ cell development, we have identified a list of lncRNA candidates that demonstrate specific expression in the major developmental stages, including type A spermatogonia, pachytene spermatocytes and round spermatids. The data provides a new and substantially different approach in addressing novel developmental mechanisms in spermatogenesis.
Purine biosynthesis and the case of the “missing” genes
Authors: Sarisky Catherine Ann , Smee Kevin M , Brown Anne M
Published: June 2021

This work is a survey of known and suspected purine biosynthesis genes in sequenced archaea. We searched existing sequence databases for genes for the 11 steps (17 distinct genes) of de novo purine biosynthesis in 65 sequenced archaea, and carefully inspected each hit for the presence of active site residues and other residues known or suspected to be required for function. We found a number of problems with existing annotations, such as missing active site residues, fusions, and incorrect initiation sites. There is also evidence that the pathway has been shaped by horizontal gene transfer, duplication, and gene loss. Two groups of archaea lack a gene normally required for purine biosynthesis, suggesting that there are at least two more variants in this pathway to discover. We propose the existence of a gene encoding a GAR transformylase enzyme that is structurally unrelated to either known GAR transformylase, expected to be present in seven of the 65 archaea studied. A second proposed gene encodes a novel IMP cyclohydrolase function and is expected to be present in the Crenarchaea and Pyrococci.

Functional characterization of some purine biosynthesis enzymes will also be reported.
Structural studies on P6 protein suggest it may not be a good vaccine candidate against Nontypable Haemophilus Influenzae
Authors: Snyder Joy , Kalmeta Breanna , Sharma Sharad , Pichichero Michael , Michel Lea Vacca
Published: June 2021

Nontypable Haemophilus influenzae (NTHi) is the cause of several serious diseases, including pneumonia and otitis media; thus, the development of a vaccine against NTHi would be highly beneficial. The outer membrane protein P6 is currently a top vaccine candidate for NTHi. Past studies have demonstrated that P6 can interact with molecules both inside AND outside of the cell, but its structure suggests that it does not cross the outer cellular membrane. This apparent contradiction points to the urgent need for further studies on P6 in order to better understand its structure and membrane orientation. The overall goal of this project was to determine if P6 is surface exposed and able to interact with extracellular antibodies. To accomplish our goal, we utilized recombinant DNA technologies, ELISA experiments and flow cytometry to examine wild‐type NTHi, a P6 knock‐out and a P6 single‐mutant. The results of our preliminary experiments suggest that P6 monoclonal antibodies may be promiscuously binding to proteins other than P6. Further studies on P6's orientation in NTHi are imperative for future vaccine development. This study was funded by NIH NIDCD RO1 08671 and the Rochester Institute of Technology.
Enzyme Kinetics of Native and Recombinant Secondary Alcohol Dehydrogenase originated from Micrococcus leuteus WIUJH20
Authors: Huang Jenq‐Kuen , Youngblut Nick D , Shoger Nicholas J , Takkalpalli Vamshi , Lin Chi‐Tsai , Wen Lisa
Published: June 2021

A novel secondary alcohol dehydrogenase (2°‐ADH) from M. luteus WIUJH20 has been characterized and its gene cloned & expressed in this lab previously. This 2°‐ADH belongs to NAD+‐dependent short‐chain L‐3‐hydroxyacyl‐CoA dehydrogenase (SCADH) based on amino acid sequence homology. The native and recombinant 2°‐ADH have been purified to homogeneity. Both forms of the enzyme were soluble and were stable at −20°C which facilitated our kinetic studies toward various substrates. Our results showed that the 2°‐ADH has low substrate specificity: it catalyzed either the hydroxyl fatty acids or straight chain aliphatic secondary alcohols with hydroxyl group in either D or L form on a number of carbon positions; hydroxyl fatty esters were poor substrates; and cyclic aliphatic secondary alcohols and hydroxyacyl‐CoA were not substrates for both forms of 2°‐ADHs. To address the substrate specificities between the 2°‐ADH and SCADH, we have created three amino acids substitution mutants by site‐directed mutagenesis. The kinetic properties of the mutant proteins toward various substrates are compared to that of the wild‐type 2°‐ADH.

This research is supported in part by Western Illinois University (WIU) Foundation and the College of Arts and Sciences.
Biochemical characterization of an N‐terminally histidine tagged styrene oxide isomerase from Pseudonomas putida (S12)
Authors: Liao Sindy , Gassner George
Published: June 2021

In the first step of the styrene catabolic and detoxification pathway styrene is transformed to styrene oxide, a biochemical alkylating agent and carcinogen. In Pseudomonas putida (S12), Styrene Oxide Isomerase (SOI) performs the critical role of catalyzing the isomerization of styrene oxide to phenylacetaldehyde, a less toxic intermediate. The detailed kinetic mechanism of SOI is a subject of current investigation. Conditions have been identified that allow an N‐terminally histidine‐tagged version of Styrene Oxide Isomerase (N‐SOI) to be over‐expressed and partially purified by Ni2+‐affinity chromatography in a catalytically active form for biochemical characterization. An enzyme‐coupled assay with phenylacetaldehyde dehydrogenase was developed to indirectly monitor the activity of N‐SOI and kinetically characterize the enzyme by using a microplate reader. By using this assay steady‐state kinetic parameters of N‐SOI with (S)‐styrene oxide were determined to be Vmax = (4.6 ± 0.5) uM/min and KM = (1.6 ± 0.7) uM at pH 8 and 25°C. The substrate specificity and catalytic role of protein‐protein interactions of N‐SOI with the other components of the styrene degradation pathway will be reported.
Exploring the Mechanism of Acyl Carrier Protein Interactions and Acyl Transfer
Authors: Murphy Peter W , Volkman Gerrit , Rowland Elden E , Liu Xiang‐Qin , Byers David M
Published: June 2021

As an essential protein in bacterial fatty acid synthesis, acyl carrier protein (ACP) is an attractive anti‐microbial drug target. The three‐dimensional structure of ACP is comprised of three main parallel α–helices, which create a hydrophobic binding pocket that encloses a fatty acyl chain when attached. ACP interacts with most partner enzymes through the acidic helix II, but the exact mechanism of interaction and acyl transfer to a partner enzyme is poorly understood. For acyl transfer to occur, we hypothesize that a conformational change is required. To examine this requirement, we have analyzed a cyclic version of Vibrio harveyi ACP created by split‐intein technology (Volkmann, et al. (2010) JBC 285: 8605) using molecular dynamics, mass spectrometry, tryptophan fluorescence, and other biophysical techniques. We have demonstrated that cyclic ACP is more thermodynamically stable than wild‐type linear ACP. Furthermore, we show that cyclic ACP is able to act as a substrate for holo‐ACP synthase, yielding a cyclic ACP capable of carrying fatty acids. (Supported by NSERC and an NSHRF Student Research Award)
Peroxiredoxin 6 targeting to lysosomal organelles requires MAPK activity and binding to a chaperone protein 14‐3‐3 epsilon in lung epithelial cells
Authors: Sorokina Elena , Feinstein Sheldon , Fisher Aron B.
Published: June 2021

Peroxiredoxin 6 (Prdx6), a bifunctional protein with both GSH peroxidase and phospholipase A2 (aiPLA2) activities, has been localized to cytosolic and acidic compartments (lamellar bodies and lysosomes) in lung alveolar epithelium. We propose that Prdx6 targeting to lysosomal organelles favors its PLA2 activity which requires an acidic pH. We now show that ERK and p38 MAPK regulate Prdx6 targeting to lysosomal organelles in MLE12 and A549 lung epithelial cell lines. Co‐localization of Prdx6 with lysosomal marker LAMP1 showed a decrease of Prdx6 lysosomal targeting in cells treated with ERK or p38 MAPK inhibitors, or an inhibitor of PKC, an upstream kinase activator of MAPKs. Immunocytochemistry, immunoprecipitation followed by PAGE, and an in situ proximity ligation assay (Duolink) showed interaction between Prdx6 and 14‐3‐3ε, a member of the 14‐3‐3 chaperone system which is known to facilitate transport of signaling proteins in the early secretory pathway. Moreover, inhibition of PKC, ERK or p38 abolished the interaction between Prdx6 and 14‐3‐3ε. This interaction is required for Prdx6 translocation along the exocytotic pathway resulting in its sub‐cellular localization to acidic organelles and consequent activation as a PLA2. These findings suggest that ERK and p38 MAPK regulate Prdx6 targeting to lysosomal organelles by activation of 14‐3‐3ε, as a chaperone protein. [HL019737]
Alpha‐glucogenic activity of mammalian mucosal enzymes on different disaccharides
Authors: Lee Byung‐Hoo , Quexada‐Calvillo Roberto , Nichols Buford , Rose David R. , Hamaker Bruce R.
Published: June 2021

Small intestine mucosal maltase‐glucoamylase (MGAM) and sucrase‐isomaltase (SI) have two subunits each (N‐ and C‐ terminal) that are involved in the digestion of glycemic carbohydrates to glucose and fructose from diets. In this study, recombinant MGAM and SI enzymes were reacted with disaccharides, namely kojibiose, nigerose, maltose, and isomaltose each containing two glucose molecules as well as sucrose, but with different á‐linkages, to elucidate their digestion abilities. Reactions were carried at 37°C in 10 mM PBS and one unit (U) enzyme activity was defined as 1 μg of glucose released from maltose per 10 min. After enzyme reactions, released glucose was measured by the GOPOD method. Notably, each of the four subunits had á‐(1, 2), (1, 3) and (1, 4) hydrolysis activities, but N‐SI at 100 U was the only one to have á‐(1, 6) hydrolysis activity. Moreover, every subunit had á‐(1, 6) hydrolysis activity at higher enzyme concentration (above 2500 U of subunits except N‐SI). Each subunit had á‐glucosidase activity on different glycosidic linkages though maltase activity was at least 25 times greater than isomaltase activity. Also, only C‐SI and C‐MGAM showed sucrase activity with relatively lower than maltase activity. This is the first report of á‐glucogenic activity of disaccharides for each mucosal enzyme subunit and provides a new view of carbohydrate digestion model in the small intestine.

Grant Funding Source: CNRC/ARS/USDA
A potential control point of glucose delivery from starchy foods: intestinal mucosal α‐glucosidase digestion
Authors: Lin Amy Hui‐Mei , Nichols B. L. , Quezada‐Calvillo R. , Rose D. , Hamaker B. R.
Published: June 2021

To control the glucose delivery from starchy foods, much attention has been given to α‐amylase (AMY), as opposed to the contribution from mucosal α‐glucosidases. The aim of this research was to investigate the digestion capability of mucosal α‐glucosidases on various starch structures to explore their possible expanded roles in digestion. Substrates were incubated with recombinant N‐ (Nt) and C‐terminals (Ct) of maltase‐glucoamylase (MGAM) and sucrase‐isomaltase (SI) for different digestion periods, and the glucogenesis and residue structures were examined. At raw starch granule level, mucosal α‐glucosidases showed the capability to produce glucose from uncooked starch granules at a low digestion completion. At the cooked starch level, CtMGAM showed high digestion completion near 80% without AMY participation; other three subunits reached 20–30% digestion. At the α‐limit dextrin (LDx) level, the four subunits showed different roles where NtMGAM only digested short linear oligomers but the other three subunits individually digested large branched molecules with different completion percentage. Collectively, these findings suggest mucosal α‐glucosidases, particularly CtMGAM, may be potential control points for glucose delivery in the human body. This work was supported by internal funding from the Whistler Center and Children's Nutrition Research Center of the ARS/USDA.
Roles of G proteins in Human Breast Cell Invasion
Authors: Moon Aree , Kim Eun‐Sook , Kim Jong‐Sook , Jeong Jae‐Boon , Kim Seonhoe , Lee Kyung‐Min , Ko Eunyoung , Noh Dong‐Young , Hwang Ki‐Tae , Ha Ji Hee , Lee Chang Ho , Hwang Sejin , Kim Sang Geon
Published: June 2021

Although mounting evidence suggests a role for G12 proteins, Gα12 and Gα13, in tumor progression, a direct role of G12 proteins has not been determined. This study aims to elucidate the molecular mechanism for tumorigenic/invasive potential of Gα12/13 in MCF10A human breast epithelial cells. Here we report, for the first time, that Gα12/13 induce upregulation of matrix metalloproteinase(MMP)‐2 leading to the invasive/migratory phenotypes in MCF10A cells. We further show that p53 is an important transcription factor for induction of MMP‐2 transcriptional activation by Gα12/13. Using human breast tissue samples, we demonstrate that the expression levels of Gα12 and MMP‐2 are strongly correlated with the pathogenically diagnosed cancer. To elucidate a causal relationship between inflammation and the control of invasiveness of breast cells, the effect of the inflammatory lipid sphingosine‐1‐phosphate(S1P) on the regulation of invasive/migratory phenotypes of MCF10A cells was investigated. S1P causes induction of MMP‐9 in vitro/in vivo, and enhances invasion/migration which is triggered by S1P3‐Gαq coupling. Activation of PLC‐β4 and intracellular Ca2+ release are required for S1P‐induced MMP‐9 upregulation. Taken together, this study elucidated the role of G12 proteins and S1P in regulating processes for MMP expression and malignant phenotypic conversion of breast cells.
Identification of a novel protein at the lagging edge of migrating cells
Authors: Swaney Kristen F. , Devreotes Peter N.
Published: June 2021

The amoeba Dictyostelium discoideum has been used to elucidate many of the signaling pathways that mediate the directed migration of cells up chemical gradients, or chemotaxis. Studies of Dictyostelium have shown that chemotaxis depends on the localization of signaling components specifically to the leading or lagging edge of cells. Therefore, a screen was conducted to identify novel chemotactic regulators based on protein localization. To do this, Pleckstrin homology (PH) domain‐containing proteins were tagged with GFP, expressed in Dictyostelium cells, and assessed for intracellular localization during migration. Like known PH domain‐containing proteins that bind phosphoinositide‐3,4,5‐trisphosphate, several of the screened fusion proteins localized to membrane protrusions at the leading edge. However, one protein, PH21, was found at the lagging edge. Null and overexpression mutants were generated to assess the function of this novel lagging‐edge protein in the regulation of chemotaxis. So far, it appears that PH21 plays a role in polarity, an important component of normal chemotactic behavior. By identifying new proteins at both the leading and lagging edges of cells, several potential mediators of chemotaxis have been isolated, and the function of these proteins is currently being elucidated. Funding was provided by the American Heart Association to KFS and by NIH grants GM34933 and GM28007 to PND.