bluuebunny/crossref_metadata_2025 huggingface dataset:
Hypoxia modulates lipopolysaccharide (LPS)‐induced fractalkine (CX3CL1) production by human trophoblast
Authors: Szukiewicz Dariusz , Mittal Tarun Kumar
Published: June 2021

The chemokine CX3CL1 and its receptor CX3CR1 are expressed in human trophoblast. Autoregulatory interactions between CX3CL1 and CX3CR1 are crucial for adhesive properties of trophoblastic cells. Low oxygen environment is physiological for trophoblast differentiation and regulates its invasiveness. We examined influence of hypoxia on LPS‐induced CX3CL1 production in relation to the CX3CR1 status. Placental samples were obtained after spontaneous abortions between 10–12 weeks of gestation. The cytotrophoblast cells were isolated using Kliman's method based on trypsin, DNase and a 5–70% Percoll gradient centrifugation, and cultured in vitro. After 3 days the cultures were kept under hypoxia (2% O2) for 48 hours (Group I; N=18) or cultured under normoxia (Group II; N=18), and then LPS (1 microg/ml) was added to the culture media with/without pretreatment with anti‐CX3CR1 antibody. Concentrations of CX3CL1 in culture media were estimated in consecutive days using ELISA. Hypoxia produced significant (p<0.05) but transient decrease in CX3CL1 production in response to LPS concentration. Blockade of CX3CR1 produced irreversible lowering of CX3CL1 concentration, more evident in Group II (a mean 4.4‐fold decrease vs. 2.1‐fold in Group I). The results may support standpoint that both oxygen tension and CXCR1 status may modulate human trophoblast invasion, influencing the course of pregnancy.
Dietary fat‐induced release of diamine oxidase into intestinal lymph is mediated by histamine H4 receptor
Authors: SAKATA YASUHISA , Ji Yong , Yang Qing , Tso Patrick
Published: June 2021

The release of histamine‐degrading enzyme diamine oxidase (DAO) into intestinal lymph is enhanced by the ingestion of nutrients, especially fat. Fat absorption also stimulates the release of histamine into lymph. We have previously reported that histamine H1 but not H2 or H3 receptor antagonist weakly inhibited lipid‐induced DAO release. The aim of this study was to determine the effect of histamine H4 receptor antagonist on the release of DAO by lipid ingestion. Intestinal lymph fistula rats (n=5) received a Liposyn bolus (4.4 kcal/3 ml) through the intraduodenal tube. The H4 receptor antagonist JNJ7777120 (37 mg/kg BW) was injected intraperitoneally 30 min before lipid infusion. Lymph was collected in cold and lymphatic DAO activity was measured by a radiometric assay. DAO protein was detected by western blot. JNJ7777120 treatment did not completely inhibit the release of DAO, but significantly reduced the peak of DAO activity (54.7±5.6 mU/ml) compared to controls (137.8±28.8 mU/ml, P=0.037). The DAO cumulative output in 1.5 hours was significantly decreased in JNJ7777120 treated rats (1.4±0.4 mg) compared to controls (4.0±0.9 mg, P=0.040). The activity data agree with the Western blot data which showed that JNJ7777120 treatment markedly decreased amounts of DAO in lymph. We conclude that lipid‐induced DAO release into lymph is mediated by histamine H4 receptor.

Grant Support: NIH/NIDDK DK059360, DK092138
Feedbacks In Sonic Hedgehog Circuits With Cytokines In H. pylori Mediated Gastritis
Authors: Marwaha Shruti , Schumacher Michael , Zavros Yana , Eghbalnia Hamid
Published: June 2021

Deregulation of the Sonic Hedgehog (SHH) pathway, a critical player in normal development, has been shown to be associated with gastrointestinal abnormalities and cancers. However, the underlying mechanism remains to be fully understood. Computational models are excellent tools for investigating the complex circuitry, crosstalk, and the dynamic behavior of the associated signaling pathways. However, appropriately detailed models are lacking. Using qrt‐PCR data, algebraic methods and pathway mining, we present a model that elucidates the interactions involving SHH and cytokines activated by Helicobacter pylori, the most common pathogen associated with gastric inflammation and gastric malignancy. Focusing on the relationships between SHH and key cytokines (IL1β, IL‐12, IFNγ and IL‐10) activated by H. pylori, we describe a kinetic model that examines the dynamic regulation of these cytokines by SHH during H. pylori infection. Our mathematical model predicts that SHH, IL‐1β, NFkB and IL‐10 engage in series of feedback loops that exhibit a chronic oscillatory behavior. Predictions from our model provide useful insights for designing more targeted in vivo and in vitro experiments.
Gender‐Specific Pulmonary Hypertension and Altered Skeletal Muscle Function in Lung‐Specific TNFα Overexpressing Mice
Authors: Tang Kechun , Gu Yusu , Dalton Nancy , Peterson Kirk , Wagner Peter D. , Breen Ellen C.
Published: June 2021

Chronic expression of pulmonary TNFα results in inflammation, emphysema, pulmonary hypertension and muscle wasting. This study investigates the gender‐specific effects of pulmonary TNFα expression on lung and skeletal muscle function. SP‐C regulated expression of TNFα results in a greater relative TNFα increase in male compared to female mice (lung: female 29 fold, male 74 fold, serum: female 4.6 fold, male 8.0 fold). Airspace size was increased by TNFα in both genders (male: WT 35.1 μm, TNFα 82.9 μm, female: WT 35.1 μm, TNFα 72.7 μm). Increased right ventricular pressure, implying pulmonary hypertension, was present in male SP‐C/TNFα mice. (Male: WT 27.7±6.99 mmHg, TNFα 38.8±8.03 mmHg, p=0.01, Female: 21.6±2.89 mmHg, TNFα 25.7.8±12.39 mmHg). Muscle fatigue, measured in isolated soleus and EDL, was more rapid in male SP‐C/TNFα soleus (WT 431±50 sec, TNFα 361±45 sec, p=0.03). Citrate synthase (CS) and β‐HAD enzyme activities were selectively decreased in male SP‐C/TNFα soleus (CS: WT 3.83±0.58U, TNFα 2.82±0.73U, p=0.02, β‐HAD: WT 1.22±0.22U, TNFα 0.93±0.21U, p=0.045). These pulmonary and skeletal muscle changes may contribute to the weight loss, muscle atrophy and impaired endurance observed in male but not female SP‐C/TNFα mice, and suggests female mice are partially protected from overexpression of this inflammatory cytokine.
Can a Single Anthropometric Feature of the Lumbar Vertebra Result in a Correct Identification of Sex?
Authors: Jelaca-Tavakoli Maria
Published: June 2021

This blind anthropometric study demonstrates that sexual dimorphism in the fourth (L4) and fifth (L5) human lumbar vertebra exists and that it can be used for the estimation of sex. The Maximum Lumbar Vertebral Body Diameter (MBD) from a total of 1686 vertebra from five different skeletal collections are included in the total sample studied. The vertebral standards were developed first from the MBD of 1175 vertebra (study sample) of which 574 vertebral measurements were collected from South African and 601 from North American Osteological Collections. The remaining 511 lumbar vertebra (test sample) were collected from the Terry (306) and Maxwell (205) Collections. The sex was estimated as vertebral MBD from the test sample were measured and the collected measurements compared against the standards derived from the study sample. The estimates of sex from the L4 MBD resulted in 80% and from the L5 in 86% accuracy, suggesting that L5 got slightly higher sexing potential. The fact that ethnic background, age or stature played no role in the estimates and also that durability of the lower lumbar spine and its sexually dimorphic anthropometric attributes resulted in notably high sex estimation accuracy levels demands further attention.
Bilateral Directional Assymetry in the Human Clavicle
Authors: Fatah Emam ElHak Abdel , Shirley Natalie R. , Auerbach Benjamin , Mahfouz Mohamed
Published: June 2021

This study investigates bilateral directional asymmetry (BDA) in the human clavicle using the McCormick Collection, a documented autopsy sample from 1986–98 autopsies in East Tennessee (n=1125). BDA has been examined extensively in the human skeleton, especially limb bones. Most of these studies rely on comparisons of metric measurements typically used in skeletal analyses. While most of these measurements are thought to reflect mechanically relevant morphologies, these dimensions may not reflect the most morphologically asymmetrical areas. In an effort to holistically address BDA, this study examines standard metric measurements on the entire sample, conducts a curvature analysis of the medial axis on a subset (n=33) of 3D models developed from CT scans using statistical atlases, and examines the average surface‐to‐surface differences of this subset of modeled clavicles. In males and females, mean length of the left clavicle is significantly greater than the right (α=.05). Furthermore, maximum midshaft diameter is significantly greater in the right clavicle than in the left. All p‐values are more significant in males than in females. Shape analyses indicate that magnitudes of greatest difference between the left and right surface models occur at muscle attachment sites. These preliminary results point to a strong biomechanical influence on BDA in the human clavicle in both sexes, particularly males.
Exploring the Anatomical Spatial Relationships affecting the Anterior and Posterior Ethmoid Foramina (AEF & PEF) using 3‐D Geometric Morphometric Technique
Authors: Fisher Juliya , Masters Alina , Golubovskaya Yevgeniya , Pagano Anthony S. , Caceres Noel , Marquez Samuel
Published: June 2021

Close relationships between ethmoid and maxillary sinuses, and orbital cavities have long challenged ENT surgeons. Identification of ethmoid vasculature (EV) in or adjacent to the frontal ethmoid suture line is critical in preventing intracranial entry and orbital hemorrhage during surgery. This study investigated influence of body size on spatial relationships among anterior ethmoid foramen (AEF) and posterior ethmoid foramen (PEF; which transmit arterial branches to ethmoid sinuses and meninges) and posterosuperior‐most point on the maxillary sinus roof (MSR). Ten hemi‐heads were harvested from donor bodies at SUNY Downstate. Each specimen was dissected to expose orbital cavity boundaries, infraorbital foramen, MSR, and zygomaticomaxillary suture. Landmark coordinate data were collected using a Microscribe digitizer & scaled (Procrustes scaling). Centroid size (sum of squared distances from each landmark to centroid) was used for size standardization. Results showed strong & negative correlations (p<0.05) between centroid size & distance between 1) AEF & PEF, 2) points on the orbital rim and the AEF and PEF, and 3) lateral‐most point on the orbital rim & MSR. Orbital width & height were similarly correlated with size (p<0.05) while no significance was revealed in MSR distance from the medial orbital wall. Orbital size appears to be the determining factor in changing relative positions of AEF & PEF.

Grant Funding Source : SUNY Downstate Medical Center
Tracking branching variation of the axillary artery
Authors: Hartley Benjamin Rush , Marquez Samuel
Published: June 2021

The axillary artery is divided into three parts for describing branching location. This study quantifies atypical branching in the third part, which usually gives rise to the subscapular artery and anterior and posterior circumflex humeral (CH) arteries. Rarely, the third part yields the profunda brachii artery (PBa). Tracking these branching patterns is important in accurate execution of diagnostic and surgical procedures. Further, as more arteries arise from a common trunk, more structures are at risk for ischemia via vascular insult. Axillary arterial branching was examined in 24 donors, yielding a sample size of 48 configurations. Five types of branching patterns were catalogued: Type 1 being the most common and Type 5 being the least. Pattern frequency was noted as follows: Type 1 = .563; Type 2 = .146; Type 3 = .125; Type 4 = .104; Type 5 = .063. Interestingly, the traditional “textbook” branching pattern (Type 1) was only observed in 56% percent of the sample. An unusual pattern where the PBa and both CH arteries originate from the subscapular artery was observed in over 6% of cases. As the PBa is the single source for collateral circulation of the antebrachium, disturbance to its integrity in these peculiar vascular arrangements, could have profound effects on clinical outcomes.

Grant Funding Source: SUNY Downstate College of Medicine
The Japanese macaque is an endemic species consisting of two subspecies: Macaca fuscata fuscata and Macaca fuscata yakui
Authors: Sato Iwao , Miwa Yoko , Sunohara Masataka , Mine Kazuharu , Shimada Kazuyuki
Published: June 2021

The Macaca fuscata yakui (MFY) is indigenous to Yakushima Island and is a subspecies of Macaca fuscata fuscata (MFF), which lives from Honshu to Shikoku and Kyushu, Japan. However the differences in the skull of the MFY and MFF are unknown, despite the different skull sizes. The infraorbital canal (IOC) indicates that the feature of the frontal view reflects the transversal growth of the maxilla of the skull. In this study, we show the IOC structure of the MFF and MFY using a cone‐beam computed tomography apparatus. These specimens are stored in the osteological collections of the Department of Neurology, Gross Anatomy Section, Kagoshima University Graduate School of Medical and Dental Sciences (Japan). The maximum height of the IOC, the maximum height from the palatine to the inferior border of the center orbital foramen, and the volume of the IOC of the MFF were significantly greater than those of the MFY (p<0.05). We also classified four types of IOC 3D images, a tube‐like shape, funnel shape, pinched shape, and double‐canal type, in these Japanese macaques. However, a double‐canal type separated into two lumens was found in the five cases of MFY (31.3%, 5/16)). These results suggest that the lower face of MFY with a shorter face indicates a need to prepare for a hard diet compared with the MFF and is related to the food preferences between MFF and MYF.
Comparative functional morphology of the avian integument: Implications for the evolutionary history of feathers
Authors: Orellana Elise R. , Dubansky Brooke H. , Homberger Dominique G.
Published: June 2021

The integuments of a turkey (Meleagris gallopovo) and an Amazon parrot (Amazona ventralis) differ in the arrangement of their feather muscles, which raise or depress contour feathers; the relative size of their feather tracts and apteria; and the type, distribution, and muscle attachments of their downy feathers. Common aspects, however, include smooth dermal feather and apterial muscles; dermal and subcutaneous fat layers that serve as a hydraulic skeleton for the feather follicles; and an elastic membrane that separates the fat layers and is responsible for returning the raised feathers to their resting position. Similarly, smooth dermal muscles, dermo‐subcutaneous fat tissue, and elastic membranes have been described for the integument of squamate reptiles that have imbricating scales. In contrast, the integument of crocodilian reptiles, which have plate‐like, non‐imbricating scales like dinosaurs, has lost the smooth dermal muscles and has reduced its dermo‐subcutaneous fat, but has retained an elastic layer that separates the dermis from the subcutis and is responsible for returning the stretched skin to its resting position. The comparison of avian and reptilian skin morphology illustrates an example of mosaic evolution and allows the reconstruction of the evolutionary history of the avian integument from that of a lizard‐like ancestor with imbricating scales. Funding: LSU Foundation to DGH

Grant Funding Source: N.A.
The expansion and recoil mechanism of the skin of alligators and its implications for their feeding mechanism
Authors: Dubansky Brooke H , Homberger Dominique G
Published: June 2021

Alligators are said to swallow large food items, but the assumption of an expandable throat skin has not been tested. Alligator skin can expand only through its soft‐cornified interscale skin segments. In the relaxed skin, collagenous skin ligaments in the dermis extend radially to attach to the underside of the rigid, hard‐cornified scales, which are slightly raised above the folded interscale skin. In the stretched skin, the skin ligaments assume a more circumferential orientation, thereby pulling down the scales and allowing the interscale skin to expand, while the Stratum elasticum between the dermis and subcutis is stretched. When the stretched skin is released, the Stratum elasticum returns to its resting length, thereby pushing the scales back to their raised position, allowing the interscale skin to refold, and returning the skin ligaments to their resting configuration. Maximum expansibility of the skin was found in the intermandibular region, which allows the manipulation of large food items in the oral cavity. A lack of circumferential expansibility was found in the cervical region, which limits the size of food items that can pass through the throat before they reach the narrow Isthmus thoracis. Hence, our anatomical analysis, together with observations of feeding alligators, shows that alligators do not swallow food items that are large relative to their neck size. Funding: LSU Foundation to DGH

Grant Funding Source : NA
Expression of thyroid hormone receptor in tooth germ of newt (Cynops pyrrhogaster)
Authors: MIWA Yoko , SHIMADA Kazuyuki , SATO Iwao
Published: June 2021

The jaw of Japanese newts Cynops pyrrhogaster could be fully reproduced following amputation including tooth germ. Cynops pyrrhogaster are polyphyodont and series of teeth germ have developed in their jaws. We hypothesize that the sequential developed tooth germ are induced by Thyroid hormone and its receptor network. We examined Thyroid hormone receptor(THR) gene expression in Cynops pyrrhogaster tooth from gdentine matrix formation and mineralization stage h to genameloid maturation and enamel‐like matrix formation stage h by in situ hybridization histochemistry. We demonstrated THR mRNA to be localized predominantly in the coronal side of odontoblasts at gdentine matrix formation and mineralization stage h. Histochemical analyses revealed the THR expression in odontoblasts and surrounding tissues were dependent on the stage of tooth development. This study was supported by Young Scientists Grant No. (B) 23792117 from the Ministry of Education, Culture, Sports, Science, and Technology, of Japan.
Convergence of the MicroRNA and NMD Pathways in Neurons
Authors: Shum Eleen Y , Lou Chih H , Wilkinson Miles F
Published: June 2021

Nonsense‐mediated RNA decay (NMD) was originally identified to degrade “nonsense” mRNAs, but since then it has become clear that it also regulates ~10% of wild‐type transcripts. This regulatory role suggests that NMD itself is subject to regulation. Indeed, we recently identified a conserved regulatory circuit in mature neurons that regulates NMD through the ability of the microRNA miR‐128 to repress the NMD factors UPF1 and MLN51. Here, we report the identification of another microRNA miR‐9, which is expressed during neurogenesis, that regulates NMD by targeting UPF3B. Loss of UPF3B was recently shown to cause intellectual disability (ID) in humans. First, using reporter constructs, we obtained both gain‐and loss‐of‐function evidence that the UPF3B 3′UTR is regulated by miR‐9. Second, we found that endogenous UPF3B transcripts in cell lines are regulated by miR‐9. This miR‐9‐UPF3B circuit is likely to be physiologically relevant, as we observed that miR‐9 and UPF3B are colocalized in neurons in many regions of the mouse brain. We propose that the regulation of UPF3B by miR‐9 is important in neurogenesis. This may be disrupted in several human diseases, as mutations in human UPF3B are found not only in ID patients, but those with psychological disorders, including autism and schizophrenia.

Research Supported by NIH
Analysis of Pax3 phosphorylation in regulating DNA binding in early myogenic differentiation
Authors: Hollenbach Andrew D , Loupe Jacob M , Iyengar Aditi S , Miller Patrick J
Published: June 2021

Pax3, a member of the paired‐class homeodomain family of transcription factors, is essential for regulating early myogenic differentiation. In undifferentiated cells it regulates the expression of genes that promote proliferation while simultaneously inhibiting differentiation. Upon receiving signals to differentiate, Pax3 then promotes the expression of downstream myogenic factors. Literature evidence suggests that phosphorylation of Pax3 contributes to its biological activities. Consistent with this fact we have identified three sites of phosphorylation on Pax3 (Serines 201, 205, and 209). In the present work we examine the effects of phosphorylation on the ability of Pax3 to bind to DNA and activate transcription. We demonstrate that phosphorylation of Pax3 at specific sites inhibits the ability of Pax3 to bind to DNA in vitro and alters its ability to interact with the co‐repressor hDaxx. Further, we provide evidence to suggest that the decision to enter differentiation occurs in the G1 phase of the cell cycle, for which we initiated studies to examine cell cycle‐dependent changes of Pax3 phosphorylation. Taken together, these results allow us to propose a working model to describe how phosphorylation regulates Pax3 biological activity in early myogenesis. (Funding: NCI R01 CA138656)
The N‐terminal Primase‐like Domain of the Drosophila Mitochondrial Replicative DNA Helicase Binds an Iron Sulfur Cluster: Structural and Functional Implications
Authors: Farnum Gregory A , Kaguni Laurie S
Published: June 2021

The N‐terminal region of the metazoan mitochondrial replicative DNA helicase (mtDNA helicase) shares extensive sequence conservation with the primase domain of bacteriophage T7 primase‐helicase. Primase activity has not been demonstrated in metazoan mtDNA helicases, most likely because several Mg2+‐binding residues of the primase active site in T7 primase are not conserved in Metazoa, although several conserved primase sequence motifs are well conserved. Interestingly, insects have retained the four cysteines of conserved motif I that have been shown to bind zinc in the T7 enzyme. Therefore, to study the function of the N‐terminal domain of metazoan mtDNA helicase, we designed two N‐terminal constructs of the Drosophila melanogaster protein comprising residues N24‐A333 (NTD) and N24‐P123 (ZBD). Purified fractions of the full N‐terminal domain construct (NTD) contain an iron sulfur cluster, as determined by UV‐Visible spectroscopy, as well as by iron and sulfide determination. Based on T7 homology, the NTD contains the conserved zinc binding (ZBD) and RNA polymerase (RPD) domains. Purified fractions of the ZBD construct also contained an Fe‐S cluster, suggesting the zinc‐binding site has been converted to an Fe‐S binding site in the Dm mtDNA helicase. We will present data using differential scanning fluorimetry, fluorescence resonance energy transfer and differential scanning calorimetry regarding the involvement of the Fe‐S cluster in the structural stability and in nucleic acid ligand binding by the NTD form.
Characterization of a MarR Homolog in Vibrio vulnificus
Authors: Lemon Tiffany , Grove Anne
Published: June 2021

A negative repressible operon in the genome of Vibrio vulnificus codes for a MarR family transcriptional regulator and an antibacterial efflux pump. These divergent genes are regulated by the MarR homolog, which binds to the region between the genes. The MarR homolog of V. vulnificus is predicted to display ligand‐responsiveness— specifically to urate—based on key residue conservation with HucR. HucR is a MarR homolog of Deinococcus radiodurans with a proposed mechanism for the attenuation of DNA binding by urate. We have expressed and isolated the V. vulnificus homolog using recombinant DNA techniques, and EMSA confirms the formation of protein‐DNA complexes. Now, we test urate along with other possible ligands for the attenuation DNA binding. Identifying the natural ligand of V. vulnificus's MarR homolog will stand to have major impacts on health as the unexpressed efflux pump will not impair antibacterial treatment of this species.
Rapid bacterial DNA extraction from stool samples
Authors: Duggan Megan , Parrish Torrey , Williams Patrick
Published: June 2021

Stool samples are a common specimen used in a diagnostic setting. Due to the nature of stool samples, which have a large number of PCR inhibitors, extracting PCR‐ready DNA can be difficult. We have developed a rapid, simple protocol using Evogen ONE chemistry to extract DNA from stools. In this protocol, either 100ul of liquid stool was pipetted or a sterile swab was immersed in formed stool to gather sample which was washed with phosphate‐buffered saline. The supernatant was removed. 250ul of Evogen ONE was added to the sample, vortexed and then incubated for 5 minutes at 95°C. Then the sample was centrifuged and the lysate was removed. 1ul of the lysate containing the DNA was used in the following PCR reactions. A universal bacterial primer set was used with SYBR Green (Bio‐Rad) to show a wide variety of bacterial DNA present. Stool samples that were initially spiked with Salmonella enterica to show specific target recovery were amplified with the Salmonella ttr primer set and a ttr specific HyBeacons probe. All DNA extracts demonstrated amplification products. DNA concentration was measured on a NanoDrop (ThermoFisher). The crossing threshold (Ct) for the generic bacterial primer set was on average 13 cycles, and the Ct for Salmonella‐spiked stools was 27. DNA concentration varied from 150 to 600ng/ul which is typical of the sample type. We have shown that the above protocol is fast, easy to use, and produces high‐quality DNA.
Reduced tumor load and metastasis in vivo by targeting a metalloprotease‐disintegrin, ADAM8, in highly invasive adenocarcinomas
Authors: Schlomann Uwe , Ferdous Taheera , Golfi Panagiota , Koller Garrit , Parsons Maddy , Höfling Sabrina , Hagemann Thorsten , Bossard Maud , Nimsky Christopher , Bartsch Jörg W
Published: June 2021

ADAM proteases (A Disintegrin And Metalloprotease Domain) are multi domain proteases with functions in tumor cell growth, cell adhesion and ECM remodeling. High expression levels of ADAM8 are correlated with bad patient prognosis in brain, lung, and pancreatic cancer. The objective of this study was to validate ADAM8 as drug target in adenocarcinomas. In pancreatic tumor cells Panc1, ADAM8 overexpression (Panc1/A8) leads to increased cell migration and cleavage of fibronectin and collagen I and IV, causing enhanced invasion into the ECM via ADAM8. In Panc1/A8 cells, ADAM8 interacts with β1 integrin, activating ERK, Akt and p38 pathways. Panc1/A8 cells were grafted orthotopically into pancreas of nude mice. Larger tumors were found from Panc1/A8 cells compared to control cells. More importantly, higher metastatic load from these cells in liver and spleen was observed. As therapy approach, we designed a cyclic peptide (“cycP1”) mimicking the integrin binding loop of ADAM8. CycP1 impedes ADAM8‐β1 integrin interactions causing reduced intracellular signaling. In vivo, cycP1 is very potent in reducing tumor load and formation of metastases compared to untreated mice. Our results suggest that inhibition of ADAM8 in highly invasive adenocarcinomas is feasible for therapeutic intervention and provides a solid basis for further preclinical drug development. Supported by Cancer Research UK.
VEGFR‐1/FLT‐1 is induced by protein kinase C and its Ectodomain Cleavage is regulated by glycosylation and metalloproteases
Authors: Raikwar Nandita S , Liu Kang Z , Thomas Christie P
Published: June 2021

Vascular endothelial growth factor receptor‐1/fms‐related tyrosine kinase 1 (VEGF‐1/FLT‐1) is a membrane bound receptor tyrosine kinase involved in angiogenesis and vasculogenesis. In vascular endothelial cells, the protein kinase C agonist phorbol 12‐myristate 13‐acetate (PMA) stimulates Flt1 expression and promotes the cleavage of Flt1. To explore the regulation of Flt1 biogenesis and cleavage we expressed FLT‐1 with N and C terminal epitopes in HEK 293cells. We observed that Flt1 expression is strongly induced by PMA in a time dependent manner with an N‐terminal soluble fragment in conditioned media and a~60 kDa intracellular C‐terminal fragment. Heparin treatment led to increase in the ectodomain of FLT‐1 in conditioned media suggesting that the cleaved form of Flt1 although extracellular is bound to the cell surface proteoglycans as described for soluble Flt1(sFlt1). Application of pharmacological ER and Golgi stress agents inhibited glycosylation of Flt1 and completely blocked the cleavage and release of the ectodomain of FLT‐1. Co‐expression of the metalloproteases ADAM10 or ADAM17 significantly reduced full length FLT‐1 expression consistent with enhanced cleavage. Together our data suggests that post translational proteolytic cleavage of FLT‐1 releases its extracellular domain in the media and this release is modulated in part by heparin binding, glycosylation and metalloproteases. NIDDK
Amyloid beta oligomers trigger death receptors‐mediated apoptosis in cerebral endothelial cells
Authors: Fossati Silvia , Ghiso Jorge , Rostagno Agueda
Published: June 2021

Vascular deposition of Aβ in sporadic and familial Alzheimer's disease, through poorly understood molecular mechanisms, leads to alterations in cerebral blood flow, focal ischemia, and cerebral micro‐/macro‐hemorrhages, significantly contributing to cognitive impairment. We aimed to determine the molecular mechanisms triggering apoptosis of vessel wall cells in presence of Aβ40 or its vasculotropic variants E22Q or L34V. Challenging human brain microvascular endothelial cells with both variants and wild‐type Aβ40, we showed that TRAIL death receptors DR4 and DR5 specifically mediate oligomeric Aβ induction of extrinsic apoptotic pathways. Caspase‐8 activation preceded activation of caspase‐9. The caspase‐8 inhibitor cFLIP was downregulated, and mitochondrial paths were engaged through BID cleavage. Up‐regulation of DR4 and DR5 and co‐localization with Aβ at the cell membrane suggested their involvement as initiators of the apoptotic machinery. Direct binding assays using receptor chimeras confirmed the specific interaction of oligomeric Aβ with DR4 and DR5 whereas apoptosis protection achieved through RNA silencing of both receptors highlighted their active role in downstream apoptotic pathways unveiling new targets for therapeutic intervention.

Support: NIH AG30539, NS051715, American Heart and Alzheimer's Associations.
Acupuncture ameliorated skeletal muscle atrophy induced by hindlimb suspension and spiral wire immobilization in mice
Authors: Onda Akiko , Jiao Qibin , Minamisawa Susumu , Fukubayashi Toru
Published: June 2021

Loss of skeletal muscle mass has a profound effect on daily life for the elderly and patients with serious diseases. We hypothesized that acupuncture is an alternative strategy for preventing skeletal muscle atrophy. In this study, we investigated the effect of acupuncture on two different mouse models of skeletal muscle atrophy: hindlimb suspension (HS) and spiral wire immobilization (SWI). Acupuncture was performed on the gastrocnemius muscle for 30 minutes every day. Muscle wet weight was significantly reduced by HS for two weeks. The HS‐induced reduction in muscle mass was significantly increased by acupuncture intervention in soleus. The mRNA expression levels of atrogin‐1/MAFbx and MuRF1, which play a principal role in muscle‐specific degradation as E3 ubiquitin ligases, were significantly increased by HS. Those were significantly decreased by acupuncture intervention compared to the HS group. In a protein synthesis pathway, the expression levels of Akt1 and TRPV4 mRNAs were significantly increased by acupuncture compared to the HS group. Muscle wet weight was significantly reduced by SWI for five days. The SWI ‐induced reduction was significantly increased by acupuncture intervention compared to the SWI group in soleus. These results indicate that acupuncture treatment may have an ameliorated effect on skeletal muscle atrophy through both protein synthesis and degradation pathways.
An autoregulatory feedback loop between nuclear receptor SHP and Mdm2 that controls Mdm2, SHP and p53 protein stability
Authors: yang zhihon , zhang yuxia , wang li
Published: June 2021

Methods

Immunoprecipitation, Western blots, in vitro ubiquitination assays, luciferase reporter assays, qPCR, ChIP assays, and apoptosis assays etc.

Results

Mdm2 attenuates SHP expression by promoting SHP protein ubiquitination and downregulating SHP mRNA, and SHP in turn stabilizes Mdm2 protein by abrogating Mdm2 selfubiquitination. p53 reduces SHP protein through proteasome, but not phosphorylation and acetylation, mediated SHP degradation by Mdm2, and inhibits SHP mRNA by transcriptional repression. SHP destabilizes p53 by augmentation of Mdm2 ubiquitin ligase activity toward p53. Such cross‐regulation critically depends on the physical interaction of SHP with Mdm2 through the SHPK170 residue. Consequently, SHP antagonizes p53 dependent induction of apoptosis and inhibition of tumor growth by AHPN and 3‐Cl‐AHPC in colon cancer cells.

Conclusions

The Mdm2‐SHP‐p53 interplay represents a novel component of Mdm2/p53 crosstalk that dictates p53 activity and function.

This work was supported by the National Institutes of Health (DK080440) to L.W.
Characterization of the cynomolgus monkey 20alpha hydroxysteroid dehydrogenase enzyme
Authors: MIN Kwan-Sik , Nanjidsuren Tseeleema , Park Cha-Won , Yun Seong-Jo
Published: June 2021

The 20alpha‐HSD protein predominantly converts progesterone into its biologically inactive form, 20alpha‐hydroxyprogesterone (20alpha‐OHP) and plays a crucial role in the termination of pregnancy and initiation of parturition. Our study showed that the 20alpha‐HSD mRNA and protein are coordinately expressed in the ovary at pre‐ovulation and in the placenta and oviduct at pre‐parturition. Therefore, monkey 20alpha‐HSD in the placenta, ovary and oviduct plays an important role in the estrous cycle, pregnancy, and parturition.
Role of SM22α in Angiotensin II Induced Vascular Smooth Muscle Cell Contraction
Authors: Xiao-Li Xie , Mei Han
Published: June 2021

The major function of vascular smooth muscle cells (VSMCs) is to maintain normal vascular tone via coordinated contraction‐relaxation cycling. Angiotensin II (Ang II), a vasoactive peptide, induces vascular smooth muscle cell (VSMC) contraction. SM22α, an actin binding protein, is highly expressed in contractile VSMCs. It is unclear whether SM22α plays a role in Ang II‐induced contraction. In the present study, differentiation of VSMCs in vitro was induced by all trans retinoic acid (ATRA), and the roles of SM22α in contraction of VSMCs were assessed using specific siRNA. Knockdown of SM22α significantly attenuated the contractility in redifferentiated VSMCs, which is associated with the suppression of Ang II‐induced activation of extracellular signal regulated kinase (ERK) signaling pathway. The loss of SM22α proteins inhibited the formation of actin stress fibres. Furthermore, disruption of the actin filament network by cytochalasin D impaired the activation of ERK pathway. Overall, our results suggest SM22α regulates VSMC contraction through mediates Ang II‐induced ERK activation and actin stress fibre formation.

Source of funding: This study was supported by the National Natural Science Foundation of China (31071003).
PDZ interactions between PHLPP phosphatases and the NHERF scaffold
Authors: Kunkel Maya , Garcia Erin L , Hall Randy A , Newton Alexandra C
Published: June 2021

The PHLPP family of serine/threonine protein phosphatases plays a critical role in regulating the balance between cell survival and apoptosis. The PHLPP family consists of three members, the N‐terminal alternative splice variants, PHLPP1α and PHLPP1β, and PHLPP2, a separate gene product. All isozymes contain a type I PDZ‐binding motif at their C‐terminus: TPL for PHLPP1 and TAL for PHLPP2. We sought to identify potential PDZ domain‐containing proteins that scaffold these phosphatases in order to elucidate differences in PHLPP1 and PHLPP2 signaling at spatially restricted locations within the cell. Using a proteomic array containing 96 purified PDZ domains, we have identified domains that interact with the 25 C‐terminal residues of PHLPP1 and PHLPP2. Specifically, we have observed that the PDZ ligands of both PHLPP1 and PHLPP2 can interact with the scaffold proteins NHERF‐1 and NHERF‐2. In live cells, addition of the last 10 residues of PHLPP1 or PHLPP2 to cyan fluorescent protein (CFP) is sufficient to relocalize CFP to NHERF‐1 or NHERF‐2. However, NHERF‐dependent relocalization is only observed for full‐length PHLPP2; full‐length PHLPP1 is in a conformation in which the C‐terminus is not available to interact with the NHERF PDZ domains. These results suggest differential signaling by PHLPP2 versus PHLPP1 at the NHERF scaffold.

This work was supported by NIH P01 DK54441
Glucagon‐like peptide‐1 signals to PKB and mTORC1 via the activation of the insulin‐like growth factor receptor (IGF1R) in islets of Langerhans
Authors: Mostafa Norhan Mohamed El-Sayed Badr El-Din , Moore Claire E , Herbert Terry P
Published: June 2021

Objectives

Glucagon‐like peptide‐1(GLP‐1) stimulates β‐cell proliferation and protects cells against cytokine induced apoptosis via activation of mTORC1 and PKB. However, it is not fully understood how GLP1 receptor activation leads to the activation of PKB/mTORC1 although it has been reported that this is mediated by EGFR transactivation. Therefore, study aims to investigate the transduction pathways by which GLP‐1 regulates PKB/mTORC1 activation.

Results

We showed that that AG1478 (an EGFR inhibitor), at a dose that effectively blocks EGF‐stimulated Erk phosphorylation, does not affect GLP1‐stimulated PKB or mTORC1 activation. However, diazoxide, an inhibitor of exocytosis, significantly reduced GLP1‐induced PKB and mTORC1 activation. siRNA mediated knock‐down of insulin receptor expression was unable to inhibit GLP1‐stimulated PKB or mTORC1 activation. In contrast, siRNA mediated knockdown of the IGF1 receptor effectively blocked GLP1‐stimulated PKB and mTORC1 activation.

Conclusions

Acute activation of PKB and mTORC1 by GLP1 is mediated via the activation of the IGF1 receptor, presumably through the autocrine effect of IGF.
Mechanisms of transformation by the MEK1 mutants K57N and D67N
Authors: Procaccia Shiri , Seger Rony
Published: June 2021

The MAPK kinase MEK1 is a central component of the Extracellular signal‐Regulated Kinase (ERK) cascade. Its proper function is required for the regulation of a variety of cellular processes. Recently, activating mutations of MEK1 were found in different cancers and shown to be the driver of cellular transformation in these cases. These included mainly the substitutions of Lys57 to Asn (K57N) and Asp67 to Asn (D67N). In order to understand the mechanism of transformation by MEK1 mutants, we undertook to study their signaling properties. Overexpression of both mutants resulted in increased proliferation of cells. Western blot analysis revealed that K57N and to some extent D67N mutant had increased phosphorelation of both their activatory Ser (218 and 222) and also Ser298. Interestingly, the overexpressed mutants only marginally affected the activities of downstream targets, indicating a possible ERK1/2‐independent mechanism of transformation. Indeed, the D67N mutant exhibited different subcellular localization, and was much more abundant in the nucleus, regardless of stimulation. In addition, both mutants resulted in increased phosphorylation of Elongation Factor 2 (EF2). The aberrant cross‐talk with other signaling pathways, the increased phosphorylations and the nuclear localization, are likely to contribute carcinogenesis by these mutants.

Supported by grant from the Israel Science Foundation.
Interaction of ibogaine with human a3b4 nicotinic receptors in different conformational states
Authors: Emert Ilana , Targowska-Duda Katarzyna M , Feuerbach Dominik , Jozwiak Krysztof , Arias Hugo R
Published: June 2021

The interaction of ibogaine and phencyclidine (PCP) with human (h) α3β4 nicotinic acetylcholine receptors (AChRs) in different conformational states was determined using radioligand binding assays, Ca2+ influx detections, thermodynamic and kinetic measurements. The results show that ibogaine inhibits (±)‐epibatidine‐induced Ca2+ influx in hα3β4 AChRs with ~9‐fold higher potency than PCP, [3H]ibogaine binds to a single site in hα3β4 with relatively high affinity (Kd = 0.46 ± 0.006 μM), and ibogaine has slightly higher affinity for desensitized than resting hα3β4 AChRs, compared with PCP. These results correlate with the docking studies suggesting that ibogaine and PCP interact with a binding domain between the serine (position 6′) and valine/phenylalanine (position 13′) rings. This interaction is mediated mainly by van der Waals contacts, which is in agreement with the observed enthalpic contribution determined by non‐linear chromatography. Calculated entropic contribution also indicates local conformational changes. Research support came from the Science Foundation Arizona and Stardust Foundation.
CAPS primes vesicles for regulated exocytosis through SNARE protein interactions
Authors: Daily Neil , Martin Thomas
Published: June 2021

CAPS (Ca2+‐dependent activator protein for secretion) is a 1289 amino acid protein expressed in neural and peptide hormone‐secreting endocrine cells that functions to prime vesicles for Ca2+‐triggered membrane fusion. The ability of CAPS to prime vesicles for Ca2+‐triggered fusion may be through interactions with the SNARE proteins, the core components of the membrane fusion machinery. These studies utilized membrane‐reconstituted SNARE proteins to determine the SNARE protein interactions responsible for CAPS activity. There are two SNARE proteins located on the plasma membrane; syntaxin‐1A and SNAP‐25; and one on the vesicle membrane; VAMP‐2. Liposome co‐flotation studies revealed that CAPS binds with high affinity to liposomes containing syntaxin‐1A and SNAP‐25, and moderate affinity to liposomes containing VAMP‐2. In addition, CAPS interacts with the central SNARE motifs of the SNARE proteins, which it utilizes to accelerate SNARE‐dependent liposome fusion. These studies further our understanding of how CAPS interacts with the SNARE proteins and their critical role in CAPS function in regulated exocytosis.
Identifying Chemotaxis Protein‐Protein Interactions in the Symbiotic Aquatic Bacterium Epulopiscium Sp. B
Authors: Kessler Julie A. , Piefer Andrew J.
Published: June 2021

The purpose of this project is to identify specific protein‐protein interactions that occur during chemotaxis in the symbiotic aquatic bacterium Epulopiscium. Chemotaxis is the process by which cells respond to changes in their environment by triggering movement. This is comparable to how white blood cells migrate toward sites of infection. Several protein‐protein interactions in Epulopiscium have been hypothesized based on protein sequence homology with chemotaxis proteins from other bacteria, though these interactions have never been experimentally confirmed. This project will test specific Epulopiscium chemotaxis protein‐protein interactions through a two part strategy (yeast‐2‐hybrid and GST pull‐down assay) to confirm or refute hypothesized interactions and potentially identify Epulo proteins of unknown function. PCR was performed on genomic DNA from Epulo and candidate regions of CheA and CheW orthologs were cloned to explore possible protein‐protein interactions. Expected results will shed light on chemotaxis within this organism and further the “interactome” map of Epulo which may help elucidate the symbiotic mechanism. This project was funded by a generous gift of the Freedmans to Hartwick College.
Discovery, characterization, and structural analyses of glucan phosphatases from plants to humans
Authors: Gentry Matthew S. , Meekins David A. , Taylor Adam O. , Santelia Diana , Kotting Oliver , Zeeman Samuel C. , Vander Kooi Craig W.
Published: June 2021

We initially demonstrated that the human phosphatase laforin and plant phosphatase Starch EXcess4 (SEX4) are glucan phosphatases. Mutations in the gene encoding laforin cause glycogen to become hyperphosphorylated, resulting in starch‐like glucans called Lafora bodies (LBs) and the fatal neurodegenerative disorder Lafora's disease. Plant starch contains covalently bound phosphate at the C6‐ and C3‐position of glucosyl residues. Reversible phosphorylation of starch outer glucans renders these glucans accessible to hydrolyzing enzymes. In the absence of starch phosphorylation or dephosphorylation, plants accumulate excessively large starch granules that they are unable to breakdown. SEX4 activity is a necessary step in the starch degradation cycle of glucan phosphorylation, hydrolysis, and dephosphorylation. We utilized X‐ray crystallography to determine the structure of SEX4 and this structure assisted in the identification of Like Sex Four2 (LSF2). Unlike SEX4, LSF2 lacks a carbohydrate‐binding module yet LSF2 binds starch. We found that SEX4 releases phosphate from the C3‐ and C6‐position, but prefers C6. Alternatively, LSF2 hydrolyzes phosphate from the C3‐position and plants lacking LSF2 have elevated C3‐bound phosphate. Lastly, we determined the crystal structure of LSF2 and utilize site‐directed mutagenesis with in vitro assays to define the structural elements of glucan phosphatases.
PPP1CC2 levels are highly regulated in testis to ensure normal spermatogenesis and male fertility
Authors: Dasgupta Shuvalaxmi , Sinha Nilam , Vijayaraghavan Srinivasan
Published: June 2021

PPP1CC2 is one of the four isoforms of type 1 serine/threonine protein phosphatase (PP1). PPP1CC1 and PPP1CC2 are two alternatively spliced isoforms of Ppp1cc gene. Targeted deletion of Ppp1cc gene causes male infertility; females are normal suggesting that PPP1CC isoforms are indispensable in testis.

To determine the specific role of the two PPP1CC isoforms, we transgenically expressed PPP1CC2 driven by the testis specific Pgk2 promoter. The results show that fertility as determined by sperm number, morphology and motility is restored in Ppp1cc null mice in lines where transgene expression was relatively high.

The goal of the present work was to determine if overexpression of PPP1CC2 in wildtype (+/+) testis affected spermatogenesis and sperm function. Wild type mice were crossed with Ppp1cc2 transgenic mice (+/−; Tg+) to derive lines with three copies of Ppp1cc2 gene (two endogenous and one transgenic copy; +/+, Tg+). Analysis of +/+;Tg+ adult mice show that they are phenotypically indistinguishable from +/+ and +/− control littermates in terms of sperm morphology, motility and number. Western blot analysis showed there was no increase in PPP1CC2 levels despite a dramatic increase in Ppp1cc2 mRNA levels in testis of +/+;Tg+ compared to +/+ mice. We suggest that homeostatic mechanisms operating post‐transcriptionlly limit the steady state amounts of testis PP1CC2 levels. (NIH ……..)
Open Minds, Open Opportunities
Authors: Drake Richard L
Published: June 2021

During the last decade of the 20th Century and the first decade of the 21st Century curricular reform has been a popular theme. In fact, reform on the current scale has not occurred since the early 1900's after Abraham Flexner released his landmark report “Medical Education in the United States and Canada”. His report, suggesting major changes in how physicians were educated, became the norm and has generally stayed intact until the last 20 years. During that time increased demands on medical school curriculums related to the explosion of knowledge in biomedical sciences and the pressure to add additional clinical experiences have increased the momentum for curriculum reform. For the educator in the anatomical sciences curricular reform has been a time of excitement, fear, apprehension, etc. How have these curricular changes affected education in the anatomical sciences? And, what types of innovative teaching activities have anatomists developed that mesh with the philosophy for educating physicians in the 21st century?