bluuebunny/crossref_metadata_2025 huggingface dataset:
Ca2+ signalling is important for loss of keratinocyte adhesion in pemphigus vulgaris
Authors: Schmitt Thomas , Waschke Jens
Published: April 2020

Pemphigus vulgaris (PV) is a severe blistering skin disease caused by autoantibodies (PV‐IgG) against desmogleins (Dsg)1 and 3. Ca2+ release in response to PV‐IgG has been reported. However, its contribution to loss of keratinocyte adhesion is largely unknown. Phosphatidylinositol‐4‐kinase‐a (PI4K) and phospholipase‐C‐γ1 (PLC) release inositol‐3‐phos‐phate (IP3) activating the IP3‐receptor (IP3R) and thereby causing Ca2+ flux from the endoplasmic reticulum (ER) into the cytoplasm. Low Ca2+ in the ER activates the Ca2+ release‐activated‐channel (CRAC) to replenish the Ca2+ store. Inhibitors against PI4K, PLC, IP3R or CRAC were applied under conditions under which they effectively blocked Ca2+ influx. All inhibitors ameliorated PV‐IgG‐induced alterations of Dsg1 and Dsg3 localization, keratin filament retraction and loss of cell adhesion in dissociation assays. Inhibition of PLC completely abolished PV‐IgG‐induced and also ameliorated AK23‐mediated loss of adhesion. These data demonstrate that Ca2+ signalling is important for loss of cell adhesion in PV but independent from antibodies against Dsg3 supporting the hypothesis that autoantibodies engage different signalling mechanisms to reduce cell adhesion.

Support or Funding Information

Funded by the DFG

Ca‐dependendent interacellular signalling in pemphigus vulgaris. Black arrows indicate calcium‐flux.

Figure 1
3D Kinetics, an Upgrade to the Classical Michaelis‐Menten Method
Authors: Abbasi Armina , Jones Jeff
Published: April 2020

Most of the kinetic data published nowadays are derived from different extensions of what Michaelis and Menten introduced to this field more than hundred years ago. The key assumption to Michaelis‐Menten kinetics is the steady state approximation where the change in the concentration of any enzyme‐substrate intermediate complex is assumed to be zero. This assumption has two underlying requirements with the first one being [S]≫[E] and the second being the limitation of the reaction time to the short initial linear region of the progress curve. The historical need for the steady state assumption was to simplify data treatment to such a degree that the analytical mathematics could be done without need for an abundance of computing power. However, with the computation power at our disposal today, none of the limiting assumptions are necessary anymore especially considering that many of these assumptions rarely hold true. The time course of aldehyde oxidase with one of it substrates, O6‐benzylguanine, is provided here as an example of the statement above. In addition to the obvious instant curvature in the time course plot which makes it difficult to identify the precise linear region, the simulation done on the ES complex based on the data further proves how the steady state approximation is a poor assumption to make especially during the shorter “linear” region of the progress curve as this region is the one in which the concentration of ES is changing the most (Figure
1). The easiest way to approach kinetic data today is to parameterize realistically complex kinetic reaction mechanisms through 3D kinetics. 3D numerical modeling uses the data derived from experiments that monitor the product formation as a function of both incubation time and substrate concentration. With this approach, scientists would be able to derive both the traditional Km and Vmax values as well as the micro‐rate constants related to each step of the enzymatic reaction (Figure
2).

Support or Funding Information

This work was supported by the National Institutes of Health grant: GM100874

A‐1) Numerical fitting of product formation with two different models (A‐2 and A‐3) shows that Michaelis Menten (dashed line) does not fit the data well over longer period of time. B) Simulation of the ES complex through the A‐3 model shows a significant change in this species’s concentration over the initial period of the time course where it is assumed to be a constant in deriving the Michaelis‐Menten equation.

Figure 1

A) Numerical fitting of the product formation over time of different initial substrate concentrations with the kinetic model provided on the right side of the graph to solve for the micro‐rate constants k3‐k5. B) Classical kinetic parameters such as Km and Vmax can be obtained from the same data set by plotting the rate versus substrate concentration for each time point.

Figure 2
Inhibition of Major Cytochrome P450 Enzyme Activities in Human Liver Microsomes by 9‐a and WES‐1, two Novel Carbonic Anhydrase Inhibitors.
Authors: Nguyn Van , Espiritu Michael , Eldehna Wagdy , Abdelrahman Mohamed , Abdel-Aziz Hatem , Abou-Serid Sahar , Elbarbry Fawzy
Published: April 2020

Both 9‐a (sulphonamide derivative) and WES‐1 (coumarin derivative) were designed and synthesized as potential selective Carbonic Anhydrase Inhibitors, and are currently being tested for anti‐cancer activity. This study was undertaken to investigate their potential inhibitory effects on the major Cytochrome P450 (CYP) drug metabolizing enzymes in human liver microsomes using specific CYP probe substrates and liquid chromatography‐tandem mass spectrometry. 9a potently inhibited CYP2E1‐catalyzed chlorzoxazone‐6′‐hydrolylation and CYP2C9‐catalyzed tolbutamide‐4′‐hydrolylation with IC50 values of 0.05 and 0.7 μM, respectively. CYP2A6‐catalyzed coumarin‐7′‐hydroxylation and CYP3A4‐catalyzed testosterone‐6β‐hydroxylation were moderately inhibited by 9‐a with IC50 values of 2.7 and 12 μM, respectively. The rest of the tested CYP enzymes were weakly or negligibly inhibited by 9‐a. WES‐1, on the other hand, demonstrated weak inhibitory effects on CYP2C19‐mediated [S]‐mephenytoin‐4′‐hydrolylation, CYP2C9 activity, and CYP2D6‐catalyzed Dextromethorphan dealkylation with IC50 values of 39, 42.5, and 57.5 μM, respectively. This in‐vitro data indicates that more in vivo studies should be conducted to examine the potential pharmacokinetic drug‐drug interactions with 9‐a due to its potent and moderate inhibition of CYP2A6, CYP2C9, CYP2E1, and CYP3A4.

Inhibitory effect of WES‐1 on the major CYP enzymes in human liver microsomes

Figure 1

Inhibitory effect of 9a on the major CYP enzymes in human liver microsomes

Figure 2
Flos Magnoliae and Its Constituent Linoleic Acid suppress T Lymphocyte Activation via Store‐Operated Calcium Entry.
Authors: KIM HYUN JONG , Nam Joo Hyun
Published: April 2020

Intracellular calcium signaling is crucial for type 2 helper T cell and mast cell activation, which is essential for allergic inflammation. It is initiated by antigen‐mediated receptor stimulation that triggers store‐operated calcium entry (SOCE) via ORAI1 calcium channel. Flos Magnoliae (FM) is widely used to treat allergic diseases such as allergic rhinitis and asthma. Although many studies have reported that FM regulates intracellular calcium signaling, research on the exact type of calcium channel modulated by FM is scarce. Therefore, we hypothesized that the anti‐allergic effects of FM might result from ORAI1 inhibition in T cells. We investigated whether a 70% ethanolic extract of FM (FMEtOH) and its constituents inhibit ORAI1 channel activity and subsequent T cell activation. We performed conventional whole‐cell patch clamp studies in STIM1 and ORAI1‐overexpressing HEK293T cells (HEKORAI1). Intracellular calcium concentration was determined using Fura‐2 dye and cytokine production measurement in Jurkat T lymphocytes. FMEtOH (0.03 mg/mL) and its fractions, especially hexane fraction (FMHex, 0.01 mg/mL), significantly inhibited SOCE and IL‐2 cytokine production in Jurkat T lymphocytes. GC/MS analysis showed linoleic acid (LA) as the major component of FMHex. FMHex at 0.01 mg/mL (equivalent to 10 μM LA) inhibited not only SOCE but also IL‐2 production, as well as CD3/CD28 receptor co‐stimulation induced calcium signaling in Jurkat T lymphocytes. FMEtOH and LA suppressed CD4+ T lymphocyte activation, at least in part, by inhibiting ISOCE. Thus, ISOCE inhibition may be a potential strategy to inhibit immune responses in inflammation.

Linoleic acid (LA) inhibits ISOCE and IL‐2 production to similar levels.

Figure 1

FMEtOH and its fractions inhibit store‐operated Ca2+ entry mediated current (ISOCE) in ORAI1 and STIM1 co‐overexpressing HEK293T cells (HEKORAI1).

Figure 2
Collaborating With Glycoscience Community To Improve Data Representation of Carbohydrates in the Protein Data Bank
Authors: Zardecki Christine , Shao Chenghua , Feng Zukang , Westbrook John D. , Persikova Irina , Burley Stephen K. , Young Jasmine Y
Published: April 2020

The Protein Data Bank (PDB) is the single global repository for experimentally‐determined 3D structures of biological macromolecules and their complexes with ligands. The Worldwide Protein Data Bank (wwPDB) is the international collaboration that manages the PDB archive according to the FAIR Principles: Findability, Accessibility, Interoperability, and Reusability. The PDB archive now holds more than 150,000 structures, which are all publicly accessible without restriction.

A major focus of the wwPDB is maintaining consistency and accuracy across the archive. As the PDB grows, developments in structure determination methods and technologies can challenge how all structures are represented. The wwPDB addresses these challenges with “remediation” efforts to improve data representation in support of making PDB data FAIR.

Understanding the structure and organization of carbohydrates is critical to comprehending their biological roles in health and disease. Approximately 10% of PDB structures contain carbohydrates. However, there is lack of uniform representation for carbohydrates due to complex nature of carbohydrates, which exhibit stereo‐isomers, anomeric configurations, and branched chains. As a result, glycoscientists and other experts are unable to fully utilize the rich structural information available for these structures.

Working with the glycoscience community, carbohydrate‐appropriate annotation tools are being developed and implemented within the OneDep system for deposition, validation, and biocuration of PDB structures. These software tools will provide standard nomenclature and consistent oligosaccharide representation that can be easily translated to other representations commonly used by glycobiologists.

wwPDB carbohydrate remediation efforts involve: (1) standardizing sugar nomenclature following IUPAC/IUBMB; (2) providing a uniform polymer representation for polysaccharides with appropriate descriptor(s); (3) adopting community software for reliable carbohydrate identification, assignment of standard nomenclature, and detection of intra/inter‐molecular connectivity between monosaccharides and other molecules/proteins; and (4) providing intra‐ and inter‐molecular connectivity at atom level explicitly.

Support or Funding Information

This carbohydrate remediation project is a collaborative wwPDB project carried out principally by the RCSB PDB with funding from NIGMS (U01 CA221216) in collaboration with the Complex Carbohydrate Research Center at the University of Georgia. RCSB PDB is funded by the NSF (DBI‐1832184), the US DOE (DE‐SC0019749), and the NCI, NIAID, and of the NIH under grant R01GM133198.

N‐Glycosylation of cardosin A in PDB structure 1b5f (Frazao, C. et al. (1999) J Biol Chem 274: 27694–27701)

Figure 1
Kinetic Characterization of Mutant Methylenetetrahydrofolate Reductase Enzymes His273Ala and His273Gln
Authors: Du Siyuan , Tetrick Maxwell G. , Li Richard , Chien Jason , Trimmer Elizabeth E.
Published: April 2020

Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in folate metabolism that binds flavin adenine dinucleotide (FAD) and uses NAD(P)H to catalyze the conversion of 5,10‐methylenetetrahydrofolate to 5‐methyltetrahydrofolate by a ping‐pong Bi‐Bi mechanism. The x‐ray crystal structure of the 5‐methyltetrahydrofolate and FAD
‐bound E. coli enzyme has revealed a hydrogen bonding network that involves amino acid residues Ser26, His273, Glu28, a structural water molecule, and the N10 of the folate. Based on previous studies that confirmed the importance of Glu28 in the catalysis of the folate half‐reaction, we hypothesized a proton relay mechanism that transports a proton from the aqueous solvent to the N10 of the folate through this hydrogen bonding network and catalyzes the formation of the putative 5‐iminium cation folate intermediate. In our study, we employed steady‐state oxidoreductase assays using menadione as an artificial electron receptor and measured kinetic parameters of the wild‐type E. coli enzyme and two mutants, His273Gln and His273Ala MTHFRs. We found that in the methyltetrahydrofolate‐menadione assay, where the folate half‐reaction is rate‐limiting, the kcat of the His273Ala mutant (0.047 ± 0.002 s−1) is more than 10‐fold lower than the wild‐type enzyme (kcat = 0.54 ± 0.01 s−1). His273Gln mutant showed similar activity to the wild‐type enzyme, but strong competitive substrate inhibition was observed. In the NADH‐menadione assay, both His273Ala and His273Gln showed insignificant change in kcat (1.2 – 1.3‐fold) and in Km (1.7 – 2‐fold). Together, these results suggest that the ability of the His273 residue to form hydrogen bonds is important in the catalysis of the folate half‐reaction and support the hypothetical proton relay mechanism. In addition, we report our use of Liquid Chromatography‐Mass Spectrometry (LC‐MS) to quantify the product in the folate‐menadione assay more accurately.

Support or Funding Information

Grinnell College Department of Chemistry

Hydrogen‐bonding network in the X‐ray structure of E28Q MTHFR

Figure 1

v/E vs. [substrate] plots obtained from methyltetrahydrofolate‐menadione oxidoreductase assays at 4C.

Figure 2
Zanthoxylum piperitum activates thermogenic gene expression and beige adipogenesis in white adipose tissues of mice
Authors: Ihara Hayato , Takeshima Ken , Kadowaki Akio , Gato Nobuki , Kishida Kunihiro , Akamizu Takashi
Published: April 2020

Metabolic syndrome increases the risk of cardiovascular disease, stroke, and diabetes mellitus. Because treatment of obesity is necessary to prevent multiple organ dysfunctions associated with metabolic syndrome, tolerable anti‐obesity drugs must be developed. In this study, we focused on Zanthoxylum piperitum (ZP), a uniquely flavored spice that confers a tingling sensation. We examined the effects of ZP on obesity and glucose–lipid metabolism in mice fed a standard diet (STD) or high‐fat diet (HFD) with or without ZP. Notably, body weight and fat mass markedly decreased, and glucose–lipid metabolism improved, in STD‐fed mice treated with ZP. Moreover, thermogenic gene expression was elevated in inguinal white adipose tissue (iWAT) from STD‐fed mice treated with 3% ZP. Immunohistochemistry of iWAT revealed uncoupling protein‐1 (UCP‐1)–positive multilocular adipocytes, suggesting induction of the beige adipogenesis. In conclusion, ZP administered in the diet decreases weight and fat mass, concomitant with induction of beige adipogenesis. These findings imply that identification of the components of ZP that exert anti‐obesity effects could facilitate development of novel anti‐obesity drugs originating from a natural food.

Support or Funding Information

This work was supported by JSPS KAKENHI Grant Number JP25504010 (to HI), JP16K12728 (to HI), Grants‐in‐Aid from the Consortium of Higher Education Institution in Wakayama (2012&2016 to HI, 2014&2015 to KT), Research and Development Support Project for Pioneering Industrial Technology by Wakayama Prefectural Government (to NG), and a Grant‐in‐Aid from the Regional Innovation Strategy Support Program 2011–2016 (to KT).

Visceral and subcutaneous fat mass were quantified by small‐animal CT

Figure 1

H&E staining and immunohistochemistry showed UCP‐1 positive multilocular adipocytes in iWAT from STD+3%ZP mice

Figure 2
Locating TATA Binding Protein in the RNA Polymerase I Pre‐Initiation Complex
Authors: Vo Giang , Jackobel Ashleigh , Knutson Bruce
Published: April 2020

Transcription of eukaryotic DNA into RNA is carried by three main RNA polymerases (Pols I–III). Pol I synthesizes ribosomal DNA (rDNA) into ribosomal RNA (rRNA), which has been directly linked to the rate of protein synthesis, cell growth and proliferation. The efficiency of Pols’ function is controlled by general transcription factors, which enable the enzyme to interact with promoter DNA at the initiation stage. In Saccharomyces cerevisiae, Pol I transcription initiation relies on four main factors, one of which is TATA Binding Protein (TBP). TBP is an essential universal transcription factor used by all three eukaryotic Pols. As its name eludes, TBP typically binds to TATA motifs found in promoter DNA. Interestingly, unlike Pol II and III, Pol I promoters are TATA‐less. Thus, unlike the location of TBPs in other PICs, the location and function of TBP within the Pol I PIC remains unclear.

Current Cryo‐EM structures of CF‐DNA‐Pol I initiation complex leaves little room for TBP to bind to its “typical” Pols II/III position. Therefore, we hypothesize that TBP must occupy a unique location, putatively more upstream, within the Pol I PIC. Combining available structural data with previous crosslinking data of CF and TBP, we modeled the interaction between the two proteins with varying distance constraints. In addition, we analyzed TBPs location on the rDNA promoter by site‐directed hydroxyl radical cleavage experiment, known as FeBABE, where DNA phosphothioate modification probes were constructed based on our predicted models. Our results from these combinational methods elucidated that TBP locates more upstream within Pol I PIC.

Support or Funding Information

This work was a part of SUNY Upstate Medical School’s Summer Undergraduate Research Fellowship 2019 and was supported by National Cancer Institute (NCI), National Institute for Dental and Craniofacial Research (NIDCR), and Carol Baldwin Research Foundation.

FeBABE site‐directed hydroxyl radical cleavage.

A and B. Sites of FeBABE incorporation into rDNA promoter in clusters of 5 include the “typical” location of TBP (1), upstream of the CE (2), 5′ of CE (3), and within the CE (4). C. Western blot showed FeBABE specific cleavage products. Cleavages were observed when FeBABE was incorporated at all four positions. Most intense cleavages were observed when FeBABE is incorporated upstream of the CE. D. Quantitation of TBP cleavages.

Figure 1
Disorders of the metabolic status and morphofunctional state of liver and kidneys of chicken
Authors: Anipchenko Polina , Shabunin Sergey , Kotarev Viacheslav , Mikhailov Evgenii , Stekol'nikov Anatolii
Published: April 2020

In this study we analyzed biochemical parameters of blood, tibia, histological analysis of liver and kidney tissues of 46‐days old pullets of egg‐laying chickens. The research aim is study of biochemical and morphological changes in replacement pullets to predict risks of growth weak replacement pullets. The research shows that in the serum total proteins were higher then normal range on 28, 8%, albumins on 32,0%. High level of this parameters leads to produce too much uric acid and accumulate it in the body and its higher on 21,3%. Energy change in serum is shown with satisfying range of glucose and reduced rate of total lipids. Sufficient calcium in serum 13,1% with lack of phosphorus. Ca/P ratio exceeds 1.2 times optimal value. Low alkaline phosphatase activity is typical for inadequate bone matrix formation. AST is higher on 25,1% AST and ALT parameters are both high on 25,1% and 15,4% respectively and it means liver function abnormal. There is very low vitamin B2 (42,4% from normal value) wwith sufficiently high carotene supply with vitamins A, E, C in the liver. Liver micronutrients: Zinc – 2,9 times more; Manganese – 3,1 times more with less concentration of Cobalt on 2,6 times and Selenium – 8,3 times. There are calcium 1,6 times less and phosphorus – 1,4 in tibia. Thus in the liver of chickens has focal or diffuse inflammation with blood flow disturbances and of necrosis were recorded. Macroscopic examination found that kidney are oversized, have flabby consistency with light brown color and surface look like almond nut. Microscopic examination found that Bowman's capsule of the cortex kidney is thickened, the space between the capsule and glomeruli is swollen, and the borders of the tubule epithelial cells are poorly distinguishable. The cells are swollen, enlarged and cone‐shaped into the gap make it narrow. The degree of swelling of the epithelial cells, and, consequently, the narrowing of the lumens were uneven. In some tubules, the gaps are still quite distinguishable, in others they look like gaps. There are tubules in which the gaps are almost invisible. Nuclei are not clearly expressed in all cells. The established violations in the interstitial metabolism and structural changes in the parenchymal organs (liver, kidneys) during the restoration of youth is a predisposing load that ensures the body's resistance to adverse environmental influences and loss of productive metabolism during egg production.

Micromorphology of the chickens' liver. Impaired blood circulation. Hematoxylin and eosin.X 640

Figure 1

The architectonics of the glomerular zone of the kidney in chickens 46 days old. Hematoxylin and eosin.X 640

Figure 2
Bilateral Anatomical Variation of the Upper Serratus Anterior: A Cadaveric Dissection
Authors: Etibo Kristen , Longoria Kendra , Thornton Kaitlin , Rosario Martin G.
Published: April 2020

Anatomical variation can have clinical implications in terms of pathology, musculature surgical considerations, and biomechanical function. While many anatomical variations have been reported, it is critical to continue to expand the record when deviations are found. According to most sources, the upper serratus anterior originates at the first and second ribs and inserts at the superior angle of the scapula. Some variations in attachments of the upper serratus anterior have been described, especially regarding variations in origin at the first few ribs. (Smith, et al., 2003). However, the literature does not describe the insertion of the serratus anterior muscle as extending along the superior border of the scapula.

PURPOSE

To report a bilateral anatomical variation of the upper serratus anterior discovered during routine dissection on a female specimen.

METHODS

While under the supervision of the anatomist at Texas Woman’s University Dallas, Doctor of Physical Therapy students dissected an 82‐year‐old Caucasian female. The students employed techniques described in Grant’s Dissector (Detton, 2017), to expose the shoulder and neck regions of the specimen. Blunt dissection techniques were utilized to expose the area further once they discovered the anatomical variation of the upper serratus anterior. Students measured the length of the upper serratus anterior that inserted on the superior border.

RESULTS

The cadaver with the serratus anterior variation, had the muscle attached to 56% of the superior border of her scapula. Comparatively, 3 other female cadavers of similar ages had significantly less attachment to the superior border‐‐an average of 27%, with a range from 16.7% to 32.6%.

DISCUSSION

Based on widely accepted descriptions of the serratus anterior attaching to the superior angle, without extending extensively onto the superior border of the scapula, and in comparison to other available cadavers, this subject had a serratus anterior with a significantly larger attachment to the superior border of the scapula. The specific actions of the upper serratus anterior include stabilizing the scapula during humeral elevation, and in upward rotation and abduction of the scapula. Therefore, this variation may have functional and clinical implications for the scapulothoracic and shoulder regions.

CONCLUSIONS

It appears that variations in the upper serratus anterior may not only include its rib attachments but also its scapular attachment. This may affect function including limitations in scapular rotation and shoulder elevation. Therefore, further investigation into anomalies and function of the upper serratus anterior is warranted.

SA Anatomical Variation: Serratus anterior attaching to the superior angle, without extending extensively onto the superior border of the scapula.

Figure 1
The vimentin rod domain targets P‐selectin to block leukocyte adhesion to inflamed endothelium
Authors: Lam Fong Wilson , Brown Cameron August , Brown Christian Valladolid , Emebo Dabel Cynthia , Cruz Miguel A.
Published: April 2020

The acute inflammatory process begins with leukocyte adhesion to vascular endothelium and platelets. This typically beneficial process is mediated through the binding of P‐selectin glycoprotein ligand‐1 (PSGL‐1) on leukocytes to P‐selectin (P‐sel) on both inflamed endothelium and activated platelets. In pathologic conditions, this key process may lead to pathologic inflammation and contributed to secondary organ injury, as in sepsis‐induced acute liver injury and ischemia‐reperfusion injury. Therefore, the development of novel therapies to attenuate inflammation may be beneficial in select conditions. We previously described the ability of recombinant human vimentin (rhVim) to block leukocyte adhesion to endothelium and platelets and to decrease leukocytic infiltration into the lungs after endotoxemia via binding to P‐sel. The
purpose of this study was to identify the active region within rhVim that interacts with P‐selectin to further dissect the mechanism by which rhVim blocks leukocyte adhesion. Using SPOT‐peptide arrays of both full‐length vimentin and P‐sel (20‐aa/spot), we identified that rhVim binds to P‐sel primarily through its rod domain. We created a recombinant form of the rod domain (rhRod) and found that it binds to P‐sel with high affinity (10 – 41 nM), and in silico modeling suggests that it also binds to regions that interfere with PSGL‐1‐P‐sel interactions (Figure
1). In vitro experiments show that rhRod has similar efficacy to rhVim in blocking leukocyte adhesion to P‐sel/Fc coated surfaces and IL‐1β/IL‐4‐co‐stimulated endothelium (~25 – 50% reduction). Finally, using intravital microscopy in endotoxemic C57Bl/6 mice (Figure
2), rhRod co‐localizes with P‐sel in the hepatic sinusoids and treatment with rhRod decreases neutrophil adhesion to hepatic sinusoids (~25% reduction). These data suggest a potential role of rhRod in attenuating inflammation through blocking P‐selectin‐PSGL‐1 interactions.

Support or Funding Information

NIH/NIGMS GM‐112806 & GM‐123261; NIH/NHLBI HL‐116524; NIH/NINDS NS‐094280; Dept. of Veterans Affairs I01 BX002551

(A) Three views of the lectin/EGF‐domain of P‐selectin (PDB 1G1Q) with PSGL‐1 complexed (PDB 1G1S). The colored domains signify the regions to which rhRod bound to P‐selectin on the SPOT array (magenta: aa 151–170; cyan: aa 43–65; and blue: aa 79–116). (B) Two views of an in silico prediction model of rhRod (green) binding to P‐selectin in the region correlating to aa residues 107–129.

Figure 1

(A) Intravital microscopy of mouse hepatic sinusoids after endotoxin exposure show P‐selectin (red) lining the sinusoidal vessels (gray). Atto 550‐labeled rhRod (green) also localizes to the sinusoids and colocalizes with P‐selectin (yellow). Firmly adherent neutrophils (cyan) were identified as cells which moved less than 1 cell body distance over 1 minute. (B) Mice receiving rhRod had significantly fewer neutrophils per vessel area. Scale bar = 50 μm.

Figure 2
A REVIEW: MALIGNANT HYPERTHERMIA‐A GENETIC DISEASE OF SODIUM CHANNEL FUNCTION
Authors: Williams Charles H.
Published: April 2020

Malignant Hyperthermia is inherited as a dominant gene in pigs and in humans. It is characterized by a rapidly increasing body temperature up to 118°F with a metabolic rate over 10× normal, an intense peripheral vasoconstriction with blood pressures over 400 mmHg, and a fatal outcome in most cases.

The MH syndrome is triggered by exercise, hauling to market, breeding activity, hot weather, and other stress factors. In humans, the cases are triggered by depolarizing muscle relaxants, Halothane anesthesia, and other halogenated anesthesia compounds. Non‐depolarizing muscle relaxants such as Pancuronium and Vecuronium are safer to use. Organon 9426 (Rocuronium) is safe to use and provides carry over protection against the development of MH.

The widespread use of Sevoflurane in humans has reduced the incidence of MH to 1:550,000. Only three cases of MH have been reported in the USA during the past 30 years. They developed in WI, FL and SC.

The MH susceptible pig is an outstanding animal model for research purposes and has enabled the development of new muscle relaxants and anesthesia agents that are safer for human use.

The sodium channels in MH susceptible animals are leaky and allow the influx of sodium into muscle cells which must be pumped out by sodium‐potassium ATPase, thereby using ATP and generating heat.

Malignant Hyperthermia is inherited as a dominant gene in pigs and in humans. It is characterized by a rapidly increasing body temperature up to 118°F with a metabolic rate over 10× normal, an intense peripheral vasoconstriction with blood pressures over 400 mmHg, and a fatal outcome in most cases.

The MH syndrome is triggered by exercise, hauling to market, breeding activity, hot weather, and other stress factors. In humans, the cases are triggered by depolarizing muscle relaxants, Halothane anesthesia, and other halogenated anesthesia compounds. Non‐depolarizing muscle relaxants such as Pancuronium and Vecuronium are safer to use. Organon 9426 (Rocuronium) is safe to use and provides carry over protection against the development of MH.

The widespread use of Sevoflurane in humans has reduced the incidence of MH to 1:550,000. Only three cases of MH have been reported in the USA during the past 30 years. They developed in WI, FL and SC.

The MH susceptible pig is an outstanding animal model for research purposes and has enabled the development of new muscle relaxants and anesthesia agents that are safer for human use. The sodium channels in MH susceptible animals are leaky and allow the influx of sodium into muscle cells which must be pumped out by sodium‐potassium ATPase, thereby using ATP and generating heat.

Support or Funding Information
Epithelial Mechanosensitive Ion Channel Piezo2 Contributes to Pressure‐Induced Epithelial Chloride Secretion in Mouse Colon
Authors: Knutson Kaitlyn Rebecca , Finholm Isabelle , Treichel Anthony J. , Whiteman Sara , Linden David R. , Grover Madhusudan , Farrugia Gianrico , Beyder Arthur
Published: April 2020

BACKGROUND

Mechanical stimulation of the gastrointestinal (GI) epithelium causes changes in epithelial ion transport by an unclear mechanism. We recently discovered the mechanosensitive ion channel Piezo2 is specifically expressed by the enteroendocrine cells (EECs) and is necessary for EEC mechanosensitivity. Further, in mouse small bowel, the pharmacologic inhibition or genetic knockout of the epithelial Piezo2 led to an inhibition of the pressure‐induced short‐circuit current (Isc) in the Ussing chamber. These results suggested that EEC Piezo2 plays a role in pressure‐induced epithelial ion transport, but the mechanism and whether these responses are region‐specific remains uknown.

AIM

Determine whether epithelial Piezo2‐mediated pressure‐induced Isc is due to Clsecretion in the mouse colon.

METHODS

Full‐thickness mid‐colon preparations of VilCre/+::Piezo2f/f (Piezo2CKO) or Vil+/+::Piezo2f/f (Piezo2WT) mice at 0.3 cm2 were mounted in 4mL Ussing chambers with normal Krebs solution. The epithelial side was intermittently pressure‐clamped at a range of pressures (5, 10, 20, 30, 40, 60 mmHg) for 10s each. Forskolin (FSK, 10μM) was applied at the end of the experiments, and FSK response was used to normalize the pressure‐induced responses (ΔIsc/ΔIsc,FSK).

RESULTS

Pressure applied to the mucosa of Piezo2CKO and Piezo2WTcolons caused a pressure‐dependent increase in Isc. Responses in Piezo2CKO colons were smaller than Piezo2WT at 40mmHg (0.41±0.1 vs. 0.76±0.1) and 60mmHg (0.51±0.1 vs. 0.87±0.1) (n=9–11, P<0.05, two‐way ANOVA). To determine the nature of the pressure‐induced Isc we removed Cl− from the Krebs solutions. Cl‐removal resulted in XX. We found a nearloss in the mechanically induced ΔIsc in both Piezo2WT tissues and Piezo2CKO: 5mmHg (− 88.85±0.1% and −87.44±0.1% Piezo2WT), 10mmHg (−86.46±0.1% Piezo2CKO and − 90.30±0.1% Piezo2WT), 20mmHg (−88.50±0.01% Piezo2CKO and −89.86±0.1% Piezo2WT), 40mmHg (−91.78±0.1% Piezo2CKO and −91.35±0.04% Piezo2WT), and 60mmHg (−93.93±0.04% Piezo2CKO and −92.08±0.04% Piezo2WT (n= 5–10, P <0.05, unpaired t‐test). Replacement of Cl− led to full recovery of the mechanically induced ΔIsc in both Piezo2WT and Piezo2CKO tissues when tested at 30 mmHg (Piezo2WT: 2.51±1.4 vs. 73.52±33.8, n=6 and 3, P <0.05, unpaired t‐test), (Piezo2CKO: 1.80±1.0 vs. 45.14±12.4, n=5 and 4, P <0.05, unpaired t‐test).

CONCLUSIONS

Hydrostatic pressure applied to the colon epithelium leads to an increase in epithelial secretion that depends chloride and Piezo2 contributes to this mechanosensitive chloride secretion.

Support or Funding Information

Supported by AGA RSA, NIH DK106456, NIH DK119683, NIH DK123549
Cell‐based hormone therapy prevents diastolic dysfunction after estrogen loss in the Spontaneously Hypertensive Rat (SHR)
Authors: Groban Leanne , Sun Xuming , Sivanandane Sittadjody , Wang Hao , Hodge Hunter S. , Basu Swati , Kim-Shapiro Daniel G. , Opara Emmanuel C.
Published: April 2020

Introduction. Preclinical left ventricular diastolic dysfunction (LVDD) is a forerunner to heart failure with preserved ejection fraction, which is seen more often in older women than men of the same age. While hormone therapy plays an important role in the management of vasomotor and genitourinary symptoms experienced by many women during the menopausal transition, its efficacy with regard to maintenance of cardiac structure and function and prevention of LVDD disease progression with aging has not been tested due to the concern for off‐target health risks (e.g., cancer, blood clots). We developed a cell‐based hormone therapy (cHT) approach that recapitulates native cell–cell interactions between ovarian granulosa and theca cells in a 3D bioengineered construct to mimic the dynamic release of sex hormones into the circulation. Our first report in ovariectomized rats shows that ovarian constructs are able to ameliorate various somatic adverse effects of hormone deficiency (e.g. bone loss). To extend these findings to cardiac health, we sought to determine the efficacy of cHT in preserving cardiac diastolic function after estrogen loss using a hypertensive rodent model.

Methods

14 SHR females underwent bilateral ovariectomy (OVX) while 5 SHRs received sham surgery at 12 weeks of age; 8 weeks post‐surgery, OVX rats were randomized (n=4–5/group) to vehicle, cHT, or pharmacologic hormone therapy (pHT: E2 (10mg/kg/day) and P4 (2mg/kg/day) subcutaneous pellets) and compared to gonad‐intact rats treated with vehicle (VEH). Body weights and tail cuff systolic blood pressure (SBP) were measured biweekly. Following 10 weeks of treatment, rats were lightly anesthetized (isoflurane) for Doppler measures of diastolic function. Data were analyzed by one‐way ANOVA. P<0.05 was significant.

Results

OVX rats, irrespective of treatment, had higher body weights when compared to sham (P<0.001). Uterine atrophy by OVX was minimized by pHT and cHT, reflecting modest E2‐related effects (Figure
A). SBP increased progressively over time (P<0.01), without overt differences across treatment groups. Even so, both cHT and pHT prevented adverse OVX‐related effects on myocardial relaxation (e') and Doppler‐derived filling pressures (E/e') when compared to vehicle (Figures
B, C).

Conclusion

These data show that novel cell‐based hormone therapy is effective in preventing diastolic dysfunction from surgically‐induced menopause in the SHR. Additional studies are underway to determine cHT’s long‐term (6 months) treatment efficacy in middle‐to‐advanced aged OVX rats.

Support or Funding Information

NIH AG061588

Figure 1
Dynamic Phosphorylation of Yeast eIF4A Drives Changes in Gene Expression that Couple Protein Synthesis with Cell Division
Authors: Sahoo Ansuman , Ashraf Marium , Walker Sarah E.
Published: April 2020

The eukaryotic translation initiation factor 4A (eIF4A) supports mRNA recruitment to the ribosomal preinitiation complex through activity of its conserved DEAD‐box RNA helicase motif. Interestingly, in plants eIF4A is differentially phosphorylated near the DEAD motif during various phases of the cell cycle by CDKA, lowering translation activity during mitosis (
Bush et al., 2016). The same Threonine residue of yeast eIF4A (T146) is present in a consensus CDK1/CDKA/Cdc28 motif, and is highly phosphorylated in large‐scale studies (
Soulard et al., 2010). To further dissect the molecular function of eIF4A, we are analyzing specific in vitro and in vivo changes that take place due to phosphorylation of eIF4A under normal conditions and in response to stress. We have mutated eIF4A residue T146 to phosphodeficient (Ala) and phosphomimetic (Glu, Asp) forms, and analyzed the effects of these mutations on growth and translation. Our data indicate that the phosphorylation status of eIF4A sensitizes yeast cells to membrane stressors in a manner affected by eIF4A•eIF4B coupling, and that constitutive phosphorylation of this residue is lethal due to arrest prior to cell division. Mechanistically, phosphorylation disrupts RNA binding by eIF4A. Together our results suggest that dynamic phosphorylation of eIF4A modulates translation changes that promote cell division and resistance to external stressors.

Support or Funding Information

This work was supported by NIH grant R00GM119173 and startup funds from SUNY at Buffalo‐College of Arts and Sciences
Digital radiological integration into the first year medical anatomy course: A systematic literature review
Authors: Williams Sarah M.
Published: April 2020

As diagnostic digital radiology is becoming the standard in medicine, numerus medical schools have incorporated digital radiology (i.e. CTs, MRIs, Xrays and Ultrasound) into their preclinical curriculum. There are a lack of resources that provide a comprehensive insight into the most common methods of digital radiological integration into the first year medical anatomy course. The purpose of this study is to perform a meta‐analysis on most common methods of digital radiological integration and to determine the overall trend in the usefulness of this technique as a teaching method. Utilizing the PICO (Population, Intervention, Comparison, Outcomes) reporting system, two of the following questions are proposed: (i) What is the most common method of integration of digital radiology in the first year medical anatomy course? (ii) What are the learner outcomes for assessment of anatomical knowledge using digital radiological imaging? The systematic literature review is conducted in accordance with Preferred Reporting Items for Systematic reviews and Meta‐Analysis (PRISMA) guidelines. Identification of citations (n=1688) were obtained searching keywords “radiology” AND “anatomy” AND “medical student” in the following four databases: PubMed, Embase, Web of Science, and Scopus (Table 1). Duplicates were excluded using the “find duplicates” tool in Medeley citation software and articles published prior to 2009 were excluded (n=276). A reviewer team will be assembled to analyze the degree of inclusion of remaining articles screened (n=1412). Results indicate that there are diverse ways to incorporate radiological images into a gross anatomy course and the method of incorporation may affect the successfulness of the method.

A summary of the screening process. Out of 1688 articles, 1412 advanced to be analyzed by the reviewer team based on the eligibility inclusion criteria.

Figure 1
Feline Chondroblastic Osteosarcoma: Comparative Analysis by Pathology Techniques
Authors: Takahashi Tamy Verony , de Albuquerque Lima Mobricci Luciana , Hyodo Vivian , Duarte Janaína
Published: April 2020

Even though primary bone tumors are considered uncommon in cats, osteosarcoma is the most common, with a prevalence of 70% to 80% of all primary bone tumors reported in this species. However, the number of cases reported are still scarce when compared to those reported in dogs. The goal of this study was to diagnose, through necropsy and histopathology, a case of feline chondroblastic osteosarcoma presented to a private practice in Brazil, describing the macroscopic and microscopic findings. The necropsy was performed at the Pathology Laboratory of Paulista University, located in São José dos Campos, Brazil, using an adaptation of the Ghon method, developed by Professor Janaína Duarte, DVM, PhD. Histopathology was performed by a veterinary diagnostic laboratory located in São Paulo, Brazil.

A female, spayed, 12 years old, domestic short hair cat, was presented to an ophthalmology specialist practice with a complaint of suspected glaucoma, which a skull radiograph later showed to be, in fact, exophthalmus caused by a mass in the left eyeball topography, with loss of definition of the zygomatic bone’s rostral aspect and the adjacent maxilla. Euthanasia was performed due to worsening of the general state and economic limitations to follow further diagnose and treatment. Necropsy revealed a lobulated, semi‐hard, pale red mass, measuring approximately 4.0 × 3.0 × 1.0 cm, adhered to the left mandible vertical ramus and pressing against the left eyeball. Transverse cutting showed a translucent, whitish, partially mineralized surface. Histopathology exhibited proliferation of spindle shaped cells, with marked degeneration, cytoplasmic retraction and acidophilic staining, arranged in disorganized bundles, permeated by immature and mineralized osteoid matrix. The nuclei were rounded, hyper‐chromatic with occasional nucleoli evidence. Figures of mitoses were not identifiable due to cellular degeneration. It was also seen that between the cells there were multiple focuses of marked chondroid tissue proliferation, highly suggestive of chondroblastic osteosarcoma.

Macroscopic findings during necropsy showed a mass with malignant and invasive biological behavior, affecting adjacent structures such as the eyeball and the zygomatic arch, which is consistent with those of histopathology that showed proliferation and marked degeneration of atypical spindle shaped cells with infiltration of the chondroid matrix by osteoid tissue.

Location of the mass, filling the left eye orbit (arrow).

Figure 1

Proliferation of spindle snapped cells permeated by osteoid and chondroid matrix.

Figure 2
Coordinated Gene Expression of Na+/Cl−cotransporter 1 (ncc1) and Clc family Cl−channel 2 (clcn2c) in Kidney and Urinary Bladder of Euryhaline Tilapia
Authors: Nelson Nastasia , Petro-Sakuma Cody , Celino-Brady Fritzie , Seale Andre , Breves Jason
Published: April 2020

Euryhaline fishes maintain hydromineral balance in a broad range of environmental salinities through the functional coordination of multiple tissues, including the gill, intestine, kidney, and urinary bladder. Fishes inhabiting freshwater (FW) environments are faced with the diffusive loss of ions and the osmotic gain of water. In turn, both kidney and urinary bladder support the reabsorption of Na+ and Cl− and the excretion of excess water. While functional responses by these two organs to FW conditions have been previously described, the subcellular pathways for Na+ and Cl− reabsorption are not well understood. We hypothesized that euryhaline tilapia (Oreochromis mossambicus), employ Na+/Cl− cotransporter 1 (Ncc1) and Clc family Cl− channel 2 (Clcn2c) for the active reabsorption of Na+ and Cl− and thus their respective gene transcripts are expressed under FW conditions. In the current study, we report ncc1 and clcn2c gene expression patterns in both steady‐state animals and animals abruptly transferred from seawater (SW) to FW. In kidney and urinary bladder, ncc1 and clcn2c gene expression was markedly enhanced (>30‐fold) in FW‐ versus SW‐acclimated tilapia. By 6 hours after transfer from SW to FW, ncc1 and clcn2c in both kidney and urinary bladder were coordinately elevated compared with controls continuously maintained in SW. These patterns in the kidney and urinary bladder occurred in concert with branchial processes supportive of Na+ and Cl− absorption from the external environment. Collectively, our results suggest that both Ncc1 and Clcn2c contribute to the functional plasticity of kidney and urinary bladder that underlies acclimation to variable environmental salinities.

Support or Funding Information

Supported by the National Science Foundation (IOS‐1755131 and ‐1755016).
Autophagy Inhibition Sensitizes Liver Kinase B1 (LKB1)‐Deficient Kras‐Driven Lung Tumors to MEK Inhibitor Trametinib
Authors: Bhatt Vrushank Dharmesh , Khayati Khoosheh , Kong Jerry , Raju Akash , Su Xiaoyang , Guo Jessie Yanxiang
Published: April 2020

Tumor suppressor liver kinase B1 (LKB1) activates 5’‐adenosine monophosphate protein kinase (AMPK) and plays an essential role in maintaining energy homeostasis. LKB1 mutations are the third most frequent mutation detected in non‐small cell lung cancer (NSCLC). Compared to p53 mutation, co‐mutations in LKB1 with KRAS causes aggressive tumor growth and resistance to chemotherapy as well as immunotherapy. Thus, identifying a novel target to improve LKB1‐deficient Kras‐driven NSCLC treatment is urgently needed. Our previous work demonstrates that autophagy inhibition is synthetically lethal in KrasG12D/+;Lkb1−/− (KL) mediated tumorigenesis; in contrast to intact autophagy KL lung tumors, loss of an autophagy‐essential gene Atg7 dramatically impaired both tumor initiation and tumor growth. This is in sharp contrast to wild‐type (WT) LKB1 (KrasG12D/+;p53−/−(KP)) tumors that are less sensitive to autophagy gene ablation. These observations indicate that LKB1 mutations predispose KRAS NSCLC to autophagy inhibition and that LKB1 mutations could be explored as a predictive biomarker for precision lung cancer therapy. To further value our discoveries in clinical translational ability, we treated mouse lung tumor derived cell lines (TDCLs) with FDA approved autophagy inhibitor hydroxychloroquine (HCQ) and found that the KL TDCLs were much more sensitive to HCQ‐induced cell death compared with Lkb1‐WT KP TDCLs. Furthermore, a combination treatment of HCQ with mitogen‐activated protein kinase kinase (MAPKK or MEK) inhibitor Trametinib showed synergistic anti‐proliferative effects in KL TDCLs, but not in KP TDCLs. To elucidate the underlying mechanism of increased sensitivity of KL TDCLs to Trametinib by autophagy ablation, we performed metabolomics profiling of KL TDCLs with Trametinib, HCQ alone or in combination treatment. We found that the levels of several glycolytic and TCA cycle intermediates, as well as amino acids were significantly upregulated upon treatment with Trametinib, which were significantly reduced by the combined treatment with HCQ and trametinib. Moreover, Trametinib treatment led to an increased level of ATP, which was decreased by the combination of HCQ and Trametinib. Similarly, we observed anti‐tumor synergistic effects of the combination treatment in KL allograft tumor growth. Our observations suggest that KL TDCLs resist to Trametinib treatment by upregulating autophagy to maintain energy homeostasis for cancer cell survival. Therefore, a combination therapy of autophagy and MEK inhibition could be a novel therapeutic strategy to specifically treat LKB1‐deficient NSCLC.

Support or Funding Information

This research has been supported by the research funds from NIH K22 CA190521 grant, American Cancer Society Early Investigator Pilot Award, Lung Cancer Research Foundation grant and startup funding from Rutgers Cancer Institute of New Jersey.