58 research outputs found
Binding energies of water to lithiated valine: formation of solution-phase structure in vacuo
No full textAbstractDissociation kinetics for loss of a water molecule from hydrated ions of lithiated valine, alanine ethyl ester and betaine are determined using blackbody infrared radiative dissociation at temperatures between −60 and 110 °C. From master equation modeling of these data, values of the threshold dissociation energy are obtained for clusters containing one through three water molecules. By comparing the values for valine with its two isomers, one a model for the nonzwitterion structure, the other a model for the zwitterion structure, information about the structure of valine in these hydrated clusters is inferred. Structures, relative energies, and water binding energies for these ions are also calculated at the B3LYP/6-31++G** level of theory. With one water molecule, both experiment and theory indicate that valine is not a zwitterion and that the lithium ion coordinates with the amino nitrogen and the carbonyl oxygen (NO coordinated) and the water molecule interacts directly with the lithium ion. With two water molecules, the zwitterion and nonzwitterion structures are nearly isoenergetic, but the experiment clearly indicates a NO-coordinated nonzwitterion structure. With three water molecules, both the experimental data and theory indicate that the lithium ion binds to the carboxylate group of valine, i.e., valine is zwitterionic with three water molecules. The agreement between the experimentally determined and calculated binding energies is good for all the clusters, with deviations of ≤ 0.12 eV
Extracellular vesicles released by host epithelial cells during Pseudomonas aeruginosa infection function as homing beacons for neutrophils
No full textAbstract Background Pseudomonas aeruginosa (PA) is an opportunistic pathogen that can cause sight threatening infections in the eye and fatal infections in the cystic fibrosis airway. Extracellular vesicles (EVs) are released by host cells during infection and by the bacteria themselves; however, there are no studies on the composition and functional role of host-derived EVs during PA infection of the eye or lung. Here we investigated the composition and capacity of EVs released by PA infected epithelial cells to modulate innate immune responses in host cells. Methods Human telomerase immortalized corneal epithelial cells (hTCEpi) cells and human telomerase immortalized bronchial epithelial cells (HBECs) were treated with a standard invasive test strain of Pseudomonas aeruginosa, PAO1, for 6 h. Host derived EVs were isolated by qEV size exclusion chromatography. EV proteomic profiles during infection were compared using mass spectrometry and functional studies were carried out using hTCEpi cells, HBECs, differentiated neutrophil-like HL-60 cells, and primary human neutrophils isolated from peripheral blood. Results EVs released from PA infected corneal epithelial cells increased pro-inflammatory cytokine production in naïve corneal epithelial cells and induced neutrophil chemotaxis independent of cytokine production. The EVs released from PA infected bronchial epithelial cells were also chemotactic although they failed to induce cytokine secretion from naïve HBECs. At the proteomic level, EVs derived from PA infected corneal epithelial cells exhibited lower complexity compared to bronchial epithelial cells, with the latter having reduced protein expression compared to the non-infected control. Conclusions This is the first study to comprehensively profile EVs released by corneal and bronchial epithelial cells during Pseudomonas infection. Together, these findings show that EVs released by PA infected corneal and bronchial epithelial cells function as potent mediators of neutrophil migration, contributing to the exuberant neutrophil response that occurs during infection in these tissues
Unraveling the energy dependence in large ΔE (V→RT) energy transfer: Separation of ΔE and probability in the collisional relaxation of highly vibrationally excited pyrazine (Evib=36 000 to 41 000 cm−1) by CO2
No full textAbstract 535: Selected reaction monitoring (SRM)-based assay for the quantification of biomarkers in human breast cancer tissue
No full textAuthor response
No full textCholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors using its B subunit (CTB). The ganglioside (glycolipid) GM1 is thought to be the sole CT receptor; however, the mechanism by which CTB binding to GM1 mediates internalization of CT remains enigmatic. Here we report that CTB binds cell surface glycoproteins. Relative contributions of gangliosides and glycoproteins to CTB binding depend on cell type, and CTB binds primarily to glycoproteins in colonic epithelial cell lines. Using a metabolically incorporated photocrosslinking sugar, we identified one CTB-binding glycoprotein and demonstrated that the glycan portion of the molecule, not the protein, provides the CTB interaction motif. We further show that fucosylated structures promote CTB entry into a colonic epithelial cell line and subsequent host cell intoxication. CTB-binding fucosylated glycoproteins are present in normal human intestinal epithelia and could play a role in cholera.</p
PRMT5 is an actionable therapeutic target in CDK4/6 inhibitor-resistant ER+/RB-deficient breast cancer
No full textAbstract CDK4/6 inhibitors (CDK4/6i) have improved survival of patients with estrogen receptor-positive (ER+) breast cancer. However, patients treated with CDK4/6i eventually develop drug resistance and progress. RB1 loss-of-function alterations confer resistance to CDK4/6i, but the optimal therapy for these patients is unclear. Through a genome-wide CRISPR screen, we identify protein arginine methyltransferase 5 (PRMT5) as a molecular vulnerability in ER+/RB1-knockout breast cancer cells. Inhibition of PRMT5 blocks the G1-to-S transition in the cell cycle independent of RB, leading to growth arrest in RB1-knockout cells. Proteomics analysis uncovers fused in sarcoma (FUS) as a downstream effector of PRMT5. Inhibition of PRMT5 results in dissociation of FUS from RNA polymerase II, leading to hyperphosphorylation of serine 2 in RNA polymerase II, intron retention, and subsequent downregulation of proteins involved in DNA synthesis. Furthermore, treatment with the PRMT5 inhibitor pemrametostat and a selective ER degrader fulvestrant synergistically inhibits growth of ER+/RB-deficient cell-derived and patient-derived xenografts. These findings highlight dual ER and PRMT5 blockade as a potential therapeutic strategy to overcome resistance to CDK4/6i in ER+/RB-deficient breast cancer
Hydration of Valine−Cation Complexes in the Gas Phase: On the Number of Water Molecules Necessary to Form a Zwitterion
No full textThe stepwise hydration of valine−alkali metal ion complexes, Val·M+(H2O)n, n = 2−6, M = Li, Na, and K,
is investigated using both theory and experiment. Experimentally, the rate of water loss from the valine clusters
is measured using blackbody infrared radiative dissociation. The kinetics for the loss of one water molecule
from these clusters are compared to those from model clusters of known zwitterionic vs nonzwitterionic
structure. Both theory and experiment indicate that the structure of Val·Li+(H2O)2 is very similar to that of
the singly and nonhydrated complexes investigated previously; the lithium is coordinated between the nitrogen
and carbonyl oxygen of nonzwitterionic valine, and the water molecules interact solely with the metal ion.
The third water molecule changes the structure of the Val·Li+ cluster significantly. The metal ion coordinates
to the C-terminal end of zwitterionic valine and to two of the water molecules. The third water molecule
hydrogen bonds to the protonated N terminus of valine. Thus, the third water molecule is the first one that
interacts directly with the valine, and this stabilizes the zwitterionic form of valine over the nonzwitterionic
form. The dissociation of the sixth water molecule from the valine cluster is slower than that of the fifth,
indicating that the cluster with six waters is especially stable relative to the cluster with five water molecules.
This provides further support for zwitterionic valine in the presence of only a limited number of water molecules.
For M = Na, two water molecules changes the metal binding position from NO coordination to the C terminus
of valine. The experiment is unable to distinguish the zwitterionic vs nonzwitterionic character of valine in
this complex, but theory indicates the nonzwitterion form. As is the case with lithiated clusters, Val·Na+(H2O)6 is more stable than Val·Na+(H2O)5. Computational results for M = K predict that the most stable
conformation of Val·K+(H2O)2 resembles Val·Na+(H2O)2, whereas the kinetic data for the sodiated and
potassiated clusters, although inconclusive, suggest the zwitterion form. The stepwise hydration studies presented
here indicate that very few water molecules are necessary to cause valine to adopt its solution-phase zwitterionic
structure
Hydration of Valine−Cation Complexes in the Gas Phase: On the Number of Water Molecules Necessary to Form a Zwitterion
No full textThe stepwise hydration of valine−alkali metal ion complexes, Val·M+(H2O)n, n = 2−6, M = Li, Na, and K,
is investigated using both theory and experiment. Experimentally, the rate of water loss from the valine clusters
is measured using blackbody infrared radiative dissociation. The kinetics for the loss of one water molecule
from these clusters are compared to those from model clusters of known zwitterionic vs nonzwitterionic
structure. Both theory and experiment indicate that the structure of Val·Li+(H2O)2 is very similar to that of
the singly and nonhydrated complexes investigated previously; the lithium is coordinated between the nitrogen
and carbonyl oxygen of nonzwitterionic valine, and the water molecules interact solely with the metal ion.
The third water molecule changes the structure of the Val·Li+ cluster significantly. The metal ion coordinates
to the C-terminal end of zwitterionic valine and to two of the water molecules. The third water molecule
hydrogen bonds to the protonated N terminus of valine. Thus, the third water molecule is the first one that
interacts directly with the valine, and this stabilizes the zwitterionic form of valine over the nonzwitterionic
form. The dissociation of the sixth water molecule from the valine cluster is slower than that of the fifth,
indicating that the cluster with six waters is especially stable relative to the cluster with five water molecules.
This provides further support for zwitterionic valine in the presence of only a limited number of water molecules.
For M = Na, two water molecules changes the metal binding position from NO coordination to the C terminus
of valine. The experiment is unable to distinguish the zwitterionic vs nonzwitterionic character of valine in
this complex, but theory indicates the nonzwitterion form. As is the case with lithiated clusters, Val·Na+(H2O)6 is more stable than Val·Na+(H2O)5. Computational results for M = K predict that the most stable
conformation of Val·K+(H2O)2 resembles Val·Na+(H2O)2, whereas the kinetic data for the sodiated and
potassiated clusters, although inconclusive, suggest the zwitterion form. The stepwise hydration studies presented
here indicate that very few water molecules are necessary to cause valine to adopt its solution-phase zwitterionic
structure
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