Antibody-based binding domain fused to TCRγ chain facilitates T cell cytotoxicity for potent anti-tumor response.

Chimeric antigen receptor T-cell (CAR-T) therapy has demonstrated potent clinical efficacy in the treatment of hematopoietic malignancies. However, the application of CAR-T in solid tumors has been limited due in part to the expression of inhibitory molecules in the tumor microenvironment, leading to T-cell exhaustion. To overcome this limitation, we have developed a synthetic T-cell receptor (TCR) that targets programmed death-ligand 1 (PD-L1), a molecule that is widely expressed in various solid tumors and plays a pivotal role in T-cell exhaustion. Our novel TCR platform is based on antibody-based binding domain, which is typically a single-chain variable fragment (scFv), fused to the γδ TCRs (TCRγδ). We have utilized the T-cell receptor alpha constant (TRAC) locus editing approach to express cell surface scFv of anti-PD-L1, which is fused to the constant region of the TCRγ or TCRδ chain in activated T cells derived from peripheral blood mononuclear cells (PBMCs). Our results indicate that these reconfigured receptors, both γ-TCRγδ and δ-TCRγδ, have the capability to transduce signals, produce inflammatory cytokines, degranulate and exert tumor killing activity upon engagement with PD-L1 antigen in vitro. Additionally, we have also shown that γ-TCRγδ exerted superior efficacy than δ-TCRγδ in in vivo xenograft model.

Probing coordinated co-culture cancer related motility through differential micro-compartmentalized elastic substrates.

Cell development and behavior are driven by internal genetic programming, but the external microenvironment is increasingly recognized as a significant factor in cell differentiation, migration, and in the case of cancer, metastatic progression. Yet it remains unclear how the microenvironment influences cell processes, especially when examining cell motility. One factor that affects cell motility is cell mechanics, which is known to be related to substrate stiffness. Examining how cells interact with each other in response to mechanically differential substrates would allow an increased understanding of their coordinated cell motility. In order to probe the effect of substrate stiffness on tumor related cells in greater detail, we created hard-soft-hard (HSH) polydimethylsiloxane (PDMS) substrates with alternating regions of different stiffness (200 and 800 kPa). We then cultured WI-38 fibroblasts and A549 epithelial cells to probe their motile response to the substrates. We found that when the 2 cell types were exposed simultaneously to the same substrate, fibroblasts moved at an increased speed over epithelial cells. Furthermore, the HSH substrate allowed us to physically guide and separate the different cell types based on their relative motile speed. We believe that this method and results will be important in a diversity of areas including mechanical microenvironment, cell motility, and cancer biology.

Determinants of the Inhibition of DprE1 and CYP2C9 by Antitubercular Thiophenes.

Mycobacterium tuberculosis (Mtb) DprE1, an essential isomerase for the biosynthesis of the mycobacterial cell wall, is a validated target for tuberculosis (TB) drug development. Here we report the X-ray crystal structures of DprE1 and the DprE1 resistant mutant (Y314C) in complexes with TCA1 derivatives to elucidate the molecular basis of their inhibitory activities and an unconventional resistance mechanism, which enabled us to optimize the potency of the analogs. The selected lead compound showed excellent in vitro and in vivo activities, and low risk of toxicity profile except for the inhibition of CYP2C9. A crystal structure of CYP2C9 in complex with a TCA1 analog revealed the similar interaction patterns to the DprE1-TCA1 complex. Guided by the structures, an optimized molecule was generated with differential inhibitory activities against DprE1 and CYP2C9, which provides insights for development of a clinical candidate to treat TB.

Capturing wetting states in nanopatterned silicon.

Spectacular progress in developing advanced Si circuits with reduced size, along the track of Moore's law, has been relying on necessary developments in wet cleaning of nanopatterned Si wafers to provide contaminant free surfaces. The most efficient cleaning is achieved when complete wetting can be realized. In this work, ordered arrays of silicon nanopillars on a hitherto unexplored small scale have been used to study the wetting behavior on nanomodulated surfaces in a substantial range of surface treatments and geometrical parameters. With the use of optical reflectance measurements, the nanoscale water imbibition depths have been measured and the transition to the superhydrophobic Cassie-Baxter state has been accurately determined. For pillars of high aspect ratio (about 15), the transition occurs even when the surface is grafted with a hydrophilic functional group. We have found a striking consistent deviation between the contact angle measurements and the straightforward application of the classical wetting models. Molecular dynamics simulations show that these deviations can be attributed to the long overlooked atomic-scale surface perturbations that are introduced during the nanofabrication process. When the transition condition is approached, transient states of partial imbibition that characterize intermediate states between the Wenzel and Cassie-Baxter states are revealed in our experiments.

Generalized potentials for a mean-field density functional theory of a three-phase contact line.

We investigate generalized potentials for a mean-field density functional theory of a three-phase contact line. Compared to the symmetrical potential introduced in our previous article [Phys. Rev. E 85, 011120 (2012)], the three minima of these potentials form a small triangle located arbitrarily within the Gibbs triangle, which is more realistic for ternary fluid systems. We multiply linear functions that vanish at edges and vertices of the small triangle, yielding potentials in the form of quartic polynomials. We find that a subset of such potentials has simple analytic far-field solutions and is a linear transformation of our original potential. By scaling, we can relate their solutions to those of our original potential. For special cases, the lengths of the sides of the small triangle are proportional to the corresponding interfacial tensions. For the case of equal interfacial tensions, we calculate a line tension that is proportional to the area of the small triangle.

Complete Genome Sequence of the Porcine Strain Brachyspira pilosicoli P43/6/78(T.).

Reported herein is the complete genome sequence of Brachyspira pilosicoli strain P43/6/78T, isolated from a pig with clinical disease. This sequence will aid in the study of genome-wide comparison among Brachyspira species.

Mean-field density functional theory of a three-phase contact line.

A three-phase contact line in a three-phase fluid system is modeled by a mean-field density functional theory. We use a variational approach to find the Euler-Lagrange equations. Analytic solutions are obtained in the two-phase regions at large distances from the contact line. We employ a triangular grid and use a successive overrelaxation method to find numerical solutions in the entire domain for the special case of equal interfacial tensions for the two-phase interfaces. We use the Kerins-Boiteux formula to obtain a line tension associated with the contact line. This line tension turns out to be negative. We associate line adsorption with the change of line tension as the governing potentials change.

Toxoplasma gondii triggers release of human and mouse neutrophil extracellular traps.

Neutrophils have recently been shown to release DNA-based extracellular traps that contribute to microbicidal killing and have also been implicated in autoimmunity. The role of neutrophil extracellular trap (NET) formation in the host response to nonbacterial pathogens has received much less attention. Here, we show that the protozoan pathogen Toxoplasma gondii elicits the production of NETs from human and mouse neutrophils. Tachyzoites of each of the three major parasite strain types were efficiently entrapped within NETs, resulting in decreased parasite viability. We also show that Toxoplasma activates a MEK-extracellular signal-regulated kinase (ERK) pathway in neutrophils and that the inhibition of this pathway leads to decreased NET formation. To determine if Toxoplasma induced NET formation in vivo, we employed a mouse intranasal infection model. We found that the administration of tachyzoites by this route induced a rapid tissue recruitment of neutrophils with evidence of extracellular DNA release. Taken together, these data indicate a role for NETs in the host innate response to protozoan infection. We propose that NET formation limits infection by direct microbicidal effects on Toxoplasma as well as by interfering with the ability of the parasite to invade target host cells.

MHC class I presentation and regulation by IFN in bony fish determined by molecular analysis of the class I locus in grass carp.

Beyond their sequences, little is known regarding MHC class I presentation and regulation by IFN in bony fish. In this work, the class I locus (Ctid-UBA) was isolated from a grass carp fosmid library, and its polymorphisms and tissue expression were investigated. The Ctid-UBA and Ctid-beta2-microglobulin genes then were expressed and refolded, and tetramer techniques were used to identify the CTL response. The interaction between grass carp type I IFN and Ctid-UBA genes was investigated. Two fosmids coding for Ctid-UBA *0101 and Ctid-UBA *0201 genes were sequenced. The SXY box and IFN-stimulated regulatory element motifs were located from the start codons to -800 bp in Ctid-UBA. A Southern blot showed three to four bands, suggesting that grass carp contains at least three class I loci. In addition, the Ctid-UBA allelic genes are expressed in all tissue of grass carp. The three-dimensional structure of Ctid-UBA *0102 showed that the peptide-binding domain was formed by the alpha1 and alpha2 domains, which could bind several nonapeptides of grass carp hemorrhagic virus. There were 1.60% more PE-positive cells in P1(QPNEAIRSL)-immunized fish than in blank and adjuvant control groups. Additionally, recombinant grass carp IFN could regulate the expression of Ctid-UBA. These results characterize the class I presentation, CTL response, and regulation by type I IFN in bony fish.

cDNA cloning, genomic structure and expression analysis of the goose (Anser cygnoides) MHC class I gene.

To provide data for studies on avian disease resistance, goose MHC class I cDNA (Ancy-MHC I) was cloned from a goose cDNA library, it's genomic structure and expression analysis were investigated. The mature peptides of Ancy-MHC I cDNA encoded 333 amino acids. The genomic organization is composed of eight exons and seven introns. Based on the genetic distance, six Ancy-MHC I genes from six individuals can be classified into four lineages. A total of nineteen amino acid positions in peptide-binding domain showed high scores by Wu-kabat index analysis. The Ancy-MHC I amino acid sequence displayed seven critical HLA-A2 amino acids that bind with antigen polypeptides, and have an 85.4-98.9% amino acid homology with each genes, and a 59.8-66.0% amino acid homology with chicken MHC class I. Expression analyses using Q-RT-PCR to detect the tissue-specific expression of Ancy-MHC I mRNA in an adult goose. The result appeared that Ancy-MHC I cDNA was expressed in the liver, spleen, intestine, kidney, lung, pancreas, heart, brain, and skin. The phylogenetic tree appears to branch in an order consistent with accepted evolutionary pathways.