Covalent Chemical Ligation Strategy for Mono- and Polyclonal Immunoglobulins at Their Nucleotide Binding Sites.

Nonspecific ligation methods have been traditionally used to chemically modify immunoglobulins. Site-specific ligation of compounds (toxins or ligands) to antibodies has become increasingly important in the fields of therapeutic antibody-drug conjugates and bispecific antibodies. In this present study, we took advantage of the reported nucleotide-binding pocket (NBP) in the Fab arms of immunoglobulins by developing indole-based, 5-fluoro-2,4-dinitrobenzene-derivatized OBOC peptide libraries for the identification of affinity elements that can be used as site-specific derivatization agents against both mono- and polyclonal antibodies. Ligation can occur at any one of the few lysine residues located at the NBP. Immunoconjugates resulting from such affinity elements can be used as therapeutics against cancer or infectious agents.

Versatile Nanoring Fabrication Assisted by Hole-mask Colloidal Lithography.

Nanomaterials shaped as rings are interesting nanostructures with control of the materials properties at the nanoscale. Nanoring plasmonic resonators provide tunable optical resonances in the near-infrared with application in sensing. Fabrication of nanorings can be carried out via top-down approaches based on electron beam lithography with high control of the ring size parameters but at high cost. Alternatively, fabrication via self-assembly approaches has a higher speed/lower cost but at the cost of control of ring parameters. Current colloidal lithography approaches can provide nanoring fabrication over large areas but only of specific materials and a select set of rings (large ring diameters or small rings with ultrathin walls). We extend Hole-mask Colloidal Lithography to use ring shaped holes, allow the deposition of arbitrary materials, and allow the independent tuning of ring-wall thickness over a large range of values. We present a generic approach for the fabrication of nanorings formed from a range of materials including low cost (e.g., Cu, Al) and nonplasmonic (e.g., W) materials and with control of ring wall thickness and diameter allowing tuning of ring parameters and materials for applications in nanooptics and beyond.

Aloe Vera Extract Gel-Biosynthesized Selenium Nanoparticles Enhance Disease Resistance in Lettuce by Modulating the Metabolite Profile and Bacterial Endophytes Composition.

The use of nanotechnology to suppress crop diseases has attracted significant attention in agriculture. The present study investigated the antifungal mechanism by which aloe vera extract gel-biosynthesized (AVGE) selenium nanoparticles (Se NPs) suppressed Fusarium-induced wilt disease in lettuce (Lactuca sativa). AVGE Se NPs were synthesized by utilizing sodium selenite as a Se source and AVGE as a biocompatible capping and reducing agent. Over 21 d, 2.75% of total AVGE Se NPs was dissolved into Se ions, which was more than 8-fold greater than that of bare Se NPs (0.34%). Upon exposure to soil applied AVGE Se NPs at 50 mg/kg, fresh shoot biomass was significantly increased by 61.6 and 27.8% over the infected control and bare Se NPs, respectively. As compared to the infected control, the shoot levels of citrate, isocitrate, succinate, malate, and 2-oxo-glutarate were significantly upregulated by 0.5-3-fold as affected by both Se NPs. In addition, AVGE Se NPs significantly increased the shoot level of khelmarin D, a type of coumarin, by 4.40- and 0.71-fold over infected controls and bare Se NPs, respectively. Additionally, AVGE Se NPs showed greater upregulation of jasmonic acid and downregulation of abscisic acid content relative to bare Se NPs in diseased shoots. Moreover, the diversity of bacterial endophytes was significantly increased by AVGE Se NPs, with the values of Shannon index 40.2 and 9.16% greater over the infected control and bare Se NPs. Collectively, these findings highlight the significant potential of AVGE Se NPs as an effective and biocompatible strategy for nanoenabled sustainable crop protection.

Examining the efficacy of vibrotactile displays for monitoring patient vital signs: Six laboratory studies of change detection and state identification.

Healthcare workers often monitor patients while moving between different locations and tasks, and away from conventional monitoring displays. Vibrotactile displays can provide patient information in vibrotactile patterns that are felt regardless of the worker's location. We examined how effectively participants could identify changes in vibrotactile representations of patient heart rate (HR) and oxygen saturation (SpO2). In Experiment 1, participants identified changes in HR and SpO2 with greater than 90% accuracy while using vibrotactile displays configured in either an integrated or a separated format. In Experiment 2, incidental auditory and visual cues were removed and performance was still greater than 90% for the integrated display. In Experiments 3 and 4, ongoing tasks with low or high task load were introduced; high load worsened participants' response accuracy and speed at identifying vital signs. In Experiments 5 and 6, alternative designs were tested, including a design with a seemingly more natural mapping of HR to vibrotactile stimulation. However, no design supported more accurate performance than the integrated display. Results are interpreted with respect to multiple resource theory, and constraints on conforming to design guidelines are noted. Vibrotactile displays appear to be viable and therefore potentially suitable for use in healthcare and other contexts. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

TGF-ß1 prevents up-regulation of the P2X7 receptor by IFN-γ and LPS in leukemic THP-1 monocytes.

The P2X7 receptor is an extracellular ATP-gated cation channel critical in inflammation and immunity, and can be up-regulated by IFN-γ and LPS. This study aimed to examine the effect of TGF-ß1 on the up-regulation of P2X7 function and expression in leukemic THP-1 monocytes differentiated with IFN-γ and LPS. Cell-surface molecules including P2X7 were examined by immunofluorescence staining. Total P2X7 protein and mRNA was assessed by immunoblotting and RT-PCR respectively. P2X7 function was evaluated by ATP-induced cation dye uptake measurements. Cell-surface P2X7 was present on THP-1 cells differentiated for 3days with IFN-γ and LPS but not on undifferentiated THP-1 cells. ATP induced ethidium(+) uptake into differentiated but not undifferentiated THP-1 cells, and the P2X7 antagonist, KN-62, impaired ATP-induced ethidium(+) uptake. Co-incubation of cells with TGF-ß1 plus IFN-γ and LPS prevented the up-regulation of P2X7 expression and ATP-induced ethidium(+) uptake in a concentration-dependent fashion with a maximum effect at 5ng/ml and with an IC(50) of ~0.4ng/ml. Moreover, ATP-induced YO-PRO-1(2+) uptake and IL-1ß release were abrogated in cells co-incubated with TGF-ß1. TGF-ß1 also abrogated the amount of total P2X7 protein and mRNA induced by IFN-γ and LPS. Finally, TGF-ß1 prevented the up-regulation of cell-surface CD86, but not CD14 and MHC class II, by IFN-γ and LPS. These results indicate that TGF-ß1 prevents the up-regulation of P2X7 function and expression by IFN-γ and LPS in THP-1 monocytes. This suggests that TGF-ß1 may limit P2X7-mediated processes in inflammation and immunity.

Murine epidermal Langerhans cells and keratinocytes express functional P2X7 receptors.

Extracellular ATP via the activation of purinergic P2 receptors has an emerging role in cutaneous biology; however, the distribution of these receptors in mouse skin is poorly defined. This study investigated whether murine epidermal cell subpopulations express functional purinergic P2X(7) receptors. P2X(7) expression was examined by immunoblotting and immunofluorescence staining of epidermal cells from C57Bl/6 mice. P2X(7) function was evaluated by nucleotide-induced ethidium(+) uptake measurements in epidermal cells from C57Bl/6 mice, and from P2X(7) deficient mice and wild-type littermate controls. P2X(7) was detected in whole epidermal cell preparations, and specifically on Langerhans cells (LCs) and keratinocytes (KCs). ATP induced ethidium(+) uptake into LCs and KCs, with EC(50) values of 503 and 482 microm, respectively. BzATP, and to a lesser extent ATPgammaS and ADP, also induced ethidium(+) uptake; while UTP, alphabeta-meth-ATP and NAD were ineffective. ATP-induced ethidium(+) uptake was impaired by Na(+) and Mg(2+), and the P2X(7) antagonist, A-438079 and was absent in LCs and KCs from P2X(7) deficient mice. These results demonstrate that murine LCs and KCs express functional P2X(7), and support a role for this receptor in cutaneous biology.

Formulation optimization of an ephrin A2 targeted immunoliposome encapsulating reversibly modified taxane prodrugs.

Ephrin A2 targeted immunoliposomes incorporating pH-sensitive taxane prodrugs were developed for sustained delivery of active drug to solid tumors. Here we describe the systematic formulation development and characterization of these immunoliposomes. We synthesized both paclitaxel and docetaxel prodrugs to formulate as ephrin A2-targeted liposomes stabilized in the aqueous core with sucroseoctasulfate (SOS). The optimized lipid formulation was comprised of egg-sphingomyelin, cholesterol, and polyethylene glycol distearoyl glycerol (PEG-DSG). The formulations examined had a high efficiency of prodrug encapsulation (as high as 114 mol% taxane per mole phospholipid) and subsequent stability (>3 years at 2-8 °C). The taxane prodrug was stabilized with extraliposomal citric acid and subsequently loaded into liposomes containing a gradient of SOS, resulting in highly stable SOS-drug complexes being formed inside the liposome. The internal prodrug and SOS concentrations were optimized for their impact on in vivo drug release and drug degradation. Cryo-electron microscope images revealed dense prodrug-SOS complex in the aqueous core of the immunoliposomes. Ephrin A2-targeted taxane liposomes exhibited sub-nanomolar (0.69 nM) apparent equilibrium dissociation constant toward the extracellular domain of the ephrin A2 receptor, long circulation half-life (8-12 h) in mouse plasma, a release rate dependent on intraliposomal drug concentration and stable long-term storage. At an equitoxic dose of 50 mg taxane/kg, ephrin A2-targeted liposomal prodrug showed greater antitumor activity than 10 mg/kg of docetaxel in A549 non-small cell lung, as well as MDA-MB-436 and SUM149 triple negative breast cancer xenograft models. The lead molecule entered a Phase I clinical trial in patients with solid tumors (NCT03076372).

Lentiviral vectors mediate stable and efficient gene delivery into primary murine natural killer cells.

Natural killer (NK) cells are lymphocytes that provide an important line of defense against many types of microorganisms, viruses and tumors. The development of an efficient gene transfer system for genetically modifying primary murine NK cells will facilitate the studies of NK cell differentiation, acquisition of self-tolerance, and induction of anti-tumor responses. In this study we used an enhanced green fluorescent protein (EGFP)-expressing vector to carry out a systematic evaluation of the efficiency of lentiviral transduction of primary murine NK cells with or without prior interleukin-2 (IL-2) activation. In a single-step transduction protocol, we demonstrated that human immunodeficiency virus type 1-based lentiviral vectors support an average of 40% transduction efficiency on primary NK cells. These genetically modified NK cells are found to maintain stable EGFP transgene expression in vitro, and can be further expanded in IL-2 supplemented culture medium. Lentiviral transduction does not affect NK surface phenotypes or functions (apoptosis, cytokine production and cytotoxicity). We further demonstrated efficient gene transfer into differentiating NK cells derived from the lentiviral-transduced murine hematopoietic progenitor cells in vitro. This study therefore establishes a simple and efficient approach to the genetic engineering of primary murine NK cells, and will prove useful in studying basic NK cell biology and in exploring the therapeutic potential of NK cells in inbred and transgenic mouse models.

Atypical chronic myeloid leukemia in a German Shepherd Dog.

A 4-y-old neutered male German Shepherd Dog was presented with a 3-d duration of lethargy, restlessness, and vomiting. Physical examination revealed generalized lymphadenopathy, pale mucous membranes, systolic heart murmur, dehydration, and fever. Hematologic abnormalities included moderate-to-marked leukocytosis, characterized by neutrophilia with a left shift to progranulocytes and 2% presumptive myeloid blasts, marked anemia that was nonregenerative, and marked thrombocytopenia. Dysplasia was evident in neutrophils and platelets. Bone marrow examination revealed marked myeloid and megakaryocytic hyperplasia with 7% blasts, erythroid hypoplasia, and trilineage dysplasia. Flow cytometric analysis confirmed that bone marrow cells were mostly of neutrophil lineage, with reduced expression of common leukocyte antigens (CD45, CD18) and neutrophil-specific antigen. Bone marrow cells were cytogenetically analyzed for the breakpoint cluster region-Abelson oncogene using multicolor fluorescent in situ hybridization. The genetic aberration was present in 7% of cells, which was a negative result (>10% of cells is considered positive). Euthanasia was elected. Histologic examination showed extensive infiltration of multiple organs by neoplastic myeloid cells, with effacement of lymph node and splenic architecture. The final diagnosis was atypical chronic myeloid leukemia (aCML), an uncommon myeloproliferative disorder with features of myelodysplastic syndromes (dysplasia) and chronic leukemia (neutrophilic leukocytosis with <20% marrow blasts, extramedullary infiltrates). The trilineage dysplasia, lack of monocytosis, and supporting cytogenetics distinguish aCML from CML, chronic neutrophilic leukemia, and chronic myelomonocytic leukemia.

Discovery and characterization of a high-affinity and high-specificity peptide ligand LXY30 for in vivo targeting of α3 integrin-expressing human tumors.

BACKGROUND: α3ß1 integrin is overexpressed in several types of human cancer and is associated with poor prognosis, metastasis, and resistance to cancer treatment. We previously identified a cyclic peptide ligand LXY1 that specifically binds to the α3ß1 integrin on human glioblastoma U-87MG cells. Here, we optimized LXY1 through one-bead one-compound combinatorial library screening and site-specific modifications to improve its in vivo binding property. METHODS: Three bead libraries were synthesized and whole-cell binding assays were performed. The binding capacity of individual peptide ligands against different tumor cells was determined by flow cytometry and confirmed by optical imaging. A complex joining biotinylated ligand with streptavidin-Cy5.5 was used for in vivo target imaging in both subcutaneous and orthotopic U-87MG xenograft mouse models. RESULTS: LXY30, a cyclic peptide with the sequence cdG-Phe(3,5-diF)-G-Hyp-NcR, emerged as the most potent and selective ligand for the α3 subunit of α3ß1 integrin with improved in vitro and in vivo tumor-targeting effects compared to LXY1 in U-87MG cells. LXY30 is considerably stable in plasma as demonstrated in an in vitro stability study in 90 % human plasma. LXY30 also binds to several other known α3ß1 integrin-expressing glioblastoma, lung, and breast cancer cell lines with various affinities. CONCLUSIONS: Our data support further investigating the role of LXY30 as a human tumor-targeting peptide ligand for systemic and intracranial delivery of imaging agents and cancer therapeutics.

SHP-1 phosphatase is a critical regulator in preventing natural killer cell self-killing.

Balance of signals generated from the engaged activating and inhibitory surface receptors regulates mature NK cell activities. The inhibitory receptors signal through immunoreceptor tyrosine based inhibitory motifs (ITIM), and recruit phosphatases such as SHP-1 to inhibit NK cell activation. To directly examine the importance of SHP-1 in regulating activities and cell fate of mature NK cells, we used our established lentiviral-based engineering protocol to knock down the SHP-1 protein expression in primary C57BL/6NCrl cells. Gene silencing of the SHP-1 in primary NK cells abrogated the ability of ITIM-containing NK inhibitory receptors to suppress the activation signals induced by NK1.1 activating receptors. We followed the fates of stably transduced SHP-1 silenced primary NK cells over a longer period of time in IL-2 containing cultures. We observed an impaired IL-2 induced proliferation in the SHP-1 knockdown NK cells. More interestingly, these "de-regulated" SHP-1 knockdown NK cells mediated specific self-killing in a real-time live cell microscopic imaging system we developed to study NK cell cytotoxicity in vitro. Selective target recognition of the SHP-1 knockdown NK cells revealed also possible involvement of the SHP-1 phosphatase in regulating other NK functions in mature NK cells.

Altering the specificity of NK:target cell interactions by genetic manipulation of NK receptor expression on primary mouse NK cells.

A balance of signals generated via stimulatory and inhibitory NK receptors determines both target cell specificity and the outcome of NK-target cell interactions. The feasibility of introducing naturally occurring or genetically engineered chimeric NK receptors at the effector cell level may prove useful in NK cell-based immunotherapies. Here, we utilized a previously established lentiviral transduction system to over-express a model NKR-P1B inhibitory receptor on primary mouse NK cells. These genetically engineered NK cells became more sensitive to inhibitory signals delivered by target cells expressing the cognate NKR-P1B ligand, Ocil/Clr-b. This study demonstrated the utility of lentiviral vectors as a means to stably manipulate the target cell specificity of primary NK cells.