Preparation of Monoclonal Antibody and Development of Indirect Competitive Enzyme-Linked Immunosorbent Assay and Fluorescence-Linked Immunosorbent Assay for Detecting 3-Amino-5-methylmorpholino-2-oxazolidinone (AMOZ) in Edible Animal Tissue.

To monitor the illegal used of furaltadone, a highly sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and fluorescence-linked immunosorbent assay (FLISA) based on a monoclonal antibody (mAb) were developed for the detection of 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ), the major metabolite of furaltadone in animal tissues. The highly specific mAb, which was very sensitive to a nitrophenyl derivative of AMOZ (2-NP-AMOZ) with IC50 values of 0.11 and 0.09 ng/mL for ic-ELISA and FLISA, respectively, was selected for the development of immunoassays. For both the ic-ELISA and FLISA for AMOZ-spiked experiments, acceptable recovery rates of 81.1-105.3% and coefficients of variation of 4.7-9.8% were obtained. In addition, results from both ic-ELISA and FLISA methods for spiked samples' data showed excellent correlation coefficients ranging from 0.9652 to 0.9927. Meanwhile, the proposed ic-ELISA and FLISA for thirty spiked samples were confirmed by standard LC-MS/MS with high correlation coefficients of 0.9911 and 0.9921, respectively. These results suggest that the developed ic-ELISA and FLISA are valid and cost-effective tools for high-throughput monitoring methods for AMOZ residues in animal tissues.

Morpholinos Do Not Elicit an Innate Immune Response during Early Xenopus Embryogenesis.

It has recently been reported that a common side effect of translation-blocking morpholino antisense oligonucleotides is the induction of a set of innate immune response genes in Xenopus embryos and that splicing-blocking morpholinos lead to unexpected off-target mis-splicing events. Here, we present an analysis of all publicly available Xenopus RNA sequencing (RNA-seq) data in a reexamination of the effects of translation-blocking morpholinos on the innate immune response. Our analysis does not support the authors' general conclusion, which was based on a limited number of RNA-seq datasets. Moreover, the strong induction of an immune response appears to be specific to the tbxt/tbxt2 morpholinos. The more comprehensive study presented here indicates that using morpholinos for targeted gene knockdowns remains of considerable value for the rapid identification of gene function.

Tau Oligomer Pathology in Nucleus Basalis Neurons During the Progression of Alzheimer Disease.

Although tau is the primary constituent of neurofibrillary tangles (NFTs), evidence suggests that its toxic moiety is oligomeric in Alzheimer disease (AD). In this regard, tau oligomers correlate more strongly with neuronal loss than NFTs and exhibit neurotoxicity in preclinical AD models. Here, we investigated the spatiotemporal progression of oligomeric tau accumulation within the highly vulnerable cholinergic neurons of the nucleus basalis of Meynert (nbM) in AD. Tissue from subjects who died with a clinical diagnosis of no cognitive impairment, mild cognitive impairment, or AD was immunostained with the tau oligomeric complex 1 (TOC1) antibody, a marker of tau oligomers, and p75NTR, a cholinergic cell marker. Stereological estimates revealed a significant increase in the number of TOC1 nbM immunopositive (+) neurons with a concomitant decrease in p75NTR+ nbM neurons during the transition from mild cognitive impairment to AD. Immunofluorescence identified TOC1+ neurons that colocalized with the pretangle tau marker phospho-Ser422, which persisted into late stage NFTs immunoreactive for MN423. Analysis of the nbM subfields revealed a topographical caudal to rostral gradient of TOC1+ neurons during disease progression. Taken together, these data suggest that toxic tau oligomers accumulate caudorostrally in selectively vulnerable nbM neurons during the onset of AD.

Intraperitoneal injection is not always a suitable alternative to intravenous injection for radiotherapy.

Abstract Intraperitoneal (IP) injection is frequently reported to be as effective as intravenous (IV) injection. Because it allows administering a larger volume with more radioactivity, we have investigated this route and the possibility of using it to circumvent the volume constraint we earlier experienced with pretargeting radiotherapy. Using (99m)Tc as the label, the pharmacokinetics (PK) of the cMORF effector (a DNA analogue) was evaluated after IP or IV injection in normal mice by necropsy and SPECT/CT imaging. In another experiment, nude mice bearing tumors were used and they received MORF-CC49 pretargeting antibody IV 2 days earlier than labeled cMORF IV or IP. Tumor accumulations of cMORF were measured at 6 hours after its injections. The absorbed radiation doses for (188)Re or (90)Y pretargeting were estimated using the (99m)Tc data and a self-absorbed model. Although the absorbed radiation doses to other organs were comparable, the dose to intestines after IP injection was 30-fold higher than IV injection due to the slow entry into the circulation. It had reached such a level as high as the dose to the kidneys that cleared the radioactivity and usually were at the highest level. Nevertheless, the slow entry did not reduce the tumor accumulation. In conclusion, using IP in place of IV led to an unacceptably high absorbed radiation dose to the intestines although the tumor accumulation was not compromised. This effect may be applicable to other radiotherapeutic agents as well.

Inhibition of primary effusion lymphoma engraftment in SCID mice by morpholino oligomers against early lytic genes of Kaposi's sarcoma-associated herpesvirus.

BACKGROUND: Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with several malignant diseases, including Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman's disease. The objectives of this study were to investigate the use of peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) against KSHV early lytic genes and to assess their efficacy in severe combined immunodeficiency disease (SCID) mice against PEL engraftment. PPMOs are short, single-stranded DNA analogues that contain a backbone of morpholine rings and phosphorodiamidate linkages and have high delivery efficiency into cells. METHODS: PEL cells were treated with PPMOs against viral interferon regulatory factor 1 (vIRF-1) and expression of vIRF-1 was analysed. PPMOs against vIRF-1 and viral interleukin-6 (vIL-6) were evaluated against PEL cell engraftment in SCID mice. The PPMOs were incubated with BCBL-1 cells and then introduced into the peritoneal cavities of SCID mice, followed by 9 more doses of PPMOs administered at 2-day intervals. At weeks 3 and 9 after BCBL-1 delivery, peritoneal lavage was collected and the ratio of PEL cells among total cells was determined by flow cytometry analysis. RESULTS: Treatment of PEL cells with PPMOs against vIRF-1 led to a reduction of vIRF-1 expression in a dose-dependent manner. Reduction of vIRF-1 expression resulted in higher levels of cellular interferon regulatory factor 3 and of signal transducer and activator of transcription 1. SCID mice receiving a PPMO against vIL-6 had no engraftment of PEL cells and remained healthy throughout the 120-day study. CONCLUSIONS: This study showed that PPMOs can be effective antiviral agents against KSHV. Blocking the expression of early lytic genes might be beneficial for the control of KSHV-associated malignant diseases.