p16INK4A flow cytometry of exfoliated cervical cells: Its role in quantitative pathology and clinical diagnosis of squamous intraepithelial lesions.

BACKGROUND: P16INK4A is a surrogate signature compensating for the specificity and/or sensitivity deficiencies of the human papillomavirus (HPV) DNA and Papanicolaou smear (Pap) co-test for detecting high-grade cervical squamous intraepithelial lesions or worse (HSIL+). However, traditional p16INK4A immunostaining is labour intensive and skill demanding, and subjective biases cannot be avoided. Herein, we created a high-throughput, quantitative diagnostic device, p16INK4A flow cytometry (FCM) and assessed its performances in cervical cancer screening and prevention. METHODS: P16INK4A FCM was built upon a novel antibody clone and a series of positive and negative (p16INK4A -knockout) standards. Since 2018, 24 100-women (HPV-positive/-negative, Pap-normal/-abnormal) have been enrolled nationwide for two-tier validation work. In cross-sectional studies, age- and viral genotype-dependent expression of p16INK4A was investigated, and optimal diagnostic parameter cut-offs (using colposcopy and biopsy as a gold standard) were obtained. In cohort studies, the 2-year prognostic values of p16INK4A were investigated with other risk factors by multivariate regression analyses in three cervicopathological conditions: HPV-positive Pap-normal, Pap-abnormal biopsy-negative and biopsy-confirmed LSIL. RESULTS: P16INK4A FCM detected a minimal ratio of 0.01% positive cells. The p16INK4A -positive ratio was 13.9 ± 1.8% among HPV-negative NILM women and peaked at the ages of 40-49 years; after HPV infection, the ratio increased to 15.1 ± 1.6%, varying with the carcinogenesis of the viral genotype. Further increments were found in women with neoplastic lesions (HPV-negative: 17.7 ± 5.0-21.4 ± 7.2%; HPV-positive: 18.0 ± 5.2-20.0 ± 9.9%). Extremely low expression of p16INK4A was observed in women with HSILs. As the HPV-combined double-cut-off-ratio criterion was adopted, a Youden's index of 0.78 was obtained, which was significantly higher than that (0.72) of the HPV and Pap co-test. The p16INK4A -abnormal situation was an independent HSIL+ risk factor for 2-year outcomes in all three cervicopathological conditions investigated (hazard ratios: 4.3-7.2). CONCLUSIONS: FCM-based p16INK4A quantification offers a better choice for conveniently and precisely monitoring the occurrence of HSIL+ and directing risk-stratification-based interventions.

Construction of a dual-responsive dual-drug delivery platform based on the hybrids of mesoporous silica, sodium hyaluronate, chitosan and oxidized sodium carboxymethyl cellulose.

An intelligent drug delivery platform based on the hybrids of mesoporous silica nanoparticles (MSN), sodium hyaluronate (HA), chitosan (CS) and oxidized sodium carboxymethyl cellulose (oxCMC) is developed, which can be used for dual-responsive dual-drug delivery. Hydrophilic cytarabine (Cyt) is first loaded into the mesopores of the aminated MSN (NH2-MSN), which is encapsulated by the hydrogel of HA and cystamine (Cys) crosslinked via amidation. The Cyt encapsulated hydrogel which is denoted as Cyt/NH2-MSN/HA is co-encapsulated with hydrophobic methotrexate (MTX) into the hydrogel of CS and oxCMC resulted from Schiff base reaction. Since the acylhydrazone bonds (-HC=N-) between CS and oxCMC are sensitive to pH and the disulfide bonds (-S-S-) in Cys are sensitive to glutathione (GSH), the resultant dual-drug encapsulated hydrogel, denoted as Cyt/NH2-MSN/HA/MTX/CS/oxCMC, can be used for dual-responsive (pH and GSH) drug delivery. The results of cell viability demonstrate that the developed dual-drug encapsulated hydrogel has significantly higher efficacy of chemotherapy than that of single-drug (MTX or Cyt) encapsulated hydrogel.

Dual stimuli-responsive nanoplatform based on core-shell structured graphene oxide/mesoporous silica@alginate.

A dual stimuli-responsive nanoplatform was rationally designed for controlled drug delivery. The nanosheets of graphene oxide (GO) were first modified with aminated mesoporous silica (NH2-mSiO2), and then methotrexate (MTX) was loaded into the mesopores of mSiO2. Alginate (Alg) acted as the "gatekeeper" was then anchored to the MTX-loaded GO/NH2-mSiO2 by amidation reaction, achieving the encapsulation of MTX in the core-shell structured GO/mSiO2@Alg. Due to the high pH sensitivity of amide bond and the excellent photothermal conversion ability of GO, the constructed nanoplatform could be used for pH and near-infrared (NIR) controlled delivery of MTX. The results of cell viability test demonstrate the high inhibitory rate of the dual stimuli-responsive nanoplatform toward hepatoma (HepG2) cells.

Construction of a pH- and near-infrared irradiation-responsive nanoplatform for chemo-photothermal therapy.

Au nanoclusters, decorated with graphene quantum dots (GQDs), were obtained through photocatalytic reduction of AuCl43- by UV irradiation, and then cytarabine (Cyt) was loaded to the Au/GQDs via charge-dipole interactions. Mercaptopropionic acid (MPA) was anchored to the Cyt-loaded Au/GQDs through the formation of Au-S bond, which was further encapsulated by polyethyleneimine (PEI) via charge-dipole interactions. The delivery of Cyt from the quaternary complex (Au/GQDs/MPA/PEI) is pH-sensitive and can be modulated by near-infrared (NIR) irradiation. The results of cell viability test indicate that the developed nanoplatform can be used for chemo-photothermal combination therapy of cancer cells, and the efficacy of chemo-photothermal combination therapy is significantly higher than that of the single mode of photothermal therapy (PTT) or chemotherapy.

RNAi-knockdown of the Locusta migratoria nuclear export factor protein results in insect mortality and alterations in gut microbiome.

BACKGROUND: The migratory locust Locusta migratoria is one of the most important agricultural pests worldwide. The nuclear export factor 1 (NXF1) protein plays a crucial role in mediating mRNA transport from the nucleus to the cytoplasm. This study evaluates whether NXF1 could be a potential target for RNAi-mediated pest control of L. migratoria. RESULTS: We cloned and characterized the nuclear export factor lm-nxf1 of L. migratoria. Lm-nxf1 was expressed in all tissues examined, including head, fat body, hemolymph, trunk, leg and midgut, with high expression observed in the hemolymph and fat body. Injection of lm-nxf1 dsRNA into hemolymph resulted in inhibition of mRNA export in hemocytes, which were used as a target for observing mRNA export. Total hemocyte levels were reduced by ca. 97% in lm-nxf1-dsRNA-treated locusts, and high insect mortality occurred with LT50 = 7.75 day as compared with 18.15 day for gfp-dsRNA-treated controls. Further, the locust intestine became atrophy, and the opportunistic pathogens Enterobacter aerogenes, Klebsiella pneumoniae and Enterobacter asburiae were specifically detected in midgut after lm-nxf1 dsRNA treatment. CONCLUSIONS: The results reveal that knockdown of the lm-nxf1 gene affects the survival of L. migratoria, indicating that lm-nxf1 is a potential target for RNAi-mediated pest control. © 2018 Society of Chemical Industry.