Background: Smaller nanoparticles facilitate the delivery of DNA into cells through endocytosis and improve transfection efficiency. The aim of this study was to determine whether protamine sulfate-coated calcium phosphate (PS-CaP) could stabilize particle size and enhance transfection efficiency. Methods: pEGFP-C1 green fluorescence protein was employed as an indicator of transfection efficiency. Atomic force microscopy was used to evaluate the morphology and the size of the particles, and an MTT assay was introduced to detect cell viability and inhibition. The classical calcium phosphate method was used as the control. Results: Atomic force microscopy images showed that the PS-CaP were much smaller than classical calcium phosphate particles. In 293 FT, HEK 293, and NIH 3T3 cells, the transfection efficiency of PS-CaP was higher than for the classical calcium phosphate particles. The difference in efficiencies implies that the smaller nanoparticles may promote the delivery of DNA into cells through endocytosis and could improve transfection efficiency. In addition, PS-CaP could be used to transfect HEK 293 cells after one week of storage at 4°C with a lesser extent of efficiency loss compared with classical calcium phosphate, indicating that protamine sulfate may increase the stability of calcium phosphate nanoparticles. The cell viability inhibition assay indicated that both nanoparticles show similar low cell toxicity. Conclusion: PS-CaP can be used as a better nonviral transfection vector compared with classical calcium phosphate.

The etiology and significance of genomic instability (GIN), a hallmark of human cancers, remains controversial. The paradigm that inactivation of tumor suppressors [e.g. p53 or adenomatous polyposis coli (APC) genes] leads to GIN is largely based on experiments in vitro and in animal models. It remains unclear whether GIN is a cause or a result of cancer, particularly in patients. Precancerous Barrett's esophagus (BE) provides a clinical model to investigate GIN in cancer progression. We analyzed specimens from endoscopic biopsies or esophagectomies in patients with BE (ten cases, including five cases with multilayered epithelium (ME)), BE-associated esophageal adenocarcinoma (ten cases), or with normal gastro-esophageal junction (five cases). Chromosomal enumeration probe Cep 7, 11, 12, 17 and 18 were detected by fluorescence in situ hybridization (FISH). Expression of p53 and APC were determined by immunohistochemistry. Increased p53 expression, a measurement of p53 mutations, was observed in BE with high grade dysplasia (HGD) and in BE-associated esophageal cancer (EC). The expression of wild type APC was decreased in BE with HGD and in advanced EC. Chromosomal abnormalities were found in all EC samples. Numeric changes of chromosome 7, 11 and 12 were observed in BE in 14%, 64% and 43% of cases, respectively. Aneusomy of chromosome 11 and 12 were found in ME and in BE without dysplasia, in the presence of normal expression pattern of p53 and APC. Our results suggest that GIN is an early event that occurs at precancerous stages prior to changes in tumor suppressor genes (p53 and APC) in BE-associated tumorigenesis in patients, suggesting that GIN may serve as a causative link between chronic inflammation and cancer.

Mesenchymal stromal cells (MSCs) including mesenchymal stem cells to potentially differentiate into different tissue lineages widely exist in various tissues. In recent years, the clinical research and application of MSCs have become more extensive, but no standardized guidelines for the preparation and quality control of clinical-grade MSCs currently exist. To standardize the preparation and quality control of MSCs using the human umbilical cord, placenta, bone marrow, and adipose tissue as sample sources for the Chinese Association of Neurorestoratology (CANR; Preparatory) and the China Committee of International Association of Neurorestoratology (IANR-China Committee) member units, this standard is formulated following the T11/CSSCR 001-2017 General Requirements for Stem Cells, Good Manufacturing Practice Pharmaceutical Products (2010 Edition), Pharmacopoeia of the People's Republic of China (2015 Edition), Guiding Principles for Quality Control of Stem Cell Preparations and Preclinical Research (Trial), Code for Cell Banking Facility Quality Management, Sterile Drug Appendix to Pharmaceutical Production Quality Management Regulations, GMP Appendix — Cell Therapy Products (Draft for Comment), The International Society for Cellular Therapy position statement (2006), and Clinical Cell Therapy Guidelines for Neurorestoration (IANR/CANR 2017). Moreover, this standard includes donor evaluation, sample collection, cell preparation, cell inspection, packaging, labeling, transportation and storage, and quality control. It represents the minimum requirements for clinical-grade mesenchymal stromal cell culture and quality control. Moreover, it will be further optimized following the progress of preclinical and clinical research.

Activity-dependent neuroprotective protein (ADNP) is vital for embryonic development and brain formation. Besides, the upregulated expression of ADNP enhances tumorigenesis in some human tumors like bladder cancer (BC). However, the potential roles of ADNP in drug resistance and the related mechanisms in BC is unknown. We performed this study to elucidate the influence of ADNP in the chemoresistance of BC and tried to explore the underlying molecular mechanism. The expressions of ADNP in BC from progression and non-progression patient specimens were measured by quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC). In vitro experiments including colony formation, cell counting kit-8 (CCK-8), wound healing, and in vivo tumorigenesis assay were performed to explore the effects of ADNP on chemoresistance of BC. The impacts of ADNP on TGF-β/Smad signaling pathways were explored by western blot. Our results showed that the expression of ADNP mRNA and protein were significantly upregulated in BC tissues of the patients who suffered tumor-progression via RT-PCR and western blot. Cox regression survival analysis revealed that patients with high ADNP expression closely linked to shorter tumor-free survival. ADNP downregulation in BC showed more sensitive to cisplatin in vivo, while ADNP overexpression showed the opposite results. Additionally, we confirmed that ADNP promoted cell migration and EMT, thereby inducing cisplatin resistance, which may be related to TGF-β / Smad signaling pathway.

Endocytosis is critical for normal cellular function through clearing foreign materials and protecting the host from pathogen/virus attack. Innate immune cells play important roles in specifically recognizing and degrading microbes by generating phagosomes and phagolysosomes. However, the knowledge of how innate immunity regulates endocytosis in vitro and in vivo remains limited. In this review, we attempt to systematically and comprehensively summarize our current understanding of endocytosis and the role of Rab GTPases in the innate immune system. Understanding the immunity mechanisms of endocytosis might help develop targeted therapeutics for various applications, including viral inactivation and clearance, pathogen removal and even adjuvantenhanced antibody responses.

Long fragment cloning is a challenge for its difficulty in accurate amplifying and tendency to get unwanted mutation. Here we discuss Restriction-based Multiple-fragment Assembly Strategy's advantages and limitations. In this strategy, rather than PCR amplifying the entire coding sequence (CDS) at one time, we amplified and sequenced smaller fragments which are shorter than 1.5kb spanning the CDS. After that, the sequence-proved fragments were assembled by digestion-ligation cloning to the target vector. We test its universality in our script programmed in Python. Our data shows that, among the entire human and mouse CDS, at least 70% of long CDS cloning will benefit from this strategy.

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