Transdermal microarrayed electroporation for enhanced cancer immunotherapy based on DNA vaccination.

Despite the tremendous clinical potential of nucleic acid-based vaccines, their efficacy to induce therapeutic immune response has been limited by the lack of efficient local gene delivery techniques in the human body. In this study, we develop a hydrogel-based organic electronic device (µEPO) for both transdermal delivery of nucleic acids and in vivo microarrayed cell electroporation, which is specifically oriented toward one-step transfection of DNAs in subcutaneous antigen-presenting cells (APCs) for cancer immunotherapy. The µEPO device contains an array of microneedle-shaped electrodes with pre-encapsulated dry DNAs. Upon a pressurized contact with skin tissue, the electrodes are rehydrated, electrically triggered to release DNAs, and then electroporate nearby cells, which can achieve in vivo transfection of more than 50% of the cells in the epidermal and upper dermal layer. As a proof-of-concept, the µEPO technique is employed to facilitate transdermal delivery of neoantigen genes to activate antigen-specific immune response for enhanced cancer immunotherapy based on a DNA vaccination strategy. In an ovalbumin (OVA) cancer vaccine model, we show that high-efficiency transdermal transfection of APCs with OVA-DNAs induces robust cellular and humoral immune responses, including antigen presentation and generation of IFN-γ+ cytotoxic T lymphocytes with a more than 10-fold dose sparing over existing intramuscular injection (IM) approach, and effectively inhibits tumor growth in rodent animals.

Risk of colon cancer-related death in people who had cancer in the past.

BACKGROUND: The rate of second primary malignancies (SPM) is gradually increasing. Yet, the risk of death from primary cancer vs. SPM is still not well understood. In this study, we investigated the survival of patients with colorectal cancer (as SPM) who had cancer in the past (prior cancer) and the risk factors of SPM death in this population. MATERIALS AND METHODS: Based on the Surveillance, Epidemiology, and End Results (SEER) database, we identified 1866 colon cancer patients with prior cancer in our main cohort and 43,959 colon cancer patients, including 37,440 patients with colon cancer as only malignancy and 6519 patients with colon cancer as subsequent colon cancer (SCC), in a second cohort and 3429 colon cancer patients, including 2371 patients with prior colon cancer (PCC) and 1058 patients with colon cancer as SPM, in a third cohort. After propensity score matching, 6519 pairs of subjects were identified in second cohort. RESULTS: Patients with prior prostate and breast cancer had a higher risk of developing colon cancer compared to those with gastrointestinal cancer. Also, colon cancer patients with different prior cancer had different survival rates. Furthermore, except for prior lung cancer (52.78 vs. 25.93%), most subjects died due to colon cancer complications. The ratio of colon cancer deaths to prior cancer deaths in patients with a low stage and high stage was 1.51 and 6.64, respectively. In addition, colon cancer-specific survival (CSS) and OS rates were significantly lower in subjects with colon cancer as the SPM than in those with PCC. Also, compared with PCC, SPM was associated with OS and CSS with HR 1.59 (95 CI 1.43-1.78) and HR 2.00 (95% CI 1.70-2.36). Furthermore, compared with only colon cancer, SCC was associated with OS and CSS with HR 1.23 (95 CI 1.17-1.29) and HR 1.13 (95% CI 1.06-1.21). CONCLUSIONS: Prior cancer was found to have an adverse impact on OS in patients with colon cancer (secondary cancer), most of whom died due to colon cancer as secondary cancer itself rather than prior cancer. Early detection and treatment strategies should be investigated in this population.

Compact Conjugated Polymer Dots with Covalently Incorporated Metalloporphyrins for Hypoxia Bioimaging.

Hypoxia is closely related to multiple diseases, especially in tumors, which increases the aggressiveness and drug resistance of cancer cells. Precise hypoxia imaging is of great significance for cancer diagnosis and the evaluation of therapeutic effects. A kind of hydrophobic polymer (i.e., PFPtTFPP) as an imaging probe for hypoxia with fluorene as an energy donor and an oxygen-sensitive PtII porphyrin as an energy acceptor was developed. Compact polymer dots (Pdots) with a small size were prepared by nanoprecipitation. The PFPtTFPP Pdots showed excellent hypoxia sensing in solution with high sensitivity and full reversibility. The emission intensity, quantum yields, lifetime, and single-particle brightness significantly increased under hypoxia conditions. Remarkably, hypoxia imaging in vitro and in vivo was realized, and a clear increase in brightness was observed under hypoxia conditions and in the tumor area. Excellent hypoxia imaging ability is beneficial to potential applications in cancer diagnosis.

Curcusone C induces telomeric DNA-damage response in cancer cells through inhibition of telomeric repeat factor 2.

Telomeric repeat factor 2 (known as TRF2 or TERF2) is a key component of telomere protection protein complex named as Shelterin. TRF2 helps the folding of telomere to form T-loop structure and the suppression of ATM-dependent DNA damage response activation. TRF2 has been recognized as a potentially new therapeutic target for cancer treatment. In our routine screening of small molecule libraries, we found that Curcusone C had significant effect in disrupting the binding between TRF2 and telomeric DNA, with potent antitumor activity against cancer cells. Our result showed that Curcusone C could bind with TRF2 without binding interaction with TRF1 (telomeric repeat factor 1) although these two proteins share high sequence homology, indicating that their binding conformations and biological functions in telomere could be different. Our mechanistic studies showed that Curcusone C bound with TRF2 possibly through its DNA binding site causing blockage of its interaction with telomeric DNA. Further in cellular studies indicated that the interaction of TRF2 with Curcusone C could activate DNA-damage response, inhibit tumor cell proliferation, and cause cell cycle arrest, resulting in tumor cell apoptosis. Our studies showed that Curcusone C could become a promising lead compound for further development for cancer treatment. Here, TRF2 was firstly identified as a target of Curcusone C. It is likely that the anti-cancer activity of some other terpenes and terpenoids are related with their possible effect for telomere protection proteins.

Interaction of Quindoline derivative with telomeric repeat-containing RNA induces telomeric DNA-damage response in cancer cells through inhibition of telomeric repeat factor 2.

BACKGROUND: Telomeric repeat-containing RNA (TERRA) is a large non-coding RNA in mammalian cells, which forms an integral component of telomeric heterochromatin. TERRA can bind to an allosteric site of telomeric repeat factor 2 (TRF2), a key component of Shelterin that protect chromosome termini. Both TERRA and TRF2 have been recognized as promising new therapeutic targets for cancer treatment. METHODS: Our methods include FRET assay, SPR, CD, microscale thermophoresis (MST), enzyme-linked immunosorbent assay (ELISA), chromatin immunoprecipitation (ChIP), colony formation assays, Western blot, immunofluorescence, cell cycle arrest and apoptosis detection, and xCELLigence real-time cell analysis (RTCA). RESULTS: In our routine screening of small molecule libraries, we found that a Quindoline derivative, CK1-14 could bind to and stabilize TERRA G-quadruplex structure, which could bind more tightly with an allosteric site of a telomeric binding protein TRF2, resulting in dissociation of TRF2 from telomeric DNA. Further in cellular studies indicated that the above effect of CK1-14 on TERRA G-quadruplex could activate DNA-damage response and cause cell cycle arrest, resulting in inhibition of U2OS cell proliferation and causing cell apoptosis. CONCLUSIONS: Our mechanistic studies indicated that interaction of CK1-14 with TERRA induces telomeric DNA-damage response in U2OS cancer cells through inhibition of TRF2. CK1-14 could be further developed as a promising lead compound targeting telomere for cancer treatment. GENERAL SIGNIFICANCE: Our present study provides the first evidence that allosteric modulation of TRF2 by TERRA G-quadruplex with a binding ligand could become a promising new strategy for cancer treatment especially for ALT tumor cells.

Mapping the molecular determinants of BRAF oncogene dependence in human lung cancer.

Oncogenic mutations in the BRAF kinase occur in 6-8% of nonsmall cell lung cancers (NSCLCs), accounting for more than 90,000 deaths annually worldwide. The biological and clinical relevance of these BRAF mutations in NSCLC is incompletely understood. Here we demonstrate that human NSCLC cells with BRAF(V600E), but not other BRAF mutations, initially are sensitive to BRAF-inhibitor treatment. However, these BRAF(V600E) NSCLC cells rapidly acquire resistance to BRAF inhibition through at least one of two discrete molecular mechanisms: (i) loss of full-length BRAF(V600E) coupled with expression of an aberrant form of BRAF(V600E) that retains RAF pathway dependence or (ii) constitutive autocrine EGF receptor (EGFR) signaling driven by c-Jun-mediated EGFR ligand expression. BRAF(V600E) cells with EGFR-driven resistance are characterized by hyperphosphorylated protein kinase AKT, a biomarker we validated in BRAF inhibitor-resistant NSCLC clinical specimens. These data reveal the multifaceted molecular mechanisms by which NSCLCs establish and regulate BRAF oncogene dependence, provide insights into BRAF-EGFR signaling crosstalk, and uncover mechanism-based strategies to optimize clinical responses to BRAF oncogene inhibition.

Synthesis and characterization of 1H-phenanthro[9,10-d]imidazole derivatives as multifunctional agents for treatment of Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative brain disorder that is characterized by dementia, cognitive impairment, and memory loss. Diverse factors are related to the development of AD, such as increased level of ß-amyloid (Aß), acetylcholine, metal ion deregulation, hyperphosphorylated tau protein, and oxidative stress. METHODS: The following methods were used: organic syntheses of 1H-phenanthro[9,10-d]imidazole derivatives, inhibition of self-mediated and metal-induced Aß1-42 aggregation, inhibition studies for acetylcholinesterase and butyrylcholinesterase, anti-oxidation activity studies, CD, MTT assay, transmission electron microscopy, dot plot assay, gel electrophoresis, Western blot, and molecular docking studies. RESULTS: We synthesized and characterized a new type of 1H-phenanthro[9,10-d]imidazole derivatives as multifunctional agents for AD treatment. Our results showed that most of these derivatives exhibited strong Aß aggregation inhibitory activity. Compound 9g had 74% Aß1-42 aggregation inhibitory effect at 10µM concentration with its IC50 value of 6.5µM for self-induced Aß1-42 aggregation. This compound also showed good inhibition of metal-mediated (Cu(2+) and Fe(2+)) and acetylcholinesterase-induced Aß1-42 aggregation, as indicated by using thioflavin T assay, transmission electron microscopy, gel electrophoresis, and Western blot. Besides, compound 9g exhibited cholinesterase inhibitory activity, with its IC50 values of 0.86µM and 0.51µM for acetylcholinesterase and butyrylcholinesterase, respectively. In addition, compound 9g showed good anti-oxidation effect with oxygen radical absorbance capacity (ORAC) value of 2.29. CONCLUSIONS: Compound 9g was found to be a potent multi-target-directed agent for Alzheimer's disease. GENERAL SIGNIFICANCE: Compound 9g could become a lead compound for further development as a multi-target-directed agent for AD treatment.

Association between TRP channels and glutamatergic synapse gene polymorphisms and migraine and the comorbidities anxiety and depression in a Chinese population.

Background: Genetic and environmental factors contribute to migraine and the comorbidities of anxiety and depression. However, the association between genetic polymorphisms in the transient receptor potential (TRP) channels and glutamatergic synapse genes with the risk of migraine and the comorbidities of anxiety and depression remain unclear. Methods: 251 migraine patients containing 49 comorbidities with anxiety and 112 with depression and 600 controls were recruited. A customized 48-plex SNPscan kit was used for genotyping 13 SNPs of nine target genes. Logistic regression was conducted to analyze these SNPs' association with the susceptibility of migraine and comorbidities. The generalized multifactor dimension reduction (GMDR) was applied to analyze the SNP-SNP and gene-environment interactions. The GTEx database was used to examine the effects of the significant SNPs on gene expressions. Results: The TRPV1 rs8065080 and TRPV3 rs7217270 were associated with an increased risk of migraine in the dominant model [ORadj (95% CI): 1.75 (1.09-2.90), p = 0.025; 1.63 (1.02-2.58), p = 0.039, respectively]. GRIK2 rs2227283 was associated with migraine in the edge of significance [ORadj (95% CI) = 1.36 (0.99-1.89), p = 0.062]. In migraine patients, TRPV1 rs222741 was associated with both anxiety risk and depression risk in the recessive model [ORadj (95% CI): 2.64 (1.24-5.73), p = 0.012; 1.97 (1.02-3.85), p = 0.046, respectively]. TRPM8 rs7577262 was associated with anxiety (ORadj = 0.27, 95% CI = 0.10-0.76, p = 0.011). TRPV4 rs3742037, TRPM8 rs17862920 and SLC17A8 rs11110359 were associated with depression in dominant model [ORadj (95% CI): 2.03 (1.06-3.96), p = 0.035; 0.48 (0.23-0.96), p = 0.042; 0.42 (0.20-0.84), p = 0.016, respectively]. Significant eQTL and sQTL signals were observed for SNP rs8065080. Individuals with GRS (Genetic risk scores) of Q4 (14-17) had a higher risk of migraine and a lower risk of comorbidity anxiety than those with Genetic risk scores scores of Q1 (0-9) groups [ORadj (95% CI): 2.31 (1.39-3.86), p = 0.001; 0.28 (0.08-0.88), p = 0.034, respectively]. Conclusion: This study suggests that TRPV1 rs8065080, TRPV3 rs7217270, and GRIK2 rs2227283 polymorphism may associate with migraine risk. TRPV1 rs222741 and TRPM8 rs7577262 may associate with migraine comorbidity anxiety risk. rs222741, rs3742037, rs17862920, and rs11110359 may associate with migraine comorbidity depression risk. Higher GRS scores may increase migraine risk and decrease comorbidity anxiety risk.

Synthesis and anti-hyperglycemic activity of hesperidin derivatives.

A series of hesperidin derivatives were prepared and identified by IR, (1)H NMR, and MS spectra. These compounds were evaluated in vitro and in vivo based on α-glucosidase inhibition, glucose consumption of HepG2 cells, and blood glucose level in streptozotocin-induced diabetic mice. The results revealed that all the compounds exhibited anti-hyperglycemic activities. The inhibition at 10(-3) M of compounds 3 and 7a on α-glucosidase were 55.02% and 53.34%, respectively, as compared to 54.80% by acarbose. Treated by compound 3 and the reference drug metformin, glucose consumption of HepG2 cell were 1.78 and 2.11 mM, respectively. After the streptozotocin-induced diabetic mice were oral administrated with compound 3 at 100 mg kg(-1) d(-1) for 10 days, the blood glucose level of 3 treated mice (13.23 mM, P<0.05) showed significant difference when compared to model control (23.03 mM). Thus, compound 3 exhibited promising anti-hyperglycemic activity.

Discrete single-cell microRNA analysis for phenotyping the heterogeneity of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a highly heterogenous cancer in hematopoiesis, and its subtype specification is greatly important in the clinical practice for AML diagnosis and prognosis. Increasing evidence has shown the association between microRNA (miRNA) phenotype and AML therapeutic outcomes, emphasizing the need for novel techniques for convenient, sensitive, and efficient miRNA profiling in clinical practices. Here, we describe a nanoneedle-based discrete single-cell microRNA profiling technique for multiplexed phenotyping of AML heterogeneity without the requirement of sequencing or polymerase chain reaction (PCR). In virtue of a biochip-based and non-destructive nature of the assay, the expression of nine miRNAs in large number of living AML cells can be simultaneously analyzed with discrete single-cell level information, thus providing a proof-of-concept demonstration of an AML subtype classifier based on the multidimensional miRNA data. We showed successful analysis of subtype-specific cellular composition with over 90% accuracy and identified drug-responsive leukemia subpopulations among a mixed suspension of cells modeling different AML subtypes. The adoption of machine learning algorithms for processing the large-scale nanoneedle-based miRNA data shows the potential for powerful prediction capability in clinical applications to assist therapeutic decisions. We believe that this platform provides an efficient and cost-effective solution to move forward the translational prognostic usage of miRNAs in AML treatment and can be readily and advantageously applied in analyzing rare patient-derived clinical samples.

Dietary Intake of Calcium and Magnesium in Relation to Severe Headache or Migraine.

Background: Migraine is a common neurological disorder and is affected by nutrients. Calcium and magnesium are essential minerals that play an important role in nerve function. So we investigated the association between dietary calcium and magnesium and migraine. Methods: We extracted 10,798 adults from the National Health and Nutrition Examination Surveys (NHANES) of America in 1999 to 2004. We classified patients who reported having severe headache or migraine as having possible migraine. Multivariable logistic regression and restricted cubic spline regression were conducted to determine the association between dietary calcium and magnesium and migraine. Results: We found that the adjusted ORs of the association between dietary calcium and magnesium and migraine for comparing the highest quintile intake with the lowest quintile intake were 0.77 (95% CI: 0.63-0.93, P = 0.008) and 0.69 (95% CI: 0.55-0.86, P = 0.001), respectively. For women, the adjusted ORs of dietary calcium and magnesium were 0.72 (95% CI: 0.56-0.93, P = 0.009) and 0.62 (95% CI: 0.47-0.83, P = 0.001), respectively. For men, the adjusted OR was 0.71 (95% CI: 0.52-0.97, P = 0.028) comparing the highest and the lowest quintile of calcium intake, but there was no statistically significant association between dietary magnesium intake and migraine. Joint analyses showed that the OR in the high-calcium and high-magnesium group was 0.74 (95% CI: 0.60-0.92, P = 0.006) compared with the low-calcium and low-magnesium group in women. Conclusions: High dietary intake of calcium and magnesium, independently or in combination, were inversely associated with migraine in women. For men, high dietary calcium was negatively related to migraine, but magnesium was not associated with migraine.

Association Between Dietary Iron Intake and Serum Ferritin and Severe Headache or Migraine.

Background: Dietary iron intake and serum ferritin in relation to severe headache or migraine remain largely unknown. Therefore, we investigated the associations between dietary iron intake and serum ferritin with severe headache or migraine among American adults. Methods: This cross-sectional study included 7,880 adults (≥20 years) from the National Health and Nutrition Examination Surveys (NHANES) of America from 1999 to 2004. We performed multivariable logistic regression and restricted cubic spline (RCS) regression to assess the association of dietary iron and serum ferritin with severe headache or migraine. Results: Most women aged 20-50 years consumed less dietary iron than their recommended dietary allowances. Dietary iron intake was inversely associated with severe headache or migraine in women aged 20-50 years. For women over 50 years, serum ferritin was negatively associated with severe headache or migraine. For men, there was no significant relationship between dietary iron and serum ferritin, and severe headache or migraine. Conclusions: Dietary iron intake has different effects on migraine in women of different ages, and this different effect may be due to age-related menstrual changes. Women aged 20-50 years should have a higher awareness of RDA and increase their dietary iron intake if needed, which may play an important role in preventing severe headache or migraine. Higher serum ferritin levels in women aged 50 and above may have a protective effect against migraine.

COPB2 promotes metastasis and inhibits apoptosis of lung adenocarcinoma cells through functioning as a target of miR-216a-3p.

BACKGROUND: Lung adenocarcinoma is a non-small cell lung cancer with a high mortality. There is little published data on the role of coatomer protein complex subunit ß (COPB2) in lung adenocarcinoma. The current study aimed to explore the effects of COPB2 on lung adenocarcinoma cells. METHODS: The differential expression of COPB2 in normal cells and lung adenocarcinoma cells was detected by quantitative real time-polymerase chain reaction (qRT-PCR) and Western blotting. Then, cell viability assay, flow cytometry and Transwell experiments were performed to study the effects of COPB2 on cell growth, apoptosis, migration and invasion. MiRNA targeting COPB2 was predicted by TargetScan and validated by luciferase assay, qRT-PCR and Western blotting. The effects of miRNA inhibitor on siCOPB2 were analyzed by rescue experiments. Finally, apoptosis and metastatic marker proteins were detected by Western blotting. RESULTS: COPB2 was high-expressed in lung adenocarcinoma cells. Silencing COPB2 inhibited cell viability and cell metastasis, and significantly increased apoptosis. MiR-216a-3p was predicted to be able to target COBP2. Rescue experiment showed that miR-216a-3p inhibitor promoted cell viability, migration and invasion, and inhibited apoptosis of lung adenocarcinoma cells, partly reversed the effects of siCOPB2. Moreover, Western blotting showed that siCOPB2 up-regulated expressions of cleaved Caspase-3, Caspase-3, BCL2 associated X (Bax), and E-Cadherin, and down-regulated expressions of BCL2 apoptosis regulator (Bcl-2), N-Cadherin, and Vimentin, and the above effects were also partly reversed by miR-216a-3p inhibitor. CONCLUSIONS: High-expressed COPB2 promotes metastasis and inhibits apoptosis of lung adenocarcinoma cells through functioning as a target of miR-216a-3p.

Establishing reference intervals for urine and serum iodine levels: A nationwide multicenter study of a euthyroid Chinese population.

CONTEXT: Urinary iodine (UI) is commonly used for evaluating iodine status, whereas serum iodine (SI) is more closely correlated with bioavailable iodine. However, no reliable reference intervals (RIs) for clinical use are available. We aimed to establish RIs for SI, UI, and a ratio of UI to urinary creatinine (U-Cre) applicable to the Chinese population. METHODS: This multicenter cross-sectional study enrolled 930 apparently healthy adults from six representative cities in China (Beijing, Dongying, Guiyang, Urumqi, Shenzhen, and Qiqihar) in 2017. Thyroid ultrasonography and thyroid function tests, including antithyroid antibody tests, were performed to exclude individuals with latent thyroid diseases. An iodine intake-related questionnaire survey was performed. SI and UI were measured using inductively coupled plasma-mass spectrometry. Possible influencing factors of iodine levels were evaluated using multiple regression analysis. RESULTS: Post-exclusion, the final analysis included 894 individuals. Seafood intake frequency was positively correlated with SI (standardized partial regression coefficient = 0.23) but not with UI and UI/U-Cre. SI was positively correlated with serum TT4 (Spearman correlation coefficient: 0.40), TT3 (0.23), and FT4 (0.18). SI and UI showed no age- or sex-specific variations. Significantly higher UI/U-Cre values were observed in Qiqihar than in Beijing, Guizhou, and Shenzhen. Shenzhen showed the lowest UI levels among all evaluated cities. With application of latent abnormal values exclusion procedurere, the RIs for SI, UI, and UI/U-Cre in the population were 36.0-79.3 µg/L, 19-385 µg/L, 22-450 µg/g, respectively. CONCLUSIONS: We established RIs for UI and SI among healthy Chinese individuals with no thyroid nodule or dysfunction.

Synthesis and electrochemical performances of Na3V2(PO4)2F3/C composites as cathode materials for sodium ion batteries.

Na3V2(PO4)2F3 (NVPF) with NASCION (Na superionic conductor) is recognized as a potential cathode material owing to its high theoretical capacity. However, the electronic conductivity of NVPF is much lower than its ionic conductivity, which seriously affects the properties of this material. The carbon layer can be used as the conductive medium to enhance the conductivity of NVPF. In this study, we propose a single-step solid-state reaction method based on mechanical activation with pitch as the carbon source to synthesize NVPF/C composites. The crystallographic structure and morphology of all as-prepared samples were investigated by XRD, Raman spectroscopy, BET measurement, thermal analysis, SEM and TEM. Furthermore, the electrochemical performance and kinetic properties were analyzed by CV, galvanostatic charge-discharge and EIS tests. These tests outcomes demonstrated that the NVPF/C-2 composite with a carbon content of 12.14 wt% showed an excellent rate performance and cycle stability. It presented reversible capacities of 103 and 95 mA h g-1 at 0.2 and 10C, respectively, and an outstanding retention of 91.9% after 500 cycles at 5C. These excellent properties of the NVPF/C-2 composite are attributed to its high ion diffusion coefficient and small charge transfer impedance.

Monomethyl auristatin E-conjugated anti-EGFR antibody inhibits the growth of human EGFR-positive non-small cell lung cancer.

PURPOSE: Epidermal growth factor receptor (EGFR) is highly expressed on non-small cell lung cancers (NSCLC) and a valuable therapeutic target. This study aimed at producing and characterizing monomethyl auristatin E (MMAE)-conjugated anti-EGFR antibody as a novel EGFR-targeting therapy for NSCLC. METHODS: A humanized anti-EGFR monoclonal antibody (named RC68) was purified and conjugated with MMAE using a MC-VC-PAB or PY-VC-PAB linker. The in vitro and in vivo antitumor activity of RC68-MC-VC-PAB-MMAE and RC68-PY-VC-PAB-MMAE were characterized. RESULTS: The RC68 was generated from RC68-expressing cells and had a purity of > 99.0%. The RC68 recognized EGFR on tumor cells, particularly for higher EGFR expressing H125, A431, HCC827 and H1975 cells. The RC68 was conjugated with an average of 4 MMAE molecules to generate RC68-MC-VC-PAB-MMAE and RC68-PY-VC-PAB-MMAE, respectively. The RC68-MC-VC-PAB-MMAE, RC68-PY-VC-PAB-MMAE and RC68 displayed similar binding affinity to EGFR on tumor cells, and RC68-MC-VC-PAB-MMAE and RC68-PY-VC-PAB-MMAE were effectively internalized by H125 cells. The RC68-MC-VC-PAB-MMAE and RC68-PY-VC-PAB-MMAE inhibited the growth of H125 cells in vitro with an IC50 7.37-8.04 ng/mL and implanted H125 tumors in vivo, but did not affect body weights of mice. The antitumor effect of RC68-MC-VC-PAB-MMAE was stronger than RC68-PY-VC-PAB-MMAE, which was also stronger than docetaxel in vivo. CONCLUSIONS: These novel antibody-drug conjugates, particularly for RC68-MC-VC-PAB-MMAE, may be a potential candidate for treatment of EGFR + NSCLC.

Risk factors for delirium: are therapeutic interventions part of it?

Background: Delirium is associated with increased morbidity and mortality in critically ill patients. Research on risk factors for delirium allows clinicians to identify high-risk patients, which is the basis for early prevention and diagnosis. Besides the risk factors for delirium that are commonly studied, here we more focused on the less-studied therapeutic interventions for critically ill patients which are potentially modifiable. Materials and methods: A total of 320 non-comatose patients admitted to the ICU for more than 24 hrs during 9 months were eligible for the study. Delirium was screened once daily using the CAM-ICU. Demographics, admission clinical data, and daily interventions were collected. Results: Ninety-two patients (28.75%) experienced delirium at least once. Delirious patients were more likely to have longer duration of mechanical ventilation, ICU stay, and hospital stay. Most of the less-studied therapeutic interventions were linked to delirium in the univariate analysis, including gastric tube, artificial airway, deep intravenous catheter, arterial line, urinary catheter, use of vasoactive drugs, and sedative medication. After adjusting with age and ICU length of stay, mechanical ventilation (OR: 5.123; 95% CI: 2.501-10.494), Acute Physiology and Chronic Health Evaluation (APACHE) II score≥20 at admission (OR: 1.897; 95% CI: 1.045-3.441), and gastric tube (OR: 1.935, 95% CI: 1.012-3.698) were associated with increased risk of delirium in multivariate analysis. Conclusion: Delirium was associated with prolonged mechanical ventilation, ICU stay, and hospital stay. Multivariate risk factors were gastric tube, mechanical ventilation, and APACHE II score. Although being a preliminary study, this study suggests the necessity of earliest removal of tubes and catheters when no longer needed.

Development of a Novel EGFR-Targeting Antibody-Drug Conjugate for Pancreatic Cancer Therapy.

BACKGROUND: Overexpression of epidermal growth factor receptor (EGFR) is common in pancreatic cancer and associated with the poor prognosis of this malignancy. OBJECTIVE: To develop anti-EGFR antibody-drug conjugates (ADCs) for use in a novel EGFR-targeting approach to treat pancreatic cancer. METHODS: A humanized anti-EGFR monoclonal antibody (RC68) was generated by mouse immunization and complementary-determining region grafting technology. Two RC68-based ADCs, RC68-MC-VC-PAB-MMAE and RC68-PY-VC-PAB-MMAE, were synthesized by conjugating monomethyl auristatin E (MMAE), a small-molecule cytotoxin, to RC68 through two distinct linkers (MC and PY). Internalization of the RC68-based ADCs was examined by flow cytometry. The in vitro and in vivo antitumor activities of RC68-based ADCs were evaluated in human pancreatic cancer cells and in a BXPC-3 xenograft nude mouse model, respectively. RESULTS: The RC68-based ADCs bound to EGFR on the surface of tumor cells and were effectively internalized, resulting in the death of EGFR-positive cancer cell lines. The RC68-based ADCs (at 5 or 10 mg/kg) were more potent than gemcitabine hydrochloride (60 mg/kg) at inhibiting the growth of BXPC-3 xenografts. Moreover, RC68-PY-VC-PAB-MMAE was found to have superior stability in human plasma compared with RC68-MC-VC-PAB-MMAE. CONCLUSION: A novel EGFR-targeting ADC, RC68-PY-VC-PAB-MMAE, shows promise as an effective, selective, and safe therapeutic agent for EGFR-positive pancreatic cancer.

[The efficiently expression of soluble CD20 in HEK293T cells].

Objective To construct the eukaryotic expression vector of CD20 and overexpress soluble CD20 proteins in HEK293T cells. Methods The total RNA from human peripheral blood mononuclear cells (PBMCs) was used to amplify the coding sequence of CD20 by reverse transcription PCR, and then the CD20 gene was cloned into the expression vector pCMV3-C-His. After PCR and sequencing validation, the recombinant CD20 protein was transiently expressed in HEK293T cells and detected by ELISA and Western blot analysis. Moreover, the expression conditions were optimized to improve the expression level of CD20. Results The coding sequence of CD20 was successfully cloned into eukaryotic expression vector pCMV3-C-His. After 72 hours of transfection, the expression of CD20 was detected both in intracellular and supernatant by Western blot analysis. The passage number of HEK293T cells was related to the expression level of CD20 in supernatant. Conclusion The coding sequence of CD20 was successfully cloned into the eukaryotic expression vector, and CD20 was over expressed in HEK293T cells.

Development of novel anti-CD19 antibody-drug conjugates for B-cell lymphoma treatment.

B-cell lymphoma remains one of the most refractory tumors, and as such the development of novel treatment approaches, such as antibody-drug conjugates (ADCs), is required. To improve the stability and homogeneity of the ADCs, a humanized anti-CD19 monoclonal antibody (RC58) was developed in the present study. RC58 was based on the CD19 antigen as a potential molecular target of human B-cell lymphomas. RC58 has high CD19-binding affinity and can be internalized in CD19-positive cells through endocytosis. Furthermore, three types of RC58-based ADCs (ADC-1, ADC-2, and ADC-3) were generated using three kinds of Maleimide-PEG-based linkers with two different cytotoxins. The anti-tumor activities of the ADCs were confirmed by in vitro and in vivo experiments. The stability of the ADCs was also evaluated by incubation in human plasma for 10 days. In vitro experiments showed that the three ADCs had distinct inhibitory effects on several B-lymphoma cell lines. Meanwhile, a close correlation between efficacy and drug concentration was found in a nude mouse xenograft model of human B-cell lymphoma, after treatment with RC58-based ADCs. Our results suggest that ADC-1, with high efficiency, could be used as a potential therapeutic agent for human B-cell malignancies.