Enzymatic Synthesis of Aptamer-Polynucleotide Nanoparticles with High Anticancer Drug Loading for Targeted Delivery.

We report a targeted prodrug delivery platform that can deliver a cytostatic nucleobase analog with high drug loading. We chose fluorouracil (5FU), a drug used to treat various cancers, whose active metabolite 5-fluorodeoxyuridine monophosphate (5-FdUMP) is the antineoplastic agent. We use terminal deoxynucleotidyl transferase (TdT) to polymerize 5-fluorodeoxyuridine triphosphate (5-FdUTP) onto the 3'-end of an aptamer. We find that (i) addition of hydrophobic, unnatural nucleotides at the 3'-end of the 5-FdU polynucleotide by TdT leads to their spontaneous self-assembly into nuclease resistant micelles, (ii) aptamers presented on the micelle corona retain specificity for their cognate receptor on tumor cells, and (iii) the micelles deliver 5FU to tumor cells and exhibit greater cytotoxicity than the free drug. The modular design of our platform, consisting of a targeting moiety, a polynucleotide drug, and a self-assembly domain, can be adapted to encompass a range of polymerizable therapeutic nucleotides and targeting units.

Finite element analysis comparison of Type 42A2 fracture fixed with external titanium alloy locking plate and traditional external fixation frame.

BACKGROUND: At present, not all Type AO/OTA 42A2 open fractures can be treated by external fixation brackets, not to mention the inconvenience of this technique in clinical practice. External titanium alloy locking plates, which are lightweight and easy-to-operate, can be used as an alternative treatment option for such patients. However, there are few reports of finite element biomechanical analysis on the titanium alloy locking plates and fixation brackets being placed on the medial side of the tibial fracture. In this study, the biomechanical properties of titanium alloy locking plates and fixation brackets for treating Type AO/OTA 42A2 fractures were compared by applying the finite element method, and the results provided data support for the clinical application of the external titanium alloy locking plate technique. METHODS: Type AO/OTA 42A2 fracture models were constructed using CT data of a male volunteer for two external fixation techniques, namely the external titanium alloy locking plate technique and the external fixation bracket technique, according to commonly-used clinical protocols. Then, the four-point bending, axial compression, clockwise rotation and counterclockwise rotation tests under the maximum load were simulated in finite element analysis software. The stress distribution, peak stress and overall tibial displacement data for the two different external fixation techniques were obtained and compared. RESULTS: In the four different test conditions (i.e., four-point bending, axial compression, clockwise torsion, counterclockwise torsion) under the maximum load, the two external fixation techniques showed obvious von Mises stress concentration at the contacts between the screw and tibia, between the screw and titanium alloy locking plate, between the self-tapping self-drilling needle and tibia, between the self-tapping self-drilling needle and the external fixation device, as well as around the fracture end and around the cortical bone at the upper and lower ends of the tibia. The peak stress was ranged 26.67-558.77 MPa, all below the yield stress strength of titanium alloy. The peak tibial displacement of the external titanium alloy locking plate model was smaller than that of the fixation bracket model. In terms of structural stability, the external titanium alloy locking plate technique was superior to the external fixation bracket technique. CONCLUSIONS: When fixing Type AO/OTA 42A2 fractures, external titanium alloy locking plates are not only lightweight and easy-to-operate, but also have better performance in terms of axial compression, bending and torsion resistance. According to the finite element biomechanical analysis, external titanium alloy locking plates are superior to traditional external fixation brackets in treating Type AO/OTA 42A2 fractures and can better meet the needs of clinical application.

Toxic effects of ship exhaust gas closed-loop scrubber wash water.

To meet the strict requirements of reducing sulfur emissions, an increasing number of commercial ships have installed exhaust gas cleaning systems (EGCSs). However, wash water produced during the cleaning process is discharged back to the marine environment. We investigated the effects of closed-loop scrubber (natrium-alkali method) wash water on three trophic species. Severe toxic effects were found when Dunaliella salina, Mysidopsis bahia, and Mugilogobius chulae were exposed to 0.63-6.25, 0.63-10, and 1.25-20% concentrations of wash water, respectively. The 50% effective concentration in 96 h (EC50-96 h) for D. salina was 2.48%, and the corresponding total polycyclic aromatic hydrocarbons (PAHs) and heavy metals were 22.81 and 23.67 µg L-1. The 50% lethal concentration in 7 d (LC50-7 d) values for M. bahia and M. chulae were 3.57% and 20.50%, respectively. The lowest observed effect concentration (LOEC) values for M. bahia and M. chulae were 1.25% and 2.5%, respectively, and the corresponding total PAHs and heavy metals were 11.50 and 11.93 and 22.99 and 23.86 µg L-1. M. bahia's body weight was negatively correlated with the amount of wash water. Low concentrations of wash water (0-5%) had no significant effect on the reproduction of M. bahia. Although concentrations of 16 PAHs and 8 heavy metals are known, different compounds might react with each other and form more unknown toxic substances, and the measured toxicity comes from synergistic effects between various pollutants. Therefore, future work is needed to clarify other more toxic contaminants in wash water. We highly recommend that wash water be treated before being discharged to the marine environment.

N-linoleyltyrosine resisted the growth of non-small cell lung cancer cells via the regulation of CB1 and CB2 involvement of PI3K and ERK pathways.

Background: N-linoleyltyrosine (NITyr), one of the anandamide analogs, exerts activity via the endocannabinoid receptors (CB1 and CB2), which showed anti-tumor effects in various tumors. Therefore, we speculated that NITyr might show anti-non-small cell lung cancer (NSCLC) effects via the CB1 or CB2 receptor. The purpose of the investigation was to reveal the anti-tumor ability of NITyr on A549 cells and its mechanisms. Methods: The viability of A549 cells was measured by MTT assay, and the cell cycle and apoptosis were both examined by flow cytometry; in addition, cell migration was tested by wound healing assay. Apoptosis-related markers were measured by immunofluorescence. The downstream signaling pathways (PI3K, ERK, and JNK) of CB1 or CB2 were examined through Western blotting. The expressions of CB1 and CB2 were detected by immunofluorescence. Finally, the AutoDock software was used to validate the binding affinity between the targets, such as CB1 and CB2, with NITyr. Results: We found that NITyr inhibited cell viability, hindered the cell cycle, resulted in apoptosis, and inhibited migration. The CB1 inhibitor, AM251, and the CB2 inhibitor, AM630, weakened the aforementioned phenomenon. The immunofluorescence assay suggested that NITyr upregulated the expression of CB1 and CB2. Western blot analysis indicated that NITyr upregulated the expression of p-ERK, downregulated the expression of p-PI3K, and did not affect p-JNK expression. In conclusion, NITyr showed a role in inhibiting NSCLC through the activation of CB1 and CB2 receptors involved in PI3K and ERK pathways.

Clinical implications of conflicting variant interpretations in the cancer genetics clinic.

PURPOSE: The aim of this study was to describe the clinical impact of commercial laboratories issuing conflicting classifications of genetic variants. METHODS: Results from 2000 patients undergoing a multigene hereditary cancer panel by a single laboratory were analyzed. Clinically significant discrepancies between the laboratory-provided test reports and other major commercial laboratories were identified, including differences between pathogenic/likely pathogenic and variant of uncertain significance (VUS) classifications, via review of ClinVar archives. For patients carrying a VUS, clinical documentation was assessed for evidence of provider awareness of the conflict. RESULTS: Fifty of 975 (5.1%) patients with non-negative results carried a variant with a clinically significant conflict, 19 with a pathogenic/likely pathogenic variant reported in APC or MUTYH, and 31 with a VUS reported in CDKN2A, CHEK2, MLH1, MSH2, MUTYH, RAD51C, or TP53. Only 10 of 28 (36%) patients with a VUS with a clinically significant conflict had a documented discussion by a provider about the conflict. Discrepant counseling strategies were used for different patients with the same variant. Among patients with a CDKN2A variant or a monoallelic MUTYH variant, providers were significantly more likely to make recommendations based on the laboratory-reported classification. CONCLUSION: Our findings highlight the frequency of variant interpretation discrepancies and importance of clinician awareness. Guidance is needed on managing patients with discrepant variants to support accurate risk assessment.

Oncologic safety of the pedicled submental island flap for reconstruction in oral tongue squamous cell carcinoma: An analysis of 101 cases.

OBJECTIVE: To evaluate whether the pedicle submental island flap (SIF) can be safely used in the oral tongue squamous cell carcinoma (OTSCC) patients with pathologically node-positive (pN+) neck, especially pN+ at level I. METHODS: Retrospectively, 101 OTSCC patients with SIF reconstruction were enrolled. Oncological outcomes included the total locoregional recurrence, the SIF related locoregional recurrence (SRLR) which referred to the local recurrence at flap and ipsilateral neck recurrence at level I, recurrence free survival (RFS), overall survival (OS), and disease specific survival (DSS). RESULTS: Sixty-one patients were pathologically node-negative (pN0) and 40 were pN+. Thirteen patients experienced locoregional recurrence, of which 5 had a SRLR. The pN+ group had a significantly higher locoregional recurrence rate, lower 5-year RFS, OS and DSS than pN0 group (P < 0.05). Patients with pN0 had a significantly higher neck RFS when compared to those with pN+ either at level I (P = 0.005) or at other levels (P < 0.001). However, the neck RFS was similar between the two subgroups of pN+ (P = 0.550). Especially, patients with pN+ at level I had a significantly higher SRLR rate (P = 0.006) compared to those with pN0 at level I. Multivariate analysis showed that pN+ was an unfavorable factor for tumor recurrence and OS. CONCLUSION: Our data did not support the use of SIF in OTSCC patients with pN+ neck at level I due to an significantly increased SRLR rate compared to those with pN0 neck at level I.

Extended Maxillary Osteotomy Guide: A Design That Allows Manipulation of the Osteotomy Direction on the Posterior and Inner Walls of the Maxilla.

PURPOSE: Design an extended osteotomy guide (EOG) for Le Fort I osteotomy to improve the safety of surgery. MATERIALS AND METHODS: The digital Le Fort I osteotomy guide was designed in MIMICS 23.0. Twenty-eight patients were randomized into 2 groups. Patients in the experimental group used EOG, and patients in the control group used a traditional osteotomy guide (TOG). Virtual designs and actual postoperative outcomes were compared by cone-beam computed tomography. The safety of the operation was confirmed by the accuracy of the osteotomy direction and depth on the inner and posterior walls of the maxilla. RESULTS: All positioning deviations of both osteotomy guides were <0.3 mm (P>0.05). The osteotomy depths on the inner and posterior walls with the EOG and TOG deviated by 0.789±1.179 and 1.811±1.345 mm (P=0.004) and 0.648±0.999 and 1.262±0.942 mm (P=0.030), respectively. The angles of deviation of the osteotomy direction on the inner and posterior walls by the EOG and TOG were 2.025±2.434 and 5.069±2.391 degrees (P<0.001) and 2.772±2.979 and 8.653±4.690 degrees (P<0.001), respectively. CONCLUSIONS: The EOG was more accurate than TOG for manipulating osteotomy direction and depth on the inner and posterior maxillary walls. Thus, EOG could ensure higher surgical safety than TOG.

Lipopolysaccharide/D-galactosamine-induced acute liver injury could be attenuated by dopamine receptor agonist rotigotine via regulating NF-κB signaling pathway.

The pathological of lipopolysaccharide (LPS)/D-galactosamine (D-Gal)-induced acute liver injury is similar to what is seen clinically, and be mediated by the release of pro-inflammatory mediators. A growing body of studies have shown that dopamine (DA) and DA receptor agonist are associated with inflammation and immune response. Rotigotine, a non-ergoline dopamine receptor agonist, is a drug for the treatment of Parkinson's disease. Rotigotine-loaded microspheres (RoMS) is an intramuscular extended-release agent, which can steadily release rotigotine for more than 7 days after a single administration. The present study aimed to investigate the effects of rotigotine and RoMS on inflammation and acute liver injury induced by LPS/D-Gal in mice. The LPS/D-Gal-induced liver injury was evidenced by increases of serum aminotransferases activities and liver histological lesions. Pretreatment with rotigotine or RoMS not only ameliorated the liver histologic lesions, but also reduced the activities of serum aminotransferases and the production of TNF-α. It also showed that rotigotine and RoMS increased DA receptor 2 (DRD2) expression in LPS/D-Gal-exposed mice. Rotigotine and RoMS activated ß-arrestin 2, inhibited the phosphorylation of Akt, IκB and the transposition of NF-κB. In line with the above findings, the protective effects of rotigotine and RoMS were abrogated by haloperidol, a DA receptor antagonist. In conclusion, dopamine receptor agonist can regulate NF-κB inflammatory signaling pathway and exert protective effects in LPS/D-Gal-induced liver injury.

Enhanced immunogenicity of foot and mouth disease DNA vaccine delivered by PLGA nanoparticles combined with cytokine adjuvants.

Although the immunogenicity of DNA vaccines is nonideal, they are still considered as potential alternative vaccine candidates to conventional vaccines. Various DNA delivery systems, including nanoparticles, have been extensively explored and validated to further enhance the immunogenicity of DNA vaccines. DNA vaccines are considered as alternative vaccine candidates. Various DNA delivery systems, including nanoparticles, have been extensively explored to enhance the immunogenicity of DNA vaccines. In this study, positively charged Poly (D, l-lactide-co-glycolic acid) (PLGA) nanoparticles were generated and characterized as a delivery system for O-serotype foot-and-mouth DNA vaccine. A recombinant plasmid encoding swine interleukin (IL)-18, IL-2, or granulocyte-macrophage colony-stimulating factor (GM-CSF) gene was introduced into the DNA vaccine to further improve its immunogenicity, which was evaluated in a guinea pig model. PLGA-pVAX-VP013/IL-18 elicited significantly (P = 0.0149) higher FMDV-specific antibody levels than naked DNA before the challenge. The level of neutralizing antibodies induced by PLGA-pVAX-VP013/IL-18, PLGA-pVAX-VP013/IL-2, and PLGA-pVAX-VP013/GM-CSF significantly increased compared with that induced by naked DNA (P < 0.0001). The lymphocyte proliferation assay showed that cellular immunity induced by PLGA-pVAX-VP013/IL-18 and PLGA-pVAX-VP013/GM-CSF was dramatically enhanced compared with that induced by the inactivated vaccine. The protection by PLGA-pVAX-VP013/IL-18 was consistent with that by the inactivated vaccine post-challenge and was followed by PLGA-pVAX-VP013/GM-CSF. Therefore, cationic PLGA nanoparticles can deliver DNA vaccines and induce humoral and cellular immune responses. The co-administration of FMD DNA vaccine with IL-18 formulated with PLGA nanoparticles was the optimal strategy to improve the immunogenicity of FMD DNA vaccines.

A likelihood-based approach to assessing frequency of pathogenicity among variants of unknown significance in susceptibility genes.

Commercialized multigene panel testing brings unprecedented opportunities to understand germline genetic contributions to hereditary cancers. Most genetic testing companies classify the pathogenicity of variants as pathogenic, benign, or variants of unknown significance (VUSs). The unknown pathogenicity of VUSs poses serious challenges to clinical decision-making. This study aims to assess the frequency of VUSs that are likely pathogenic in disease-susceptibility genes. Using estimates of probands' probability of having a pathogenic mutation (ie, the carrier score) based on a family history probabilistic risk prediction model, we assume the carrier score distribution for probands with VUSs is a mixture of the carrier score distribution for probands with positive results and the carrier score distribution for probands with negative results. Under this mixture model, we propose a likelihood-based approach to assess the frequency of pathogenicity among probands with VUSs, while accounting for the existence of possible pathogenic mutations on genes not tested. We conducted simulations to assess the performance of the approach and show that under various settings, the approach performs well with very little bias in the estimated proportion of VUSs that are likely pathogenic. We also estimate the positive predictive value across the entire range of carrier scores. We apply our approach to the USC-Stanford Hereditary Cancer Panel Testing cohort, and estimate the proportion of probands that have VUSs in BRCA1/2 that are likely pathogenic to be 10.12% [95%CI: 0%, 43.04%]. This approach will enable clinicians to target high-risk patients who have VUSs, allowing for early prevention interventions.

Concentrically Integrative Bioassembly of a Three-Dimensional Black Phosphorus Nanoscaffold for Restoring Neurogenesis, Angiogenesis, and Immune Homeostasis.

Black phosphorus is well known for its excellent electromechanical properties. Although it has previously been used for therapeutic drug delivery in cancer, it has never been applied as an electroactive polymer for post-trauma tissue regeneration (e.g., in cardiac muscles and neurons). The major concern currently preventing such applications is its controversial biosafety profile in vivo. Here, we demonstrate the production of a concentrically integrative layer-by-layer bioassembled black phosphorus nanoscaffold. This scaffold has remarkable electrical conductivity, permitting smooth release into the surrounding microenvironment. We confirmed that, under mild oxidative stress, our black phosphorus nanoscaffold induced angiogenesis and neurogenesis and stimulated calcium-dependent axon regrowth and remyelination. Long-term in vivo implantation of this nanoscaffold during severe neurological defect regeneration induced negligible toxicity levels. These results provide new insight into the regenerative capability of manufactured 3D scaffolds using neuroengineered 2D black phosphorus nanomaterials.

Biodegradable Carriers for Delivery of VEGF Plasmid DNA for the Treatment of Critical Limb Ischemia.

The safe and efficient delivery of therapeutic nucleic acid is a prerequisite for an effective DNA therapy. In this study, we condensed the low molecular weight polyethylenimine (PEI, 1.8k Da) with 2,6-pyridinedicarboxaldehyde (PDA), both of which are degradable in vivo, to synthesize a biodegradable polycationic material (PDAPEI) to deliver vascular endothelial growth factor (VEGF) plasmid DNA (pDNA). Particle size and zeta potential of this novel degradable PEI derivatives-pDNA nanoparticle were investigated and in vitro cytotoxicity was estimated on human umbilical vein endothelial cells (HUVECs). Using pDNA-encoding VEGF-A and green fluorescence protein (GFP), we also checked transfection efficiency of the vector (PDAPEI) and found its excellent performance at 40 w/w ratio. We successfully established peripheral ischemia animal model on C57/BL6J mice to evaluate the therapeutic effect of PDAPEI/pVEGF-A polyplex system on ischemic disease and a conclusion was made that PDAPEI is a promising gene vector in the treatment of peripheral ischemic artery disease (PAD).