Search

Driven by the need for simulating compressible flows, Germano identity-based [Z. Yin and P. A. Durbin, “An adaptive DES model that allows wall-resolved eddy simulation,” Int. J. Heat Fluid Flow 62, 499–509 (2016)] and Vreman operator-based [Bader et al., “A hybrid model for turbulence and transition, with a locally varying coefficient,” Flow, Turbul. Combust. 108, 935–954 (2022)] dynamic ℓ2−ω delayed detached eddy simulation (DDES) formulations are constructed on the k−ω shear stress transport (SST) model. The Bachalo–Johnson transonic axisymmetric bump is simulated to assess the models’ capability in handling the compressible boundary layers under pressure gradient and transonic shock–boundary layer interaction. The new dynamic ℓ2−ω DDES formulation based on k−ω SST overcomes the issues of freestream sensitivity and inaccurate compressible boundary layer profile observed in the original k−ω (88) based model. The new SST-based dynamic model using the Vreman operator to compute the model coefficient (Vreman-dynamic model) has superior performance against Germano identity-based model due to its capability of suppressing the subgrid viscosity during the initial development of a separating shear layer. The Vreman-dynamic model predicts a reattachment location similar to the zonal improved-DDES/direct numerical simulation approach by Spalart et al. [“Large-eddy and direct numerical simulations of the bachalo-johnson flow with shock-induced separation,” Flow, Turbul. Combust. 99, 865–885 (2017)] on a much coarser mesh demonstrating its potential for application in industrial flows.

Background: Arthritis is a cartilage degenerative disease that is mainly induced by the degradation of the cartilage extracellular matrix (ECM), which is found to be regulated by the expression level of a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMT-5), an enzyme degrading Aggrecans in the ECM. Feprazone is a classic nonsteroidal anti-inflammatory drug with promising efficacy in arthritis. The present study aims to investigate the protective effect of Feprazone on the degraded Aggrecan in the human chondrocytes induced with tumor necrosis factor-α (TNF-α) and to clarify the underlying mechanism. Methods: To investigate the effect of Feprazone, the CHON-001 chondrocytes were stimulated with TNF-α (10 ng/mL) in the presence or absence of Feprazone (3, 6 μM) for 24 h. Mitochondrial membrane potential was evaluated using the Rhodamine 123 assay. The gene expressions of interleukin-1β (IL-1β), interleukin-8 (IL-8), monocyte chemotactic protein 1 (MCP-1), and ADAMTS-5 in the treated chondrocytes were detected using real-time quantitative polymerase chain reaction (qRT-PCR), and the protein levels of these targets were determined using enzyme-linked immunosorbent assay (ELISA). SOX-4 was knocked down by transfecting the siRNA into the chondrocytes. Western blot analysis was utilized to evaluate the expression levels of SOX-4, Aggrecan, and protein kinase C (PKCα). Results: First, the reduced mitochondrial membrane potential (ΔΨm) and secretion of proinflammatory factors (IL-1β, IL-8, and MCP-1) induced by TNF-α were significantly reversed by treatment with Feprazone. Second, the expression of Aggrecan was significantly decreased by stimulation with TNF-α via upregulation of ADAMTS-5 but was dramatically reversed by the introduction of Feprazone. Third, we found that TNF-α elevated the expression of ADAMTS-5 by upregulating SOX-4, which was observed to be related to the activation of PKCα. Lastly, the elevated expression of SOX-4 induced by TNF-α was significantly reversed by Feprazone. Conclusions: Feprazone might ameliorate TNF-α-induced loss of Aggrecan via the inhibition of the SOX-4/ADAMTS-5 signaling pathway.

OBJECTIVE: To design a new type of three-dimensional honeycomb guide for percutaneous cannulated screw placement in femoral neck fracture and evaluate its effectiveness. METHODS: The clinical data of 40 patients with femoral neck fracture who met the selection criteria between June 2019 and December 2020 were retrospectively analyzed. According to different intraoperative positioning methods, they were divided into control group (20 cases, free hand positioning screws) and study group (20 cases, new guide assisted positioning screws). There was no significant difference in gender, age, side, cause of injury, Garden classification, and time from injury to operation between the two groups (P>0.05). The operation time, fluoroscopy times, guide needle puncture times, and fracture healing time of the two groups were recorded. The hip function was evaluated by Harris score at last follow-up. At immediate after operation, the following imaging indexes were used to evaluate the accuracy of screw implantation distribution: screw spacing, screw coverage area, distance from screw to cervical cortex, parallelism between screws, and screw to cervical axial deviation. RESULTS: All operations were successfully completed, and the guide needle did not penetrate the femoral neck cortex. There was no significant difference in operation time and fluoroscopy times between the two groups (P>0.05); the guide needle puncture times in the study group was significantly less than that in the control group (t=8.209, P=0.000). Imaging detection at immediate after operation showed that the screw spacing and screw coverage area in the study group were significantly greater than those in the control group (P0.05). CONCLUSION: The new three-dimensional honeycomb guide has the advantages of simple structure and convenient use. It can reduce the puncture times of the guide needle and effectively improve the accuracy distribution of cannulated screw implantation.

Background. Knee synovitis is a common sports injury. We proposed the use of UTVOR, which is a combination of the use of volatile oil of Olibanum (VOO) and volatile oil of Chuanxiong Rhizoma (VOCR) and conventional ultrasound (US) therapy, to treat knee synovitis. Design, Setting, Participants, and Interventions. Participants were randomly assigned into a control group (conventional US therapy group) and a test group (UTVOR group). The control group received conventional US therapy with a coupling agent as the medium. The test group received a revised US therapy with VOO and VOCR as media. Both groups were treated once per day for three consecutive days. Main Outcome Measures. The subjects’ Visual Analogue Scale (VAS) pain score, Lysholm knee score, knee swelling degree, circumference, and range of motion of the knee joint were evaluated before the first treatment and 24 h after the third treatment. The VAS pain score was considered the primary outcome, while the three other measurements were regarded as the secondary outcomes. An adverse event was reported subjectively and recorded. Results. A total of 116 participants were included in the analysis (test group: n = 64; control group: n = 52). The evaluation results showed that the VAS pain scores of the male and female participants in both groups decreased after treatment ( P < 0.001 ), but only the difference among the male sub-group had significant between-group difference ( P < 0.001 ). After treatment, the Lysholm scores in both groups increased significantly (all P < 0.001 ), the range of motion and the circumference of the injured knee decreased significantly ( P < 0.001 ), while no between-group difference was observed in general or in the gender sub-groups (all P > 0.025 ). No side effect or complication was reported during the treatment. Conclusion. UTVOR had a superior analgesic effect to conventional US therapy in the male population, but its effects on alleviating joint function, swelling, and range of motion were comparable to that of conventional US therapy. Our study found that UTVOR can be an effective method to reduce pain and treat knee synovitis, and it is subjectively safe. Trial registration. This study was registered under the Chinese Clinical Trial Registry (Trial Registration Number: ChiCTR2000035671).

The adaptive, ℓ2–ω delayed detached eddy simulation model was selected to simulate the flow in the V103 linear compressor cascade. The Reynolds number based on axial chord length is 138, 500. Various inflow turbulent intensities from 0% to 10% were tested to evaluate the performance of the adaptive model. The adaptive model is capable of capturing the laminar boundary layer and the large-scale perturbations inside it. The instability of large-scale disturbances signals the switch to a hybrid simulation of the turbulent boundary layer—thus, the transition front is predicted. In the case of separation-induced transition, the adaptive model, which uses eddy simulation in separated flow, can predict the separation bubble size accurately. Generally, the adaptive ℓ2–ω model can simulate the transitional separated flow in a linear compressor cascade, with a correct response to varying turbulent intensities.

Mycoplasma infection is the most prevalent contamination in cell culture. Analysis of cell culture in laboratories from different countries shows that mycoplasma contamination ranges from 15% to 80% and, in some cases, even reaches 100% (Chernov et al., 2014). Whilst mycoplasma infection is not visible to the naked eye in cell culture, the consequences of mycoplasma contamination have been shown to induce a number of cellular changes, for example, increased resistance to chemotherapeutic drugs. Therefore, any results obtained from tissue culture studies, in the presence of mycoplasma contamination, potentially render the data invalid (Kim et al., 2015; Gedye et al., 2016). As such, mycoplasmas are not harmless bystanders and cannot be ignored in in vitro studies.

In this study, we investigated whether melittin could suppress hypoxia-induced vasculogenic mimicry (VM) formation in liver cancer and explored the underlying mechanisms. Melittin significantly inhibited the proliferation of liver cancer cells with or without CoCl2presence. Melittin also significantly inhibited CoCl2-induced migration, invasion, and VM formation of liver cancer cells. CoCl2treatment suppressed the expression of E-cadherin and elevated the expression of N-cadherin and Vimentin. Melittin reversed the changes in the protein and mRNA levels of these epithelial-mesenchymal transition (EMT) markers. CoCl2-induced accumulation of HIF-1αincreased the level of phosphorylated Akt and upregulated the expression of VEGF and MMP-2/9. Melittin decreased the HIF-1αlevel and thereby suppressed the levels of p-Akt, VEGF, and MMP-2/9. In addition, the inhibitor of PI3K/Akt also suppressed CoCl2-induced EMT and liver cancer cells migration, and the activator of Akt, SC-79, partly blocked the effect of melittin on CoCl2-induced EMT and liver cancer cells migration. In the xenograft tumor model in nude mice, melittin treatment significantly suppressed the tumor growth, VM formation, and HIF-1αexpression in the tumor. In conclusion, this study indicates melittin may inhibit hypoxia-induced VM formation and EMT in liver cancer through inhibiting HIF-1α/Akt pathway.

This study aimed to investigate the underlying molecular pathogenic mechanism of Sec62 in hepatocellular carcinoma (HCC). Microarray analysis was conducted to profile the global gene expression in the HCC cell line Huh7 cells transfected with Sec62high vs. NC and Sec62low vs. NC. Ingenuity pathway analysis and gene set enrichment analysis were used to perform Sec62-related signaling pathway analysis from screened differentially expressed genes (DEGs). A protein-protein interaction network was constructed. Experimental validation of the expression of key DEGs was conducted. Hypoxia-induced tube formation was undertaken to investigate the role of Sec62 in angiogenesis. A total of 74 intersected DEGs were identified from Huh7 cells with Sec62high vs. NC and Sec62low vs. NC. Among them, 65 DEGs were correlated with the expression of Sec62. The P53 signaling pathway was found to be enriched in Huh7 cells with Sec62high vs. NC, while the acute phase response signaling pathway was enriched in Huh7 cells with Sec62low vs. NC. DEGs, such as serine protease inhibitor E (SERPINE) and tumor necrosis factor receptor superfamily, member 11B (TNFRSF11B), were not only identified as the lead genes of these enriched pathways, but were also found to be closely related to Sec62. Moreover, knockdown of Sec62 decreased the expression of SERPINE1 (plasminogen activator inhibitor type 1 (PAI-1)) and TNFRSF11B, whereas overexpression of Sec62 had the opposite effects. In addition, knockdown of Sec62 inhibited hypoxia-induced tube formation via PAI-1. Sec62 promoted pro-angiogenesis of HCC under hypoxia by regulating PAI-1, and it may be a crucial angiogenic switch in HCC.

Delayed detached eddy simulations and wall-modeled eddy simulations using the adaptive DES model were performed to simulate corner separation in the Ecole Centrale de Lyon linear compressor cascade. The adaptive DES model directly uses length scale to define eddy viscosity, which makes it nature to compute the model constant CDES dynamically. The dynamic procedure adapts viscosity to local flow and grid. Delayed detached eddy simulations, with and without the dynamic procedure, were performed to demonstrate the benefit of adapting viscosity to local flow. Recycling method was adopted to generate inflow unsteady turbulent boundary layer for wall-modeled eddy simulations. The wall-modeled eddy simulation showed improvement over delayed-DES, in terms of static pressure coefficient around the blade and total pressure loss at downstream locations.

The success of gene and cell therapy in clinic during the past two decades as well as our expanding ability to manipulate these biomaterials are leading to new therapeutic options for a wide range of inherited and acquired diseases. Combining conventional therapies with this emerging field is a promising strategy to treat those previously-thought untreatable diseases. Traditional Chinese medicine (TCM) has evolved for thousands of years in China and still plays an important role in human health. As part of the active ingredients of TCM, proteins and peptides have attracted long-term enthusiasm of researchers. More recently, they have been utilized in gene and cell therapy, resulting in promising novel strategies to treat both cancer and non-cancer diseases. This manuscript presents a critical review on this field, accompanied with perspectives on the challenges and new directions for future research in this emerging frontier.

Postsurgical recurrence within 2 years is the major cause of poor survival of hepatocellular carcinoma (HCC) patients. However, the molecular mechanism underlying HCC recurrence remains unclear. Here, we distinguish the function and mechanism of Sec62 in promoting HCC recurrence. The correlation between Sec62 and early recurrence was demonstrated in 60 HCC samples from a prospective study. HCC cells with Sec62 knockdown (Sec62KD) or overexpression (Sec62OE) were used to determine the potential of Sec62 in cell migration in vitro. Microarray analysis comparing Sec62KD or Sec62OE to their control counterparts was used to explore the mechanisms of Sec62-induced recurrence. A luciferase-labelled orthotopic nude mouse model of HCC with Sec62KD or Sec62OE was used to validate the potential of Sec62 in early HCC recurrence in vivo. We found that high expression of Sec62 was positively correlated with surgical recurrence in clinical HCC samples. Multivariate analysis revealed that Sec62 was an independent prognostic factor for early recurrence in postoperative HCC patients. Moreover, Sec62 promoted migration and invasion of HCC cells in vitro and postsurgical recurrence in vivo. Mechanically, integrinα/CAV1 signalling was identified as one of the targets of Sec62 in cell movement. Overexpression of integrin α partially rescued the Sec62 knockdown-induced inhibition of cell migration. Sec62 is a potentially prognostic factor for early recurrence in postoperative HCC patients and promotes HCC metastasis through integrinα/CAV1 signalling. Sec62 might be an attractive drug target for combating HCC postsurgical recurrence.

In turbomachinery CFD simulations, Reynolds-Averaged Navier-Stokes (RANS) based approaches tend to under-predict turbulent mixing, which is of particular importance in the endwall region. Detached Eddy Simulation (DES), solving Reynolds-averaged equations near the wall and partially resolving turbulent content elsewhere, bypasses the difficulties in statistic modeling. Previous work using Delayed-DES (DDES) in a linear compressor cascade [6] showed improved loss profile prediction owing to better representation of flow structures. The observation was that certain grid refinement near separated shear layer is necessary for resolving unsteadiness and further more, to reveal a bimodal behavior of the separation. Recent development in DES, the Shear-Layer-Adapted (SLA) modification [5], is designed to accelerate the growth of resolved turbulence downstream of the RANS-to-LES interface. In the current work, the impact of SLA modification on the DES simulation of linear compressor cascade flow is investigated. We incorporated the SLA modification into the SST-DDES [7] model and quantified the benefit of it in turbomachinery applications.

A modification to the Adaptive-DES method of Yin et al. (2015) is proposed to improve its near-wall behavior. The modification is to the function (Clim) that imposes a lower limit on the dynamically evaluated coefficient (CDES). The modification allows Adaptive-DES to converge to wall-resolved eddy simulation, when grid resolution supports it. On coarse grids, or at high Reynolds number, it reverts to shielded DES — that is to DDES. The new formulation predicts results closer to wall-resolved LES than the previous formulation. It provides an ability to simulate transition: it is tested in both orderly and bypass transition. In fully turbulent, attached flow, the modification has little effect. Any improvement in predictions stem from better near-wall behavior of the adaptive method.

Emodin, an anthraquinone derivative from the root and rhizome of Rheum palmatum L., was found to have antitumor effects in different types of cancer by regulating multi-molecular targets. The aim of the present study was to explore the effect of emodin on the migration and invasion of MHCC-97H human hepatocellular carcinoma cells and the underlying molecular mechanisms. Firstly, it was demonstrated that emodin can inhibit cell proliferation and induce apoptosis of cells in a time- and dose-dependent manner, using a MTT assay and flow cytometry, respectively. However, when emodin concentration was

A transport model for hybrid RANS/LES simulation of passive scalars is proposed. It invokes a dynamically computed subgrid Prandtl number. The method is based on computing test-filter fluxes. The formulation proves to be especially effective on coarse grids, as occur in DES. After testing it in a wall resolved LES, the present formulation is applied to the Adaptive DDES model of Yin et al. (Phys. Fluids 27, 025105 2015). It is validated by turbulent channel flow and turbulent boundary layer computations.

Background The choice between unicondylar knee arthroplasty (UKA) and total knee arthroplasty (TKA) is likely to have long-term implications for patient-reported health outcomes. However, high-quality studies that compare the outcomes of TKA and UKA and their effects are still lacking in the literature. Thus, the aim of the present study was to compare the UKA and TKA techniques with regard to functional outcomes and perioperative complications in patients who had isolated medial osteoarthritis. Methods This was a retrospective, single-center, matched-controlled study performed with approval of our hospital (Kunshan hospital of Traditional Chinese Medicine affiliated to Nanjing University of Traditional Chinese Medicine), with the ethics number KZY2020-37. To reduce the effect of selection bias and potential confounding in this observational study, a 1:1 matching algorithm was applied. The groups were split by sex, age to within 6 years, and body mass index within 5 kg/m. Thus, we retrospectively reviewed the records of 240 consecutively enrolled patients who underwent UKA and 240 patients who underwent TKA from January 2013 to June 2015 from the database of our institution. Written informed consent was obtained from all subjects participating in the trial. Clinical outcomes included range of motion, Short Form 12 score, new Knee Society Score, Western Ontario and McMaster Universities Arthritis Index, and the complications. The outcome measures were evaluated by a physiotherapist and were assessed preoperatively and postoperatively at 6 months and 2 years. The mean follow-up time was 3 years. Conclusion We hypothesized that there was no significant difference between the 2 groups in terms of postoperative outcomes. Trial registration Our study was registered in Research Registry (researchregistry5828).

Background: Postsurgical recurrence within 2 years occurs in 30-50% of Hepatocellular carcinoma (HCC) patients, being the major cause of mortality. We have reported that high expression of Sec62 was found in surgical recurrence patients of HCC from a retrospective study. However, the role and mechanism of Sec62 in HCC recurrence remains poorly understand. Here we distinguish the the function of Sec62 in early HCC recurrence. Methods: A luciferase-labeled orthotopic model of HCC with Sec62 overexpressed or downregulated, seperately was used to validate the potential of Sec62 in early HCC recurrence in vivo. The microarray analysis comparing Sec62low or Sec62high and their control counterparts was used to explore targets in Sec62 induced recurrence. The role of Sec62 was further proved in 60 HCC patients undergoing surgical resection from a prospective study. Findings: We found that Sec62 overexpression promoted postsurgical recurrence in vivo and the migration and invasion of HCC cells in vitro. Mechanically, integrin α/CAV1 signaling was identified as a target of Sec62 for cells movement. Up-regulation of integrin α and CAV1 rescued the decreased cell migration and invasion, which were induced by Sec62 knockdown. Furthermore, high expression of Sec62 was correlated with surgical recurrence in clinical HCC samples and positively correlated with Integrin α/CAV1 signaling. Multivariate analysis revealed that Sec62 was an independent prognostic indicator for cumulative recurrence in this cohort of HCC patients post-surgical. Interpretation: Overexpression of Sec62 was implicated in recurrence of HCC after surgical and Sec62 overexpression may be a potential molecular therapeutic targets for HCC. Funding: This work was supported by National Nature Science Foundation of China (grant number: 81473484) and Nature Science Foundation of Shanghai (grant number: 14ZR1408400). Declaration of Interest: The authors declare that they have no conflict of interest. Ethical Approval: The animal protocols were performed in agreement with SIBS Guide for the Care and Use of Laboratory Animals and were approved by Animal Ethics Committee of Zhongshan Hospital, Fudan University.

Background: Postsurgical recurrence within 2 years is the major cause of poor survival of hepatocellular carcinoma (HCC) patients. However, the molecular mechanism underlying HCC recurrence remains unclear. Here, we distinguish the function and mechanism of Sec62 in promoting HCC recurrence. Methods: The correlation between Sec62 and early recurrence was demonstrated in 60 HCC samples from a prospective study. HCC cells with Sec62 knockdown (Sec62KD) or overexpression (Sec62OE) were used to determine the potential of Sec62 in cell migration in vitro. Microarray analysis comparing Sec62KD or Sec62OE to their control counterparts was used to explore the mechanisms of Sec62-induced recurrence. A luciferase-labelled orthotopic nude mouse model of HCC with Sec62KD or Sec62OE was used to validate the potential of Sec62 in early HCC recurrence in vivo. Findings: High expression of Sec62 was positively correlated with surgical recurrence in clinical HCC samples. Multivariate analysis revealed that Sec62 was an independent prognostic factor for early recurrence in postoperative HCC patients. Moreover, Sec62 promoted migration and invasion of HCC cells in vitro and postsurgical recurrence in vivo. Mechanically, integrinα/CAV1 signalling was identified as one of the targets of Sec62 in cell movement. Overexpression of integrinα partially rescued the Sec62 knockdown-induced inhibition of cell migration. Interpretation: Sec62 is a potentially prognostic factor for early recurrence in postoperative HCC patients and promotes HCC metastasis through integrinα/CAV1 signalling. Sec62 might be an attractive drug target for combating HCC postsurgical recurrence. Funding Statement: This work was supported by the National Nature Science Foundation of China (grant number: 81473484) and the Nature Science Foundation of Shanghai (grant number: 14ZR1408400). Declaration of Interests: The authors declare that they have no conflict of interest. Ethics Approval Statement: The animal protocols were performed in agreement with the SIBS Guide for the Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of Zhongshan Hospital, Fudan University.

To summarize the effectiveness of 3.5 mm T support plate fixation for simple postero-lateral tibial plateau fractures by posterolateral inverted L-shaped approach.Between March 2011 and January 2016, 13 patients with simple posterolateral tibial plateau fracture were treated with 3.5 mm T support plate fixation by posterolateral inverted L-shaped approach. Of 13 cases, 6 were male and 7 were female, aged 28 to 52 years (mean, 43 years). The left side was involved in 5 cases and the right side in 8 cases. The causes of injury were traffic accidents in 6 cases, falls in 3 cases, and falling from height in 4 cases. All of patients had fresh closed fracture by X-ray, CT three dimensional reconstruction, and MRI. According to Schatzker classification, 4 cases were rated as type II and 9 cases as type III. The time between injury and operation was 5-9 days (mean, 7 days).One case had incision skin necrosis, which was cured after debridement and skin grafting; and primary healing was obtained in the other cases. The patients were followed up 10-16 months (mean, 13 months). The X-ray film showed that the fracture line was blurred at 3 months after operation, and disappeared at 12 months after operation. There was no complications of wound infection, major neurovascular injury, loosening or breakage of internal fixation, and dislocation of joint surface. The Hospital for Special Surgery (HSS) knee function score was 94 (range, 89-97) at last follow-up; all were excellent.The 3.5 mm T support plate fixation by posterolateral inverted L-shaped approach is one of preferred surgical method for the treatment of simple posterolateral tibial plateau fractures.总结后外侧倒 L 形入路 3.5 mm T 形支撑钢板治疗单纯后外侧胫骨平台骨折的临床疗效。.2011 年 3 月—2016 年 1 月，采用后外侧倒 L 形入路 3.5 mm T 形支撑钢板内固定治疗单纯后外侧胫骨平台骨折 13 例。男 6 例，女 7 例；年龄 28～52 岁，平均 43 岁。左侧 5 例，右侧 8 例；致伤原因：交通事故伤 6 例，摔伤 3 例，高处坠落伤 4 例。术前行 X 线片、CT 三维重建和 MRI 检查，均为单纯后外侧胫骨平台骨折；Schatzker 分型：Ⅱ型 4 例，Ⅲ型 9 例。受伤至手术时间 5～9 d，平均 7 d。.术后 1 例患者切口皮肤坏死，给予清创植皮后创面愈合；余均Ⅰ期愈合。13 例患者均获随访，随访时间 10～16 个月，平均 13 个月。所有患者术后 3 个月 X 线片示骨折线均模糊，术后 12 个月骨折线均消失。无切口感染、重要血管神经损伤、内固定物松动或断裂以及关节面再次移位塌陷等并发症发生。末次随访时按改良美国特种外科医院（HSS）膝关节功能评分评价膝关节功能，获 89～97 分，平均 94 分；均达优。.后外侧倒 L 形入路 3.5 mm T 形支撑钢板内固定可作为治疗单纯后外侧胫骨平台骨折的首选手术方式之一。.

Viral vectors-based gene therapy is an attractive alternative to common anti-cancer treatments. In the present studies, AAV serotype 6 vectors were identified to be particularly effective in the transduction of human prostate (PC3), breast (T47D) and liver (Huh7) cancer cells. Next, we developed chimeric AAV vectors with Arg-Gly-Asp (RGD) peptide incorporated into the viral capsid to enable specific targeting of integrin-overexpressing malignant cells. These AAV6-RGD vectors improved transduction efficiency approximately 3-fold compared with wild-type AAV6 vectors by enhancing the viral entry into the cells. We also observed that transduction efficiency significantly improved, up to approximately 5-fold, by the mutagenesis of surface-exposed tyrosine and threonine residues involved in the intracellular trafficking of AAV vectors. Therefore, in our study, the AAV6-Y705-731F+T492V vector was identified as the most efficient. The combination of RGD peptide, tyrosine and threonine mutations on the same AAV6 capsid further increased the transduction efficiency, approximately 8-fold in vitro. In addition, we mutated lysine (K531E) to impair the affinity of AAV6 vectors to heparan sulfate proteoglycan. Finally, we showed a significant increase in both specificity and efficiency of AAV6-RGD-Y705-731F+T492V+K531E vectors in a xenograft animal model in vivo. In summary, the approach described here can lead to the development of AAV vectors with selective tropism to human cancer cells.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. In China, traditional Chinese herb medicine has been widely used in the treatment of HCC. Jiedu Recipe (JR) is a common used prescription which has shown good results against HCC. However, the exact mechanisms of JR are still unknown. Therefore, we investigated the efficacy of JR on HCC in the current study. JR inhibited the cell viability of both SMMC-7721 and Huh7 cells in both time- and dose-dependent manners. Transwell assay revealed that JR decreased the number of migrated cells of SMMC-7721 cells. JR treatment increased the E-cadherin expression level and decreased the levels of p-Smad2/3 and Smad2/3. Further study showed that JR reversed the effect of TGFβ1 on the expression of E-cadherin, vimentin, N-cadherin, and MMP2/9. JR also significantly inhibited TGFβ1-induced migration and invasion of SMMC-7721 and Huh7 cells determined by wound healing assay and transwell assay. TGFβ1 treatment increased the phosphorylation of Smad2/3, p38 MAPK, JNK, ERK1/2, and Akt in SMMC-7721 cells and pretreatment with JR blocked TGFβ1-induced activation of Smad2/3 and Akt and MAPKs. In conclusion, JR inhibits liver cancer cells migration and invasion through epithelial mesenchymal transition (EMT) inhibition via Smad2/3 dependent and independent pathways, suggesting it is an effective therapeutic strategy against HCC metastasis.

This study is a continuation of our previous work on microwave-assisted membrane distillation. The crystal morphology of sodium chloride and calcium carbonate in the process of membrane distillation and microwave membrane distillation was investigated in this work. The effect of salt concentration on the ability of absorbing microwave of solution and the mass transfer of microwave membrane distillation were also analyzed. The results show that the ionic conduction is the main between two factors (ionic conduction and dipole rotation) which have effect on absorbing microwave. When the mass concentration of sodium chloride solution is up to 5%, the microwave energy absorbed by solution is almost five times as much as pure water. Moreover, the increase in salt concentration has no significant extra influence on the mass transfer of microwave membrane distillation. The crystallization experiments confirm that microwave irradiation do not affect membrane fouling or worsen it. The different effect of microwave irradiation on crystallization of two typical inorganic salts is observed in this study. For sodium chloride, the microwave heating leads to the decline of the total particle number, while the total deposition keeps constant and the size distribution of crystal particles becomes more uniform, compared with conventional heating. As for calcium carbonate, microwave irradiation not only increases the total deposition and total particle number, but also induces the crystals growth. Especially, it is found that microwave irradiation induces the crystals to grow toward aragonite. The ratios of the aragonite number to the total particle number increase from 28.0% and 56.5% to 81.1% and 85.8% at the two higher feed mass concentrations, respectively.

The field of gene therapy has been increasingly studied in the last four decades, and its clinical application has become a reality in the last 15 years. Traditional Chinese medicine (TCM), an important component of complementary and alternative medicine, has evolved over thousands of years with its own unique system of theories, diagnostics and therapies. TCM is well-known for its various roles in preventing and treating infectious and chronic diseases, and its usage in other modern clinical practice. However, whether TCM can be applied alongside gene therapy is a topic that has not been systematically examined. Here we provide an overview of TCM theories in relation to gene therapy. We believe that TCM theories are congruent with some principles of gene therapy. TCM-derived drugs may also act as gene therapy vehicles, therapeutic genes, synergistic therapeutic treatments, and as co-administrated drugs to reduce side effects. We also discuss in this review some possible approaches to combine TCM and gene therapy.

In this paper, a vacuum membrane distillation (VMD) system equipped with a microwave source was designed. And the polyvinylidene fluoride (PVDF) hollow fiber hydrophobic membranes were selected to carry out microwave vacuum membrane distillation (MWVMD). The effects of feed temperature, vacuum degree and feed velocity on microwave strengthening mass transfer process were preliminarily investigated. The results showed that microwave irradiation could effectively induce uniform heating in the radial direction of the membrane module, and significantly improve the mass transfer process of vacuum membrane distillation. The maximum membrane distillation mass transfer coefficient increasing rate in this study was obtained as about 27.7% in the conditions of the temperature of 60 °C, the feed velocity of 0.14 m/s and the vacuum degree of −96 kPa. And the increment was largened with the decrease of the feed temperature and feed velocity as well as the increase of the vacuum degree. The influences of microwave irradiation on the membrane properties were also investigated. The results indicated that microwave irradiation had no significant effect on the mechanical properties and hydrophobicity of membrane materials which were situated in microwave field for more than 120 h. The influence of microwave irradiation on the membrane fouling was also analyzed. For the solution which contains 30 mg/L Ca 2+ , microwave irradiation aggravated the deposition of calcium carbonate to some extent.

Traditional Chinese medicine formulates treatment according to body constitution (BC) differentiation. Different constitutions have specific metabolic characteristics and different susceptibility to certain diseases. This study aimed to assess the characteristic genes of gan-shen Yin deficiency constitution in different diseases. Fifty primary liver cancer (PLC) patients, 94 hypertension (HBP) patients, and 100 diabetes mellitus (DM) patients were enrolled and classified into gan-shen Yin deficiency group and non-gan-shen Yin deficiency group according to the body constitution questionnaire to assess the clinical manifestation of patients. The mRNA expressions of 17 genes in PLC patients with gan-shen Yin deficiency were different from those without gan-shen Yin deficiency. However, considering all patients with PLC, HBP, and DM, only MLH3 was significantly lower in gan-shen Yin deficiency group than that in non-gen-shen Yin deficiency. By ROC analysis, the relationship between MLH3 and gan-shen Yin deficiency constitution was confirmed. Treatment of MLH3 (−/− and −/+) mice with Liuweidihuang wan, classical prescriptions for Yin deficiency, partly ameliorates the body constitution of Yin deficiency in MLH3 (−/+) mice, but not in MLH3 (−/−) mice. MLH3 might be one of material bases of gan-shen Yin deficiency constitution.

Oxidative stress, which occurs after ultraviolet (UV) radiation, usually results in Glucocorticoid (GC) resistance and the subsequent development of skin inflammation. One approach to protecting the skin against UV radiation is the use of antioxidants. The ginsenoside Rg1 is a novel natural antioxidant isolated from the medicinal plant Panax ginseng C.A. Mey. We demonstrated that UVB exposure exacerbated inflammation and reduced both the level of the glucocorticoid receptor (GR) and the efficacy of dexamethasone (Dex) in human keratinocytes (HaCaT cells). Pretreatment with Rg1 increased the expression of GR and restored Dex responsiveness to inflammation in UVB-irradiated HaCaT cells. Mechanistically, Rg1 rescued UVB-induced HDAC2 degradation. HDAC2 knockdown partially abolished the Rg1-induced up-regulation of GR and the enhancement of GC sensitivity. In addition, Rg1 reduced the production of reactive oxygen species (ROS), which preceded the up-regulation of HDAC2, and consequent sensitization of cells to Dex. Moreover, Rg1 treatment promoted the translocation and activation of Nrf2. Nrf2 knockdown partially abolished the Rg1-induced decrease of ROS production and increase of HDAC2. Rg1 also potentiated the anti-inflammatory effects of Dex in UVB-irradiated mouse skin. In conclusion, we demonstrated that Rg1 attenuated UVB-induced GC insensitivity. Notably, these effects were partially mediated by the Nrf2/HDAC2 pathway.

An adaptive method for detached-eddy simulation (DES) is tested by simulations of flow in a family of three-dimensional (3D) diffusers. The adaptive method either adjusts the model constant or defaults to a bound if the grid is too coarse. On the present grids, the adaptive method adjusts the model constant over most of the flow, without resorting to the default. Data for the diffuser family were created by wall-resolved, large-eddy simulation (LES), using the dynamic Smagorinsky model, for the purpose of testing turbulence models. The family is a parameterized set of geometries that allows one to test whether the pattern of separation is moving correctly from the top to the side wall as the parameter increases. The adaptive DES model is quite accurate in this regard. It is found to predict the mean velocity accurately, but the pressure coefficient is underpredicted. The latter is due to the onset of separation being slightly earlier in the DES than in the LES.

Magnetic-luminescent nanocomposites have obtained a great deal of attention due to their potential applications in magnetic separation of cells, magnetic resonance imaging and luminescence imaging, and so on. Herein, a new and facile two-step synthetic strategy was developed to prepare magnetic Fe3O4 modified single crystalline up-conversion luminescent NaYF4: Yb3+, Er3+ nanocrystals using Fe3O4 nanoparticles as the seeds. Acting as the "nuclei" in the reaction, the presence of Fe3O4 nanoparticles facilitates the formation of up-conversion NaYF4: Yb3+, Er3+ nanocrystals on their surfaces through heterogeneous nucleation process. The structure and properties of these multifunctional nanocrystals were characterized by transmission electron microscope (TEM), X-ray photoelectron spectroscopy (XPS), photo luminescence (PL) and X-ray diffraction (XRD). The results revealed that the as-prepared multifunctional nanocrystals show dual distinct properties of up-conversion fluorescent and superparamagnetism, which were cubic in shape and very uniform in size with an average size ranging 30–40 nm. These unique functionalities could be very attractive for biological systems, and some biological applications such as cell-labelling were also demonstrated.

We showed that WT adeno-associated virus serotype 2 (AAV2) genome devoid of a conventional polyadenylation [poly(A)] signal underwent complete genome replication, encapsidation and progeny virion production in the presence of adenovirus. The infectivity of the progeny virion was also retained. Using recombinant AAV2 vectors devoid of a human growth hormone poly(A) signal, we also demonstrated that a subset of mRNA transcripts contained the inverted terminal repeat (ITR) sequence at the 3′ end, which we designated ITR in RNA (ITRR). Furthermore, AAV replication (Rep) proteins were able to interact with the ITRR. Taken together, our studies suggest a new function of the AAV2 ITR as an RNA element to mediate transgene expression from poly(A)-deleted mRNA.

Several groups have demonstrated that a number of naturally occurring compounds can significantly enhance the therapeutic efficacy of viral vectors, especially recombinant AAV vectors (Ren et al., Neurosci Lett. 479(3): 187-91, 2010; Zhang et al., Gene Ther. 18(2): 128-34, 2011; Mitchell et al., J Virol. 87(8): 4571-83, 2013; Wang et al., J Integr Med. 12(1): 20-34, 2014). In our recently published studies (Ling et al., Hum Gene Ther. 25(12): 1023-34, 2014), we provided further evidence of enhanced efficacy of a combinatorial approach involving drug-mediated chemotherapy and rAAV vector-mediated cancer gene therapy to target human hepatocellular carcinoma. In these studies, we identified that shikonin, a naphthoquinone, inhibits human liver tumor growth as well as significantly enhances the efficacy of rAAV vectors carrying a therapeutic gene, tricosanthin (TCS), in a murine xenograft model in vivo. Although our initial mechanistic studies suggested that shikonin-mediated enhancement of rAAV vector transduction was through its proteasome inhibitory activity, which was similar to that of MG132, a specific proteasome inhibitor. However, no further increase was observed when cells were co-treated with both shikonin and MG132.Since shikonin is a multifunctional compound, we wished to evaluate its effect on rAAV vector-mediated transgene expression in additional human cell types. In a human erythroleukemia cell line, K562, frequently used as a model for hematopoietic cell transduction studies, shikonin enhanced the transduction efficiency of various rAAV serotype vectors by up to 10-fold. Surprisingly, however, MG132 had no effect in K562 cells, even at the highest concentration that did not induce cytotoxicity. Thus, in a systematic study, various proteasome and protease inhibitors were evaluated. Western blot assays for the detection of polyubiquitinated total cellular proteins were performed to confirm the effectiveness of these drugs in K562 cells. Whereas carfilzomib only had a modest effect (

Cancer is a leading cause of human death worldwide. Commonly used methods of treatment such as surgery, radiation and chemotherapy tend to be largely successful but are extremely invasive and have serious toxic side effects. Thus, oncolytic viruses represent an attractive alternative to currently used anti-cancer treatments. Among different viral methods for gene delivery, vectors based on a human parvovirus, the adeno-associated virus (AAV) has attracted much attention mainly because of its non-pathogenic nature, and its ability to mediate long-term, sustained therapeutic gene expression.In current studies, AAV serotype 6 was identified to be particularly effective in transduction of several human prostate (PC3), breast (T47D) and liver (Huh7) cancer cell. We also found that transduction efficiency of these vectors can be significantly improved, up to 5-fold, by site-direct mutagenesis of surface exposed residues involved in intracellular trafficking. Over thirty single and multiple tyrosine (Y), serine (S), threonine (T) capsid mutants were developed and AAV6-Y705-731F+T492V was identified the most efficient.However, promiscuity of the AAV vectors makes it difficult to use in vivo. The recent studies on AAV crystal structure have identified specific regions of the capsid which are surface-exposed and tolerant to insertion of the peptides. In the current studies, we developed novel AAV-based vectors for targeting of human cancer cells by incorporating Arg-Gly-Asp (RGD) containing peptide to enable AAV to infect integrin-expressing cells. These AAV6-RGD vectors improve transduction efficiency approximately 3-fold compared with wild-type AAV6 vectors by enhancing viral entry to the cells. We correlated increased transduction efficiency with levels of expression of avβ5 integrin.The combination of RGD-peptide and mutations of surface-exposed residues on a single AAV6 capsid, further increases transduction efficiency up to approximately 10-fold. We next used qPCR analysis to quantitate the vector genomes in cytoplasmic and nuclear fractions of cells infected with the WT and the mutant AAV6 vectors at different time points. We showed that the increase in the transduction efficiency of the AAV6-Y705-731F+T492V vector correlated well with the improvement of both the entry and nuclear translocation of the vectors, compared with AAV6-WT.To conduct animal studies, we also introduced an additional mutation at lysine (K531E) since this was shown to impair the ability of vectors to bind to heparan sulfate proteoglycan and thus decrease the affinity to mice hepatocytes. We used a xenograft animal model in immune-deficient mice to prove the specificity and high transduction efficiency of AAV6-RGD-Y705-731F+T492V-K531E vectors in vivo.In summary, this approach can potentially lead to the development of therapeutic AAV vectors with selective tropism and high transduction efficiency of human cancer cells.

We first reported that of the 10 most commonly used AAV serotype vectors, AAV6 is the most efficient in transducing primary human bone marrow-derived CD34+ hematopoietic stem/progenitor cells (HSPCs), both in vitro and in murine xenograft models in vivo (Cytotherapy, 15: 986-998, 2013; PLoS One, 8(3): e58757, 2013). More recently, two independent groups also reported successful transduction of primary human CD34+ cells using the wild-type (WT) AAV6 vectors (Sci. Transl. Med., 7: 307ra156, 2015; Nat. Biotechnol., 33: 1256-1263, 2015), except that multiplicities of infection (MOIs) ranging from 100,000-200,000 vgs/cell were used to achieve ~40-55% transduction efficiency. Furthermore, the transduction efficiency of the WT AAV6 vector varies greatly in HSPCs from different donors, ranging between ~6-87%. Here we report two distinct strategies to further increase the transduction efficiency in HSPCs from donors that are transduced poorly with the WT AAV6 vectors. The first strategy involved modification of the viral capsid proteins where specific surface-exposed tyrosine (Y) and threonine (T) residues were mutagenized to generate a triple-mutant (Y705F+Y731F+T491V) AAV6 vector. The second strategy involved the use of ex vivo transduction at high cell density, which revealed a novel mechanism, which we have termed, ‘cross-transduction’. The combined use of these strategies resulted in transduction efficiency exceeding 90% at an MOI of 20,000 vgs/cell in primary human cord blood-derived HSPCs at day 4 (Fig. 1AFig. 1A). scAAV6 vectors were more efficient than ssAAV6 vectors, but at high cell density, there was a modest enhancement in EGFP-positivity even with ssAAV6 vectors. However, 14 days post-transduction, virtually no EGFP-positive cells could be detected (Fig. 1BFig. 1B), suggesting the loss of vector genomes, and hence, the lack of stable integration of vector genomes in HSPCs. Our studies have significant implications in the optimal use of capsid-optimized AAV6 vectors in genome editing in HSPCs. *These authors contributed equally to this work #Co-corresponding authorsView Large Image | Download PowerPoint Slide

There are two challenges in adeno-associated virus (AAV)-mediated cytotoxic gene therapy: i) overexpression of the cytotoxic transgene in the producer cell lines, such as HEK293, hampers efficient AAV vector production; and ii) most, if not all, AAV serotype vectors possess hepatic tropism since liver is the major tissue for cellular metabolism. These two challenges can be partially resolved by tissue specific promoters. However, in the cases that a specific promoter is not available, constitutively active promoters, such as the chicken β-actin (CBA) promoter, have to be used. For example, there is no well-studied specific promoter for hepatic satellite cells, which are the major cell type involved in liver fibrosis. In addition, most tissue-specific are weak promoters and are often too large to be packaged into AAV vectors. To address the above problems simultaneously, we explored the use of miR122 and its target sequence for the conditional regulation of transgene expression not only in the producer cells during AAV production, but also in the liver after systemic delivery. We first established a HEK293 cell line that overexpresses miR122. The functional miR122 expression was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) as well as Western blot assays against cellular proteins that are known to be regulated by miR122, such as c-Met. The expression of miR122 showed little effect on HEK293 cell growth and AAV protein expression. Next, we generated rAAV plasmids harboring suicide genes under the control of CBA promoter and a miR122 target (miR122T) sequence in the 3’-UTR. Two strong cytotoxic genes were selected, which encodes trichosanthin (a type 1 ribosome-inactivating protein) and diphtheria toxin (an exotoxin causing diphtheria). The presence of miR122T sequence had little effect on the protein functions in miR122-free cells, but significantly reduced the protein expression in the cells overexpressing miR122. Consequently, the AAV8 vectors carrying suicide genes and miR122T sequence yielded significantly increased production, up to 75-fold, in the HEK293-miR122 cells, when compared to that in the parental HEK293 cells (Table 1). Finally, we showed that AAV8 vectors that were produced from the HEK293-miR122 cells preserved the same tropisms and full bioactivity in vivo. Most importantly, the AAV8 vectors carrying a miR122T sequence mediated little transgene expression in normal liver. Taken together, we conclude that the use of HEK293-miR122 cells and a miR122T sequence should be applied to attenuate the transgene cytotoxicity during AAV vector production and infection of normal liver tissues.HEK293-miR122 and miR122T sequence together increase yields of AAV vectors carrying suicide genes.HEK293Batch 1Batch 2Batch 3AveSDssAAV8-Fluc2.68E + 129.96E + 112.78E + 122.15E + 121.00E + 12ssAAV8-Fluc-122T8.72E + 112.28E + 121.34E + 121.50E + 127.17E + 11ssAAV8-TCS6.23E + 081.34E + 093.75E + 087.79E + 085.01E + 08ssAAV8-TCS-122T2.34E + 091.68E + 094.27E + 081.48E + 099.72E + 08HEK293-miR122Batch 1Batch 2Batch 3AveSDssAAV8-FLuc4.04E + 126.60E + 121.05E + 123.90E + 122.78E + 12ssAAV8-FLuc-122T5.61E + 122.74E + 127.80E + 117.80E + 112.43E + 12ssAAV8-TCS3.45E + 091.39E + 098.50E + 081.90E + 091.37E + 09ssAAV8-TCS-122T1.66E + 118.50E + 109.30E + 101.15E + 114.46E + 10 View Table in HTML

Current challenges for recombinant adeno-associated virus (rAAV) vector–based cancer treatment include the low efficiency and the lack of specificity in vivo. rAAV serotype 3 (rAAV3) vectors have previously been shown to be ineffective in normal mouse tissues following systemic administration. In the present study, we report that rAAV3 vectors can efficiently target and transduce various human liver cancer cells in vivo. Elimination of specific surface-exposed serine and threonine residues on rAAV3 capsids results in further augmentation in the transduction efficiency of these vectors, without any change in the viral tropism and cellular receptor interactions. In addition, we have identified a potential chemotherapy drug, shikonin, as a multifunctional compound to inhibit liver tumor growth as well as to significantly enhance the efficacy of rAAV vector-based gene therapy in vivo. Furthermore, we also document that suppression of tumorigenesis in a human liver cancer xenograft model can be achieved through systemic administration of the optimized rAAV3 vectors carrying a therapeutic gene, and shikonin at a dose that does not lead to liver damage. Our research provides a novel means to achieve not only targeted delivery but also the potential for gene therapy of human liver cancer.

The human embryonic kidney cell line, HEK293, is readily transduced by the most commonly used AAV2 serotype vectors because these adherent cells abundantly express the cell surface receptor, heparan sulfate proteoglycan (HSPG), and one of the co-receptors, human fibroblast growth factor receptor 1 (FGFR1) for AAV2. However, a human erythroleukemia cell line, K562, commonly used as a model for human hematopoietic cell transduction, is not transduced as efficiently, although these cells grown in suspension, express both HSPG and FGFR1 only at modestly lower levels. We reasoned that the lack of proximity of HSPG and FGFR1 on K562 cells might account for the suboptimal transduction of these cells, and hypothesized that if the transduction was performed at high cell density, presumably allowing for HSPG on one cell to come in close proximity to FGFR1 on the neighboring cell, then AAV2 bound to HSPG on one cell could utilize FGFR1 on the neighboring cell to gain entry in the latter, and vice versa, thus leading to increased transduction. To test this hypothesis, HEK293 and K562 cells were transduced with 3×103 vgs/cell of scAAV2-EGFP vectors either at low-density (60,000 cells in 50 ml) or at high-density (480,000 cells in 50 ml). Transgene expression was evaluated by flow cytometry 48 hrs post-transduction. Whereas ~90% of HEK293 cells expressed the transgene at low-density, the transduction efficiency at high-density increased up to ~95%. On the other hand, only ~35% of K562 cells were transduced at low-density, but interestingly, the transduction efficiency at high-density increased to ~65%, thus corroborating that close cell-cell contact facilitated vector entry into these cells. That the initial cell-cell contact was critical in achieving improved transduction, was further corroborated by experiments in which cells were transduced at low-density, and subsequently pooled together to reach high-density, and conversely, cells were transduced at high-density, and soon after transduction, were diluted to low-density. The increased transduction was observed only under the latter condition. Similar results were obtained with two additional human cell lines, M07e and Raji, which express low to extremely low levels of HSPG and FGFR1, respectively, and consequently, are transduced extremely poorly by AAV2 vectors. These studies were also extended to include primary human CD34+ hematopoietic stem/progenitor cells (HSPCs), also grown in suspension, and known to be transduced sub-optimally by AAV2 vectors. In these studies also, whereas only~5% of these cells were transduced at low-density, the transduction efficiency increased up to ~20% at high-density. Taken together, our studies have revealed a novel mechanism, which we have termed “cross-transduction”, which AAV vectors exploit to gain entry into target cells, and which may have implications in the optimal use of AAV vectors in human gene therapy applications.

We have reported that of the 10 commonly used AAV serotype vectors, AAV6 is the most efficient in transducing primary human hematopoietic stem/progenitor cells (HSPCs), both in vitro and in murine xenograft models in vivo (Cytotherapy, 15: 986-998, 2013; PLoS One, 8(3): e58757, 2013). However, the transduction efficiency of these vectors ranged between ~6-87% in HSPCs obtained from several different donors (n=11). Such a wide range of transduction efficiency of AAV6 vectors is presumably due to different levels of expression of the putative receptors and/or co-receptors on these cells. In our more recent studies, we observed that the transduction efficiency of AAV2 vectors could be augmented both by performing transduction of HSPCs with the wild-type (wt)-AAV2 vectors at high cell density, or by using capsid-modified Y444F+Y500F+Y731F+T491V-mutant AAV2 vectors. In the present studies, we examined whether similar strategies could also be employed to increase the transduction efficiency of HSPCs from donors that are not transduced efficiently by wt-AAV6 vectors. Primary human HSPCs were transduced with either 3×103 or 3×104 vgs/cell of scAAV6-EGFP vectors either at low-density (20,000 cells in 50 ml) or at high-density (200,000 cells in 50 ml). scAAV2-EGFP vectors were also used for comparison. Transgene expression was evaluated by flow cytometry 48 hrs post-transduction. These results showed that whereas only ~14% of the cells transduced at low-density with high moi expressed the transgene, the transduction efficiency at high-density increased up to ~20% and 25%, at low, and high mois, respectively, also with a significant increase in the mean fluorescence intensity, thus corroborating that the initial cell-cell contact was a critical factor in achieving increased transduction. Next, the transduction efficiencies of the wt- and the capsid-modified triple-mutant (Y705F+Y731F+T492V) AAV6 vectors were compared. Again, the wt- and the capsid-modified quadruple-mutant (y444F+Y500f+Y731F+T491V) AAV2 vectors were used for comparison. HSPCs were transduced with 1×104 vgs/cell at high-density (200,000 cells in 50 ml), and transgene expression was evaluated by flow cytometry 48 hrs post-transduction. These results also clearly documented that the ~27% transduction efficiency of the wt-AAV6 vectors was increased by up to ~45% with the capsid-modified AAV6 vectors, with a concomitant increase in the mean fluorescence intensity. Taken together, these studies further corroborate the novel mechanism of “cross-transduction” by recombinant AAV vectors of human cells in general, and HSPCs in particular. Additional human HSPC transplant studies in a murine xenograft model in vivo are currently underway, the successful completion of which is likely to have significant implications of these strategies in the optimal use of AAV6 vectors in the potential gene therapy of human disorders involving the hematopoietic system.