Human Bocavirus 1 NP1 acts as an ssDNA-binding protein to help AAV2 DNA replication and cooperates with RPA to regulate AAV2 capsid expression.

Adeno-associated virus (AAV) requires co-infection with helper virus for efficient replication. We previously reported that Human Bocavirus 1 (HBoV1) genes, including NP1, NS2, and BocaSR, were critical for AAV2 replication. Here, we first demonstrate the essential roles of the NP1 protein in AAV2 DNA replication and protein expression. We show that NP1 binds to single-strand DNA (ssDNA) at least 30 nucleotides (nt) in length in a sequence-independent manner. Furthermore, NP1 colocalized with the BrdU-labeled AAV2 DNA replication center, and the loss of the ssDNA-binding ability of NP1 by site-directed mutation completely abolished AAV2 DNA replication. We used affinity-tagged NP1 protein to identify host cellular proteins associated with NP1 in cells cotransfected with the HBoV1 helper genes and AAV2 duplex genome. Of the identified proteins, we demonstrate that NP1 directly binds to the DBD-F domain of the RPA70 subunit with a high affinity through the residues 101-121. By reconstituting the heterotrimer protein RPA in vitro using gel filtration, we demonstrate that NP1 physically associates with RPA to form a heterologous complex characterized by typical fast-on/fast-off kinetics. Following a dominant-negative strategy, we found that NP1-RPA complex mainly plays a role in expressing AAV2 capsid protein by enhancing the transcriptional activity of the p40 promoter. Our study revealed a novel mechanism by which HBoV1 NP1 protein supports AAV2 DNA replication and capsid protein expression through its ssDNA-binding ability and direct interaction with RPA, respectively.IMPORTANCERecombinant adeno-associated virus (rAAV) vectors have been extensively used in clinical gene therapy strategies. However, a limitation of these gene therapy strategies is the efficient production of the required vectors, as AAV alone is replication-deficient in the host cells. HBoV1 provides the simplest AAV2 helper genes consisting of NP1, NS2, and BocaSR. An important question regarding the helper function of HBoV1 is whether it provides any direct function that supports AAV2 DNA replication and protein expression. Also of interest is how HBoV1 interplays with potential host factors to constitute a permissive environment for AAV2 replication. Our studies revealed that the multifunctional protein NP1 plays important roles in AAV2 DNA replication via its sequence-independent ssDNA-binding ability and in regulating AAV2 capsid protein expression by physically interacting with host protein RPA. Our findings present theoretical guidance for the future application of the HBoV1 helper genes in the rAAV vector production.

A variant of the venom allergen-like protein, DdVAP2, is required for the migratory endoparasitic plant nematode Ditylenchus destructor parasitism of plants.

The potato rot nematode, Ditylenchus destructor, poses a serious threat to numerous root and tuber crops, yet the functional characterization of effectors from this migratory endoparasitic plant nematode remains limited. Despite inhabiting distinct habitats, sedentary and migratory plant parasitic nematodes share the structurally conserved effectors, such as venom allergen-like proteins (VAPs). In this study, a variant of DdVAP2 was cloned from D. destructor. The transcription profile analysis revealed that DdVAP2 was higher expressed in D. destructor feeding on either potato or sweet potato compared to on fungus via qRT-PCR. And DdVAP2 was highly expressed at all life stages feeding on sweet potato, except for eggs. DdVAP2 was confirmed to be specifically expressed in the subventral esophageal glands of D. destructor through in situ hybridization assays. Combined with functional validation of the signal peptide of DdVAP2, it suggested that DdVAP2 could be secreted from nematode into host. Heterologous expression of DdVAP2 in Nicotiana benthamiana revealed that the protein localized in both cytosol and nuclei of plant cells. Knocking down DdVAP2 by RNAi in D. destructor resulted in infection and reproduction defects on plants. All the results suggest that DdVAP2 plays a crucial role in the interaction between D. destructor and plants by facilitating the nematode infection.

Recent advances in small molecule and peptide inhibitors of glucose-regulated protein 78 for cancer therapy.

Glucose-regulated protein 78 (GRP78) is one of key endoplasmic reticulum (ER) chaperone proteins that regulates the unfolded protein response (UPR) to maintain ER homeostasis. As a core factor in the regulation of the UPR, GRP78 takes a critical part in the cellular processes required for tumorigenesis, such as proliferation, metastasis, anti-apoptosis, immune escape and chemoresistance. Overexpression of GRP78 is closely correlated with tumorigenesis and poor prognosis in various malignant tumors. Targeting GRP78 is regarded as a potentially promising therapeutic strategy for cancer therapy. Although none of the GRP78 inhibitors have been approved to date, there have been several studies of GRP78 inhibitors. Herein, we comprehensively review the structure, physiological functions of GRP78 and the recent progress of GRP78 inhibitors, and discuss the structures, in vitro and in vivo efficacies, and merits and demerits of these inhibitors to inspire further research. Additionally, the feasibility of GRP78-targeting proteolysis-targeting chimeras (PROTACs), disrupting GRP78 cochaperone interactions, or covalent inhibition are also discussed as novel strategies for drugs discovery targeting GRP78, with the hope that these strategies can provide new opportunities for targeted GRP78 antitumor therapy.

Retrospective Study on Left-Sided Breast Radiotherapy: Dosimetric Results and Correlation with Physical Factors for Free Breathing and Breath Hold Irradiation Techniques.

Objectives: In breast radiotherapy, the proximity of the target to sensitive structures together with the uncertainty introduced by respiratory movement, make this treatment one of the most studied to increase its effectiveness. Dosimetric and physical variables play an important role and the study of their correlation and impact on treatment is fundamental. This retrospective study aims to highlight the dosimetric differences of 2 different clinical data sets of patients receiving left-sided breast irradiation in free breathing (FB) or breath hold (BH). Methods: A total of 155 left breast carcinoma patients receiving whole-breast irradiation in FB (73 patients) and BH (82 patients) were enrolled in this study. The dosimetric parameters of the target, heart, left and right lung and right breast were evaluated and compared, and possible correlations were studied in both groups. Results: No significant difference (P > .05) was found in the target dosimetry; a clear advantage in BH for both high and low doses received by the heart, with reductions of the dosimetric parameters between 27.1% and 100% (P < .003); for the left lung reductions decreased with increasing dose (-22.4% and -13.4% for doses of 5 and 20 Gy, respectively, P < .003). Conclusion: Significant correlations for BH treatments were registered between the volumes of the target and left lung, and the dosimetric parameters of the heart and left lung. BH treatment brings significant dosimetric advantages to organs at risk for a wide range of patients with different anatomy, target volumes and lung capacity, with additional benefits for small-sized breasts and important lung capacity.

Impact of Positioning Errors on the Dosimetry of Breath-Hold-Based Volumetric Arc Modulated and Tangential Field-in-Field Left-Sided Breast Treatments.

Heart diseases and cardiovascular events are well-known side effects in left-sided breast irradiation. Deep inspiration breath hold (BH) combined with fast delivery techniques such as volumetric modulated arc therapy (VMAT) or tangential field-in-field (TFiF) can serve as a valuable solution to reduce the dose to the heart. This study aims to compare the impact of positioning errors in VMAT and TFiF plans for BH left-sided breast treatments. Fifteen left-sided breast patients treated in BH with TFiF technique were included in this retrospective study. For each patient, a second plan with VMAT technique was optimized. Eighteen setup variations were introduced in each of these VMAT and TFiF reference plans, shifting the isocenter along six different directions by 3, 5, and 10 mm. A total of 540 perturbed plans, 270 for each technique, were recalculated and analyzed. The dose distributions on the target and organs at risk obtained in the different perturbed scenarios were compared with the reference scenarios, using as dosimetric endpoints the dose-volume histograms (DVH). The results were compared using the Wilcoxon test. Comparable plan quality was obtained for the reference VMAT and TFiF plans, except for low doses to organs at risk for which higher values (p < 0.05) were obtained for VMAT plans. For TFiF plans, perturbations of the isocenter position of 3, 5, or 10 mm produced mean deviations of the target DVH dosimetric parameters up to -0.5, -1.0, and -5.2%, respectively; VMAT plans were more sensitive to positioning errors resulting in mean deviations up to -0.5, -4.9, and -13.9%, respectively, for the same magnitude of the above mentioned perturbations. For organs at risk, only perturbations along the left, posterior, and inferior directions resulted in dose increase with a maximum deviation of +2% in the DVH dosimetric parameters. A notable exception were low doses to the left lung and heart for 10 mm isocenter shifts for which the mean differences ranged between +2.7 and +4.1%. Objective information on how external stresses affect the dosimetry of the treatment is the first step towards personalized radiotherapy.

Impact of positioning errors in the dosimetry of VMAT left-sided post mastectomy irradiation.

BACKGROUND: Volumetric modulated arc therapy (VMAT) adopted in post-mastectomy radiation therapy (PMRT) has the capacity to achieve highly conformal dose distributions. The research aims to evaluate the impact of positioning errors in the dosimetry of VMAT left-sided PMRT. METHODS: A total of 18 perturbations where introduced in 11 VMAT treatment plans that shifted the isocenter from its reference position of 3, 5, 10 mm in six directions. The thoracic wall and supraclavicular clinical target volumes (CTVs), the heart and the left lung dose volume histograms (DVHs) of 198 perturbed plans were calculated. The absolute differences (∆) of the mean dose (Dm) and DVH endpoints Vx and Dy (percentage volume receiving x Gy, and dose covering y% of the volume, respectively) were used to compare the dosimetry of the reference vs perturbed plans. RESULTS: Isocenter shifts in the anterior and lateral directions lead to maximum disagreement between the CTVs dosimetry of perturbed vs reference plans. Isocenter shifts of 10 mm shown a decrease of D95, D98 and Dm of 12.8, 18.0, and 2.9% respectively, for the CTVs. For 5 mm isocenter shifts, these differences decreased to 3.2, 5.2, and 0.9%, respectively, and for 3 mm shifts to 1.0, 1.7, and 0.6%, respectively. For the organs at risk (OARs), only isocenter shifts in the right, posterior and inferior directions worsen the plan dosimetry, nevertheless not negligible lung ∆ V20 of + 2.6%, and heart ∆ V25 of + 1.6% persist for 3 mm shifts. CONCLUSIONS: Inaccuracy in isocenter positioning for VMAT left-sided PMRT irradiation may impact the dosimetry of the CTVs and OARs to a different extent, depending on the directions and magnitude of the perturbation. The acquired information could be useful for planning strategies to guarantee the accuracy of the treatment delivered.

Conjugation Site Analysis by MS/MS Protein Sequencing.

In-depth knowledge about the site of drug-linker conjugation is important for the understanding of the conjugation efficiency and the exact locations of payloads for antibody-drug conjugates (ADCs). Here we describe a peptide mapping-based protocol, covering sample preparation procedure, LC-MS/MS setup, and data processing (auto and manual), to determine the locations of drug-linker attachment on mAbs. In comparison with classical mAb peptide mapping, some improvements will be highlighted for maintaining hydrophobic drug-loaded peptides in solution, enabling efficient chromatographic separation and mass spectrometric detection, and allowing for their unambiguous identification in LC-MS/MS map by using diagnostic fragmentation ions of the payload.

Ginkgo biloba extract improves brain uptake of ginsenosides by increasing blood-brain barrier permeability via activating A1 adenosine receptor signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginkgo biloba leaves and Panax ginseng are Chinese medicine commonly used in combination for cerebral disease. AIM OF THE STUDY: To investigate the effect of standard extract of Ginkgo biloba leaves (EGb) on facilitating brain uptake of ginsenoside and its underlying mechanisms. MATERIALS AND METHODS: The increasing uptake of ginsenosides in the brain of rats by EGb were detected by LC-MS/MS analysis. Evans blue and FITC-dextran leakage were determined to evaluate blood-brain barrier (BBB) permeability in vivo. Transendothelial electrical resistance (TEER) and Na-F penetration rate were measured with a co-culture of the human cerebral microvascular endothelial cell line (hCMEC/D3) and human normal glial cell line (HEB) in vitro BBB model. WB were used to analyzed the expression of BBB tight junctions (TJs) related protein (ZO-1, Occludin, Claudin-3, p-ERM, and p-MLC), ultrastructure of TJs was determined by transmission electron microscope. RESULTS: LC-MS/MS analysis demonstrated that EGb could improve brain uptake of ginsenoside Rg1, Re, Rd and Rb1. In vivo study showed that, BBB permeability was significantly increased after EGb administration, evidenced by the markedly increased penetration of FITC-dextran and Evans Blue into the mice brain parenchyma. In the in vitro BBB model, reduced TEER and increased Na-F penetration rate was observed in EGb group, which was associated with alteration of TJs ultrastructure. Furthermore, the expression of p-ERM and p-MLC in hCMEC/D3 as well as mice brain microvessels were significantly upregulated, but no significant change on the expression of TJs proteins (ZO-1, Occludin and Claudin-3). Moreover, the effect of EGb on in vitro BBB permeability and ERM, MLC phosphorylation was counteracted by DPCPX, an A1 adenosine receptor (A1R) antagonist. CONCLUSIONS: EGb might induce ERM/MLC phosphorylation and increase the cell-cell junction gaps to cause a reversible increase of the BBB permeability via A1R signaling pathway. Our results may contribute to better use of EGb in the treatment of brain diseases.

Automatic measurement of air gap for proton therapy using orthogonal x-ray imaging with radiopaque wires.

PURPOSE: The main objective of this study was to develop a technique to accurately determine the air gap between the end of the proton beam compensator and the body of the patient in proton radiotherapy. METHODS: Orthogonal x-ray image-based automatic coordinate reconstruction was used to determine the air gap between the patient body surface contour and the end of beam nozzle in proton radiotherapy. To be able to clearly identify the patient body surface contour on the orthogonal images, a radiopaque wire was placed on the skin surface of the patient as a surrogate. In order to validate this method, a Rando® head phantom was scanned and five proton plans were generated on a Mevion S250 Proton machine with various air gaps in Varian Eclipse Treatment Planning Systems (TPS). When setting up the phantom in a treatment room, a solder wire was placed on the surface of the phantom closest to the beam nozzle with the knowledge of the beam geometry in the plan. After the phantom positioning was verified using orthogonal kV imaging, the last pair of setup kV images was used to segment the solder wire and the in-room coordinates of the wire were reconstructed using a back-projection algorithm. Using the wire as a surrogate of the body surface, we calculated the air gaps by finding the minimum distance between the reconstructed wire and the end of the compensator. The methodology was also verified and validated on clinical cases. RESULTS: On the phantom study, the air gap values derived with the automatic reconstruction method were found to be within 1.1 mm difference from the planned values for proton beams with air gaps of 85.0, 100.0, 150.0, 180.0, and 200.0 mm. The reconstruction technique determined air gaps for a patient in two clinical treatment sessions were 38.4 and 41.8 mm, respectively, for a 40 mm planned air gap, and confirmed by manual measurements. There was strong agreement between the calculated values and the automatically measured values, and between the automatically and manually measured values. CONCLUSIONS: An image-based automatic method has been developed to conveniently determine the air gap of a proton beam, directly using the orthogonal images for patient positioning without adding additional imaging dose to the patient. The method provides an objective, accurate, and efficient way to confirm the target depth at treatment to ensure desired target coverage and normal tissue sparing.

Fufang-Zhenzhu-Tiaozhi Capsule reduces restenosis via the downregulation of NF-kappaB and inflammatory factors in rabbits.

BACKGROUND: To investigate the effects of a Chinese herbal medicine Fufang-Zhenzhu Tiaozhi Capsule (FTZ) on restenosis and elucidate the mechanism of action. METHODS: A restenosis model was established by balloon rubbing the endothelium of the abdominal aorta followed by high fat diet. Rabbits were divided into blank control group, restenosis group, FTZ group (0.66 mg/kg/day), atorvastatin group (5 mg/kg/day) and FTZ + atorvastatin group (n = 8). Vascular stenosis was analyzed by X-ray. Serum levels of chemokines and cytokines interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-12 (IL-12), C-reactive protein (CRP), tumor necrosis factor-alpha (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and intercellular adhesion molecule-1 (ICAM-1) were measured by ELISA. The levels of NF-κB, IκB-α, P-IκBα, IKK-α, and P-IKKα/ß from injured abdominal arteries were detected by Western blotting. RESULTS: Restenosis was induced successfully via abdominal artery balloon injuries and high fat diet. Restenosis was significantly decreased in FTZ group compared with restenosis group (P < 0.05). FTZ group had markedly reduced serum lipid levels (P < 0.05). In addition, the levels of TNF-α, IL-1, IL-6, IL-8, IL-12, ICAM-1 and MCP-1 decreased by FTZ treatment (P < 0.05). The expression of NF-κB in the atherosclerotic lesions was significantly attenuated in FTZ group (P < 0.05). CONCLUSION: FTZ could reduce restenosis via reducing NF-κB activity and inflammatory factor expression within the atherosclerotic lesion in a rabbit restenosis model. FTZ may be a new therapeutic agent for restenosis.

Left atrial myxoma complicated with multi-system embolization.

BACKGROUND: Atrial myxoma accounts for approximately 50% of all cardiac tumors. The majority of myxomas are located in the left atrium and present variable clinical manifestation. CASE PRESENTATION: A young man was transferred to our hospital with sudden onset of resting pain, pallor and numb in right leg. An atrial mobile mass was detected by transthoracic echocardiography. Anticoagulant and antithrombotic therapy were administered, a timely surgery was performed and the mass was confirmed as a myxoma. The patient did not discharge any discomfort post-operation. CONCLUSION: For patients with atrial myxoma, early diagnosis is essential, anticoagulant or antithrombotic therapy and surgery have a great importance to prevent further embolism.

A clinical study of lung cancer dose calculation accuracy with Monte Carlo simulation.

BACKGROUND: The accuracy of dose calculation is crucial to the quality of treatment planning and, consequently, to the dose delivered to patients undergoing radiation therapy. Current general calculation algorithms such as Pencil Beam Convolution (PBC) and Collapsed Cone Convolution (CCC) have shortcomings in regard to severe inhomogeneities, particularly in those regions where charged particle equilibrium does not hold. The aim of this study was to evaluate the accuracy of the PBC and CCC algorithms in lung cancer radiotherapy using Monte Carlo (MC) technology. METHODS AND MATERIALS: Four treatment plans were designed using Oncentra Masterplan TPS for each patient. Two intensity-modulated radiation therapy (IMRT) plans were developed using the PBC and CCC algorithms, and two three-dimensional conformal therapy (3DCRT) plans were developed using the PBC and CCC algorithms. The DICOM-RT files of the treatment plans were exported to the Monte Carlo system to recalculate. The dose distributions of GTV, PTV and ipsilateral lung calculated by the TPS and MC were compared. RESULT: For 3DCRT and IMRT plans, the mean dose differences for GTV between the CCC and MC increased with decreasing of the GTV volume. For IMRT, the mean dose differences were found to be higher than that of 3DCRT. The CCC algorithm overestimated the GTV mean dose by approximately 3% for IMRT. For 3DCRT plans, when the volume of the GTV was greater than 100 cm(3), the mean doses calculated by CCC and MC almost have no difference. PBC shows large deviations from the MC algorithm. For the dose to the ipsilateral lung, the CCC algorithm overestimated the dose to the entire lung, and the PBC algorithm overestimated V20 but underestimated V5; the difference in V10 was not statistically significant. CONCLUSIONS: PBC substantially overestimates the dose to the tumour, but the CCC is similar to the MC simulation. It is recommended that the treatment plans for lung cancer be developed using an advanced dose calculation algorithm other than PBC. MC can accurately calculate the dose distribution in lung cancer and can provide a notably effective tool for benchmarking the performance of other dose calculation algorithms within patients.