A polymeric 1H/19F dual-modal MRI contrast agent with a snowman-like Janus nanostructure.

Magnetic resonance imaging (MRI) has emerged as a pivotal tool in contemporary medical diagnostics, offering non-invasive and high-resolution visualization of internal structures. Contrast agents are essential for enhancing MRI resolution, accurate lesion detection, and early pathology identification. While gadolinium-based contrast agents are widely used in clinics, safety concerns have prompted exploration of metal-free alternatives, including fluorine and nitroxide radical-based MRI contrast agents. Fluorine-containing compounds exhibit excellent MRI capabilities, with 19F MRI providing enhanced resolution and quantitative assessment. Nitroxide radicals, such as PROXYL and TEMPO, offer paramagnetic properties for MRI contrast. Despite their versatility, nitroxide radicals suffer from lower relaxivity values (r1) compared to gadolinium. Dual-modal imaging, combining 1H and 19F MRI, has gained prominence for its comprehensive insights into biological processes and disease states. However, existing dual-modal agents predominantly utilize gadolinium-organic ligands without incorporating nitroxide radicals. Here, we introduce a novel dual-modal MRI contrast agent (J-CA) featuring a Janus asymmetric nanostructure synthesized via seeded emulsion polymerization and post-modification. J-CA demonstrates excellent in vitro and in vivo performance in both 19F and 1H MRI, with a T2 relaxation time of 5 ms and an r1 value of 0.31 mM-1 s-1, ensuring dual-modal imaging capability. Moreover, J-CA exhibits superior biocompatibility and organ targeting, making it a promising candidate for precise lesion imaging and disease diagnosis. This work introduces a new avenue for metal-free dual-modal MRI, addressing safety concerns associated with traditional contrast agents.

Theoretical Study on the O-H Fracture of Methanol on PtnCu4-n (n = 1, 2, 3) Catalysts with Different Coverages.

Direct methanol fuel cells (DMFCs) have attracted increasing attention as a very promising and important energy source. In this paper, density functional theory (DFT) is used to study the structure and O-H fracture mechanism of methanol adsorption on PtnCu4-n (111) (n = 1, 2, 3) binary metal catalyst surfaces under different coverages. By comparing the adsorption energy and dehydrogenation energy barriers of methanol, it is found that the adsorption strength and dehydrogenation energy barriers of methanol on Pt and Cu sites decreased with increasing coverage. At the same Pt and Cu ratio, methanol is more easily adsorbed on Cu sites. When Pt/Cu = 3:1 and 1:3, the PtCu binary catalyst has a significant impact on the energy barrier of breaking the O-H bond in methanol with the increase of coverage. Especially when Pt/Cu = 1:3 and the coverage is 1/4 ML, the energy barriers of O-H bond breaking in methanol on Pt and Cu sites are 0.63 and 0.61 eV, respectively, which are lower than that on pure Pt. It means that the Cu sites played a very important role in reducing the O-H fracture energy barrier of methanol. When Pt/Cu = 1:1, the change in the dehydrogenation energy barrier of methanol on Pt sites and Cu sites is not significant, indicating that the coverage has little effect on it.

Imidazolium-Based Main-Chain Copolymers With Alternating Sequences for Broad-Spectrum Bactericidal Activity and Eradication of Bacterial Biofilms.

In response to the escalating challenge of bacterial drug resistance, the imperative to counteract planktonic cell proliferation and eliminate entrenched biofilms underscores the necessity for cationic polymeric antibacterials. However, limited efficacy and cytotoxicity challenge their practical use. Here, novel imidazolium-based main-chain copolymers with imidazolium (PIm+) as the cationic component are introduced. By adjusting precursor molecules, hydrophobicity and cationic density of each unit are fine-tuned, resulting in broad-spectrum bactericidal activity against clinically relevant pathogens. PIm+1 stands out for its potent antibacterial performance, with a minimum inhibitory concentration of 32 µg mL-1 against Methicillin-resistant Staphylococcus aureus (MRSA), and substantial biofilm reduction in Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) biofilms. The bactericidal mechanism involves disrupting the outer and cytoplasmic membranes, depolarizing the cytoplasmic membrane, and triggering intracellular reactive oxygen species (ROS) generation. Collectively, this study postulates the potential of imidazolium-based main-chain copolymers, systematically tailored in their sequences, to serve as a promising candidate in combatting drug-resistant bacterial infections.

Nomogram predicting long-term overall and cancer-specific survival of patients with buccal mucosa cancer.

BACKGROUND: Few models about the personalized prognosis evaluation of buccal mucosa cancer (BMC) patients were reported. We aimed to establish predictive models to forecast the prognosis of BMC patients. METHODS: The complete clinicopathological information of BMC patients from the surveillance, epidemiology and end results program was collected and reviewed retrospectively. Two nomograms were established and validated to predict long-term overall survival (OS) and cancer-specific survival (CSS) of BMC patients based on multivariate Cox regression survival analysis. RESULTS: 1155 patients were included. 693 and 462 patients were distributed into modeling and validation groups with 6:4 split-ratio via a random split-sample method. Based on the survival analysis, independent prognostic risk factors (variables that can be used to estimate disease recovery and relapse chance) influencing OS and CSS were obtained to establish nomograms. Then, we divided the modeling group into high- and low-risk cohorts. The low-risk cohort had improved OS and CSS compared to the high-risk cohort, which was statistically significant after the Log-rank test (p < 0.05). Furthermore, we used the concordance index (C-index), calibration curve to validate the nomograms, showing high accuracy. The decision curve analyses (DCA) revealed that the nomograms had evident clinical value. CONCLUSIONS: We constructed two credible nomogram models, which would give the surgeons reference to provide an individualized assessment of BMC patients.

Biomechanical Evaluation of Patient-Specific Polymethylmethacrylate Cranial Implants for Virtual Surgical Planning: An In-Vitro Study.

Cranioplasty with freehand-molded polymethylmethacrylate implants is based on decades of experience and is still frequently used in clinical practice. However, data confirming the fracture toughness and standard biomechanical tests are rare. This study aimed to determine the amount of force that could be applied to virtually planned, template-molded, patient-specific implants (n = 10) with an implant thickness of 3 mm, used in the treatment of a temporoparietal skull defect (91.87 cm2), until the implant cracks and finally breaks. Furthermore, the influence of the weight and porosity of the implant on its force resistance was investigated. The primary outcome showed that a high force was required to break the implant (mean and standard deviation 1484.6 ± 167.7 N), and this was very strongly correlated with implant weight (Pearson's correlation coefficient 0.97; p < 0.001). Secondary outcomes were force application at the implant's first, second, and third crack. Only a moderate correlation could be found between fracture force and the volume of porosities (Pearson's correlation coefficient 0.59; p = 0.073). The present study demonstrates that an implant thickness of 3 mm for a temporoparietal skull defect can withstand sufficient force to protect the brain. Greater implant weight and, thus, higher material content increases thickness, resulting in more resistance. Porosities that occur during the described workflow do not seem to reduce resistance. Therefore, precise knowledge of the fracture force of polymethylmethacrylate cranial implants provides insight into brain injury prevention and serves as a reference for the virtual design process.

Fabrication and Characterization of PCL/HA Filament as a 3D Printing Material Using Thermal Extrusion Technology for Bone Tissue Engineering.

The most common three-dimensional (3D) printing method is material extrusion, where a pre-made filament is deposited layer-by-layer. In recent years, low-cost polycaprolactone (PCL) material has increasingly been used in 3D printing, exhibiting a sufficiently high quality for consideration in cranio-maxillofacial reconstructions. To increase osteoconductivity, prefabricated filaments for bone repair based on PCL can be supplemented with hydroxyapatite (HA). However, few reports on PCL/HA composite filaments for material extrusion applications have been documented. In this study, solvent-free fabrication for PCL/HA composite filaments (HA 0%, 5%, 10%, 15%, 20%, and 25% weight/weight PCL) was addressed, and parameters for scaffold fabrication in a desktop 3D printer were confirmed. Filaments and scaffold fabrication temperatures rose with increased HA content. The pore size and porosity of the six groups' scaffolds were similar to each other, and all had highly interconnected structures. Six groups' scaffolds were evaluated by measuring the compressive strength, elastic modulus, water contact angle, and morphology. A higher amount of HA increased surface roughness and hydrophilicity compared to PCL scaffolds. The increase in HA content improved the compressive strength and elastic modulus. The obtained data provide the basis for the biological evaluation and future clinical applications of PCL/HA material.

A tumor-specific pro-IL-12 activates preexisting cytotoxic T cells to control established tumors.

It is a challenge to effectively reactivate preexisting tumor-infiltrating lymphocytes (TILs) without causing severe toxicity. Interleukin-12 (IL-12) can potently activate lymphocytes, but its clinical use is limited by its short half-life and dose-related toxicity. In this study, we developed a tumor-conditional IL-12 (pro-IL-12), which masked IL-12 with selective extracellular receptor­binding domains of the IL-12 receptor while preferentially and persistently activating TILs after being unmasked by matrix metalloproteinases expressed by tumors. Systemic delivery of pro-IL-12 demonstrated reduced toxicity but better control of established tumors compared with IL-12-Fc. Mechanistically, antitumor responses induced by pro-IL-12 were dependent on TILs and IFNγ. Furthermore, direct binding of IL-12 to IL-12R on CD8+, not CD4+, T cells was essential for maximal effectiveness. Pro-IL-12 improved the efficacy of both immune checkpoint blockade and targeted therapy when used in combination. Therefore, our study demonstrated that pro-IL-12 could rejuvenate TILs, which then combined with current treatment modalities while limiting adverse effects for treating established tumors.

Prefabricated 3D-Printed Tissue-Engineered Bone for Mandibular Reconstruction: A Preclinical Translational Study in Primate.

The advent of three dimensionally (3D) printed customized bone grafts using different biomaterials has enabled repairs of complex bone defects in various in vivo models. However, studies related to their clinical translations are truly limited. Herein, 3D printed poly(lactic-co-glycolic acid)/ß-tricalcium phosphate (PLGA/TCP) and TCP scaffolds with or without recombinant bone morphogenetic protein -2 (rhBMP-2) coating were utilized to repair primate's large-volume mandibular defects and compared efficacy of prefabricated tissue-engineered bone (PTEB) over direct implantation (without prefabrication). 18F-FDG PET/CT was explored for real-time monitoring of bone regeneration and vascularization. After 3-month's prefabrication, the original 3D-architecture of the PLGA/TCP-BMP scaffold was found to be completely lost, while it was properly maintained in TCP-BMP scaffolds. Besides, there was a remarkable decrease in the PLGA/TCP-BMP scaffold density and increase in TCP-BMP scaffolds density during ectopic (within latissimus dorsi muscle) and orthotopic (within mandibular defect) implantation, indicating regular bone formation with TCP-BMP scaffolds. Notably, PTEB based on TCP-BMP scaffold was successfully fabricated with pronounced effects on bone regeneration and vascularization based on radiographic, 18F-FDG PET/CT, and histological evaluation, suggesting a promising approach toward clinical translation.

3D-printed titanium implant combined with interleukin 4 regulates ordered macrophage polarization to promote bone regeneration and angiogenesis.

AIMS: The use of 3D-printed titanium implant (DT) can effectively guide bone regeneration. DT triggers a continuous host immune reaction, including macrophage type 1 polarization, that resists osseointegration. Interleukin 4 (IL4) is a specific cytokine modulating osteogenic capability that switches macrophage polarization type 1 to type 2, and this switch favours bone regeneration. METHODS: IL4 at concentrations of 0, 30, and 100 ng/ml was used at day 3 to create a biomimetic environment for bone marrow mesenchymal stromal cell (BMMSC) osteogenesis and macrophage polarization on the DT. The osteogenic and immune responses of BMMSCs and macrophages were evaluated respectively. RESULTS: DT plus 30 ng/ml of IL4 (DT + 30 IL4) from day 3 to day 7 significantly (p < 0.01) enhanced macrophage type 2 polarization and BMMSC osteogenesis compared with the other groups. Local injection of IL4 enhanced new bone formation surrounding the DT. CONCLUSION: DT + 30 IL4 may switch macrophage polarization at the appropriate timepoints to promote bone regeneration. Cite this article: Bone Joint Res 2021;10(7):411-424.

Nanobody Nb6 fused with porcine IgG Fc as the delivering tag to inhibit porcine reproductive and respiratory syndrome virus replication in porcine alveolar macrophages.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly contagious virus that has led to enormous economic loss worldwide because of ineffective prevention and treatment. In view of their minimized size, high target specificity and affinity, nanobodies have been extensively investigated as diagnostic tools and treatments of many diseases. Previously, a PRRSV Nsp9-specific nanobody (Nb6) was identified as a PRRSV replication inhibitor. When it was fused with cell-penetrating peptide (CPP) TAT, Nb6-TAT could enter the cells for PRRSV suppression. However, delivery of molecules by CPP lack cell specificity and have a short duration of action. PRRSV has a tropism for monocyte/macrophage lineage, which expresses high levels of Fcγ receptors. Herein, we designed a nanobody containing porcine IgG Fc (Fcγ) to inhibit PRRSV replication in PRRSV permissive cells. Fcγ fused Nb6 chimeric antibody (Nb6-pFc) was assembled into a dimer with interchain disulfide bonds and expressed in a Pichia pastoris system. The results show that Nb6-pFc exhibits a well-binding ability to recombinant Nsp9 or PRRSV-encoded Nsp9 and that FcγR-mediated endocytosis of Nb6-pFc into porcine alveolar macrophages (PAM) was in a dose-dependent manner. Nb6-pFc can inhibit PRRSV infection efficiently not only by binding with Nsp9 but also by upregulating proinflammatory cytokine production in PAM. Together, this study proposes the design of a porcine IgG Fc-fused nanobody that can enter PRRSV susceptible PAM via FcγR-mediated endocytosis and inhibit PRRSV replication. This research reveals that nanobody-Fcγ chimeric antibodies might be effective for the control and prevention of monocyte/macrophage lineage susceptible pathogeneses.

Quality Characteristics and Clinical Relevance of In-House 3D-Printed Customized Polyetheretherketone (PEEK) Implants for Craniofacial Reconstruction.

Additive manufacturing (AM) of patient-specific implants (PSIs) is gradually moving towards in-house or point-of-care (POC) manufacturing. Polyetheretherketone (PEEK) has been used in cranioplasty cases as a reliable alternative to other alloplastic materials. As only a few fused filament fabrication (FFF) printers are suitable for in-house manufacturing, the quality characteristics of the implants fabricated by FFF technology are still under investigated. This paper aimed to investigate PEEK PSIs fabricated in-house for craniofacial reconstruction, discussing the key challenges during the FFF printing process. Two exemplary cases of class III (Group 1) and class IV (Group 2) craniofacial defects were selected for the fabrication of PEEK PSIs. Taguchi's L9 orthogonal array was selected for the following nonthermal printing process parameters, i.e., layer thickness, infill rate, number of shells, and infill pattern, and an assessment of the dimensional accuracy of the fabricated implants was made. The root mean square (RMS) values revealed higher deviations in Group 1 PSIs (0.790 mm) compared to Group 2 PSIs (0.241 mm). Horizontal lines, or the characteristic FFF stair-stepping effect, were more perceptible across the surface of Group 1 PSIs. Although Group 2 PSIs revealed no discoloration, Group 1 PSIs displayed different zones of crystallinity. These results suggest that the dimensional accuracy of PSIs were within the clinically acceptable range; however, attention must be paid towards a requirement of optimum thermal management during the printing process to fabricate implants of uniform crystallinity.

In Vitro Mechanical and Biological Properties of 3D Printed Polymer Composite and ß-Tricalcium Phosphate Scaffold on Human Dental Pulp Stem Cells.

3D printed biomaterials have been extensively investigated and developed in the field of bone regeneration related to clinical issues. However, specific applications of 3D printed biomaterials in different dental areas have seldom been reported. In this study, we aimed to and successfully fabricated 3D poly (lactic-co-glycolic acid)/ß-tricalcium phosphate (3D-PLGA/TCP) and 3D ß-tricalcium phosphate (3D-TCP) scaffolds using two relatively distinct 3D printing (3DP) technologies. Conjunctively, we compared and investigated mechanical and biological responses on human dental pulp stem cells (hDPSCs). Physicochemical properties of the scaffolds, including pore structure, chemical elements, and compression modulus, were characterized. hDPSCs were cultured on scaffolds for subsequent investigations of biocompatibility and osteoconductivity. Our findings indicate that 3D printed PLGA/TCP and ß-tricalcium phosphate (ß-TCP) scaffolds possessed a highly interconnected and porous structure. 3D-TCP scaffolds exhibited better compressive strength than 3D-PLGA/TCP scaffolds, while the 3D-PLGA/TCP scaffolds revealed a flexible mechanical performance. The introduction of 3D structure and ß-TCP components increased the adhesion and proliferation of hDPSCs and promoted osteogenic differentiation. In conclusion, 3D-PLGA/TCP and 3D-TCP scaffolds, with the incorporation of hDPSCs as a personalized restoration approach, has a prospective potential to repair minor and critical bone defects in oral and maxillofacial surgery, respectively.

Effects of Steam Sterilization on 3D Printed Biocompatible Resin Materials for Surgical Guides-An Accuracy Assessment Study.

Computer-assisted surgery with three-dimensional (3D) printed surgical guides provides more accurate results than free-hand surgery. Steam sterilization could be one of the factors that affect the dimensions of surgical guide resin materials, leading to inaccuracies during surgeries. The purpose of this study was to evaluate the effects of steam sterilization on the dimensional accuracy of indication-specific hollow cube test bodies, manufactured in-house using Class IIa biocompatible resin materials (proprietary and third-party). To evaluate the pre- and post-sterilization dimensional accuracy, root mean square (RMS) values were calculated. The results indicate that, in all the groups, steam sterilization resulted in an overall linear expansion of the photopolymeric resin material, with an increase in outer dimensions and a decrease in inner dimensions. The effects on the dimensional accuracy of test bodies were not statistically significant in all the groups, except PolyJet Glossy (p > 0.05). The overall pre- and post-sterilization RMS values were below 100 and 200 µm, respectively. The highest accuracies were seen in proprietary resin materials, i.e., PolyJet Glossy and SLA-LT, in pre- and post-sterilization measurements, respectively. The dimensional accuracy of third-party resin materials, i.e., SLA-Luxa and SLA-NextDent, were within a comparable range as proprietary materials and can serve as an economical alternative.

Author Correction: A next-generation tumor-targeting IL-2 preferentially promotes tumor-infiltrating CD8+ T-cell response and effective tumor control.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Evaluation of the Dimensional Accuracy of 3D-Printed Anatomical Mandibular Models Using FFF, SLA, SLS, MJ, and BJ Printing Technology.

With the rapid progression of additive manufacturing and the emergence of new 3D printing technologies, accuracy assessment is mostly being performed on isosymmetric test bodies. However, the accuracy of anatomic models can vary. The dimensional accuracy of root mean square values in terms of trueness and precision of 50 mandible replicas, printed with five common printing technologies, were evaluated. The highest trueness was found for the selective laser sintering printer (0.11 ± 0.016 mm), followed by a binder jetting printer (0.14 ± 0.02 mm), and a fused filament fabrication printer (0.16 ± 0.009 mm). However, highest precision was identified for the fused filament fabrication printer (0.05 ± 0.005 mm) whereas other printers had marginally lower values. Despite the statistically significance (p < 0.001), these differences can be considered clinically insignificant. These findings demonstrate that all 3D printing technologies create models with satisfactory dimensional accuracy for surgical use. Since satisfactory results in terms of accuracy can be reached with most technologies, the choice should be more strongly based on the printing materials, the intended use, and the overall budget. The simplest printing technology (fused filament fabrication) always scored high and thus is a reliable choice for most purposes.

A next-generation tumor-targeting IL-2 preferentially promotes tumor-infiltrating CD8+ T-cell response and effective tumor control.

While IL-2 can potently activate both NK and T cells, its short in vivo half-life, severe toxicity, and propensity to amplify Treg cells are major barriers that prevent IL-2 from being widely used for cancer therapy. In this study, we construct a recombinant IL-2 immunocytokine comprising a tumor-targeting antibody (Ab) and a super mutant IL-2 (sumIL-2) with decreased CD25 binding and increased CD122 binding. The Ab-sumIL2 significantly enhances antitumor activity through tumor targeting and specific binding to cytotoxic T lymphocytes (CTLs). We also observe that pre-existing CTLs within the tumor are sufficient and essential for sumIL-2 therapy. This next-generation IL-2 can also overcome targeted therapy-associated resistance. In addition, preoperative sumIL-2 treatment extends survival much longer than standard adjuvant therapy. Finally, Ab-sumIL2 overcomes resistance to immune checkpoint blockade through concurrent immunotherapies. Therefore, this next-generation IL-2 reduces toxicity while increasing TILs that potentiate combined cancer therapies.

Particulate Coral Hydroxyapatite Sheltered by Titanium Mesh for Localized Alveolar Rehabilitation After Onlay Graft Failure: A Case Report.

Reconstruction of bone loss in the alveolar ridge has long been challenging. Autologous bone grafts are considered as the "golden standard," while little research has focused on how to repair pronounced alveolar bone defects after autologous bone graft failure. The aim of this study was to detail a method based on the titanium mesh technique coupled with particulate coral hydroxyapatite to solve the onlay graft failure. With bone deficiency in the No. 11 and No. 24-25 regions, we harvested 2 autologous bone blocks for reconstruction. Two weeks after transplantation, the graft in the No. 11 region had healed uneventfully, while the graft in the anterior mandible became infected because of soft tissue dehiscence. After removal of the failed autologous bone block, pure coral hydroxyapatite stabilized within titanium mesh was used for alveolar rehabilitation. Six months later, the width of the local alveolar bone was evaluated. After the titanium mesh was removed, a biopsy was performed to study bone regeneration by micro computerized tomography and histology, following by a standard Straumann implant insertion. Although there was wound dehiscence 14 days after bone augmentation, repeated local rinsing and anti-inflammation therapy controlled the inflammatory reaction. The total horizontal bone gain was 4.2 ± 0.5 mm. Micro computerized tomography revealed that the closer the coral hydroxyapatite was to the host bone, the more was resorbed and the more bone regenerated. Histology showed mature lamellar bone structures, with evident residual coral hydroxyapatite. A 3-year follow-up revealed stable bone around the dental implant and successful function of the implant-born prosthesis. This study proposes that the method of particulate coral hydroxyapatite sheltered by titanium mesh is a promising solution in handling alveolar bone augmentation failure. More cases are needed for further research to form an efficient treatment procedure.

[The progress of research on three-dimensional printed jaw scaffolds].

Large defects of jaw caused by tumor, trauma and so on in oral and maxillofacial region lead to facial deformity, language and chewing dysfunction, which severely damage the patient's life quality. Three-dimensional printing (3DP) is also named additive manufacturing (AM), which can print materials layer by layer to create three-dimensional objects. The complex shape of jaw defects can be accurately reconstructed using 3DP scaffold combined with image data, computer-aided-design and manufacture. It has specific advantages compared with traditional way of jaw reconstruction and has attracted much attention in the field of jaw tissue engineering recently. This article presented the progress of 3DP scaffold and its application in jaw reconstruction, providing a new idea for jaw reconstruction.

Threonine 80 phosphorylation of non-structural protein 1 regulates the replication of influenza A virus by reducing the binding affinity with RIG-I.

Influenza A virus evades host antiviral defense through hijacking innate immunity by its non-structural protein 1 (NS1). By using mass spectrometry, threonine 80 (T80) was identified as a novel phosphorylated residue in the NS1 of the influenza virus A/WSN/1933(H1N1). By generating recombinant influenza viruses encoding NS1 T80 mutants, the roles of this phosphorylation site were characterized during viral replication. The T80E (phosphomimetic) mutant attenuated virus replication, whereas the T80A (non-phosphorylatable) mutant did not. Similar phenotypes were observed for these mutants in a mouse model experiment. In further study, the T80E mutant decreased the binding capacity between NS1 and viral nucleoprotein (NP), leading to impaired viral ribonucleoprotein (vRNP)-mediated viral transcription. The T80E mutant was also unable to inhibit interferon (IFN) production by reducing the binding affinity between NS1 and retinoic acid-induced gene 1 protein (RIG-I), causing attenuation of virus replication. Taken together, the present study reveals that T80 phosphorylation of NS1 reduced influenza virus replication through controlling RIG-I-mediated IFN production and vRNP activity.

[Comparison of cellular localization of NS1 from different subtypes of influenza A virus].

The non-structural (NS1) protein is a multifunctional molecular protein encoded by influenza A virus genome. NS1 plays an important role in inhibition of host immune responses. In order to assess the cellular localization of NS1 in different influenza A virus subtypes, we performed the immunofluorescence assay to observe the cellular location of NS1 after infection with influenza A virus WSN (H1N1), PR8 (H1N1), CA04 (H1N1), SD (H9N2) and AH01 (H7N9) in A549 cells and MDCK cells respectively. According to the results, NS1-WSN and NS1-PR8 accumulated mainly in cytoplasm at 24 h post infection, while the NS1-CA04 and NS1-SD appeared major in the nucleus. We also observed localization of NS1 by transfected 293T cells with plasmids which encoding the full-length NS1 from WSN, SD and AH01. The key sites which might determine the different cellular localization of NS1 were chosen by sequence alignment, and seven residues which were different between WSN, PR8 and CA04, SD and AH01 were finally focused. However, we found that single mutation of these residues could not alter the localization of NS1. The data indicated that the difference of location might not be caused by substitution of a single site, which contributes to our understanding of the diverse regulation of host factors during different subtypes of influenza virus infection.