Carbene Footprinting Directs Design of Genetically Encoded Proximity-Reactive Protein Binders.

Genetically encoding proximal-reactive unnatural amino acids (PrUaas), such as fluorosulfate-l-tyrosine (FSY), into natural proteins of interest (POI) confer the POI with the ability to covalently bind to its interacting proteins (IPs). The PrUaa-incorporated POIs hold promise for blocking undesirable POI-IP interactions. Selecting appropriate PrUaa anchor sites is crucial, but it remains challenging with the current methodology, which heavily relies on crystallography to identify the proximal residues between the POIs and the IPs for the PrUaa anchorage. To address the challenge, here, we propose a footprinting-directed genetically encoded covalent binder (footprinting-GECB) approach. This approach employs carbene footprinting, a structural mass spectrometry (MS) technique that quantifies the extent of labeling of the POI following the addition of its IP, and thus identifies the responsive residues. By genetically encoding PrUaa into these responsive sites, POI variants with covalent bonding ability to its IP can be produced without the need for crystallography. Using the POI-IP model, KRAS/RAF1, we showed that engineering FSY at the footprint-assigned KRAS residue resulted in a KRAS variant that can bind irreversibly to RAF1. Additionally, we inserted FSY at the responsive residue in RAF1 upon footprinting the oncogenic KRASG12D/RAF1, which lacks crystal structure, and generated a covalent binder to KRASG12D. Together, we demonstrated that by adopting carbene footprinting to direct PrUaa anchorage, we can greatly expand the opportunities for designing covalent protein binders for PPIs without relying on crystallography. This holds promise for creating effective PPI inhibitors and supports both fundamental research and biotherapeutics development.

Recent advances in nanotechnology approaches for non-viral gene therapy.

Gene therapy has shown great potential in the treatment of many diseases by downregulating the expression of certain genes. The development of gene vectors as a vehicle for gene therapy has greatly facilitated the widespread clinical application of nucleic acid materials (DNA, mRNA, siRNA, and miRNA). Currently, both viral and non-viral vectors are used as delivery systems of nucleic acid materials for gene therapy. However, viral vector-based gene therapy has several limitations, including immunogenicity and carcinogenesis caused by the exogenous viral vectors. To address these issues, non-viral nanocarrier-based gene therapy has been explored for superior performance with enhanced gene stability, high treatment efficiency, improved tumor-targeting, and better biocompatibility. In this review, we discuss various non-viral vector-mediated gene therapy approaches using multifunctional biodegradable or non-biodegradable nanocarriers, including polymer-based nanoparticles, lipid-based nanoparticles, carbon nanotubes, gold nanoparticles (AuNPs), quantum dots (QDs), silica nanoparticles, metal-based nanoparticles and two-dimensional nanocarriers. Various strategies to construct non-viral nanocarriers based on their delivery efficiency of targeted genes will be introduced. Subsequently, we discuss the cellular uptake pathways of non-viral nanocarriers. In addition, multifunctional gene therapy based on non-viral nanocarriers is summarized, in which the gene therapy can be combined with other treatments, such as photothermal therapy (PTT), photodynamic therapy (PDT), immunotherapy and chemotherapy. We also provide a comprehensive discussion of the biological toxicity and safety of non-viral vector-based gene therapy. Finally, the present limitations and challenges of non-viral nanocarriers for gene therapy in future clinical research are discussed, to promote wider clinical applications of non-viral vector-based gene therapy.

Investigating the depolarization property of skin tissue by degree of polarization uniformity contrast using polarization-sensitive optical coherence tomography.

The depolarization property of skin has been found to be important for skin cancer detection. Previous techniques based on light polarization lack the capability of depth differentiation. Polarization-sensitive optical coherence tomography (PS-OCT) has the advantage of both depth-resolved 3D imaging and high sensitivity to polarization. In this study, we investigate the depolarization property of skin tissue using PS-OCT, especially with the degree of polarization uniformity (DOPU) contrast. Well designed skin phantoms with various surface roughness levels and optical properties mimicking skin are imaged by PS-OCT and the DOPU values are quantified. The result shows a correlation between DOPU and surface roughness, where a higher roughness corresponds to a lower DOPU value. An index matching experiment with a water layer confirms the impact of surface condition on light depolarization. Refraction of backscattered photons on the surface boundary is attributed to the broadening of backscattering angle and thus depolarization. To the best of our knowledge, this is the first time the impact of surface roughness on DOPU is reported and its mechanism explained. Furthermore, through preliminary in vivo skin imaging, the capability of DOPU in detecting depolarization in skin is demonstrated. By utilizing the 3D imaging from PS-OCT, DOPU can offer a high-resolution depth differentiation and quantification of depolarization in skin tissue.

Application of Polarization Sensitive-Optical Coherence Tomography to the Assessment of Phase Retardation in Subpleural Cancer in Rabbits.

BACKGROUND: Polarization sensitive-optical coherence tomography (PS-OCT) provides the unique advantage of being able to measure the optical characteristics of tissues by using polarized light. Although the well-organized fibers of healthy muscle can change the polarization states of passing light, damaged tissue has different behaviors. There are studies on optical imaging methods applied to the respiratory organs; however, they are restricted to structural imaging. In particular, the intercostal muscle situated under the pleura is very challenging to visualize due to the difficulty of access. METHOD: In this study, PS-OCT was used to identify subpleural cancer in male New Zealand white rabbits (3.2-3.4 kg) and to assess the phase retardation changes in normal and cancerous chest walls. VX2 cell suspension was injected between the intercostal muscle and parietal pleura and a tented area was observed by thoracic scope. A group of rabbits (n = 3) were sacrificed at day 7 after injection and another group (n = 3) at day 14. RESULTS: In the PS-OCT images, pleura thickness changes and muscle damage were criteria to understand the stages of the disease. The results of image and phase retardation analysis matched well with the pathologic examinations. CONCLUSION: We were able to visualize and analyze subpleural cancer by PS-OCT, which provided structural and functional information. The measured phase retardation could help to identify the margin of the tumor. For further studies, various approaches into other diseases using polarization light are expected to have positive results.

Investigation of the Clinical Potential of Polarization-Sensitive Optical Coherence Tomography in a Laryngeal Tumor Model.

BACKGROUND: The vocal cord tissue consists of three anatomical layers from the surface to deep inside: the epithelium that contains almost no collagen, the lamina propria that is composed of abundant collagen, and the vocalis muscle layer. It is clinically important to visualize the tissue microstructure using a non-invasive method, especially in the case of vocal cord nodules or cancer, since histological changes in each layer of the vocal cord cause changes in the voice. Polarization-sensitive optical coherence tomography (PS-OCT) enables phase retardation measurement to evaluate birefringence of tissue with varied organization of collagen fibers in different tissue layers. Therefore, PS-OCT can visualize structural changes between normal and abnormal vocal cord tissue. METHOD: A rabbit laryngeal tumor model with different stages of tumor progression was investigated ex-vivo by PS-OCT. A phase retardation slope-based analysis, which quantifies the birefringence in different layers, was conducted to distinguish the epithelium, lamina propria, and muscle layers. RESULTS: The PS-OCT images showed a gradual decrease in birefringence from normal tissue to advanced tumor tissue. The quantitative analysis provided a more detailed comparison among different stages of the rabbit laryngeal tumor model, which was validated by the corresponding histological findings. CONCLUSION: Differences in tissue birefringence was evaluated by PS-OCT phase retardation measurement. It is also possible to indirectly infer the dysplastic changes based on the mucosal and submucosal alterations.

New Mechanism of Gemcitabine and Its Phosphates: DNA Polymerization Disruption via 3'-5' Exonuclease Inhibition.

Gemcitabine (dFdC), a modified deoxycytidine (dC) widely used in tumor treatment, is a prodrug that is phosphorylated to generate mono-, di-, and triphosphates. The triphosphate (dFdCTP) is incorporated into DNA to terminate DNA synthesis in cancer. Some incorporated dFdC nucleotides can be partially removed by the 3'-5' exonuclease activity, namely its editing function, and the others escape the editing. However, whether there is an active mechanism for dFdC to escape the editing remains unclear. We have first discovered that unlike dFdC, its mono-, di-, and triphosphates can inhibit the 3'-5' exonuclease of DNA polymerase I, suppress editing, and allow the active escaping mechanism, whereas its polymerase activity is not remarkably affected. As such, these phosphates can prevent the removal of the incorporated dFdC residue, thereby actively blocking DNA extension and synthesis. The inhibition efficiency of these phosphates follows the increased order of the mono-, di-, and triphosphates of gemcitabine (dFdC < dFdCMP < dFdCDP < dFdCTP). In addition, after the deletion of the 3'-5' exonuclease of cellular DNA polymerase I, the Escherichia coli mutant is more sensitive to dFdCTP than is wild-type E. coli. Our new discovery of the ability of these dFdC phosphates to inhibit exonuclease activity suggests a novel anticancer mechanism of gemcitabine and its phosphate derivatives.

Discovery and Characterization of 1H-1,2,3-Triazole Derivatives as Novel Prostanoid EP4 Receptor Antagonists for Cancer Immunotherapy.

The prostanoid EP4 receptor is one of the key receptors associated with inflammatory mediator PGE2-elicited immunosuppression in the tumor microenvironment. Blockade of EP4 signaling to enhance immunity-mediated tumor elimination has recently emerged as a promising strategy for cancer immunotherapy. In our efforts to discover novel subtype-selective EP4 antagonists, we designed and synthesized a class of 1H-1,2,3-triazole-based ligands that display low nanomolar antagonism activity toward the human EP4 receptor and excellent subtype selectivity. The most promising compound 59 exhibits single-digit nanomolar potency in the EP4 calcium flux and cAMP-response element reporter assays and effectively suppresses the expression of multiple immunosuppression-related genes in macrophage cells. On the basis of its favorable ADMET properties, compound 59 was chosen for further in vivo biological evaluation. Oral administration of compound 59 significantly inhibited tumor growth in the mouse CT26 colon carcinoma model accompanied by enhanced infiltration of cytotoxic T lymphocytes in the tumor tissue.

Comparison of Clinical and Biomechanical Outcomes between Partial Fibulectomy and Drug Conservative Treatment for Medial Knee Osteoarthritis.

BACKGROUND: Upper partial fibulectomy has been preliminarily proved to have the efficacy for pain alleviation and improvement of function in patients with mild to moderate medial compartment knee osteoarthritis (KOA). However, the previous studies lack the control group with other treatments. The aim of this prospective, randomized controlled study is to compare the clinical and biomechanical effects between upper partial fibulectomy and drug conservative treatment on improvement of clinical pain, function, and gait for patients with mild to moderate medial knee osteoarthritis (KOA) and further discuss its biomechanical mechanism. METHODS: From August 2016 to February 2017, 49 and 48 patients with mild to moderate medial KOA were allocated to fibulectomy and drug groups. We assessed the patients' visual analog scale (VAS) pain score, Hospital for Special Surgery (HSS) knee score, limb alignment, passive flexion/extension range of motion (ROM) of the knee, and 3D gait kinematics and kinetics parameters before and after intervention. Repeated-measures ANOVA with Dunnett's post hoc assessment and multivariate analysis of variance were applied for intragroup and intergroup comparisons, respectively. RESULTS: The improvement in the fibulectomy group on the VAS pain score, HSS knee score, walking speed, and walking knee range of motion (ROM) was statistically better than that in the drug group. The decreased overall peak knee adduction moment (KAM) (decreased by 16.1%) and hip-knee-ankle (HKA) angle (decreased by 0.99° from a more varus alignment to a more neutral alignment) of the affected and operated side 1 year after surgery were observed in the fibulectomy group. CONCLUSION: This research demonstrated that as a biomechanical intervention, upper partial fibulectomy can be a better choice in pain relief and function and gait improvement than drug conservative treatment for patients with early-stage knee OA. The long-term clinical outcomes, indication, and rationale for the improvement in clinical symptoms should be investigated further.

Functionalization of Novel Theranostic Hydrogels with Kartogenin-Grafted USPIO Nanoparticles To Enhance Cartilage Regeneration.

Here, kartogenin (KGN), an emerging stable nonprotein compound with the ability to promote differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into chondrocytes, was grafted onto the surface of modified ultrasmall superparamagnetic iron-oxide (USPIO) and then integrated into cellulose nanocrystal/dextran hydrogels. The hydrogels served as a carrier for the USPIO-KGN and a matrix for cartilage repair. We carried out in vitro and in vivo studies, the results of which demonstrated that KGN undergoes long-term stable sustained release, recruits endogenous host cells, and induces BMSCs to differentiate into chondrocytes, thus enabling in situ cartilage regeneration. Meanwhile, the USPIO-incorporated theranostic hydrogels exhibited a distinct magnetic resonance contrast enhancement and maintained a stable relaxation rate, with almost no loss, both in vivo and in vitro. According to noninvasive in vivo observation results and immunohistochemistry analyses, the regenerated cartilage tissue was very similar to natural hyaline cartilage. This innovative diagnosis and treatment system increases the convenience and effectiveness of chondrogenesis.

Curcumin Attenuates Asthmatic Airway Inflammation and Mucus Hypersecretion Involving a PPARγ-Dependent NF-κB Signaling Pathway In Vivo and In Vitro.

Asthma is characterized by airway inflammation and mucus hypersecretion. Curcumin possessed a potent anti-inflammatory property involved in the PPARγ-dependent NF-κB signaling pathway. Then, the aim of the current study was to explore the value of curcumin in asthmatic airway inflammation and mucus secretion and its underlying mechanism. In vivo, mice were sensitized and challenged by ovalbumin (OVA) to induce chronic asthma. Airway inflammation and mucus secretion were analyzed. In vitro, BEAS-2B cells were obtained. MCP-1, MUC5AC, and PPARγ expression and the phosphorylation of NF-κB p65 and NF-κB p65 DNA-binding activity were measured in both the lungs and BEAS-2B cells. shRNA-PPARγ was used to knock down PPARγ expression. We found that OVA-induced airway inflammation and mucus hypersecretion in mice, OVA and IL-4-induced upregulation of MCP-1 and MUC5AC, suppression of PPARγ, and activation and translocation of NF-κB p65 were notably improved by curcumin both in vivo and in vitro. Our data also showed that these effects of curcumin were significantly abrogated by shRNA-PPARγ. Taken together, our results indicate that curcumin attenuated OVA-induced airway inflammation and mucus hypersecretion in mice and suppressed OVA- and IL-4-induced upregulation of MCP-1 and MUC5AC both in vivo and in vitro, most likely through a PPARγ-dependent NF-κB signaling pathway.

Photoacoustic tomography for imaging the prostate: a transurethral illumination probe design and application.

In vivo imaging of prostate cancer with photoacoustic tomography is currently limited by the lack of sufficient local fluence for deep tissue penetration and the risk of over-irradiation near the laser-tissue contact surface. We propose the design of a transurethral illumination probe that addresses those limitations. A high energy of 50 mJ/pulse is coupled into a 1000-µm-core diameter multimode fiber. A 2 cm diffusing end is fabricated, which delivers light in radial illumination. The radial illumination is then reflected and reshaped by a parabolic cylindrical mirror to obtain nearly parallel side illumination with a doubled fluence. The fiber assembly is housed in a 25 Fr cystoscope sheath to provide protection of the fiber and maintain a minimal laser-tissue contact distance of 5 mm. A large laser-tissue contact surface area of 4 cm2 is obtained and the fluence on the tissue surface is kept below the maximum permissible exposure. By imaging a prostate mimicking phantom, a penetration depth of 3.5 cm at 10 mJ/cm2 fluence and 700 nm wavelength is demonstrated. The results indicate that photoacoustic tomography with the proposed transurethral probe has the potential to image the entire prostate while satisfying the fluence maximum permissible exposure and delivering a high power to the tissue.

Sulfated modification of arabinogalactans from Larix principis-rupprechtii and their antitumor activities.

A highly branched arabinogalactan isolated from Larix principis-rupprechtii and subjected to sulfation derivatization to promote their antitumor bioactivity. Several structural features of the sulfated arabinogalactans (S-LAG) were investigated: molecular weight, monosaccharide constitution, and chemical structures. Spectral analysis indicated that sulfate groups were successfully introduced on arabinogalactan. Sulfated products showed different degrees of substitution (DS) ranging from 0.61 to 0.80, and different Mw ranging from 19.24 to 22.03 kDa. Monosaccharide composition before and after sulfation indicated some level of derivatization selectivity. In vitro cancer cell tests demonstrated that S-LAGs were effective inhibitors to cancer cell growth depending on their dosage. The toxicity mechanisms were further investigated, and assay results revealed that S-LAGs mainly induced cancer cellular apoptosis to promote atrophy and inhibit cell proliferation. The results obtained in this work offer strong demonstration of modified arabinogalactans as a potential medical substance for treating different forms of cancer.

Construction of arabinogalactans/selenium nanoparticles composites for enhancement of the antitumor activity.

Selenium nanoparticles (SeNPs) were synthesized using arabinogalactans (LAG) as a formation scaffolding and particle stabilizer to investigate their anti-tumor properties. The formation, morphology, size, and in vitro biological activity of LAG-SeNPs were characterized by UV-vis, FT-IR, scanning electron microscopy (SEM), transmission electron microscopy (TEM), dynamic light scattering (DLS) and cell toxicity assays. SEM and TEM of LAG-SeNPs visualized the individual spherical nanoparticles, while the spectroscopic characterization revealed the modes of interaction which lead to the stable particle properties of the LAG-SeNPs. Cell toxicity assays indicated that the products had significant inhibitory effect on A549, HepG-2 and MCF-7 cells with a dose-dependent effect. The toxicity mechanisms of LAG-SeNPs were further investigated, and assay results revealed that LAG-SeNPs mainly induced cancer cellular apoptosis to promote atrophy and inhibit cell proliferation. The sum of these findings demonstrates the positive effects that a biomass-derived polysaccharide exert upon non-metal/metalloid-based nanoparticles and the ensuing material's viability as treatment against human cancers.

Bioinert Control of Zwitterionic Poly(ethylene terephtalate) Fibrous Membranes.

Poly(ethylene terephtalate) (PET)-based materials face general biofouling issues that we addressed by grafting a copolymer of glycidyl methacrylate and sulfobetaine methacrylate, poly(GMA- r-SBMA). The grafting procedure involved a dip-coating step followed by UV-exposure and led to successful grafting of the copolymer as evidenced by X-ray photoelectron spectroscopy and zeta potential measurements. It did not modify the pore size nor the porosity of the PET membranes. In addition, their surface hydrophilicity was considerably improved, with a water contact angle falling to 30° in less than 20 s and 0° in less than 1 min. The effect of copolymer concentration in the coating bath (dip-coating procedure) and UV exposure time (UV step) were scrutinized during biofouling studies involving several bacteria such as Escherichia coli and Stenotrophomonas maltophilia, but also whole blood and HT1080 fibroblasts cells. The results indicate that if all conditions led to improved biofouling mitigation, due to the efficiency of the zwitterionic copolymer and grafting procedure, a higher concentration (15 mg/mL) and longer UV exposure time (at least 10 min) enhanced the grafting density which reflected on the biofouling results and permitted a better general biofouling control regardless of the nature of the biofoulant (bacteria, blood cells, fibroblasts).

Modelling Thermally Induced Non-Equilibrium Gas Flows by Coupling Kinetic and Extended Thermodynamic Methods.

Thermally induced non-equilibrium gas flows have been simulated in the present study by coupling kinetic and extended thermodynamic methods. Three different types of thermally induced gas flows, including temperature-discontinuity- and temperature-gradient-induced flows and radiometric flow, have been explored in the transition regime. The temperature-discontinuity-induced flow case has shown that as the Knudsen number increases, the regularised 26 (R26) moment equation system will gradually loss its accuracy and validation. A coupling macro- and microscopic approach is employed to overcome these problems. The R26 moment equations are used at the macroscopic level for the bulk flow region, while the kinetic equation associated with the discrete velocity method (DVM) is applied to describe the gas close to the wall at the microscopic level, which yields a hybrid DVM/R26 approach. The numerical results have shown that the hybrid DVM/R26 method can be faithfully used for the thermally induced non-equilibrium flows. The proposed scheme not only improves the accuracy of the results in comparison with the R26 equations, but also extends their capability with a wider range of Knudsen numbers. In addition, the hybrid scheme is able to reduce the computational memory and time cost compared to the DVM.

Toward Intra-Operative Prostate Photoacoustic Imaging: Configuration Evaluation and Implementation Using the da Vinci Research Kit.

We compare different possible scanning geometries for prostate photoacoustic tomography (PAT) while considering a realistic reconstruction scenario in which the limited view of the prostate and the directivity effect of the transducer are considered. Simulations and experiments confirm that an intra-operative configuration in which the photoacoustic signal is received by a pickup transducer from the anterior surface of the prostate provides the best approach. We propose a PAT acquisition system that includes a da Vinci system controlled by the da Vinci Research Kit, an illumination laser, and an ultrasound machine with parallel data acquisition. The robot maneuvers the pickup transducer to form a cylindrical detection surface around the prostate. The robot is programmed to acquire trajectories in which the transducer face is parallel to and oriented toward a rotational tomography axis, while the laser is fired and PAT data are collected at regular intervals. We present our initial images acquired with this novel system.

Extent of Lung Involvement and Serum Cryptococcal Antigen Test in Non-Human Immunodeficiency Virus Adult Patients with Pulmonary Cryptococcosis.

BACKGROUND: Serum cryptococcal antigen (CrAg) test is the most used noninvasive method to detect cryptococcal infection. However, false-negative CrAg test is not uncommon in clinical practice. Then, the aim of this study was to investigate the factors associated with false-negative CrAg test among non-human immunodeficiency virus (HIV) adult patients with pulmonary cryptococcosis and its clinical features. METHODS: One hundred and fourteen non-HIV adult patients with pulmonary cryptococcosis, proven by biopsy, were retrospectively reviewed. Finally, 85 patients were enrolled; 56 were CrAg positive (CrAg+ group) and 29 were negative (CrAg- group). It was a cross-sectional study. Then, baseline characteristics, underlying diseases, clinical symptoms, laboratory findings, and chest radiological findings were reviewed and analyzed. Chi-square test was used to analyze categorical variable. Odds ratio (OR) was used to measure correlation. Student's t- test was obtained to analyze continuous variable. RESULTS: No difference in baseline characteristics, underlying diseases, clinical symptoms, and laboratory findings were found between two groups (P > 0.05 in all). Nevertheless, diffuse extent lesion was 82.1% in CrAg+ group and 10.3% in CrAg- group (χ2 = 40.34, P < 0.001; OR = 39.87). CONCLUSIONS: Among patients with limited pulmonary involvement, a negative serum CrAg does not preclude the diagnosis of pulmonary cryptococcosis. However, among patients with extensive pulmonary involvement, serum CrAg is a useful diagnostic tool for pulmonary cryptococcosis. Furthermore, we also noticed that the untypical and mild presentations with extensive pulmonary lesion might be the features of pulmonary cryptococcosis, which needs further investigation.

Characterization of arabinogalactans from Larix principis-rupprechtii and their effects on NO production by macrophages.

Arabinogalactans are a source of dietary fiber with health benefits. In this work, two arabinogalactans assigned as AGW and AGS were isolated from Larix principis-rupprechtii, and characterized by gel permeation chromatography (GPC), monosaccharide analysis, methylation analysis and NMR spectroscopy analysis. The average molecular weights of AGW and AGS were 1.53 × 104 and 1.84 × 104 Da, respectively. Methylation analysis and NMR spectra suggested that AGW and AGS have a 1,3-linked Galp backbone, branched at C-6 with 1,6-linked Galp side residues. The Ara residues were substituted at C-6 of 1,6-linked Galp consisting of α-L-Araf-(1→3)-α-L-Araf-(1→6)-ß-D-Galp-(1→ and ß-L-Arap-(1→6)-ß-D-Galp-(1→. Significantly, AGS (0.74%) was shown to contain 25 times more uronic acid than AGW (0.03%), which demonstrated a polyelectrolyte effect. Application of these two polysaccharides to macrophage RAW 264.7 cells was shown to increase nitric oxide (NO) production. These results provide a basis for studying the relationship between the structure and biological activity of arabinogalactans.