Tannic acid modified keratin/sodium alginate/carboxymethyl chitosan biocomposite hydrogels with good mechanical properties and swelling behavior.

Natural polymer-based hydrogels have demonstrated great potential as wound-healing dressings. They help to maintain a moist wound environment as well as promote faster healing. In this work, a multifunctional hydrogel was prepared using keratin, sodium alginate, and carboxymethyl chitosan with tannic acid modification. Micro-morphology of hydrogels has been performed by scanning electron microscopy. Fourier Transform Infrared Spectroscopy reveals the presence of hydrogen bonding. The mechanical properties of the hydrogels were examined using a universal testing machine. Furthermore, we investigated several properties of the modified hydrogel. These properties include swelling rate, water retention, anti-freezing properties, antimicrobial and antioxidant properties, hemocompatibility evaluation and cell viability test in vitro. The modified hydrogel has a three-dimensional microporous structure, the swelling rate was 1541.7%, the elastic modulus was 589.74 kPa, the toughness was 211.74 kJ/m3, and the elongation at break was 75.39%, which was similar to the human skin modulus. The modified hydrogel also showed inhibition of S. aureus and E. coli, as well as a DPPH scavenging rate of 95%. In addition, the modified hydrogels have good biological characteristics. Based on these findings, the K/SA/CCS hydrogel holds promise for applications in biomedical engineering.

rAAV capsid mutants eliminate leaky expression from DNA donor template for homologous recombination.

Precise genomic editing through the combination of CRISPR/Cas systems and recombinant adeno-associated virus (rAAV)-delivered homology directed repair (HDR) donor templates represents a powerful approach. However, the challenge of effectively suppressing leaky transcription from the rAAV vector, a phenomenon associated to cytotoxicity, persists. In this study, we demonstrated substantial promoter activities of various homology arms and inverted terminal repeats (ITR). To address this issue, we identified a novel rAAV variant, Y704T, which not only yields high-vector quantities but also effectively suppresses in cis mRNA transcription driven by a robust promoter. The Y704T variant maintains normal functionality in receptor interaction, intracellular trafficking, nuclear entry, uncoating, and second-strand synthesis, while specifically exhibiting defects in transcription. Importantly, this inhibitory effect is found to be independent of ITR, promoter types, and RNA polymerases. Mechanistic studies unveiled the involvement of Valosin Containing Protein (VCP/p97) in capsid-mediated transcription repression. Remarkably, the Y704T variant delivers HDR donor templates without compromising DNA replication ability and homologous recombination efficiency. In summary, our findings enhance the understanding of capsid-regulated transcription and introduce novel avenues for the application of the rAAV-CRISPR/Cas9 system in human gene therapy.

Fibrinogen and tumors.

Elevated plasma fibrinogen (Fg) levels consistently correlate with an unfavorable prognosis in various tumor patient cohorts. Within the tumor microenvironment, aberrant deposition and expression of Fg have been consistently observed, interacting with multiple cellular receptors and thereby accentuating its role as a regulator of inflammatory processes. Specifically, Fg serves to stimulate and recruit immune cells and pro-inflammatory cytokines, thereby contributing to the promotion of tumor progression. Additionally, Fg and its fragments exhibit dichotomous effects on tumor angiogenesis. Notably, Fg also facilitates tumor migration through both platelet-dependent and platelet-independent mechanisms. Recent studies have illuminated several tumor-related signaling pathways influenced by Fg. This review provides a comprehensive summary of the intricate involvement of Fg in tumor biology, elucidating its multifaceted role and the underlying mechanisms.

Melittin analog p5RHH enhances recombinant adeno-associated virus transduction efficiency.

OBJECTIVE: Melittin and its derivative have been developed to support effective gene delivery systems. Their ability to facilitate endosomal release enhances the delivery of nanoparticle-based gene therapy. Nevertheless, its potential application in the context of viral vectors has not received much attention. Therefore, we would like to optimize the rAAV vector by Melittin analog to improve the transduction efficiency of rAAV in liver cancer cells and explore the mechanism of Melittin analog on rAAV. METHODS: Various melittin-derived peptides were inserted into loop VIII of the capsid protein in recombinant adeno-associated virus vectors. These vectors carrying either gfp or fluc genes were subjected to quantitative polymerase chain reaction assays and transduction assays in human embryonic kidney 293 (HEK293T) cells to investigate the efficiency of vector production and gene delivery. In addition, the ability of a specific p5RHH-rAAV vector to deliver genes was examined through in vitro transduction of different cultured cells and in vivo tail vein administration to C57BL/6 mice. Finally, the intricate details of the vector-mediated transduction mechanisms were explored by using pharmacological inhibitors of every stage of the rAAV2 intracellular life cycle. RESULTS: A total of 76 melittin-related peptides were identified from existing literature. Among them, CMA-3, p5RHH and aAR3 were found to significantly inhibit transduction of rAAV2 vector crude lysate. The p5RHH-rAAV2 vectors efficiently transduced not only rAAV-potent cell lines but also cell lines previously considered resistant to rAAV. Mechanistically, bafilomycin A1, a vacuolar endosome acidification inhibitor, completely inhibited the transgene expression mediated by the p5RHH-rAAV2 vectors. Most importantly, p5RHH-rAAV8 vectors also increased hepatic transduction in vivo in C57BL/6 mice. CONCLUSION: The incorporation of melittin analogs into the rAAV capsids results in a significant improvement in rAAV-mediated transgene expression. While further modifications remain an area of interest, our studies have substantially broadened the pharmacological prospects of melittin in the context of viral vector-mediated gene delivery. Please cite this article as: Meng J, He Y, Yang H, Zhou L, Wang S, Feng X, Al-shargi OY, Yu X, Zhu L, Ling, C. Melittin analog p5RHH enhances recombinant adeno-associated virus transduction efficiency. J Integr Med. 2024; 22(1): 72-82.

The optimal design and three-dimensional finite element analysis of CAD/CAM integrated roach attachment.

Objectives: To investigate the effect of different designs of movable parts and prosthetic materials on the stress distribution of supporting tissues in mandibular free end dentition defects using three-dimensional finite element analysis of digital Roach attachments. Material and methods: A 3D model of a patient with Kennedy class I mandibular edentulous conditions was generated, and twelve prosthesis models were applied, combining two designs of removable parts and six types of CAD/CAM restorative materials with different elastic modulus (conventional zirconia, ultra-translucent zirconia, Polyetheretherketone (PEEK), Lithium disilicate, Nanoceramic resin, and resin composite (Paradigm MZ100, 3 M ESPE)). The stress distribution of abutment periodontal ligament, edentulousmucosa, and junction of attachment were analyzed using finite element analysis. Results: The stress value of the buccal neck of the periodontal ligament and the maximum compressive stress of the distal periodontal ligament of the design with clasp arms were higher than those without clasp arms, while the stress on the junction of attachment and the displacement of the mucosa in the edentulous area were smaller. Restorative materials with high elastic modulus, such as conventional zirconia and ultra-translucent zirconia, are recommended to be used as the fixed part of Roach attachment. Conclusion: CAD/CAM Roach attachments with clasp arms are recommended for the protection of mucosal soft tissue. Restorative materials with high elastic modulus, such as conventional zirconia and ultra-translucent zirconia, are recommended as the fixed part of Roach attachment for patients with free end defect of mandibular dentition. Clinical significance: This study provides references for the design with clasp arms and the selection of clinical fixed-movable prosthetic materials. Clinicians should consider the design of attachments and selection of appropriate manufacturing materials carefully to avoid negative impacts on patients' periodontal support tissues.

Rydberg State Single-Mode Polariton Lasing with Ultralow Threshold via Symmetry Engineering.

Symmetry plays an essential role in the fundamental properties of a physical system. In this work, we report on the realization of tunable single-mode polariton lasing from highly excited Rydberg states via symmetry engineering. By breaking the symmetry of the polaritonic wave function through potential wells and controlling the spatial overlap between the gain region and the eigen mode, we are able to generate single-mode polariton lasing, reversibly and dynamically, from quantized polariton states. Increasing the asymmetry of the potential well, single-mode lasing can be achieved even for the highly excited Rydberg state with a principle quantum number of N = 14. Moreover, as a result of the excellent reservoir-eigen mode overlap and efficient spatial confinement, the threshold of lasing can be reduced up to 6 orders of magnitude, compared with those conventional pumping schemes. Our results present a new strategy toward the realization of thresholdless polariton lasing with dynamical tunability.

Association of emotional and behavioral problems with sleep disturbance among Chinese adolescents: The moderation effect of academic performance.

BACKGROUND: This study aimed to explore the association of emotional and behavioral problems with sleep disturbance among Chinese adolescents, and to test whether these associations vary with adolescents' academic performance. METHODS: Data were from the 2021 School-based Chinese Adolescents Health Survey using a multistage, stratified-cluster, and random-sampling method to collect information from 22,684 middle school students in Guangdong Province, China. RESULTS: Emotional problems (aOR = 1.34, 95 % CI = 1.32-1.36), conduct problems (aOR = 1.19, 95 % CI = 1.16-1.21), hyperactivity (aOR = 1.35, 95 % CI = 1.33-1.37), and peer problems (aOR = 1.06, 95 % CI = 1.04-1.09) were significantly associated with the increased risk of sleep disturbance among middle school students in Guangdong Province. The prevalence of sleep disturbance among adolescents was 29.4 %. There were significant interactions of emotional problems/conduct problems/peer problems/prosocial behavior with academic performance on sleep disturbance. Further stratification analyses by academic performance showed that adolescents with self-reported good academic performance had a higher risk of sleep disturbance than those students with self-reported average or poor academic performance. LIMITATIONS: This study only included school students and used the cross-sectional design to preclude causality determination. CONCLUSIONS: Our findings suggest that emotional and behavioral problems elevate the risk of sleep disturbance in adolescents. Adolescent academic performance plays a modulating role in these above-mentioned significant associations for sleep disturbance.

Comparative study on the impact-sliding wear behaviour of CAD/CAM resin-ceramic materials and tooth enamel.

OBJECTIVES: To compare the impact-sliding wear of different CAD/CAM resin-ceramic materials and tooth enamel, and explore the corresponding wear damage mechanism. METHODS: Human tooth enamel (EN), Vita ENAMIC (Vita, VE), Lava Ultimate (3 M, LU), and GC CERASMART (GC, CS) were used in this study. The hardness, elastic modulus, and roughness values of the samples were measured. Further, impact-sliding wear tests were performed in a ball-on-flat configuration with spherical zirconia antagonists and the coefficients of friction (CoF) were recorded simultaneously. Additionally, a white light interferometer was used to determine the volume losses and scanning electron microscopy was used to observe the wear morphology of the wear scars and the damage feature in the vertical sections to clarify the damage mechanism during the impact-sliding wear test. RESULTS: EN exhibited the highest elastic modulus and CoF, followed by VE, LU, and CS. The hardness and roughness of EN and VE were similar and were higher than those of LU and CS. Throughout the wear tests, VE exhibited the highest volume loss, whereas CS exhibited the lowest. The wear damage characteristics of VE were similar to those of EN, displaying brittle fractures of inorganic substances and plastic deformation of organic substances in the impact part, exhibiting plough marks in the sliding parts. In the case of LU and CS, the entire wear areas displayed plastic deformation of the resin matrix, exfoliation of the filler particles, and plough marks. SIGNIFICANCE: Enamel and polymer-infiltrated ceramic network materials exhibit similar wear damage modes. Additionally, the high-density nanocomposite resin material is the most resistant to impact-sliding wear from a tribological perspective.

The use of melittin to enhance transgene expression mediated by recombinant adeno-associated virus serotype 2 vectors both in vitro and in vivo.

OBJECTIVE: Melittin, a cell-penetrating peptide, improves the efficiency of many non-viral gene delivery vectors, yet its application in viral vectors has not been well studied. The non-pathogenic recombinant adeno-associated virus (rAAV) vector is an ideal in vivo gene delivery vector. However, its full potential will only be achieved after improvement of its transduction efficiency. To improve the transduction efficiency of rAAV2 vectors, we attempted to develop a melittin-based rAAV2 vector delivery strategy. METHODS: The melittin peptide was inserted into the rAAV2 capsid either in the loop VIII of all viral proteins (VPs) or at the N terminus of VP2. Various rAAV2-gfp or -fluc vectors were subjected to quantitative real-time polymerase chain reaction and Western blot assays to determine their titers and integrity of capsid proteins, respectively. Alternatively, the vectors based on wild-type capsid were pre-incubated with melittin, followed by transduction of cultured cells or tail vein administration of the mixture to C57BL/6 and BALB/c nude mice. In vivo bioluminescence imaging was performed to evaluate the transgene expression. RESULTS: rAAV2 vectors with melittin peptide inserted in the loop VIII of VPs had low transduction efficiency, probably due to dramatically reduced ability to bind to the target cells. Fusing the melittin peptide at the N-terminus of VP2 produced vectors without the VP2 subunit. Interestingly, among the commonly used rAAV vectors, pre-incubation of rAAV2 and rAAV6 vectors with melittin significantly enhanced their transduction efficiency in HEK293 and Huh7 cells in vitro. Melittin also had the ability to increase the rAAV2-mediated transgene expression in mouse liver in vivo. Mechanistically, melittin did not change the vector-receptor interaction. Moreover, cell counting kit-8 assays of cultured cells and serum transaminase levels indicated melittin had little cytotoxicity. CONCLUSION: Pre-incubation with melittin, but not insertion of melittin into the rAAV2 capsid, significantly enhanced rAAV2-mediated transgene expression. Although further in vivo evaluations are required, this research not only expands the pharmacological potential of melittin, but also provides a new strategy to improve gene therapy mediated by rAAV vectors.

Associations of Gut Microbiota and Fatty Metabolism With Immune Thrombocytopenia.

Objective: To determine whether gut microbiota, fatty metabolism and cytokines were associated with immune thrombocytopenia (ITP). Methods: In total, 29 preliminarily diagnosed ITP patients and 33 healthy volunteers were enrolled. Fecal bacterial were analyzed based on 16S rRNA sequencing. Plasma cytokines and motabolites were analyzed using flow cytometry and liquid chromatography-mass spectrometry (LC-MS), respectively. Results: Bacteroides, Phascolarctobacterium, and Lactobacillus were enriched at the genus level in ITP patients, while Ruminococcaceae UCG-002, Eubacterium coprostanoligeues, Megamonas, and Lachnospiraceae NC2004 were depleted. At the phylum level, the relative abundance of Proteobacteria and Chloroflexi increased in ITP patients, while Firmicutes, Actinobacteria, and the Firmicutes/Bacteroidetes ratio decreased. Plasma levels of 5-hydroxyeicosatetraenoic acid (5-HETE), 6-trans-12-epi-leukotriene B4 (6t,12e-LTB4), and resolvin D2 (RvD2) were upregulated, and stachydrine, dowicide A, dodecanoylcarnitine were downregulated in ITP patients. Furthermore, RvD2 is positively correlated with order Bacteroidetes VC2.1 Bac22, 5-HETE is positively correlated with genus Azospirillum, and 6t,12e-LTB4 is positively correlated with genus Cupriavidus. In addition, stachydrine is positively correlated with family Planococcaceae, dowicide A is positively correlated with class MVP-15, and dodecanoylcarnitine is positively correlated with order WCHB1-41. Plasma levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were upregulated in ITP patients. Conclusion: Our study revealed a relationship between microbiota and fatty metabolism in ITP. Gut microbiota may participate in the pathogenesis of ITP through affecting cytokine secretion, interfering with fatty metabolism.

Relaxation Oscillations of an Exciton-Polariton Condensate Driven by Parametric Scattering.

We report the observation of coherent oscillations in the relaxation dynamics of an exciton-polariton condensate that were driven by parametric scattering processes. As a result of the interbranch scattering scheme and the nonlinear polariton-polariton interactions, such parametric scatterings exhibit a high scattering efficiency that leads to the fast depletion of the polariton condensate and the periodic shut-off of the bosonic stimulation processes, eventually causing relaxation oscillations. Employing polariton-reservoir interactions, the oscillation dynamics in the time domain can be projected onto the energy space. In theory, our simulations using the open-dissipative Gross-Pitaevskii equation are in excellent agreement with experimental observations. Surprisingly, the oscillation patterns, including many excitation pulses, are clearly visible in our time-integrated images, implying the high stability of the relaxation oscillations driven by polariton parametric scatterings.

Effect of prenatal nutritional intervention on foetal growth restriction: a real-world study in Shenzhen, China.

OBJECTIVE: Fetal growth restriction (FGR) is the primary cause of infant morbidity and mortality. Although nutritional intervention is generally used to cure FGR, its effects on early- and late-onset FGR have not been reported. This study aimed to resolve this issue in a real-world setting. STUDY DESIGN: We collected the data of pregnant women whose fetuses were diagnosed with FGR and subsequently born at Nanshan Women and Children's Care Hospital in Shenzhen, China. We conducted a MANOVA and series of Cox regression analyses to evaluate the effects of a prenatal nutritional intervention on early and late FGR after adjusting for potential confounders. RESULTS: Our results demonstrated that the average birth weights in the four sub-intervention groups were 50.36-160.05 g higher than those in the nonintervention group. These differences were insignificant with respect to early-onset FGR. In late-onset FGR, however, the interventions led to birth weight increases of 164.95-244.45 g greater than those in the nonintervention group, and these differences were significant. During early-onset FGR, four different nutritional interventions reduced the incidence of small-for-gestational age by 8.00-13.76% relative to the incidence in the nonintervention group, while in late-onset FGR, the incidence decreased by 11.37-17.39%. CONCLUSIONS: Our results based on a real-world setting reaffirmed that a prenatal nutritional intervention could improve the birth outcomes in cases of FGR and further suggested a better effectiveness on late-onset FGR.

Study on complex dynamics for the waste electrical and electronic equipment recycling activities oligarchs closed-loop supply chain.

Nowadays, with more and more WEEE (waste electrical and electronic equipment) being abandoned, WEEE recycling activities are increasingly popular. In this paper, we build a closed-loop supply chain model and focus on the recycling behaviors of the members in this supply chain, which contains two manufacturers, the retailer, and the consumer. In the reverse chain, we set up dual channels and design two recycling methods: sell-back method and trade-in method. We use classical backward induction to run the model. And then we analyze the stability of the system and the impacts of some essential parameters by numerical simulation. The speed of the manufacture's decision adjustment has a significant effect on the stability of the model. Manufacturers should adopt a stable rather than radical price adjustment strategy in order to achieve stable profit growth in the whole supply chain and avoid a chaotic price war. Manufacturers should increase innovation investment in recycling technology to reduce the cost of remanufacturing products and improve the initiative in market competition. In the end, we adopt the parameter control method and the decision-making method to control chaos, and they both have a good control effect.

Effects of loupes and microscopes on a dental technician's working posture from ergonomic aspects.

OBJECTIVES: This work aimed to study the effects of loupes and microscopes on a dental technician's working posture during practical operation from ergonomic aspects. The technician's working postures under the conditions of the naked eye, loupes, and microscopes were compared. The practical value of loupes and microscopes was assessed based on the evaluation index of working posture from ergonomic aspects. METHODS: Three dental technicians who were skilled in using loupes and microscopes from West China Stomatology Technology Department of Sichuan University were involved in this prospective rando-mized controlled trial. Before the operation, cameras were installed in the sagittal position, top-view position, and dorsal position of the operation. Each technician made five porcelain veneers of the right maxillary central incisor following the standard process. A chairside computer-aided design and computer-aided manufacturing (CAD/CAM) system was used to mill and layer the ceramic block under the naked eye (A: control group), 3.5× headwear loupes (B: loupe group), and 9× desktop microscopes (C: microscope group). The working posture was recorded by videos throughout the entire process. After each operation, the investigator used OpenPose to recognize the working posture. The joint angles of the arm, elbow, wrist, neck, and trunk, as well as their corresponding rapid upper limb assessment (RULA) scores, were calcula-ted by MATLAB. The working posture was assessed from ergonomic aspects based on the joint angles, RULA scores, and operation time. Statistical analysis was performed using SPSS 26.0. RESULTS: The RULA score of upper limb joint angles showed that the angles of the arm, elbow, wrist, neck, and trunk under the naked eye were 14.93°±9.44°, 120.19°±2.99°, 23.97°±2.84°, 47.27°±5.72°, and 7.76°±2.30°, respectively. All of the joint angles were significantly different among the three groups (P<0.05). Compared with the control group, the angles of the neck and trunk in the loupe group were reduced by 29.09% and 42.53%, respectively, whereas those in the microscope group were significantly reduced by 43.99% and 87.11%, respectively. Multiple comparisons by LSD for the angles of neck and trunk revealed that the loupe group and the microscope group were significantly different from the control group (P<0.05), and they were also significantly different from each other (P<0.05). The mean RULA scores were 6.24±0.34 in the control group, 5.53±0.35 in the loupe group, and 3.31±0.19 in the microscope group. Compared with the control group, the mean RULA score in the loupe group was lower, and that in the microscope group was significantly lower. The differences between every two groups were statistically significant (P<0.05). The mean RULA score in the microscope group was significantly lower than that in the loupe group (P<0.05). The average operation times of the control group, loupe group, and microscope group were (50.69±36.78), (52.01±34.65), and (59.44±35.81) min, respectively. No significant difference was found among the three groups (P>0.05). CONCLUSIONS: Use of loupes and microscopes showed an improvement in ergonomics and working posture of dental technicians. Microscopes had a better effect in the ergonomic convenience of the technician than loupes.

Review: Radionuclide Molecular Imaging Targeting HER2 in Breast Cancer with a Focus on Molecular Probes into Clinical Trials and Small Peptides.

As the most frequently occurring cancer worldwide, breast cancer (BC) is the leading cause of cancer-related death in women. The overexpression of HER2 (human epidermal growth factor receptor 2) is found in about 15% of BC patients, and it is often associated with a poor prognosis due to the effect on cell proliferation, migration, invasion, and survival. As a result of the heterogeneity of BC, molecular imaging with HER2 probes can non-invasively, in real time, and quantitatively reflect the expression status of HER2 in tumors. This will provide a new approach for patients to choose treatment options and monitor treatment response. Furthermore, radionuclide molecular imaging has the potential of repetitive measurements, and it can help solve the problem of heterogeneous expression and conversion of HER2 status during disease progression or treatment. Different imaging probes of targeting proteins, such as monoclonal antibodies, antibody fragments, nanobodies, and affibodies, are currently in preclinical and clinical development. Moreover, in recent years, HER2-specific peptides have been widely developed for molecular imaging techniques for HER2-positive cancers. This article summarized different types of molecular probes targeting HER2 used in current clinical applications and the developmental trend of some HER2-specific peptides.

A fecal-based test for the detection of advanced adenoma and colorectal cancer: a case-control and screening cohort study.

BACKGROUND: Colorectal cancer (CRC) is the leading cause of cancer death worldwide. Screening is a confirmed way to reduce the incidence and mortality rates of CRC. This study aimed to identify a fecal-based, noninvasive, and accurate method for detection of colorectal cancer (CRC) and advanced adenoma (AA). METHODS: Through detection in tissue (n = 96) and fecal samples (n = 88) and tested in an independent group of fecal samples (n = 294), the methylated DNA marker ITGA4 and bacterial markers Fusobacterium nucleatum (Fn) and Pepetostreptococcusanaerobius (Pa) were identified from the candidate biomarkers for CRC and AA detection. A prediction score (pd-score) was constructed using the selected markers and fecal immunochemical test (FIT) for distinguishing AA and CRC from healthy subjects by logistic regression method. The diagnostic performance of the pd-score was compared with FIT and validated in the external validation cohort (n = 117) and in a large CRC screening cohort. RESULTS: The pd-score accurately identified AA and CRC from healthy subjects with an area under the curve (AUC) of 0.958, at a specificity of 91.37%; the pd-score showed sensitivities of 95.38% for CRC and 70.83% for AA, respectively. In the external validation cohort, the sensitivities of the pd-score for CRC and AA detection were 94.03% and 80.00%, respectively. When applied in screening, the pd-score identified 100% (11/11) of CRC and 70.83% (17/24) of AA in participants with both colonoscopy results and qualified fecal samples, showing an improvement by 41.19% compared to FIT. CONCLUSIONS: The current study developed a noninvasive and well-validated approach for AA and CRC detection, which could be applied widely as a diagnostic and screening test.

The use of miR122 and its target sequence in adeno-associated virus-mediated trichosanthin gene therapy.

OBJECTIVE: Plant-derived cytotoxic transgene expression, such as trichosanthin (tcs), regulated by recombinant adeno-associated virus (rAAV) vector is a promising cancer gene therapy. However, the cytotoxic transgene can hamper the vector production in the rAAV producer cell line, human embryonic kidney (HEK293) cells. Here, we explored microRNA-122 (miR122) and its target sequence to limit the expression of the cytotoxic gene in the rAAV producer cells. METHODS: A miR122 target (122T) sequence was incorporated into the 3' untranslated region of the tcs cDNA sequence. The firefly luciferase (fluc) transgene was used as an appropriate control. Cell line HEK293-mir122 was generated by the lentiviral vector-mediated genome integration of the mir122 gene in parental HEK293 cells. The effects of miR122 overexpression on cell growth, transgene expression, and rAAV production were determined. RESULTS: The presence of 122T sequence significantly reduced transgene expression in the miR122-enriched Huh7 cell line (in vitro), fresh human hepatocytes (ex vivo), and mouse liver (in vivo). Also, the normal liver physiology was unaffected by delivery of 122T sequence by rAAV vectors. Compared with the parental cells, the miR122-overexpressing HEK293-mir122 cell line showed similar cell growth rate and expression of transgene without 122T, as well as the ability to produce liver-targeting rAAV vectors. Fascinatingly, the yield of rAAV vectors carrying the tcs-122T gene was increased by 77.7-fold in HEK293-mir122 cells. Moreover, the tcs-122T-containing rAAV vectors significantly reduced the proliferation of hepatocellular carcinoma cells without affecting the normal liver cells. CONCLUSION: HEK293-mir122 cells along with the 122T sequence provide a potential tool to attenuate the cytotoxic transgene expression, such as tcs, during rAAV vector production.

Low endotoxin E. coli strain-derived plasmids reduce rAAV vector-mediated immune responses both in vitro and in vivo.

The major challenge of recombinant adeno-associated virus (rAAV) vectors is host immunological barriers. Compared to the neutralizing antibody and the cytotoxic T lymphocyte response, the host immune responses induced by unsatisfactory rAAV manufacturing were largely ignored previously. rAAV vector production usually requires large amounts of plasmid DNAs. The DNA are commonly isolated from the DH5α bacterial strain, which contains lipopolysaccharide (LPS) contamination. LPS, also named endotoxin, in plasmid DNA is intractable, and residual endotoxin in the subsequent rAAV vectors may result in substantial host immune response. Recently, a ClearColi K12 bacterial strain is commercially available, with genetically modified LPS that does not trigger endotoxic response in mammalian cells. Here, we produced rAAV-DJ vectors by plasmids yielded from either DH5α or ClearColi K12 bacterial strains. Our data indicated that the ClearColi K12 strain had satisfactory protection for the rAAV inverted terminal repeat (ITR) sequence. As expected, the ClearColi K12-derived rAAV-DJ vectors had lower endotoxin levels. The physical and biological equivalency of the purified viral stocks were confirmed by electron micrographs, Coomassie blue staining, and transduction assays. Most importantly, the ClearColi K12-derived rAAV-DJ vectors triggered reduced nuclear factor-kappa B (NF-κB) signaling pathway both in cell cultures in vitro and in C57BL/6 mice retinas in vivo. We believe that the use of the ClearColi K12 bacterial strain could eliminate the LPS in the purified vector stock at the source. Our data indicate its promising use in future clinical development.

Paeoniflorin improves functional recovery through repressing neuroinflammation and facilitating neurogenesis in rat stroke model.

BACKGROUND: Microglia, neuron, and vascular cells constitute a dynamic functional neurovascular unit, which exerts the crucial role in functional recovery after ischemic stroke. Paeoniflorin, the principal active component of Paeoniae Radix, has been verified to exhibit neuroprotective roles in cerebralischemic injury. However, the mechanisms underlying the regulatory function of Paeoniflorin on neurovascular unit after cerebral ischemia are still unclear. METHODS: In this study, adult male rats were treated with Paeoniflorin following transient middle cerebral artery occlusion (tMCAO), and then the functional behavioral tests (Foot-fault test and modified improved neurological function score, mNSS), microglial activation, neurogenesis and vasculogenesis were assessed. RESULTS: The current study showed that Paeoniflorin treatment exhibited a sensorimotor functional recovery as suggested via the Foot-fault test and the enhancement of spatial learning as suggested by the mNSS in rat stroke model. Paeoniflorin treatment repressed microglial cell proliferation and thus resulted in a significant decrease in proinflammatory cytokines IL-1ß, IL-6 and TNF-α. Compared with control, Paeoniflorin administration facilitated von Willebrand factor (an endothelia cell marker) and doublecortin (a neuroblasts marker) expression, indicating that Paeoniflorin contributed to neurogenesis and vasculogenesis in rat stroke model. Mechanistically, we verified that Paeoniflorin repressed JNK and NF-κB signaling activation. CONCLUSIONS: These results demonstrate that Paeoniflorin represses neuroinflammation and facilitates neurogenesis in rat stroke model and might be a potential drug for the therapy of ischemic stroke.

Profiling of gut microbial dysbiosis in adults with myeloid leukemia.

Dysregulation of gut microbiota is implicated in the pathogenesis of various diseases, including metabolic diseases, inflammatory diseases, and cancer. To date, the link between gut microbiota and myeloid leukemia (ML) remains largely unelucidated. Herein, a total of 29 patients with acute myeloid leukemia (AML), 17 patients with chronic myeloid leukemia (CML), and 33 healthy subjects were enrolled, and gut microbiota were profiled via Illumina sequencing of the 16S rRNA. We evaluated the correlation between ML and gut microbiota. The microbial α-diversity and ß-diversity exhibited significant differences between ML patients and healthy controls (HCs). Compared to healthy subjects, we found that at the phylum level, the relative abundance of Actinobacteria, Acidobacteria, and Chloroflexi was increased, while that of Tenericutes was decreased. Correspondingly, at the genus level in ML, Streptococcus were increased, especially in AML patients, while Megamonas (P = 0.02), Lachnospiraceae NC2004 group, and Prevotella 9 (P = 0.007) were decreased. Moreover, ML-enriched species, including Sphingomonas, Lysobacyer, Helicobacter, Lactobacillus, Enterococcus, and Clostridium sensu stricto 1, were identified. Our results indicate that the gut microbiota was altered in ML patients compared to that of healthy subjects, which could contribute to the elucidation of microbiota-related pathogenesis of ML, and the development of novel therapeutic strategies in the treatment of ML.