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As the major energy supplier in cells, mitochondria play a significant role in regulating cellular processes. The pathogenesis of various diseases is found to be associated with dysfunctional mitochondria, and supplement of functional mitochondria has been regarded as a potential therapeutic strategy. To achieve mitochondrial replenishment, transplantation of isolated mitochondria or utilization of cells as selective mitochondrial carriers have been developed. On the one hand, isolated mitochondria can be internalized into injured cells to restore impaired functions. On the other hand, the natural process of intercellular mitochondrial transfer can replace the dysfunctional mitochondria with functional mitochondria, providing a safe and effective way to rescue damaged tissues. Cell mediated mitochondrial transfer can serve as a promising targeted therapy with mitochondria being high-efficient biotherapeutics. In this review, we summarize the updated findings of mitochondrial delivery strategies, offering an overview of the role of mitochondria, mechanisms of intercellular mitochondrial transfer, therapeutic benefits, challenges and prospects of mitochondrial delivery. The understanding of mitochondrial delivery helps to improve the therapeutic outcomes of mitochondrial dysfunctional diseases in the future.

To evaluate the effectiveness of brace treatment in patients with Chiari malformation type 1 (CM-1) or syringomyelia associated scoliosis without neurosurgical intervention. This was a retrospective case–control study. 34 CM-1 or syringomyelia (CMS) patients who received brace treatment without neurosurgical intervention were recruited. Another 68 matched patients with idiopathic scoliosis who received bracing served as the control group. The matching criteria included gender, age (± 1 years), Risser sign (± 1 grade), initial curve magnitude (± 5°), curve patterns and follow-up time (± 6 months). Patients who encountered curve progression and scoliosis surgery were compared between different groups. Until the last visit, 16 (47%) patients in CMS group and 18 (26%) patients in IS group occurred curve progression; 9 (26%) patients and 15 (22%) patients underwent scoliosis surgery, respectively. Compared to idiopathic scoliosis, patients with CMS-associated scoliosis had a significantly higher rate of curve progression (P = 0.038). However, no significant difference was observed between two groups regarding to the rate of surgery (P = 0.867). Patients with combined CM-1 and syringomyelia had a higher rate of surgery than patients with isolated CM-1 or syringomyelia (P = 0.049). The double major curve pattern was identified as the risk factor for curve progression. Brace treatment is effective for CMS-associated scoliosis without neurosurgical intervention. Compared to idiopathic scoliosis, brace can provide similar prevention for scoliosis surgery in CMS patients, but slight or moderate curve progression may occur. Specifically, patients with combined CM-1 and syringomyelia should be followed closely with a higher expectation of curve progression.

Acute respiratory distress syndrome (ARDS) is a severe respiratory disease associated with high morbidity and mortality in the clinic. In the face of limited treatment options for ARDS, extracellular vesicles derived from mesenchymal stem cells (MSC-EVs) have recently shown promise. They regulate levels of growth factors, cytokines, and other internal therapeutic molecules. The possible therapeutic mechanisms of MSC-EVs include anti-inflammatory, cell injury repair, alveolar fluid clearance, and microbe clearance. The potent therapeutic ability and biocompatibility of MSC-EVs have enabled them as an alternative option to ameliorate ARDS. In this review, recent advances, therapeutic mechanisms, advantages and limitations, as well as improvements of using MSC-EVs to treat ARDS are summarized. This review is expected to provide a brief view of the potential applications of MSC-EVs as novel biodrugs to treat ARDS.

Vaccines based on live attenuated Chlamydia elementary bodies (EBs) can cause disease in vaccinated animals and the comparably safer inactivated whole EBs are only marginally protective. Recent studies show that a vaccine formulation comprising UV-inactivated EBs (EB) and appropriate mucosal delivery systems and/or adjuvants induced significant protective immunity. We tested the hypothesis that intranasal delivery of UV-inactivated C. psittaci EB formulated in Vibrio cholerae ghosts (VCG)-chitosan nanoparticles will induce protective immunity against intranasal challenge in SPF chickens. We first compared the impact of VCG and CpG adjuvants on protective immunity following IN mucosal and IM systemic delivery of EB formulated in chitosan hydrogel/microspheres. Immunologic analysis revealed that IN immunization in the presence of VCG induced higher levels of IFN-γ response than IM delivery or the CpG adjuvanted groups. Also, vaccine efficacy evaluation showed enhanced pharyngeal bacterial clearance and protection against lung lesions with the VCG adjuvanted vaccine formulation, thereby establishing the superior adjuvanticity of VCG over CpG. We next evaluated the impact of different concentrations of VCG on protective immunity following IN mucosal immunization. Interestingly, the adjuvanticity of VCG was concentration-dependent, since protective immunity induced following IN mucosal immunization showed dose-dependent immune responses and protection. These studies reveal that formulation of inactivated chlamydial antigens with adjuvants, such as VCG and chitosan increases their ability to induce protective immune responses against challenge.

BackgroundThe management of latent tuberculosis infection (LTBI) is a key action for the realization of the “End tuberculosis (TB) Strategy” worldwide, and it is important to identify priority populations. In this prospective cohort study, we evaluated the prevalence of LTBI and incidence of active TB among close contacts and explored the suitable TB control strategy in schools.MethodsWe designed a cohort with 2 years of follow-up, recruiting freshman/sophomore TB patients’ close contacts from three administrative districts in Shanghai. These were chosen based on different levels of TB incidence reported in 2019. Questionnaires were included and all participants received both tuberculin skin test (TST) and QuantiFERON-TB Gold (QFT) at baseline, then tracked the outcomes of them during the follow-up period.ResultsThe prevalence of LTBI was 4.8% by QFT. Univariate analysis showed that the risk of LTBI was higher in those contacting bacteriologically confirmed patients or did not have BCG scars, including smokers. The risk increased with poor lighting and ventilation conditions at contact sites. Multivariate analysis showed that those contacting with bacteriologically confirmed patients (OR=4.180; 95%CI, 1.164-15.011) or who did not have BCG scars (OR=5.054; 95%CI, 2.278-11.214) had a higher risk of being LTBI, as did the current smokers (OR=3.916; 95%CI, 1.508-10.168) and those who had stopped smoking (OR=7.491; 95%CI, 2.222-25.249). During the 2-year follow-up period, three clinically diagnosed cases of TB were recorded, the 2-year cumulative incidence was 0.4% (95%CI 0.1-1.2), the median duration for TB occurrence was 1 year, the incidence rate of active TB was 2.0 per 1000 person-years with a total of 1497.3 observation person-years. For those LTBI, no one initiated preventive treatment, in the QFT (+) cohort, 1 TB case was observed, 71 person-years with an incidence rate of 14.1 14.1 (95%CI 2.5-75.6) per 1000 person-years, in the TST (+++) cohort, 2 TB cases were observed 91.5 person-years with an incidence rate of 21.9 (95%CI 6.0-76.3) per 1000 person-years.ConclusionsThe results suggest that school close contacts are one of the key populations for LTBI management. Measures should be taken to further reduce the prevalence of LTBI and the incidence of active TB among them.

Blood–tumor barrier (BTB) presents a major obstacle to brain drug delivery. Therefore, it is urgent to enhance BTB permeability for the treatment of glioma. In this study, we demonstrated that MIAT, ZAK, and phosphorylated NFκB-p65 (p-NFκB-p65) were upregulated, while miR-140-3p was downregulated in glioma-exposed endothelial cells (GECs) of BTB compared with those in endothelial cells cocultured with astrocytes (ECs) of blood–brain barrier (BBB). MIAT inhibited miR-140-3p expression, increased the expression of ZAK, enhanced the ratio of p-NFκB-p65:NFκB-p65, and promoted the endothelial leakage of BTB. Our current study revealed that miR-140-3p was complementary to the ZAK 3′untranslated regions (3′-UTR), and luciferase activity of ZAK was inhibited by miR-140-3p in 293T cells. MiR-140-3p silencing resulted in an increase in BTB permeability by targeting ZAK, while overexpression of miR-140-3p had the opposite results in GECs of BTB. Overexpression of ZAK induced an increase in BTB permeability, and this effect was related to ZAK’s ability to mediate phosphorylation of NFκB-p65. Conversely, ZAK silencing get opposite results in GECs of BTB. As a molecular sponge of miR-140-3p, MIAT attenuated its negative regulation of the target gene ZAK by adsorbing miR-140-3p. P-NFκB-p65 as a transcription factor negatively regulated the expression of TJ-associated proteins by means of chip assay and luciferase assay. Single or combined application of MIAT and miR-140-3p effectively promoted antitumor drug doxorubicin (Dox) across BTB to induce apoptosis of glioma cells. In summary, MIAT functioned as a miR-140-3p sponge to regulate the expression of its target gene ZAK, which contribution to phosphorylation of NFκB-p65 was associated with an increase in BTB permeability by down-regulating the expression of TJ associated proteins, thereby promoting Dox delivery across BTB. These results might provide a novel strategy and target for chemotherapy of glioma.

To assess the value of copy number variation sequencing (CNV-seq) and karyotyping in the prenatal diagnosis for carriers of balanced translocations.Clinical records of 135 amniocentesis samples of balanced translocation carriers undergoing simultaneous CNV-seq and karyotyping were analyzed. Chromosomal aberrations were defined as those can definitely lead to birth defects definitely, which included chromosomal numerical abnormality, large deletion/duplication and pathogenic copy number variations (pCNVs).The detection rates for karyotyping and CNV-seq were 4.44% (6/135) and 5.93% (8/135) respectively, and the latter had a detection rate of 1.48(2/135) higher than the former. A total of 68 fetal chromosomal translocations were detected by karyotying analysis.For couples carrying a balanced translocation, simultaneous CNV-seq and karyotyping is conducive to the detection of fetal chromosomal abnormalities and genetic counseling.

Background: Acute ischemic stroke (AIS) is a leading cause of disability and mortality worldwide. Prediction of penumbra existence after AIS is crucial for making decision on reperfusion therapy. Yet a fast, inexpensive, simple, and noninvasive predictive biomarker for the poststroke penumbra with clinical translational potential is still lacking. We aim to investigate whether the CircOGDH (circular RNA derived from oxoglutarate dehydrogenase) is a potential biomarker for penumbra in patients with AIS and its role in ischemic neuronal damage. Methods: CircOGDH was screened from penumbra of middle cerebral artery occlusion mice and was assessed in plasma of patients with AIS by quantitative polymerase chain reaction. Magnetic resonance imaging was used to examine the penumbra volumes. CircOGDH interacted with miR-5112 (microRNA-5112) in primary cortical neurons was detected by fluorescence in situ hybridization, RNA immunoprecipitation, and luciferase reporter assay. Adenovirus-mediated CircOGDH knockdown ameliorated neuronal apoptosis induced by COL4A4 (Gallus collagen, type IV, alpha IV) overexpression. Transmission electron microscope, nanoparticle tracking analysis, and Western blot were performed to confirm exosomes. Results: CircOGDH expression was dramatically and selectively upregulated in the penumbra tissue of middle cerebral artery occlusion mice and in the plasma of 45 patients with AIS showing a 54-fold enhancement versus noncerebrovascular disease controls. Partial regression analysis revealed that CircOGDH expression was positively correlated with the size of penumbra in patients with AIS. Sequestering of miR-5112 by CircOGDH enhanced COL4A4 expression to elevate neuron damage. Additionally, knockdown of CircOGDH significantly enhanced neuronal cell viability under ischemic conditions. Furthermore, the expression of CircOGDH in brain tissue was closely related to that in the serum of middle cerebral artery occlusion mice. Finally, we found that CircOGDH was highly expressed in plasma exosomes of patients with AIS compared with those in noncerebrovascular disease individuals. Conclusions: These results demonstrate that CircOGDH is a potential therapeutic target for regulating ischemic neuronal viability, and is enriched in neuron-derived exosomes in the peripheral blood, serving as a predictive biomarker of penumbra in patients with AIS.

Exosomes and biomimetic vesicles are widely used for gene delivery because of their excellent gene loading capacity and stability and their natural targeting delivery potential. These vesicles take advantages of both cell-based bioactive delivery system and synthetical lipid-derived nanovectors to form crossover characteristics. To further optimize the specific targeting properties of crossover vesicles, studies of their in vivo fate and various engineering approaches including nanobiotechnology are required. This review describes the preparation process of exosomes and biomimetic vesicles, and summarizes the mechanism of loading and delivery of nucleic acids or gene editing systems. We provide a comprehensive overview of the techniques employed for preparing the targeting crossover vesicles based on their cellular uptake and targeting mechanism. To delineate the future prospects of crossover vesicle gene delivery systems, various challenges and clinical applications of vesicles have also been discussed.

Aim: This study aimed to evaluate the effect of miR-24-3p in Alzheimer’s disease (AD). Materials & methods: A total of 198 participants were recruited in this study, including 104 AD patients and 94 healthy controls. Expression of miR-24-3p was detected using quantitative real-time PCR. Receiver-operating characteristic curve was used to assess the diagnostic value of miR-24-3p. In vitro AD model was established to evaluate the effect of miR-24-3p. The downstream target was detected by luciferase reporter gene assay. Results: Expression of miR-24-3p showed 1.6-fold increase in AD group compared with healthy controls, and a negative correlation of miR-24-3p with mini-mental state examination score was obtained. Receiver-operating characteristic curve showed satisfactory diagnostic accuracy. Downregulation of miR-24-3p promoted cell proliferation and inhibited cell apoptosis. KLF8 is a target gene of miR-24-3p. Conclusion: MiR-24-3p has a certain value in the diagnosis of AD and may be a potential biomarker.

To explore the genetic basis of three families with recurrence of non-immune hydrops fetalis (NIHF) but negative result by copy number variation sequencing (CNV-seq).Amniotic fluid sample and/or abortive tissues of the fetuses were collected and subjected to CNV-seq analysis. Peripheral blood samples of the parents were also taken for trio whole exome sequencing (trio WES).Fetus 1 was found to harbor heterozygous c.976GT(p.Glu326*) variant of the SOX18 gene in addition with compound heterozygous variants c.844CT(p.Arg282Trp) and c.9472+1GA of the RYR1 gene. The three variants were all inherited from its parents and have been associated with the etiology of NIHF. Based on the American College of Medical Genetics and Genomics (ACMG) standards and guidelines, the c.976GT variant of SOX18 gene and c.9472+1GA of RYR1 gene were predicted to be pathogenic (PVS1+PM2+PP3+PP4, PVS1+PM2+PP3), and c.844CT variant of RYR1 gene to be likely pathogenic (PM1+PM2+PP3). Fetus 2 was found to harbor compound heterozygous variants c.6682CT(p.Gln2228*) and c.4373_4383del(p.Val1458Alafs*63) of the PIEZO1 gene. Both variants were also inherited from its parents and are associated with the etiology of NIHF. Based on ACMG standards and guidelines, both c.6682CT and c.4373_4383del variants of PIEZO1 gene were predicted to be pathogenic (PVS1+PM2+PP4, PVS1+PM2). Fetus 3 was found to harbor compound heterozygous variants of the TTN gene c.29860GC(p.Asp9954His) and c.21107AT(p.Asp7036Val), which were respectively inherited from its parents. Both variants have been strongly associated with the phenotype, though the connection between the etiology of NIHF and variants of the TTN gene remains elusive. Based on ACMG standards and guidelines, the c.29860GC and c.21107AT variants of TTN gene were predicted to be likely pathogenic (PM1+PM2+PP3).Trio WES can improve the diagnosis rate of NIHF with a negative result by CNV-seq. Considering the urgency of prenatal diagnosis, CNV-seq and trio WES should be carried out at the same time for fetuses with NIHF.

Blood-brain barrier dysfunction is recognized as a precursor of Alzheimer's disease development. Endothelial cells as structural basis of blood-brain barrier were observed tight junction failure in amyloid-β(1-42)-stimulated environment. In this study, we found NOVA2, PPP2R3A were down-regulated while PART1, p-NFκB-p65 were up-regulated in amyloid-β(1-42)-incubated endothelial cells. Knockdown of either NOVA2 or PPP2R3A and overexpression of PART1 all increased blood-brain barrier permeability. Lower blood-brain barrier permeability was observed in overexpression of NOVA2 and PPP2R3A and knockdown of PART1 and NFκB-p65. Same tendencies were found in the tight junction-related proteins expressions. Furthermore, overexpression and knockdown of NOVA2 and PART1 had no effect on cell viability. Mechanistically, NOVA2 overexpression was confirmed to reduce half-life of PART1. PART1 could destabilize PPP2R3A messenger RNA (mRNA) by interacting with STAU1. In addition, p-NFκB-p65 functioning as transcription factor reduced the expression of tight junction-related proteins, which was prompted by low protein level of PPP2R3A. Our study highlights the crucial role of NOVA2/PART1/PPP2R3A/p-NFκB-p65 pathway in amyloid-β(1-42)-incubated endothelial cells to modulating blood-brain barrier permeability through STAU1-mediated messenger RNA degradation, implying a potential mechanism of lncRNA and protein interaction in pathogenesis of Alzheimer's disease.

The success of stem cells therapy to treat neurodegenerative diseases is currently restricted by the lack of suitable stem cells. Mesenchymal stem cells (MSCs) have demonstrated several advantages as seed-cells for the stem cells therapy. In particular, the low immunogenicity and multiple lineages differentiation capability enables the possibility of using MSCs to treat neurodegenerative diseases. However, a more potent neuronal differentiation capacity of MSCs is required during a success treatment against neurodegenerative diseases. Bioengineering using small molecules to boost the neuronal differentiation of MSCs has been proposed as a promising strategy. Herein, we developed a new series of (2-phenylthiazol-4-yl)urea derivatives and one of them, 18g were observed to successfully promote neuronal differentiation of MSCs after culturing MSCs with 18g for 4 days. In addition, neither significant cytotoxicity nor cell cycle altering were found after the incubation. Interestingly, the osteogenic differentiation potential of MSCs was not affected after 18g treatment. The present study provides a promising small molecule to boost the innate neuronal differentiation capacity of MSCs with no serious detrimental effects.

The strategy of using mesenchymal stem cells (MSCs) as a living carrier for active delivery of therapeutic agents targeting tumor sites has been attempted in a wide range of studies to validate the feasibility and efficacy for tumor treatment. This approach reveals powerful tumor targeting and tumor penetration. In addition, MSCs have been confirmed to actively participate in immunomodulation of the tumor microenvironment. Thus, MSCs are not inert delivery vehicles but have a strong impact on the fate of tumor cells. In this review, these active properties of MSCs are addressed to highlight the advantages and challenges of using MSCs for tumor-targeted delivery. In addition, some of the latest examples of using MSCs to carry a variety of anti-tumor agents for tumor-targeted therapy are summarized. Recent technologies to improve the performance and safety of this delivery strategy will be introduced. The advances, applications, and challenges summarized in this review will provide a general understanding of this promising strategy for actively delivering drugs to tumor tissues.

Pulmonary diseases are currently one of the major threats of human health, especially considering the recent COVID-19 pandemic. However, the current treatments are facing the challenges like insufficient local drug concentrations, the fast lung clearance and risks to induce unexpected inflammation. Cell-derived membrane biomimetic nanocarriers are recently emerged delivery strategy, showing advantages of long circulation time, excellent biocompatibility and immune escape ability. In this review, applications of using cell-derived membrane biomimetic nanocarriers from diverse cell sources for the targeted therapy of pulmonary disease were summarized. In addition, improvements of the cell-derived membrane biomimetic nanocarriers for augmented therapeutic ability against different kinds of pulmonary diseases were introduced. This review is expected to provide a general guideline for the potential applications of cell-derived membrane biomimetic nanocarriers to treat pulmonary diseases.

STUDY DESIGN A retrospective cross-sectional study. OBJECTIVE This study aims to determine whether the sacroiliac (SI) joint motion correlated to pelvic incidence (PI) change from standing to supine position in patients with degenerative spinal diseases. SUMMARY OF BACKGROUND DATA PI was found an unstable parameter after adolescence as the fixed nature of PI was challenged by several studies. The SI joint has been shown to have some motion, age-related degenerative changes of cartilage and SI ligaments contribute to SI joint instability. METHODS The study contains both specimen study and radiographic study. One human specimen was acquired, on which PI was measured with different sacrum-ilium positions. In radiographic study, patients with old thoracolumbar fracture, lumbar disc herniation, stenosis, and spondylolisthesis were included. Ankylosing spondylitis (AS) patients were also included as control group. PI was measured on standing x-rays and scanogram of computed tomography images in supine position. RESULTS Specimen study result revealed that SI motion would lead to the change of PI with fixed pelvic thickness. In radiographic study, 101 patients with different etiology and 30 AS patients were included. After stratifying into different age groups, standing PI was significantly larger than supine PI in each age groups (P = 0.002

BACKGROUND: DNA methylation plays a vital role in modulating genomic function and warrants evaluation as a biomarker for the diagnosis and treatment of lung squamous cell carcinoma (LUSC). OBJECTIVE: In this study, we aimed to identify effective potential biomarkers for predicting prognosis and drug sensitivity in LUSC. METHODS: A univariate Cox proportional hazards regression analysis, a random survival forests-variable hunting (RSFVH) algorithm, and a multivariate Cox regression analysis were adopted to analyze the methylation profile of patients with LUSC included in public databases: The Cancer Genome Atlas (TCGA), and the Gene Expression Omnibus (GEO). RESULTS: A methylated region consisting of 3 sites (cg06675147, cg07064331, cg20429172) was selected. Patients were divided into a high-risk group and a low-risk group in the training dataset. High-risk patients had shorter overall survival (OS) (hazard ratio [HR]: 2.72, 95% confidence interval [CI]: 1.82–4.07, P< 0.001) compared with low-risk patients. The accuracy of the prognostic signature was validated in the test and validation cohorts (TCGA, n= 94; GSE56044, n= 23). Gene set variation analysis (GSVA) showed that activity in the cell cycle/mitotic, ERBB, and ERK/MAPK pathways was higher in the high-risk compared with the low-risk group, which may lead to differences in OS.Interestingly, we observed that patients in the high-risk group were more sensitive to gemcitabine and docetaxel than the low-risk group, which is consistent with results of the GSVA. CONCLUSION: We report novel methylation sites that could be used as powerful tools for predicting risk factors for poorer survival in patients with LUSC.

With an incidence of 1/800 - 1/600, Down syndrome (DS) is the most common chromosomal disorder in humans. Whilst most DS patients has trisomy 21, a small proportion may carry translocations or mosaicisms involving chromosome 21. The main characteristics of DS include mental retardation, peculiar facies, growth retardation, congenital heart disease, duodenal stenosis, Alzheimer's disease, leukemia, and immunodeficiency, which may be due to increased dosage of critical genes. Recent studies also showed that epigenetic changes may also occur in DS. For research on patients with DS or other trisomies have been restricted by ethical considerations, and commonly used mouse models cannot fully replicate the characteristic features of DS, pluripotent stem cells induced from fetal samples or biopsy tissues from DS patients may generate models with the same genetic content, which may provide idea materials for studying the pathogenesis of DS and customized cell and/or gene therapies.

To validate the predictability of S-line in Lenke 5C patients. Lenke 5C patients with a minimum 2-year follow-up and with the lowest instrumented vertebra (LIV) at lower end vertebra were included. The S-line was defined as a line connecting the centers of concave-side pedicles of upper instrumented vertebra (UIV) and LIV on baseline films. The S-line tilt to right was defined as positive S-line status (S-line +) and tilt to left as S-line − status. Statistical analysis was performed between different subgroups. Among the 92 patients, 69 patients had a left lumbar curve and 23 patients had a right lumbar curve. For left curves, the S-line + status had a significantly higher incidence of both proximal and distal decompensation. However, for right curves, the S-line − status was the risk factor. Thus, we modified the definition of S-line: The value of S-line tilt for right curves was opposite to that for left curves. Patients with modified S-line + showed a significantly higher incidence of both proximal and distal decompensation in Lenke 5C patients with both left and right curves (p

Mesenchymal stem cells (MSCs) have the inherent tumor-homing ability with the attraction of multiple chemokines released by tumor tissues or tumor microenvironments, which can be utilized as promising cellular carriers for targeted delivery of anti-tumor drugs and genes. In most circumstances, large amount of systemicly administrated MSCs will be firstly trapped by lungs, following with re-distribution and homing to tumor tissues after lung clearance. Several approaches like enhanced interactions between chemokines and receptors on MSCs or reducing the retention of MSCs by changes of administration methods are firstly reviewed for improving the homing of MSCs towards tumor tissues. Additionally, the potentials and gains of utilizing MSCs to carry several chemotherapeutics, such as doxorubicin, paclitaxel and gemcitabine are summarized, showing the advantages of overcoming the short half-life and poor tumor targeting of these chemotherapeutics. Moreover, the applications of MSCs to protect and deliver therapeutic genes to tumor sites for selectively tumor cells eliminating or promoting immune system are highlighted. In addition, the potentials of using MSCs for tumor-targeting delivery of diagnostic and therapeutic agents are addressed. We believed that the continuous improvement and optimization of this stem cells-based cellular delivery system will provide a novel delivery strategy and option for tumor treatment.

Study design A prospective analysis. Objective To investigate whether the hyper-selective posterior fusion (upper instrumented vertebra [UIV] as the vertebra one level below the upper end vertebra [UEV], lower instrumented vertebra [LIV] as the lower end vertebra [LEV]) was applicable in posterior fusion of Lenke 5C adolescent idiopathic scoliosis (AIS) patients and what could be the indication of hyper-selective fusion. Summary of background data The improper UIV selection in selective fusion could lead to progressive thoracic compensatory curve, shoulder imbalance, and even coronal imbalance. However, few studies analyzed the clinical outcome of hyper-selective fusion. Methods A prospective analysis of 80 patients with Lenke 5C AIS who underwent selective fusion was performed. According to the relationship between UEV and UIV, the patients were divided into UEV group (UIV = UEV) and UEV-1 group (UIV = UEV-1). Radiographic parameters and the incidence of postoperative proximal decompensation were compared. The Scoliosis Research Society (SRS)-22 scores were used to evaluate clinical outcomes between two groups. Results Thirteen patients (27%) in UEV group and six (18.75%) in UEV-1 group showed proximal decompensation during follow-up, and the incidence was equivalent (P = 0.280). Within the UEV-1 group, the patients with proximal decompensation showed similar Risser grade, baseline thoracic Cobb angle, and main Cobb angle (P = 0.611, 0.435, 0.708, respectively). However, the baseline L-T apical vertebral translation (AVT) ratio was significantly larger in patients with proximal decompensation (P = 0.028). Meanwhile, patients with proximal decompensation in UEV group showed significantly smaller preoperative UIV translation and lumbar AVT but similar postoperative UIV tilt. Conclusion Hyper-selective posterior fusion strategy could be performed in Lenke 5C patients with Risser more than grade 2 and with thoracic compensatory curve over 15°. The UIV in patients with small baseline thoracic curve, represented by larger baseline lumbar-thoracic AVT ratio, should be selected as UEV to prevent proximal decompensation. Level of evidence 4.

Due to insufficient understanding of human-water interaction, many water-related problems arise in watersheds, posing severe threats to the sustainability of watershed development. Although water environmental carrying capacity (WECC) is a powerful tool to support sustainable development of watersheds, few studies considered aquatic ecological factors and uncertainty in indicator values, leading to losses of sample information in the evaluation of WECC. This paper developed a systematic framework for comprehensive WECC assessment that included the indicator system and a novel variable fuzzy pattern recognition (VFPR) approach. The WECC index system incorporated aquatic ecological factors, and addressed uncertainties associated with the indicator values. The proposed VFPR-based assessment model could realize successive evaluation to retain more original information of the sample and distinguish similar result values by treating the sample as having a continuous degree of membership instead of the traditional point form. In addition, it could be more adaptable to various circumstances including extreme cases, and closely reflect the impacts of indicator changes on the results. The established evaluation framework has been applied to Dongjiang River Basin in Guangdong Province. The spatial differences and main influencing factors of WECC in the study area were analyzed. Results show that 50% and 16.7% of the sub-regions in the study area would be subject to a poor level of WECC under pessimistic and optimistic circumstances, respectively. WECCs in the upper and lower reaches are the best and worst, respectively, which is in line with the levels of economic development in the Dongjiang River Basin. The proposed method can also be applicable to many other problems involving numerous indicators.

STUDY DESIGN A retrospective analysis. OBJECTIVE To evaluate whether the rotation of the presumed lower instrumented vertebra (LIV) on baseline convex side-bending (SB) radiographs leads to distal adding-on in Lenke 5C adolescent idiopathic scoliosis (AIS) patients. SUMMARY OF BACKGROUND DATA In Lenke 1A patients, derotation of the presumed LIV on baseline bending films is important owing to the association between LIV rotation and postoperative distal adding-on. However, the relationship between distal adding-on and derotation of the presumed LIV in Lenke 5C patients remains unknown. METHODS Eighty-five Lenke 5C patients with a minimum of 2-year follow-up for posterior fusion were enrolled. L3 was selected as the LIV in all patients. Patients were divided into the presumed LIV derotation (DR group) and nonderotation (NDR group) groups according to derotation of the presumed LIV on SB films. Radiographic parameters, including Cobb angle, coronal balance, lower disc angle of LIV, LIV tilt, and LIV translation, were measured pre- and postoperatively. RESULTS Distal adding-on occurred in 16 patients (18.8%) at the final follow-up: 2 patients (10%) in the DR group and 14 patients (21.5%) in the NDR group (P = 0.248). Distal adding-on incidence was not greatly reduced with derotation of the presumed LIV on SB films at baseline. In the NDR group, the immediately postoperative lower disc angle of the LIV, LIV tilt, and LIV translation were significantly smaller in patients without than in those with distal adding-on (all P

To analyze a family with recurrent fetal copy number variations (microdeletion and microduplication, respectively) of 1p31.1 using single nucleotide polymorphism-based array (SNP-array) and G banding chromosomal karyotyping.Amniocentesis and chorionic villus sampling were performed for a woman during the two pregnancies. Whole genome SNP-array was used to detect genomic imbalance of the fetus. The couple was also subjected to G-banding chromosomal analysis and SNP-array analysis.SNP-array showed a 1p31.1 (70 164 686-83 474 843) ×1 and a 1p31.1 (70 164 686-83 479 747) ×3 in the fetuses during the two pregnancies, respectively. SNP array results of the couple appeared to be normal. The mother of the fetuses had a 46,XX,inv(1)(p31.1p32.1) karyotype.The paracentric inversion in chromosome 1 in the gravida probably underlies the recurrent 1p31.1 copy number variations in the fetuses. SNP-array combined with G banding chromosomal analysis are suitable for prenatal diagnosis for recurrent microdeletion and microduplication in the same chromosomal region, and can provide detailed information for genetic counseling.

The genital pathogen Chlamydia is known to colonize the gastrointestinal tract. Orally delivered Chlamydia muridarum can reach the colon and maintain a long-lasting colonization there. However, C. muridarum with mutations in chromosomal genes tc0237 and tc0668 (designated a chromosomal mutant) or deficient in plasmid-encoded pGP3 (designated a plasmid mutant) is unable to do so. We now report that the chromosomal mutant is still able to reach the colon while the plasmid mutant fails to do so following an oral delivery, suggesting that lack of colon colonization by different mutants may involve distinct mechanisms. Consistently, a direct intracolonic delivery selectively restored the ability of the plasmid mutant, but not the chromosomal mutant, to colonize the colon. The chromosomal mutant was rescued only in the colon of mice deficient in gamma interferon (IFN-γ). Thus, the chromosomal mutant’s deficiency in colonizing colonic mucosal tissue is likely due to its increased susceptibility to IFN-γ-mediated immunity. Furthermore, IFN-γ deficiency was sufficient for rescuing colon colonization of an orally delivered chromosomal mutant but not plasmid mutant while mice deficient in gastric acid production rescued the plasmid mutant but not the chromosomal mutant. Both mutants are attenuated in inducing genital tract pathology. Thus, we propose that chlamydial chromosomal-gene-encoded genital tract virulence factors may be essential for Chlamydia to maintain long-lasting colonization in the colon while the plasmid may enable Chlamydia to reach the colon by promoting evasion of gastric barriers.

Chlamydia has been detected in the gastrointestinal tracts of both animals and humans. However, it remains unclear whether the chlamydial organisms can be introduced into the gastrointestinal tract via pathways independent of the oral and anal routes. We have recently shown that Chlamydia muridarum spreads from the genital tract to the gastrointestinal tract potentially via the circulatory system. To test whether hematogenous C. muridarum can spread to and establish a long-lasting colonization in the mouse gastrointestinal tract, we inoculated mice intravenously with a luciferase-expressing C. muridarum strain and monitored its distribution. After tail vein inoculation, most luciferase-generated bioluminescence signals were detected in the mouse abdominal area throughout the experiment. The ex vivo imaging revealed that the abdominal signals came from the gastrointestinal tract tissues. Simultaneous monitoring of chlamydial organisms in individual organs or tissues revealed an initial stage of systemic spreading followed by a long-lasting infection in the gastrointestinal tract. A retro-orbital vein inoculation of the C. muridarum organisms at a lower dose in a different mouse strain also led to colonization of the gastrointestinal tract. We have demonstrated that intravenous C. muridarum inoculation can result in colonization of the gastrointestinal tract, suggesting that the chlamydial organisms may use the sexual behavior-independent circulation pathway to infect the gastrointestinal tract.

Although Chlamydia trachomatis is a human genital tract pathogen, chlamydial organisms have frequently been detected in both vaginal and rectal swab samples of animals and humans. The plasmid-encoded pGP3, a genital tract virulence factor, is essential for Chlamydia muridarum to colonize the mouse gastrointestinal tract. However, intracolon inoculation to bypass the gastric barrier rescued the colonization ability of a pGP3-deficient C. muridarum mutant, suggesting that pGP3 is required for C. muridarum to reach but not to colonize the large intestine. The pGP3-deficient mutant was rapidly cleared in the stomach and was 100-fold more susceptible to gastric killing. In mice genetically deficient in gastrin, a key regulator for gastric acid production, or pharmacologically treated with a proton pump inhibitor, the ability of pGP3-deficient C. muridarum to colonize the gastrointestinal tract was rescued. The pGP3-dependent resistance was further recapitulated in vitro with treatments with HCl, pepsin, or sarkosyl. In the genital tract, deficiency in pGP3 significantly reduced C. muridarum survival in the mouse vagina and increased C. muridarum susceptibility to vaginal killing by ∼8 times. The pGP3-deficient C. muridarum was more susceptible to lactic acid killing, and the pGP3 deficiency also significantly increased C. trachomatis susceptibility to lactic acid. The above-described observations together suggest that Chlamydia may have acquired the plasmid-encoded pGP3 to overcome the gastric barrier during its adaptation to the gastrointestinal tract and the pGP3-dependent resistance may enable chlamydial evasion of the female lower genital tract barrier during sexual transmission.

The cryptic plasmid is essential for Chlamydia muridarum dissemination from the genital tract to the gastrointestinal (GI) tract. Following intravaginal inoculation, a C. muridarum strain deficient in plasmid-encoded pGP3 or pGP4 but not pGP5, pGP7, or pGP8 failed to spread to the mouse gastrointestinal tract, although mice infected with these strains developed productive genital tract infections. pGP3- or pGP4-deficient strains also failed to colonize the gastrointestinal tract when delivered intragastrically. pGP4 regulates pGP3, while pGP3 does not affect pGP4 expression, indicating that pGP3 is critical for C. muridarum colonization of the gastrointestinal tract. Mutants deficient in GlgA, a chromosome-encoded protein regulated by pGP4, also consistently colonized the mouse gastrointestinal tract. Interestingly, C. muridarum colonization of the gastrointestinal tract positively correlated with pathogenicity in the upper genital tract. pGP3-deficient C. muridarum strains did not induce hydrosalpinx or spread to the GI tract even when delivered to the oviduct by intrabursal inoculation. Thus, the current study not only has revealed that pGP3 is a novel chlamydial colonization factor in the gastrointestinal tract but also has laid a foundation for investigating the significance of gastrointestinal Chlamydia .

Despite the extensive in vitro characterization of CPAF ( c hlamydial p rotease/proteasome-like a ctivity f actor), its role in chlamydial infection and pathogenesis remains unclear. We now report that a Chlamydia trachomatis strain deficient in expression of CPAF (L2-17) is no longer able to establish a successful infection in the mouse lower genital tract following an intravaginal inoculation. The L2-17 organisms were cleared from the mouse lower genital tract within a few days, while a CPAF-sufficient C. trachomatis strain (L2-5) survived in the lower genital tract for more than 3 weeks. However, both the L2-17 and L2-5 organisms maintained robust infection courses that lasted up to 4 weeks when they were directly delivered into the mouse upper genital tract. The CPAF-dependent chlamydial survival in the lower genital tract was confirmed in multiple strains of mice. Thus, we have demonstrated a critical role of CPAF in promoting C. trachomatis survival in the mouse lower genital tracts. It will be interesting to further investigate the mechanisms of the CPAF-dependent chlamydial pathogenicity.

A high-density cell culture method was successfully established in P. pastoris with the alcohol oxidase I (AOXI) promoter in order to produce large quantities of recombinant human angiostatin (AS) which has been reported to have antiangiogenic activity. A preliminary study on fermentation conditions in shaking flasks indicated that adequacy of biomass is beneficial to obtain more products. The fermentation was carried out in a 10 l bioreactor with 5 l modified growth medium recommended by Invitrogen at 30 degrees C. The cells were first grown in glycerol-PTM4 trace salts for 24 h. When the cell density reached A(600) = 125, methanol-PTM4 trace salts was added to induce the expression of AS. During the fermentation, dissolved oxygen level was maintained at 20-30%, pH was controlled at 5 by the addition of 7 M NH(4)OH and the biomass was maintained at about A(600) = 200. After 60 h of induction, the secreted AS was 153 mg/l. The recombinant AS inhibited the angiogenesis on CAM and suppressed the growth of B16 melanoma in C57BL/6J mice (P \0.01).

A high-density cell culture method to produce human angiostatin has been successfully established by constitutive expression of the protein in Pichia pastoris. The fermentation was carried out in a 20 l bioreactor with a 10 l working volume, using a high-density cell culture method by continuously feeding with 50% glycerol-0.8% PTM4 to the growing culture for 60 h at 30 degrees C. Dissolved oxygen level was maintained at 25-30% and pH was controlled at 5 by the addition of 7 M NH4OH. Angiostatin was constitutively expressed during the fermentation by linking its expression to the P. pastoris constitutive GAP promoter (pGAP). But after 36 h of fermentation, the peak biomass growth was 305 as measured by absorption of 600 nm, while the peak angiostatin expression was 176 mg/l. Similar to the product expressed from inducible system [24], angiostatin produced from constitutive system also inhibited the angiogenesis on the CAM and suppressed the growth of B16 melanoma in C57BL/6J mouse. The above results suggest that GAP promoter is more efficient than AOX1 promoter for the expression of angiostatin in P. pastoris by shake flask culture or high-density cell fermentation and is likely to be an alternative to AOX1 promoter in large-scale expression of angiostatin and other heterologous proteins.

Endostatin (EDN), an endogenous angiogenesis inhibitor of 20 kDa, was originally isolated from the supernatant of culture murine hemangioendothelioma cell line. Interest in EDN arises from its therapeutic potential as anti-tumor and anti-angiogenesis agents. However, it is difficult to obtain sufficient quantities of native EDN from its natural resources. We report here the construction of a pMIRH-type vector pLRG29 by introducing the 25s rDNA of Hansenua wingei and the Km(R) gene into the YIp vector pSML12 and the EDN expression vector pLRG-EDN by cloning the human EDN cDNA into pLRG29. Human EDN was expressed in Hansenula polymorpha (H.p.) A16 (pLRG-EDN) as a secreted soluble protein. The yield of the secreted EDN was 65 mg/L in shake flask. The secreted EDN was purified to a purity of 98 % by the use of SP Sepharose FF ion-exchange chromatography and Sepharose-heparin Hi Trap affinity chromatography. The MTT and chicken embryo chorioallantoic membrane assay demonstrated that the human EDN produced from H. polymorpha inhibited in vitro the proliferation of human umbilical vein endothelial cells and in vivo the neovascularization induced by bFGF.

Fibroblast growth factor 21 (FGF21), a recently identified member of the FGF superfamily, is mainly secreted from the liver and adipose tissues and plays an important role in improving metabolic syndrome and homeostasis. The aim of this study is to evaluate the role of FGF21 in alcoholic fatty liver disease (AFLD) and to determine if it has a therapeutic effect on AFLD. In this paper, we tested the effect of FGF21 on alcohol-induced liver injury in a murine model of chronic ethanol gavage and alcohol-treated HepG2 cells. Male KM mice received single dose of 5 g/kg ethanol gavage every day for 6 weeks, which induced significant fatty liver and liver injury. The alcohol-induced fatty liver cell model was achieved by adding ethanol into the medium of HepG2 cell cultures at a final concentration of 75 mM for 9 days. Results showed that treatment with recombinant FGF21 ameliorated alcoholic fatty liver and liver injury both in a murine model of chronic ethanol gavage and alcohol-treated HepG2 cells. In addition, FGF21 treatment down-regulated the hepatic expression of fatty acid synthetic key enzyme, activated hepatic AMPK-SIRT1 pathway and significantly down-regulated hepatic oxidative stress protein. Taken together, FGF21 corrects multiple metabolic parameters of AFLD in vitro and in vivo by activation of the AMPK-SIRT1 pathway.

Canstatin-N DNA fragment amplified from human genome was inserted into the MCS of pGAP9K*, an intracellular expression vector of Pichia pastoris, to generate pGAP9K*-can-N which was then transformed into P. pastoris GS115 by electroporation. A transformant was chosen as an engineering strain from the plate containing G418 (700 μg/ml). D-sorbitol was selected as the only carbon source. The fermentation was carried out in a 50 L bioreactor at a 20 L working volume. After 48 h fermentation with continuous feeding of 25% (w/v) D-sorbitol and 0.8% PTM4, the cell grew to A(600)=178 and intracellularly expressed Canstatin-N reached 780 mg/L. Snail enzyme was combined with water to crack P. pastoris and to release intracellular proteins. The purified recombinant Canstatin-N inhibited CAM angiogenesis and induced significant apoptosis of the human umbilical vein endothelial cell (EVC340).

A structurally novel acetyl-CoA carboxylase (ACC) inhibitor is identified from high-throughput screening. A preliminary structure-activity relationship study led to the discovery of potent dual ACC1/ACC2 and ACC2 selective inhibitors against human recombinant ACC1 and ACC2. Selective ACC2 inhibitors exhibited IC5020 nM and1000-fold selectivity against ACC1. (S)-Enantiomer 9p exhibited high ACC2 activity and lowered muscle malonyl-CoA dose-dependently in acute rodent studies, whereas (R)-enantiomer 9o was weak and had no effect on the malonyl-CoA level.

Total RNA was extracted from placenta umbilical tissue and the canstatin cDNA was amplified from total RNA by net-RT-PCR technique. The amplified cDNA was cloned into pSP72 and sequenced. The canstatin cDNA was cut down from pSP72C with BamH I / Nde I and ligated into the vector pET-3c. The resultant plasmid pETC was then transformed into E. coli BL21 (DE3). The canstatin gene was efficiently expressed after IPTG induction as a 25 kD band on SDS-PAGE. The expressed product constituted approximately 27.9% of the total bacterial proteins estimated by densitometry and existed mainly as inclusion body. The inclusion bodies were washed, lysed, refolded and purified on the Sephadex G-75 gel filtration column to a purity of 91.4%. CAM assay showed that 10 microg purified canstatin is enough to inhibit the angiogenesis of chichen embryo microcapillary vessel.