Clathrin Light Chains negatively regulate plant immunity by hijacking the autophagy pathway.

The crosstalk between clathrin-mediated endocytosis (CME) and autophagy pathway has been reported in mammals. However, the interconnection of CME with autophagy has not been established in plants. In this report, we showed that Arabidopsis CLATHRIN LIGHT CHAIN (CLC) subunit 2 and 3 double mutant, clc2-1 clc3-1, phenocopied the Arabidopsis AUTOPHAGY-RELATED GENE (ATG) mutants both in auto-immunity and nutrient sensitivity. Accordingly, the autophagy pathway was significantly compromised in the clc2-1 clc3-1 mutant. Interestingly, we demonstrated with multiple assays that CLC2 directly interacted with ATG8h/ATG8i in a domain-specific manner. As expected, both GFP-ATG8h/GFP-ATG8i and CLC2-GFP were subjected to autophagic degradation and the degradation of GFP-ATG8h was significantly reduced in the clc2-1 clc3-1 mutant. Notably, simultaneously knocking out ATG8h and ATG8i by the CRISPR/CAS9 resulted in an enhanced resistance against Golovinomyces cichoracearum, supporting the functional relevance of the CLC2-ATG8h/8i interactions. In conclusion, our results uncovered a link between the function of CLCs and the autophagy pathway in Arabidopsis.

Partially knocking out NtPDK1a/1b/1c/1d simultaneously in Nicotiana tabacum using CRISPR/CAS9 technology results in auxin-related developmental defects.

The eukaryotic AGC protein kinase subfamily (protein kinase A/ protein kinase G/ protein kinase C-family) is involved in regulating numerous biological processes across kingdoms, including growth and development, and apoptosis. PDK1(3-phosphoinositide-dependent protein kinase 1) is a conserved serine/threonine kinase in eukaryotes, which is both a member of AGC kinase and a major regulator of many other downstream AGC protein kinase family members. Although extensively investigated in model plant Arabidopsis, detailed reports for tobacco PDK1s have been limited. To better understand the functions of PDK1s in tobacco, CRISPR/CAS9 transgenic lines were generated in tetraploid N. tabacum, cv. Samsun (NN) with 5-7 of the 8 copies of 4 homologous PDK1 genes in tobacco genome (NtPDK1a/1b/1c/1d homologs) simultaneously knocked out. Numerous developmental defects were observed in these NtPDK1a/1b/1c/1d CRISPR/CAS9 lines, including cotyledon fusion leaf shrinkage, uneven distribution of leaf veins, convex veins, root growth retardation, and reduced fertility, all of which reminiscence of impaired polar auxin transport. The severity of these defects was correlated with the number of knocked out alleles of NtPDK1a/1b/1c/1d. Consistent with the observation in Arabidopsis, it was found that the polar auxin transport, and not auxin biosynthesis, was significantly compromised in these knockout lines compared with the wild type tobacco plants. The fact that no homozygous plant with all 8 NtPDK1a/1b/1c/1d alleles being knocked out suggested that knocking out 8 alleles of NtPDK1a/1b/1c/1d could be lethal. In conclusion, our results indicated that NtPDK1s are versatile AGC kinases that participate in regulation of tobacco growth and development via modulating polar auxin transport. Our results also indicated that CRISPR/CAS9 technology is a powerful tool in resolving gene redundancy in polyploidy plants.

Silencing GmATG7 Leads to Accelerated Senescence and Enhanced Disease Resistance in Soybean.

Autophagy plays a critical role in nutrient recycling/re-utilizing under nutrient deprivation conditions. However, the role of autophagy in soybeans has not been intensively investigated. In this study, the Autophay-related gene 7 (ATG7) gene in soybeans (referred to as GmATG7) was silenced using a virus-induced gene silencing approach mediated by Bean pod mottle virus (BPMV). Our results showed that ATG8 proteins were highly accumulated in the dark-treated leaves of the GmATG7-silenced plants relative to the vector control leaves (BPMV-0), which is indicative of an impaired autophagy pathway. Consistent with the impaired autophagy, the dark-treated GmATG7-silenced leaves displayed an accelerated senescence phenotype, which was not seen on the dark-treated BPMV-0 leaves. In addition, the accumulation levels of both H2O2 and salicylic acid (SA) were significantly induced in the GmATG7-silenced plants compared with the BPMV-0 plants, indicating an activated immunity. Consistently, the GmATG7-silenced plants were more resistant against both Pseudomonas syringae pv. glycinea (Psg) and Soybean mosaic virus (SMV) compared with the BPMV-0 plants. However, the activated immunity in the GmATG7-silenced plant was not dependent upon the activation of MPK3/MPK6. Collectively, our results demonstrated that the function of GmATG7 is indispensable for autophagy in soybeans, and the activated immunity in the GmATG7-silenced plant is a result of impaired autophagy.

[Retracted] Isopsoralen­mediated suppression of bone marrow adiposity and attenuation of the adipogenic commitment of bone marrow­derived mesenchymal stem cells.

Following the publication of the above article, an interested reader drew to the authors' attention that Figs. 1C and 2 in the paper appeared to contain instances of duplicated data. The authors were able to consult their original data files, and realized that these figures had indeed been assembled incorrectly. Moreover, they identified further errors with a number of the other figures in their published formats (specifically, Figs. 3, 4, 6 and 7), and requested that a corrigendum be published to take account of all the errors that were made during the compilation of these figures. The Editor of International Journal of Molecular Medicine has considered the authors' request to publish a corrigendum, but has declined this request on account of the large number of errors that have been identified, and subsequently determined that this article should be retracted from the Journal on the basis of an overall lack of confidence in the presented data. Upon receiving this decision from the Editor, the authors were in agreement that the article should be retracted. The Editor apologizes to the readership of the Journal for any inconvenience caused. [International Journal of Molecular Medicine 39: 527­538, 2017; DOI: 10.3892/ijmm.2017.2880].

Insights into the ecological roles and evolution of methyl-coenzyme M reductase-containing hot spring Archaea.

Several recent studies have shown the presence of genes for the key enzyme associated with archaeal methane/alkane metabolism, methyl-coenzyme M reductase (Mcr), in metagenome-assembled genomes (MAGs) divergent to existing archaeal lineages. Here, we study the mcr-containing archaeal MAGs from several hot springs, which reveal further expansion in the diversity of archaeal organisms performing methane/alkane metabolism. Significantly, an MAG basal to organisms from the phylum Thaumarchaeota that contains mcr genes, but not those for ammonia oxidation or aerobic metabolism, is identified. Together, our phylogenetic analyses and ancestral state reconstructions suggest a mostly vertical evolution of mcrABG genes among methanogens and methanotrophs, along with frequent horizontal gene transfer of mcr genes between alkanotrophs. Analysis of all mcr-containing archaeal MAGs/genomes suggests a hydrothermal origin for these microorganisms based on optimal growth temperature predictions. These results also suggest methane/alkane oxidation or methanogenesis at high temperature likely existed in a common archaeal ancestor.

Disseminated cysticercosis in China with complex and variable clinical manifestations: a case series.

BACKGROUND: Cysticercosis is an emerging and neglected tropical disease (NTD) that poses a serious public health concern worldwide. Disseminated cysticercosis (DCC) is an uncommon manifestation of cysticercosis, also found in China. CASE PRESENTATION: We report three cases of DCC in patients living in China, with different clinical and radiological presentations. All three patients had DCC with active ocular cysticercosis, including one patient with widespread DCC caused by direct ingestion of Taenia solium eggs. The intravitreal cysticercus cyst in this patient was completely extracted entirely by 23-gauge pars plana vitrectomy, and the cyst was oval in shape on the flat mount preparation. CONCLUSION: The clinical presentation of DCC is highly sophisticated. The diagnosis depended on the typical radiological presentations, biopsy and flat mount preparations of the cyst.

Identification of potential whole blood MicroRNA biomarkers for the blood stage of adult imported falciparum malaria through integrated mRNA and miRNA expression profiling.

Correlating aberrant miRNA levels with imported malaria during the blood stage is required to better understand the in vivo biological and molecular processes that are involved in the response to Plasmodium falciparum infection and to find new biomarkers and diagnosis tools. We used a parallel microarray-based approach to investigate an integrated view of how the host miRNA expression profile changes in response to P. falciparum infection in whole blood obtained from six subjects with adult imported falciparum malaria (AIFM) and six normal subjects. We first investigated the functions of 1007 significantly differentially expressed genes from microarrays and predicted 56 putative pathways participating in malaria pathogenesis, which reflected systemic changes in the host under falciparum infection. Then, we validated the in silico-predicted targets of 50 differentially modulated miRNAs (7 upregulated and 43 downregulated) by examining the actual mRNA expression of these particular genes in our gene expression profile database; putative target gene sets were grouped into functional categories to investigate the roles of differentially expressed miRNAs. We considered 36 intersecting putative pathways, most of which were involved in multiple immune response processes, such as cell defense response, immune response, TNF signaling pathway, and T cell receptor signaling pathway, which may actually be regulated by differentially expressed miRNAs. Additionally, we identified five enriched upregulated miRNA sets (miR-3135b, miR-6780b-5p, miR-1246, miR-6126, and miR-3613-5p) as potential blood biomarkers of immunopathological status and prediction of early host responses, disease prognosis, and severe outcomes in AIFM.

Isopsoralen-mediated suppression of bone marrow adiposity and attenuation of the adipogenic commitment of bone marrow-derived mesenchymal stem cells.

Osteoporosis (OP) increases the risk of bone fractures and other complications, and is thus a major clinical problem. In this study, we examined the effect of isopsoralen on the differentiation of bone-derived marrow mesenchymal stem cells (BMSCs) into osteoblasts and adipocytes, as well as bone formation under osteoporotic conditions. Primary femoral BMSCs isolated from C57BL/6 mice were used to evaluate the isopsoralen-mediated regulation of the expression of alkaline phosphatase (ALP), osteocalcin (OCN) and runt-related transcription factor 2 (RUNX2) during osteogenesis 2 weeks. We also examined the expression of peroxisome proliferator-activated receptor Î³ (PPARγ) and CCAAT/enhancer binding protein ß (C/EBPß) under adipogenic conditions for 1 and 2 weeks. In addition, ovariectomized (OVX) mice were used to examine the effects of isopsoralen on bone formation for 2 months. Finally, mammalian target of rapamycin complex 1 (mTORC1) signaling was examined under osteogenic and adipogenic conditions. We found that following treatment with isopsoralen, the expression levels of ALP, OCN and RUNX2 were upregulated, whereas those of PPARγ and C/EBPß were downregulated. mTORC1 signaling was also inhibited in vitro and in vivo. In the OVX mice that were intragastrically administered isopsoralen, bone parameters (trabecular thickness, bone volume/total volume and trabecular number) in the distal femoral metaphysis were significantly increased and the adipocyte number was decreased. On the whole, our findings demonstrate that isopsoralen promoted BMSC differentiation into osteoblasts and suppressed differentiation into adipocytes.

Tenuigenin promotes the osteogenic differentiation of bone mesenchymal stem cells in vitro and in vivo.

Osteoporosis, which is a systemic skeletal disease characterized by low bone mineral density and microarchitectural deterioration of bone quality, is a global and increasing public health problem. Recent studies have suggested that Tenuigenin (TEN), a class of native compounds with numerous biological activities such as anti-resorptive properties, exerts protective effects against postmenopausal bone loss. The present study aims to investigate the osteogenic effects of TEN on bone mesenchymal stem cells (BMSCs) in vitro and in vivo. Alkaline phosphatase (ALP) activity/staining, Alizarin red staining and the expression of osteogenic markers, including runt-related transcription factor 2, osterix, osteocalcin, collagen Iα1, ß-catenin and glycogen synthase kinase-3ß were investigated in primary femoral BMSCs from C57/BL6 mice cultured under osteogenic conditions for 2 weeks to examine the effects of TEN. An ovariectomized (OVX) mouse model was used to investigate the effect of TEN treatment for 3 months in vivo. We found that ALP activity, mineralized nodules and the expression of osteogenic markers were increased and WNT/ß-catenin signaling was enhanced in vitro and in vivo. Bone parameters, including trabecular thickness, trabecular number and bone mineral density were higher in the OVX+TEN group than in control OVX mice. Our results suggest the therapeutic potential of TEN for the treatment of patients with postmenopausal osteoporosis.

Regulation of bone formation by baicalein via the mTORC1 pathway.

Osteoporosis is a systemic skeletal disease that is characterized by low bone density and microarchitectural deterioration of bone tissue. The increasing prevalence of osteoporosis has attracted much attention. In this study, MC3T3-E1 pre-osteoblasts were treated with the natural compound, baicalein (0.1 µmol/L, 1 µmol/L, 10 µmol/L), to stimulate differentiation over a 14-day period. In addition, a canonical ovariectomized (OVX) mouse model was used to investigate the effect of 3-month baicalein treatment (10 mg/kg per day) in preventing postmenopausal osteoporosis. In vitro, we found that baicalein induced activation of alkaline phosphatase, stimulated the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway, and induced expression of osteoblast differentiation markers, ie, osteocalcin, osterix, collagen Iα1, and runt-related transcription factor 2 (RUNX2), in osteoblasts. In vivo, several bone parameters, including trabecular thickness, trabecular bone mineral density, and trabecular number, in the distal femoral metaphysis were significantly increased in OVX mice treated intragastrically with baicalein for 3 months compared with OVX mice that were not treated with baicalein. We also found that expression of osteocalcin and RUNX2 was decreased in primary ossified tissue from the OVX group, and baicalein increased the levels of osteocalcin and RUNX2 in OVX mice. These data suggest that baicalein can stimulate MC3T3-E1 cells to differentiate into osteoblasts via activation of the mTORC1 signaling pathway, which includes protein kinases and transcription factors such as P-4E/BP1 and P-S6K1.

[Interaction between Toxoplasma GRA7 dense granule protein and the protein from host macrophages].

OBJECTIVE: To identify the protein from host macrophages which interacted with GRA7 dense granule protein of Toxoplasma gondii, and reveal the relationship between protein interaction and infection process. METHODS: The recombinant GRA7 protein with N-terminal GST tag were used as a bait in in vitro GST Pull-down experiment, the proteins of THP-1 monocytic macrophage cell line were captured and identified by LC-MS/MS proteomics method. The in vivo protein interaction was verified by Co-IP experiment The overexpression of the target host protein by pcDNA3.1 (+) vector in THP-1 macrophage was further used to analyze the relationship between protein interaction and infection process. RESULTS: The captured THP-1 cell protein was about Mr 29000, which was identified as human carbonic anhydrase 1 (hCA1). The significant in vivo protein-protein interaction between GRA7 and hCA1 was verified by Co-IP assay. The overexpression of hCA1 gene in THP-1 macrophage induced a higher propagation speed of Tgondii and the formation of the parasitophorous vacuole, but did nmt influence the number of the parasite. CONCLUSION: There is a significant protein interaction between Toxoplasma GRA7 dense granule protein and hCA1 enzyme from host macrophages, which is positively related with the propagation speed of T. gondii.

Enhancement of the synthesis of n-3 PUFAs in fat-1 transgenic mice inhibits mTORC1 signalling and delays surgically induced osteoarthritis in comparison with wild-type mice.

BACKGROUND: An exogenous supplement of n-3 polyunsaturated fatty acids (PUFAs) has been reported to prevent osteoarthritis (OA) through undefined mechanisms. OBJECTIVE: This study investigated the effect of alterations in the composition of endogenous PUFAs on OA, and associations of PUFAs with mammalian target of rapamycin complex 1 (mTORC1) signalling, a critical autophagy pathway in fat-1 transgenic (TG) mice. METHODS: fat-1 TG and wild-type mice were used to create an OA model by resecting the medial meniscus. The composition of the endogenous PUFAs in mouse tissues was analysed by gas chromatography, and the incidence of OA was evaluated by micro-computed tomography (micro-CT), scanning electron microscopy and histological methods. Additionally, primary chondrocytes were isolated and cultured. The effect of exogenous and endogenous PUFAs on mTORC1 activity and autophagy in chondrocytes was assessed. RESULTS: The composition of endogenous PUFAs of TG mice was optimised both by increased n-3 PUFAs and decreased n-6 PUFAs, which significantly alleviated the articular cartilage destruction and osteophytosis in the OA model (p<0.01), decreased protein expression of matrix metalloproteinase-13 (MMP-13) and ADAMTS-5 (a disintegrin and metalloproteinase with thrombospondin motifs) in the articular cartilage (p<0.01) and reduced chondrocyte number and loss of cartilage extracellular matrix. Both exogenous and endogenous n-3 PUFAs downregulated mTORC1 activity and promoted autophagy in articular chondrocytes. Conversely, mTORC1 pathway activation suppressed autophagy in articular chondrocytes. CONCLUSIONS: Enhancement of the synthesis of endogenous n-3 PUFAs from n-6 PUFAs can delay the incidence of OA, probably through inhibition of mTORC1, promotion of autophagy and cell survival in cartilage chondrocytes. Future investigation into the role of the endogenous n-6/n-3 PUFAs composition in OA prevention and treatment is warranted.

Endogenous n-3 polyunsaturated fatty acids (PUFAs) mitigate ovariectomy-induced bone loss by attenuating bone marrow adipogenesis in FAT1 transgenic mice.

AIM: To investigate the effect of endogenous n-3 polyunsaturated fatty acids (PUFAs) on bone marrow adipogenesis under osteoporosis conditions. METHODS: A mouse osteoporosis model overexpressing the FAT1 gene from Caenorhabditis elegans and converting n-6 PUFAs to n-3 PUFAs endogenously was used. RESULTS: The mice presented significantly lower bone marrow adiposity (adipocyte volume/tissue volume, mean adipocyte number) but increased the bone parameters (bone mineral density, bone mineral content, bone volume/total volume) in the distal femoral metaphysis. CONCLUSION: Endogenous n-3 PUFAs protect bone marrow adipogenesis, which provides a novel drug target.

[Separation and identification of the surface and secreted proteins in Toxoplasma gondii by biotin labeling and proteomics methods].

OBJECTIVE: To separate and identify the surface proteins and secreted proteins of Toxoplasma gondii tachyzoites of RH strain. METHODS: T. gondii tachyzoites were cultured in Vero cells, and purified by filtration and Percoll cell separation solution. The biotin-labeled tachyzoites were lysed, and the surface and secreted proteins were separated by NeutrAvidin agarose beads. After condensation and SDS-PAGE, the protein were collected, digested and identified by LC/MS-MS. RESULTS: A total of 785 T. gondii proteins were identified, 81 (10.3%) of which were originally annotated as the surface or secreted proteins. Among the highly-expressed (PSM>10) 65 proteins, 43 (66%) were originally annotated as surface or secreted proteins, while the others were predicted unknown proteins. CONCLUSION: The surface and secreted proteins of T. gondii are separated by biotin labeling and avidin chromatography, among which some potential new surface or secreted proteins of T. gondii are identified.

Establishing a disc degeneration model using computed tomography-guided percutaneous puncture technique in the rabbit.

BACKGROUND: Various animal models have been developed to investigate the complex mechanisms leading to intervertebral disc disorders and to evaluate the different therapeutic options. The needle puncture technique is commonly used to induce intervertebral degeneration in animal models. The present study aimed to establish a rabbit model of intervertebral disc degeneration using a simple, minimally invasive procedure. METHODS AND MATERIALS: The animal model was created in the rabbit using computed tomography-guided percutaneous puncture technology. An 18-gauge needle was used to induce a disc injury with a 5-mm puncture depth. Radiographic, histologic, and biochemical analyses and magnetic resonance imaging were performed to assess the consequent disc degeneration. RESULTS: Significant disc space narrowing was observed as early as 4 wk, and osteophytes were formed at 12 wk after puncture. The magnetic resonance imaging assessment demonstrated a progressive loss of T2-weighted signal intensity at the stabbed discs throughout the 12-wk period. The histologic analysis showed a progressive loss of the normal architecture from 4 wk to the end point. The biochemical assays suggested that the expression of proteoglycan decreased progressively with increasing time. CONCLUSIONS: A simple, but minimally invasive, intervertebral disc degeneration model was established successfully using computed tomography-guided percutaneous puncture technology in the rabbit. The puncture procedure can be performed with minimal damage and handling of the other structures, ensuring a uniform reproducible disc degeneration model.

Could heterotopic ossification be prevented by varying dietary n-3/n-6 polyunsaturated fatty acid ratio: a novel perspective to its treatment?

Heterotopic ossification (HO) is a common complication following with musculoskeletal trauma and surgical procedures. It usually decreases joint mobility and eventually causes loss of joint function. Despite nonsteroidal anti-inflammatory drugs (NSAIDs), the inhibitor of cyclooxygenase(COX), have been proven to prevent HO effectively via prostaglandin E2 synthesis regulation and modulation of tissue responsiveness to pro-inflammatory signaling, HO prevention is still a matter of debate for clinicians to avoid the side effect of NSAIDs. Interestingly, it is suggested that PGE2 production and pro-inflammatory microenvironment in body could be modified by varying the ratio of the precursor fatty acids in the diet. On account of the effect of dietary (n-6)/(n-3) PUFAs ratio on both COX metabolism and pro-inflammatory cytokines mediated biological responsiveness, we hypothesized lowering dietary (n-6)/(n-3) PUFAs ratio may not only directly reduce the substrate of COX-2 and COX-2 activity, but also partially ameliorate tissue inflammatory responsiveness to cytokines correlated with HO development,exerting an inhibitory effect on PGE2 synthesis to prevent HO formation. The negative role of lowering dietary (n-6)/(n-3) PUFAs ratio on angiogenesis, cytokines-induced apoptosis, inflammatory responsiveness and osteogenesis could also contribute to its action on HO development. If our hypothesis is proved to be corrected, it could be an innovative method to treat HO.

Risedronate inhibits bone marrow mesenchymal stem cell adipogenesis and switches RANKL/OPG ratio to impair osteoclast differentiation.

BACKGROUND: Osteoporosis is accompanied by an increase in bone marrow adipose tissue. Bone marrow adipogenesis has emerged as a therapeutic target for prevention of bone loss. Amino-bisphosphonates have been widely used for treatment of osteoporosis, but the mechanism through which amino-bisphosphonates inhibit osteoporosis remains unclear. The purpose of this study is to investigate the effects of bisphosphonates on bone marrow adipogenesis and the pro-osteoclastic factors produced by adipocytes in bone marrow microenvironment. MATERIALS AND METHODS: Human mesenchymal stem cells were obtained and purified from six volunteer donors. Each sample of cells was treated by increasing concentrations of risedronate with or without adipogenic induction for 14 d, and then droplets of the differentiated adipocytes were analyzed. The level of receptor activator of nuclear factor-κB ligand and osteoprotegerin, as well as pro-osteoclastic inflammatory factors interleukin-1, interleukin-6, and tumor necrosis factor α produced by adipocytes were evaluated by Western blot and ELISA assay. Moreover, the effect of risedronate on the activity of mammalian target of rapamycin complex 1, a key Ser/Thr kinase for initiation of adipocyte differentiation, was investigated. RESULTS: Risedronate not only dose-dependently inhibited the bone marrow adipogenesis from human mesenchymal stem cells but also suppressed receptor activator of nuclear factor-κB ligand, not osteoprotegerin, expression in differentiated adipocytes, as well as pro-osteoclastic inflammatory factors. Furthermore, the activity of mammalian target of rapamycin complex 1 was suppressed by risedronate. CONCLUSION: Our findings that risedronate influences the crosstalk between bone marrow adipocyte-osteoclast represent a novel mechanism for the anti-osteoporotic effects of risedronate.

Intra-articular lentivirus-mediated insertion of the fat-1 gene ameliorates osteoarthritis.

Osteoarthritis (OA) is a gradually progressive degenerative disease characterized by gradual inflammatory loss of articular cartilage caused by increased proteolytic catabolism, mediated by interleukin-1 (IL-1), tumor necrosis factor α (TNF-α), matrix metalloproteinase (MMPs), aggrecanases and other proteinases, and reduced anabolism of cartilage components, contributed by interleukin-4 (IL-4), interleukin-10 (IL-10), insulin-like growth factor 1 (IGF-1), transforming growth factor ß (TGF-ß), and bone morphogenetic proteins (BMPs). Substantial studies showed n-3 polyunsaturated fatty acids (n-3 PUFAs) exhibit a powerful anti-inflammatory effects in and ex vivo through reducing the production of IL-1 and TNF-α and increasing the expression of IL-4, IL-10, TGF-ß and IGF-1 in OA. Meanwhile, more convincing results are observed in the fat-1 transgenic mice, which are exogenously inserted in a fat-1 gene from Caenorhabditis elegans, which can endogenously convert n-6 polyunsaturated fatty acids (n-6 PUFAs) to n-3 PUFAs. Taken together, it has long been realized that dietary supplementation with fish oils that are plentiful of n-3 PUFAs can bring benefits in the treatment of osteoarthritis. Previously two phase I human studies based on in vitro transfer of the cDNA via lentivirus to arthritic joints have confirmed its feasibility and safety in human subjects. Consequently, we hypothesis that directly infect the chondrocytes and synoviocytes with lentivirus carrying the fat-1 gene could be a well therapeutic strategy for OA in humans.

[An epidemiologic study on Pneumocystis pneumonia in non-HIV infected patients in China].

OBJECTIVE: To analyze the epidemiologic features of Pneumocystis pneumonia (PCP) among non-HIV infected patients in China. METHODS: Sputum or bronchoalveolar lavage fluid (BALF) specimens obtained from 851 pneumonia patients without HIV infection from Jan. 2006 to Oct. 2008 were detected, using PCR and Gomori' s methenamine silver (UMS) stain for Pneumocystis jirovecii. RESULTS: Of the 615 sputum specimens, P. jirovecii positive rates of PCR and GMS stain were 20.3% and 10.2% respectively (P < 0.05). Of 236 BALF specimens, P. jirovecii positive rates of PCR and GMS stain were 32.6% and 25.5% respectively (P > 0.05). Of the total 851 pneumonia cases, 123 (14.5%) were GMS positive for P. jirovecii cyst and 202 cases (23.7%) were PCR positive for P. jirovecii DNA. In those immuno-suppressed patient group including patients with connective tissue diseases, organ transplant recipients, nephrotic, hematologic diseases and malignant tumor, P. jirovecii positive rate appeared the highest, 28.2% for GMS stain and 39.4% for PCR. There were also PCP patients in the immuno-competent pneumonia patient groups including senile patients with chronic diseases and patients without clear predisposing immuno-deficiencies. The positive rates of P. jirovecii GMS were 8.7% and 10.9%, respectively and 17.5% and 19.6% for P. jirovecii under PCR. CONCLUSION: PCR assay seemed sensitive for the detection of P. jirovecii in the sputum specimens and could be used for screening PCP patients without HIV infection. Our data showed that there was high risk of PCP in non-HIV infected patients in China.