Sterile Tissue Culture Research Articles

12231 PAPP-A As A Potential Target In Thyroid Eye Disease

Abstract Disclosure: C.A. Conover: None. L.K. Bale: None. D. Hamadi: None. M. Stan: ; Tourmaline, Genentech, Third Rock Ventures, ArgenX, Septerna, OrthoDi. ; Horizon, Immunovant, Lassen, Sling. Background: Proptosis in Thyroid Eye Disease (TED) can result in facial disfigurement and visual dysfunction. Studies revealed an interaction between thyroid-stimulating hormone receptor (TSHR) and insulin-like growth factor-I receptor (IGF-IR) that is essential in promoting TED pathogenesis. Treatment with Insulin-like growth factor I receptor (IGF-IR) inhibitors has been shown to be effective in reducing proptosis but with side effects. PAPP-A is metalloprotease that can increase pericellular IGF bioavailability through specific cleavage of inhibitory IGF binding proteins, in particular IGFBP-4. We propose that inhibition of IGF-IR indirectly and more selectively with PAPP-A inhibitory antibodies attenuates IGF-IR signaling in TED with fewer side effects. Methods: Informed consent was obtained from TED patients undergoing surgery in Mayo Clinic operating suites in Rochester, MN. Retro-orbital tissue was collected from 19 TED patients and 2 control subjects for fibroblast isolation and culture and was performed in a sterile tissue culture facility. TED and control fibroblasts were treated with pro-inflammatory cytokines, and flow separation of CD34- and CD34+ orbital fibroblasts was done, the latter representing infiltrating fibrocytes into the orbit in TED. Main outcome measured were PAPP-A expression and proteolytic activity, IGF-I stimulation of phosphatidylinositol 3 kinase/Akt pathway and inhibition by immuno-neutralizing antibodies against PAPP-A, CD34+ status and associated PAPP-A and IGF-IR expression. Results: Primary cultured cells showed that PAPP-A is expressed in orbital fibroblasts from TED patients but not in control subject fibroblasts and were markedly increased by pro-inflammatory cytokines stimulation. IGF-IR expression was not affected by cytokine treatment. Inhibition of PAPP-A’s proteolytic activity suppressed IGF-IR activation in orbital fibroblasts from TED patients. CD34+ orbital fibroblasts represent 80% of cells in culture from TED patients, and approximately 70% of these cells were accountable for PAPP-A and IGF-IR expression. Conclusion: These results support a role for PAPP-A in TED pathogenesis and indicate the potential for novel therapeutic targeting of the IGF axis. Presentation: 6/2/2024

PAPP-A as a Potential Target in Thyroid Eye Disease.

Proptosis in Thyroid Eye Disease (TED) can result in facial disfigurement and visual dysfunction. Treatment with Insulin-like growth factor I receptor (IGF-IR) inhibitors has been shown to be effective in reducing proptosis but with side effects. To test the hypothesis that inhibition of IGF-IR indirectly and more selectively with PAPP-A inhibitors attenuates IGF-IR signaling in TED. Informed consent was obtained from TED patients undergoing surgery, and retro-orbital tissue collected for fibroblast isolation and culture. Surgeries were performed in Mayo Clinic operating suites. Cell culture was performed in a sterile tissue culture facility. Retro-orbital tissue was collected from 19 TED patients. Treatment of TED fibroblasts with pro-inflammatory cytokines. Flow separation of CD34- and CD34+ orbital fibroblasts, the latter representing infiltrating fibrocytes into the orbit in TED. PAPP-A expression and proteolytic activity, IGF-I stimulation of phosphatidylinositol 3 kinase/Akt pathway and inhibition by immuno-neutralizing antibodies against PAPP-A, CD34+ status and associated PAPP-A and IGF-IR expression. Pro-inflammatory cytokines markedly increased PAPP-A expression in TED fibroblasts. IGF-IR expression was not affected by cytokine treatment. Inhibition of PAPP-A's proteolytic activity suppressed IGF-IR activation in orbital fibroblasts from TED patients. TED fibroblasts that were CD34+ represented ∼80% of the cells in culture and accounted for ∼70% of PAPP-A and IGF-IR expressing cells. These results support a role for PAPP-A in TED pathogenesis and indicate the potential for novel therapeutic targeting of the IGF axis.

Outbreak of Septoria leaf spot caused by Sphaerulina musiva on Populus × euramericana in China.

In June 2023, severe leaf spots were noted in Populus × euramericana cv 'Nanlin95' plantations located in the Nanjing Baguazhou Wetland Park (32°09'16.97″N, 118°48'16.74″E) of Jiangsu Province and Populus × canadensis cv 'Sacrau 79' and Populus × canadensis cv 'Guariento' in the Liyuan Village in Nanyang City (32°53'43.70″N, 112°17'29.12″E) of Henan Province, respectively. The disease incidence in both locations could reach 97.9% (556 out of 568 trees) and 98.9% (2409 out of 2436 trees), respectively. The initial symptoms appear as numerous small and circular spots (1.59 to 3.18 mm in diameter) with gray or tan centers and dark-brown margins on the leaves. As the spots age, they sometimes enlarge, often coalesce, and may extend down the petioles. Diseased leaves and petioles were both surface sterilized with 75% ethanol for 30 seconds. With the aid of a hand lens, pycnidia (brown to black, spherical in profile, 90 to 250 µm diam) were easily picked out in the center of the spots and subsequently transferred into 1 mL sterilized water for preparing the spore suspension plated on KV8 medium amended with 100 mg/liter streptomycin sulfate and 50 mg/liter chloramphenicol. After 12 days of incubation, 86 single-spore isolates were obtained and identified as typical Septoria-like fungi according to morphological features, including slow-growing, gray or black colonies with pink mucilaginous matrix and hyaline, straight or curved conidia (size = 25 to 59 × 3.5 to 4 μm; septa = 1 to 6). Species identification was further validated by PCR amplification and sequencing of the internal transcribed spacer (ITS) region with ITS1/ITS4 primer pairs. Multiple sequence alignments with ClustalW revealed that the obtained ITS sequences of 86 isolates were 100% identical to each other. A BLAST search in GenBank indicated that the selfsame sequences of two representative isolates (isolate BGZ11 of Jiangsu Province, accession no. OR660379; isolate KZB22 of Henan Province, accession no. OR711499) shared 99.8% identity (494 of 495 bp) and 100% identity (504 of 504 bp) with related sequences of Sphaerulina musiva (Peck) Quaedvlieg, Verkley, and Crous (syn. = Septoria musiva Peck) in GenBank (MN275187; KF251619), respectively. Furthermore, we used a S. musiva-specific PCR assay (Abraham et al. 2018) on symptomatic leaf samples collected from the plantation. Each sample consisted of 20 cut-out leaf spots per leaf. Eight of the 10 samples were positive for S. musiva DNA. To confirm pathogenicity, six sterile tissue culture of poplar plants (Populus trichocarpa and Populus × euramericana cv 'Nanlin895') were respectively transplanted into pots and grown in a greenhouse for a week and for a month with an 18-h photoperiod augmented with sodium lamps and a 20°C (day)/16°C (night) temperature regime. Inoculations were conducted by spraying the plants with conidia suspension (106 conidia/mL) (LeBoldus et al. 2010). Control plants were sprayed with distilled water. Leaf spots were developed on the inoculated P. trichocarpa leaves at one week and P. × euramericana cv 'Nanlin895' leaves at 10 days after inoculation while no symptoms were observed on the control plants. The fungus S. musiva was successfully reisolated from all symptomatic leaves fulfilling Koch's postulates. Sphaerulina musiva only causes an endemic leaf spot disease on its natural North American host Populus. deltoides (Feau et al. 2010; Ostry 1987). However, on susceptible Populus species (e.g., P. balsamifera, P. trichocarpa, P. maximowiczii) and hybrids, S. musiva causes not only leaf spots but also severely damaging stem and branch cankers (Jeger et al. 2018; LeBoldus et al. 2009; Sondreli et al. 2020). To our knowledge, this is the first report of S. musiva causing leaf spots on poplar in China. Large-scale timber imports (e.g., cut branches, isolated bark, wood with and without bark) potentially lead to anthropogenic-facilitated transport of this pathogen. This outbreak of Septoria leaf spot underscores the potential threat of this pathogen to P. × euramericana in China, where it is widely planted as a keystone forestry species.

Granulomatous panniculitis in the setting of histoplasmosis in a patient with dermatomyositis on immunosuppressants

This report describes a case of panniculitis in the setting of infection with histoplasma capsulatum in a patient with active dermatomyositis on immunosuppressive medications. The appearance of panniculitis on the patient’s lower extremity was interpreted as possible necrotizing cellulitis in the setting of the patient’s immunosuppressive therapy or worsening of her dermatomyositis rash. The patient was placed on multiple trials of empiric antibiotic therapy for over a month with no improvement of her plaques. The patient did not exhibit the typical clinical manifestations of histoplasmosis nevertheless, through further workup, including, histological studies and sterile tissue culture, the patient was ultimately found to be infected with H. capsulatum and initiated on antifungal therapy. The case emphasizes the importance of placing other uncommon infectious etiologies like histoplasmosis on the differential in myositis patients on immunosuppression therapy with atypical presentations. Timely consideration of such possibilities ensures that appropriate therapies are initiated without delay.

Transcriptome Analysis of the Salt-Treated Actinidia deliciosa (A. Chev.) C. F. Liang and A. R. Ferguson Plantlets

The area of saline land in the world is quite large, and there is broad room for its development and usage. 'Xuxiang' is an Actinidia deliciosa variety that is tolerant to salt and can be planted in an area of light-saline land, and has good comprehensive characteristics and high economic value. However, the molecular mechanism of salt tolerance is unknown at present. To understand the molecular mechanism of salt tolerance, the leaves of A. deliciosa 'Xuxiang' were used as explants to establish a sterile tissue culture system, and plantlets were obtained using this system. One percent concentration (w/v) of sodium chloride (NaCl) was employed to treat the young plantlets cultured in Murashige and Skoog (MS) medium, then RNA-seq was used for transcriptome analysis. The results showed that the genes related to salt stress in the phenylpropanoid biosynthesis pathway and the anabolism of trehalose and maltose pathways were up-regulated; however, those genes in the plant hormone signal transduction and metabolic pathways of starch, sucrose, glucose, and fructose were down-regulated after salt treatment. The expression levels of ten genes that were up-regulated and down-regulated in these pathways were confirmed by real-time quantitative polymerase chain reaction (RT-qPCR) analysis. The salt tolerance of A. deliciosa might be related to the expression level changes in the genes in the pathways of plant hormone signal transduction, phenylpropanoid biosynthesis, and starch, sucrose, glucose, and fructose metabolism. The increased expression levels of the genes encoding alpha-trehalose-phosphate synthase, trehalose-phosphatase, alpha-amylase, beta-amylase, feruloyl-CoA 6-hydroxylase, ferulate 5-hydroxylase, and coniferyl-alcohol glucosyl transferase might be vital to the salt stress response of the young A. deliciosa plants.

In vitro anti-oxidant and anti-adhesin assays of n-hexane and methanolic extracts of Caryota no seeds

Abstract To determine the anti-oxidant and anti-adhesin activities of the n-hexane and methanolic extracts of Caryota no (CN) seeds. For anti-adhesin properties, the wells of a sterile flat-bottomed tissue culture plate were filled with 200µL of each extract in several concentrations, incubated and washed twice. 200µL of bacterial suspension was added, incubated and washed thrice with PBS. Adherent bacteria were fixed with methanol, plates emptied, left to dry and stained with crystal violet. Excess stain was rinsed under running water, air dried, bound dye resolubilized with glacial acetic acid, and the absorbance measured using a microplate reader to calculate the percentage inhibition. For antioxidant properties, three different concentrations of the DPPH solution were prepared. 2 mg of each extract was weighed out and dissolved in 10 mL of their corresponding solvent. 1 mL of DPPH solution was added to 2 ml solution of the extracts and ascorbic acid. The reaction mixture was vortexed and left in the dark for 30 minutes then absorbance was measured against blank solution to calculate the percentage scavenging radical inhibition. Statistical difference was presumed at P < 0.05. The extracts showed significant (P < 0.05) adhesin inhibition activities, n-hexane (64%) and methanolic (46%) at 200µg/mL concentrations. The fractions of the extracts also showed various remarkable results. The anti-adhesin activity increased with increasing extract concentration in both extracts. Antioxidant assays revealed that at 200 mg/mL, both extracts were significantly (P < 0.05) lower than the control. Both the n-hexane and methanolic extracts showed remarkable adhesin inhibition activities at concentration of 200 µg/mL and antioxidant effects, with the methanolic extract demonstrating comparable levels as the standard, ascorbic acid. This implies that both extracts may serve as veritable natural sources of anti-adhesins, antioxidants and thus adjunct anti-infective agents or immune enhancers. Keywords DPPH, Caryota no, Adhesin, Reactive oxygen species, anti-oxidant

First Report of Red-Fleshed Apple Anthracnose Caused by Colletotrichum siamense.

The red-fleshed apple (Malus niedzwetzkyana) produces a colored fruit and rich anthocyanins and it has become popular among consumers in Shandong (Yang et al 2020). In recent years, anthracnose diseases have been reported in red-fleshed apple orchards and nurseries in Shandong province, China. The incidence of anthracnose in the red-fleshed apple plantings ranges from 50-90%. Initially, anthracnose lesions on fruit begin as sub-circular shaped, sunken, pale brown. Over time black lesions enlarged and coalesced into large necrotic areas. The sunken centers of mature lesion became filled with slimy pink sporulation. In September 2015, fifteen fruit with anthracnose symptoms and sporulation were collected, and 11 single-spore isolates were obtained. Three representative isolates (JNTW11, JNTW2, JNTW33) were used for morphological and molecular characterization. On PDA, the colonies were initially white and turned into pale brown in three days. Orange-brown pigmentation was produced near the center on the reverse. Aerial mycelium was cottony, dense, pale white to pale gray. Acervuli developed visible orange-pink conidial masses. Conidiophores were colorless, septate, not branched or branched at the base. Conidia were 1-celled, hyaline, subcylindrical, oblong, attenuated with blunt ends, and the average size was 16.7 ± 1.5 × 6.1 ± 0.9 μm (n = 50). Appressoria were brown, obovoid or irregular, 9.2 ± 1.6 × 8.0 ± 1.8 μm (n = 20). The morphological characters matched the descriptions of Colletotrichum gloeosporioides sensu lato (Cannon et al. 2008). Isolates JNTW11, JNTW2, and JNTW33 were subject to bioinformatic characterization by partial sequencing of 6 genetic loci including the ribosomal internal transcribed spacer (ITS), actin (ACT), beta-tub2 (TUB2), calmodulin (CAL), chitin synthase (CHS-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Weir et al, 2012). The ITS (MT577037, MT577040, MT577042), ACT (MT767712, MT767715, MT767717), TUB2 (MT767723, MT767726, MT767728), CAL (MT767689, MT767692, MT767694), CHS-1(MT767700, MT767703, MT767705), and GAPDH (MT767734, MT767737, MT767739) sequences were deposited in GenBank. The six sets of sequence data were concatenated "ITS-GAPDH-ACT-CHS-1-TUB2-CAL", and the aligned sequences (2,007 bp) had 99.0% similarity to ex-type C. siamense ICMP18578. In a maximum likelihood phylogenetic tree, the highest log likelihood was -9148.55, and the isolates tested were in the C. siamense cluster with 96 % bootstrap support. Thus, the isolates were identified as C. siamense on the basis of multilocus phylogenetic analyses and morphological characters. To complete Koch's postulates, several healthy red-fleshed apple fruit ('Jiuhong', 1 month prior to harvest) were inoculated using colonized and uncolonized hyphal plugs and a blank agar as a control. All inoculated fruit were placed in sterile tissue culture bottles containing 2 layers of wet paper towels at 28 °C under a 12 h light/dark cycle. All fruit developed anthracnose symptoms in 7 days while the controls did not develop any symptoms. The symptoms were similar to those collected from fruit in the field, and same fungus was re-isolated from the lesions. Presently it was known that C. acutatum, C. asianum, C. chrysophilum, C. cuscutae, C. fioriniae, C. fragariae, C. fructicola, C. gloeosporioides, C. godetiae, C. kahawae, C. karstii, C. limetticola, C. melonis, C. noveboracense, C. nymphaeae, C. paranaense, C. rhombiforme, C. salicis, and C. theobromicola could infect M. coronaria, M. domestica, M. prunifolia, M. pumila, and M. sylvestris worldwide. To our knowledge, this is the first report of C. siamense as a pathogen of M. niedzwetzkyana. This finding provides crucial information for the management of anthracnose disease in China.

Lost and Found: Retained Needle Causing Deep Femoral Artery Pseudo-aneurysm

A 39 year old female intravenous drug abuser was admitted with fever, severe groin pain, and a semi-flexed left hip. Four months previously, a needle broke while she was injecting cocaine into her groin. Computed tomography angiography demonstrated a large (8.2 × 7.6 cm) partially thrombosed pseudo-aneurysm of the left deep femoral artery (A, B) with a needle inside the aneurysm (arrows). During open surgery, the needle stuck in the thrombus was removed and deep femoral artery sutured from within the aneurysm sac. Despite the sterile tissue culture, vancomycin was administered for two weeks. The post-operative course was uneventful.Image 1

Methylobacterium, a major component of the culturable bacterial endophyte community of wild Brassica seed.

BackgroundPlants are commonly colonized by a wide diversity of microbial species and the relationships created can range from mutualistic through to parasitic. Microorganisms that typically form symptomless associations with internal plant tissues are termed endophytes. Endophytes associate with most plant species found in natural and managed ecosystems. They are extremely important plant partners that provide improved stress tolerance to the host compared with plants that lack this symbiosis. Plant domestication has reduced endophyte diversity and therefore the wild relatives of many crop species remain untapped reservoirs of beneficial microbes. Brassica species display immense diversity and consequently provide the greatest assortment of products used by humans from a single plant genus important for agriculture, horticulture, bioremediation, medicine, soil conditioners, composting crops, and in the production of edible and industrial oils. Many endophytes are horizontally transmitted, but some can colonize the plant’s reproductive tissues, and this gives these symbionts an efficient mechanism of propagation via plant seed (termed vertical transmission).MethodsThis study surveyed 83 wild and landrace Brassica accessions composed of 14 different species with a worldwide distribution for seed-originating bacterial endophytes. Seed was stringently disinfected, sown within sterile tissue culture pots within a sterile environment and incubated. After approximately 1-month, direct isolation techniques were used to recover bacterial endophytes from roots and shoots of symptomless plants. Bacteria were identified based on the PCR amplification of partial 16S rDNA gene sequences and annotated using the BLASTn program against the NCBI rRNA database. A diversity index was used as a quantitative measure to reflect how many different bacterial species there were in the seed-originating microbial community of the Brassica accessions sampled.ResultsBacterial endophytes were recovered from the majority of the Brassica accessions screened. 16S rDNA gene sequencing identified 19 different bacterial species belonging to three phyla, namely Actinobacteria, Firmicutes and Proteobacteria with the most frequently isolated species being Methylobacterium fujisawaense, Stenotrophomonas rhizophila and Pseudomonas lactis. Methylobacterium was the dominant genus composing 56% of the culturable isolated bacterial community and was common in 77% of accessions possessing culturable bacterial endophytes. Two selected isolates of Methylobacterium significantly promoted plant growth when inoculated into a cultivar of oilseed rape and inhibited the growth of the pathogen Leptosphaeria maculans in dual culture. This is the first report that investigates the seed-originating endophytic microorganisms of wild Brassica species and highlights the Brassica microbiome as a resource for plant growth promoting bacteria and biological control agents.

Selenate tolerance and selenium hyperaccumulation in the monocot giant reed (Arundo donax), a biomass crop plant with phytoremediation potential

The response of giant reed (Arundo donax L.) to selenium (Se), added as selenate, was studied. The development, stress response, uptake, translocation, and accumulation of Se were documented in three giant reed ecotypes STM (Hungary), BL (USA), and ESP (Spain), representing different climatic zones. Plantlets regenerated from sterile tissue cultures were grown under greenhouse conditions in sand supplemented with 0, 2.5, 5, and 10mgSekg-1 added as sodium selenate. Total Se content was measured in different plant parts using hydride generation atomic fluorescence spectroscopy. All plants developed normally in the 0-5.0mgSekg-1 concentration range regardless of ecotype, but no growth occurred at 10.0mgSekg-1. There were no signs of chlorosis or necrosis, and the photosynthetic machinery was not affected as evidenced by no marked differences in the structure of thylakoid membranes. There was no change in the maximum quantum yield of photosystem II (Fv/Fm ratio) in the three ecotypes under Se stress, except for a significant negative effect in the ESP ecotype in the 5.0mgSekg-1 treatment. Glutathione peroxidase (GPx) activity increased as the Se concentration increased in the growth medium. GPx activity was higher in the shoot system than the root system in all Se treatments. All ecotypes showed great capacity of take up, translocate and accumulate selenium in their stem and leaf. Relative Se accumulation is best described as leaf ˃˃ stem ˃ root. The ESP ecotype accumulated 1783μgg-1 in leaf, followed by BL with 1769μgg-1, and STM with 1606μgg-1 in the 5.0mgSekg-1 treatment. All ecotypes showed high values of translocation and bioaccumulation factors, particularly the ESP ecotype (10.1 and 689, respectively, at the highest tolerated Se supplementation level). Based on these findings, Arundo donax has been identified as the first monocot hyperaccumulator of selenium, because Se concentration in the leaves of all three ecotypes, and also in the stem of the ESP ecotype, is higher than 0.1% (dry weight basis) under the conditions tested. Tolerance up to 5.0mgSekg-1 and the Se hyperaccumulation capacity make giant reed a promising tool for Se phytoremediation.

Endophytic Pseudomonas induces metabolic flux changes that enhance medicinal sesquiterpenoid accumulation in Atractylodes lancea

The bacterial endophyte Pseudomonas fluorescens ALEB7B significantly enhances photosynthate accumulations in Atractylodes lancea. These carbohydrates are preferentially used by the host plant to synthesize secondary metabolites, rather than to increase plant biomass accumulation. Mechanisms underlying the allocation of endophyte–increased carbohydrate in different plant metabolic processes are largely unknown. We have studied how P. fluorescens ALEB7B enhances photosynthate accumulation and how bacterial elicitors regulate metabolic flux and increase medicinal sesquiterpenoid formation in A. lancea using the sterile tissue culture plantlets. P. fluorescens ALEB7B enhances plant photosynthate accumulation by synthesizing and secreting indole–3–acetic acid, which has been demonstrated using high–performance liquid chromatography analysis. The increased endogenous indole–3–acetic acid promotes plant root development and then assimilation. Increased carbohydrates provide the material basis for the formations of terpenoid hydrocarbon scaffolds, which has been proved using gas chromatography analysis. Further, protein and polysaccharide elicitors secreted by P. fluorescens ALEB7B have been separated and purified from the bacterial fermentation broth, which have been applied to A. lancea plantlets. Both elicitors can stimulate the conversions of terpenoid hydrocarbon scaffolds to oxygenous sesquiterpenoids, the active medicinal ingredients in A. lancea, by triggering the oxidative burst in planta. Moreover, this study separates an ABC transporter substrate–binding protein from protein elicitors secreted by P. fluorescens ALEB7B with an ÄKTA Prime Plus Purifier System and firstly shows that this protein is essential to induce oxygenous sesquiterpenoid accumulation in A. lancea. This study provides new perspectives for mechanisms of medicinal oxygenous terpenoid synthesis, which has important reference values to the cultivation of medicinal plants that have terpenoids as their active ingredients, such as Artemisia annua and Taxus chinensis.

Using a liver cell culture from Epinephelus coioides as a model to evaluate the nonylphenol-induced oxidative stress

The present study aimed to use primary liver cell culture derived from the orange-spotted grouper, Epinephelus coioides, to assess the toxic effects of nonylphenol (NP) on the hepatocyte viability and the liver antioxidant system. E. coioides was selected due to its commercial importance. NP was used in this study because of its high potential of producing oxidative stress due to increased reactive oxygen species (ROS). A liver of E. coioides was digested with PBS containing 0.1% collagenase IV. The digested cells were moved to Leibovitz L-15 culture medium with 20% fetal bovine serum (FBS), 100IUmL−1 penicillin, 100μgmL−1 streptomycin. Aliquots of cell suspension were seeded as a monolayer into sterile 25cm2 tissue culture flasks and incubated at 30°C for 14days. The medium, containing non-attached cells, was removed after 24 to 48h and a new medium was added. The IC50 of 10−4molL−1 was determined for nonylphenol using MTT assay. Cells were then incubated with L-15 medium containing 10−5, 2×10−5, 3×10−5molL−1 of NP and samples were taken after 6, 12 and 24h of incubation for analysis of LPO, SOD, CAT, GPx, LDH, AST, ALT, and ALP. Based on the results, the lowest concentration of NP was not markedly cytotoxic to primary hepatocytes and the cell sensitivity to NP increased dose-dependently. The activities of SOD, CAT and GPx decreased significantly, while activities of LPO, LDH, AST, ALT and ALP, increased significantly in a dose-related pattern in NP-treated cells. In conclusion, this study revealed that NP could induce the oxidative stress in cultivated hepatocytes of E. coioides during a short-term exposure. NP toxicity is mainly due to the induction of the reactive oxygen species (ROS), which lead to cell membrane disruption, damage of cellular metabolism, and interference with cellular macromolecules.

Transcriptomics analysis investigates sesquiterpenoids accumulation pattern in different tissues of Atractylodes lancea (Thunb.) DC. plantlet

Atractylodes lancea: (Thunb.) DC. is an endangered traditional Chinese medicinal herb with broad pharmacological activity, reflected by an abundance of sesquiterpenoids. However, the biosynthesis of these sesquiterpenoids is largely unknown due to a lack of genetic information. To understand sesquiterpenoid distribution in A. lancea, tissue-specific sesquiterpenoids accumulation patterns during different growth periods of plantlets were analyzed via gas chromatography (n = 3). Transcriptomic analyses were furthermore performed via RNA-Seq in an attempt to reveal the molecular mechanism regulating sesquiterpenoids formation in leaves, stems and roots. 85,170 unigenes with an average sequence length of 768 bp were acquired. Via analysis of differentially expressed genes, 857 unigenes were identified to be involved in sesquiterpenoids formation. Consequently, a putative sesquiterpenoid biosynthetic pathway was proposed, including mevalonate, 2-C-methyl-d-erythritol-4-phosphate, sesquiterpene synthase, cytochrome P450, and the peroxisome pathway. Moreover, the relationship between gene expression and sesquiterpenoid production in different growth periods of leaves, stems, and roots was analyzed based on Quantitative Real-Time PCR (n = 3) and gas chromatography data. In this study, the sesquiterpenoid distribution in different tissues of sterile tissue culture plantlets of A. lancea was systematically investigated via metabolite quantitative and transcriptomics analysis. This study provides a central theoretical basis for the manipulation of sesquiterpenoid biosynthesis in A. lancea.

Comparative efficacy of telavancin and daptomycin in experimental endocarditis due to multi-clonotype MRSA strains

MRSA strains of clonal complexes (CCs) 5, 8, 30 and 45 are leading causes of complicated endovascular infections associated with suboptimal clinical outcomes. Telavancin is a novel anti-MRSA agent that both inhibits bacterial cell wall synthesis and disrupts membranes by depolarization. In this study, we compared the in vitro susceptibility and in vivo efficacy of telavancin versus daptomycin in an experimental rabbit infective endocarditis (IE) model caused by four MRSA strains representing each of the above CC types. All study strains were susceptible to telavancin (MICs of ≤0.12 mg/L) and daptomycin (MICs of ≤0.5 mg/L). In vitro time-kill analyses revealed that supra-MIC levels of telavancin were effective at preventing regrowth at 24 h of incubation. In the IE animal model for all CC types, treatment with telavancin produced significantly greater reductions in MRSA counts as compared with daptomycin-treated animals in all target tissues. Moreover, telavancin-treated animals had a significantly higher percentage of sterile tissue cultures versus daptomycin-treated animals (e.g. 78%-100% versus 0% sterile vegetations and 100% versus 0%-11% sterile kidneys and spleen, in the telavancin- and daptomycin-treated animals, respectively). These results suggest that telavancin exhibits significantly greater efficacies versus daptomycin in treating experimental IE caused by MRSA clinical isolates across four common CC types.

Beneficial effects of Glycyrrhizae radix extract in preventing oxidative damage and extending the lifespan of Caenorhabditis elegans

Ethnopharmacological relevanceGlycyrrhizae radix (GR) is a medicinal herb extensively used in traditional Chinese medicine. This study aimed to evaluate the pharmacological effect of GR and the possible mechanisms of GR, to provide a pharmacological basis in traditional medicine. Materials and methodsIn the present study, C. elegans (L1-larvae to young adults) was exposed to 0.12–0.24g/mL of GR in 12-well sterile tissue culture plates at 20°C in the presence of food. Lethality, growth, lifespan, reproduction, locomotion, metabolism, intestinal autofluorescence, and reactive oxygen species (ROS) production assays were performed to investigate the possible safety profile and beneficial effects of GR in these nematodes. We found that the lifespan of nematodes exposed to 0.18–0.24g/mL of GR was extended. We then determined the mechanism of the longevity effect of GR using quantitative reverse transcription PCR and oxidative stress resistance assays induced by heat and paraquat. ResultsProlonged exposure to 0.12–0.24g/mL of GR did not induce lethality, alter body length, morphology or metabolism, affect brood size, locomotion, the development of D-type GABAergic motor neurons, or induce significant induction of intestinal autofluorescence and intestinal ROS production. In C. elegans, pretreatment with GR suppressed the damage due to heat-stress or oxidative stress induced by paraquat, a ROS generator, on lifespan, and inhibited the induction of intestinal ROS production induced by paraquat. Moreover, prolonged exposure to GR extended lifespan, increased locomotion and decreased intestinal ROS production in adult day-12 nematodes. Furthermore, prolonged exposure to GR significantly altered the expression patterns of genes encoding the insulin-like signaling pathway which had a key role in longevity control. Mutation of daf-16 gene encoding the FOXO transcription factor significantly decreased lifespan, suppressed locomotion, and increased intestinal ROS production in GR exposed adult nematodes. ConclusionsGR is relatively safe and has protective effects against the damage caused by both heat-stress and oxidative stress at the examined concentrations. Furthermore, GR is capable of extending the lifespan of nematodes, and the insulin-like signaling pathway may play a crucial role in regulating the lifespan-extending effects of GR.

Factors influencing mortality in patients undergoing surgery for acute pancreatitis

The aims of present study were to analyze the mortality risk factors in patients who had surgery for acute pancreatitis and to assess the importance of culturing peripancreatic tissue or fluid infection to ascertain the infection status. Surgery was indicated both in patients with infected severe acute pancreatitis and in those with sterile pancreatitis with an unfavorable course. During surgery, cultures were taken of tissues (pancreatic necrosis and peripancreatic fat), intra-abdominal fluid, and bile. Of 107 patients operated on, fluid culture was analyzed in 94 patients, pancreatic necrosis in 61 patients, peripancreatic fat in 39 patients, and bile in 38 patients. Sterile pancreatitis with sterile ascites was found in 17 patients, sterile pancreatitis with infected ascites in 22, and pancreatic tissue infection in 60. Multivariate analysis demonstrated that sterile tissue cultures, age over 65 years, and fewer than 12 days between the beginning of pain and surgery were risk factors for mortality. Sterile pancreatitis with sterile ascites and sterile pancreatitis with infected ascites had similar postoperative mortality (41% and 50%, respectively); the group with pancreatic tissue infection had a lower mortality (20%). Early surgery, advanced age, and sterility of tissue cultures have been demonstrated as mortality factors for acute pancreatitis. Intra-abdominal fluid may be infected in the presence of sterile necrosis.

Semi-sterilized Tissue Culture for Rapid Propagation of Grapevines (Vitis vinifera L.) Using Immature Cuttings

Rapid expansion of grapevine plantings in many parts of the world has led to increased demand for desirable planting stocks. In countries that rely on importing new varieties and have strict quarantine rules, such as Australia, vines need to stay under quarantine for ≈2 years before they are released, at which time there is very limited wood available. Hence, rapid expansion of propagating stock after release is the key to multiplying up new varieties. A novel method, referred to as Semi-sterilized Tissue Culture (SSTC) using immature single-node cuttings, was established and evaluated as a way of rapid expansion of grapevine (Vitis vinifera L.) planting stock. In the SSTC method, immature single-node cuttings were surface-sterilized using methylated spirits and then cultured in the root pulsing medium [1/2 Murashige and Skoog (MS) medium supplemented with 40 μM indole-3-butyric acid (IBA)] for 24 hours. They were then planted in sterilized aerobic rooting medium (sphagnum peat:coarse river sand:perlite = 0.5:1:2) and cultured in a tissue culture room for ≈4 weeks for root initiation and development. The rooted immature single-node cuttings were then transferred to normal propagation beds in a greenhouse and potted on for acclimatization. Tube stock generated by SSTC easily acclimatized with a 15 times higher root strike rate than cutting propagation. It also took at least 50% less time than fully sterilized micropropagation methods to produce planting stocks. The advantages of the SSTC method are that it can be conducted under semisterilized conditions, avoiding degeneration and bacterial contamination problems encountered in micropropagation methods. By removing the time-consuming steps of the explant establishment, proliferation, and maintenance in vitro, the propagation process was simplified compared with conventional sterile tissue culture procedures. The SSTC procedure removed the need for high operator skill levels, reducing expense and allowing easier commercial adoption.

A MicroRNA Processing Defect in Smokers' Macrophages Is Linked to SUMOylation of the Endonuclease DICER

Despite the fact that alveolar macrophages play an important role in smoking-related disease, little is known about what regulates their pathophysiologic phenotype. Evaluating smoker macrophages, we found significant down-regulation of multiple microRNAs (miRNAs). This work investigates the hypothesis that cigarette smoke alters mature miRNA expression in lung macrophages by inhibiting processing of primary miRNA transcripts. Studies on smoker alveolar macrophages showed a defect in miRNA maturation. Studies on the miRNA biogenesis machinery led us to focus on the cytosolic RNA endonuclease, DICER. DICER cleaves the stem-loop structure from pre-miRNAs, allowing them to dissociate into their mature 20-22-nucleotide single-stranded form. DICER activity assays confirmed impaired DICER activity following cigarette smoke exposure. Further protein studies demonstrated a decreased expression of the native 217-kDa form of DICER and an accumulation of high molecular weight forms with cigarette smoke exposure. This molecular mass shift was shown to contain SUMO moieties and could be blocked by silencing RNA directed at the primary SUMOylating ligase, Ubc9. In determining the cigarette smoke components responsible for changes in DICER, we found that N-acetylcysteine, an antioxidant and anti-aldehyde, protected DICER protein and activity from cigarette smoke extract. This massive down-regulation of miRNAs (driven in part by alterations in DICER) may be an important regulator of the disease-promoting macrophage phenotype found in the lungs of smokers.

Telavancin in Therapy of Experimental Aortic Valve Endocarditis in Rabbits Due to Daptomycin-Nonsusceptible Methicillin-Resistant Staphylococcus aureus

A number of cases of both methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) strains that have developed daptomycin resistance (DAP-R) have been reported. Telavancin (TLV) is a lipoglycopeptide agent with a dual mechanism of activity (cell wall synthesis inhibition plus depolarization of the bacterial cell membrane). Five recent daptomycin-susceptible (DAP-S)/DAP-R MRSA isogenic strain pairs were evaluated for in vitro TLV susceptibility. All five DAP-R strains (DAP MICs ranging from 2 to 4 μg/ml) were susceptible to TLV (MICs of ≤0.38 μg/ml). In vitro time-kill analyses also revealed that several TLV concentrations (1-, 2-, and 4-fold MICs) caused rapid killing against the DAP-R strains. Moreover, for 3 of 5 DAP-R strains (REF2145, A215, and B(2.0)), supra-MICs of TLV were effective at preventing regrowth at 24 h of incubation. Further, the combination of TLV plus oxacillin (at 0.25× or 0.50× MIC for each agent) increased killing of DAP-R MRSA strains REF2145 and A215 at 24 h (∼2-log and 5-log reductions versus TLV and oxacillin alone, respectively). Finally, using a rabbit model of aortic valve endocarditis caused by DAP-R strain REF2145, TLV therapy produced a mean reduction of >4.5 log(10) CFU/g in vegetations, kidneys, and spleen compared to untreated or DAP-treated rabbits. Moreover, TLV-treated rabbits had a significantly higher percentage of sterile tissue cultures (87% in vegetations and 100% in kidney and spleen) than all other treatment groups (P < 0.0001). Together, these results demonstrate that TLV has potent bactericidal activity in vitro and in vivo against DAP-R MRSA isolates.

Cytotoxic Effects of Glass Ionomer Cements on Human Dental Pulp Stem Cells Correlate with Fluoride Release

Glass ionomer cements (GICs) are commonly used as restorative materials. Responses to GICs differ among cell types and it is therefore of importance to thoroughly investigate the influence of these restorative materials on pulp stem cells that are potential source for dental tissue regeneration. Eight biomaterials were tested: Fuji I, Fuji II, Fuji VIII, Fuji IX, Fuji Plus, Fuji Triage, Vitrebond and Composit. We compared their cytotoxic activity on human dental pulp stem cells (DPSC) and correlated this activity with the content of Fluoride, Aluminium and Strontium ions in their eluates. Elution samples of biomaterials were prepared in sterile tissue culture medium and the medium was tested for toxicity by an assay of cell survival/proliferation (MTT test) and apoptosis (Annexin V FITC Detection Kit). Concentrations of Fluoride, Aluminium and Strontium ions were tested by appropriate methods in the same eluates. Cell survival ranged between 79.62% (Fuji Triage) to 1.5% (Fuji Plus) and most dead DPSCs were in the stage of late apoptosis. Fluoride release correlated with cytotoxicity of GICs, while Aluminium and Strontium ions, present in significant amount in eluates of tested GICs did not. Fuji Plus, Vitrebond and Fuji VIII, which released fluoride in higher quantities than other GICs, were highly toxic to human DPSCs. Opposite, low levels of released fluoride correlated to low cytotoxic effect of Composit, Fuji I and Fuji Triage.